Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Inhibition of STAT3 Signaling Blocks the Anti-apoptotic Activity of IL-6 in Human Liver Cancer Cells *

Inhibition of STAT3 Signaling Blocks the Anti-apoptotic Activity of IL-6 in Human Liver Cancer... THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 35, pp. 27429 –27439, August 27, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Inhibition of STAT3 Signaling Blocks the Anti-apoptotic Activity of IL-6 in Human Liver Cancer Cells Received for publication, May 9, 2010, and in revised form, June 8, 2010 Published, JBC Papers in Press, June 18, 2010, DOI 10.1074/jbc.M110.142752 ‡ § § ‡¶1 Yan Liu , Pui-Kai Li , Chenglong Li , and Jiayuh Lin From the Department of Pediatrics, Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, the § ¶ Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and the Experimental Therapeutics Program, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43205 Interleukin-6 (IL-6) is a multifunctional cytokine, which may in promoting colitis-associated cancer (CAC) tumorigenesis, block apoptosis during inflammation to protect cells under very enhancing tumor initiation cell proliferation, and preventing toxic conditions. However, IL-6 also activates STAT3 in many apoptosis of premalignant intestinal epithelial cells. High levels types of human cancer. Recent studies demonstrate that high of IL-6 have been detected in many types of human epithelial levels of IL-6 are associated with hepatocellular carcinoma, the cancers (3), and correlate with proliferation or survival of mul- most common type of liver cancer. Here we reported that IL-6 tiple myeloma, breast cancer stem cells, lung adenocarcinomas, promoted survival of human liver cancer cells through activat- prostate cancer, cervical cancer, gastric cancer, and esophageal ing STAT3 in response to doxorubicin treatment. Endogenous carcinoma (4–14). IL-6 antibody alone can induce PC-3 IL-6 levels in SNU-449 cells were higher than in Hep3B cells. xenograft regression (15). Thus, blocking IL-6-mediated signal- Meanwhile, SNU-449 cells were more resistant to doxorubicin ing cascades has a potential for treatment of these human can- than Hep3B cells. Addition of IL-6 induced STAT3 activation in cers (4, 16, 17). Hep3B cells and led to protection against doxorubicin. In con- IL-6 signals through IL-6 receptor (IL-6-R), GP130, and trast, neutralizing IL-6 with anti-IL-6 antibody decreased sur- Janus kinases (JAKs). IL-6-induced JAK family members acti- vival of SNU-449 cells in response to doxorubicin. To elucidate vate three major pathways, STAT3, MAPK, and PI3K (1, 18). the mechanism of the anti-apoptotic function of IL-6, we inves- The signal transducer and activator of transcription 3 (STAT3) tigated if STAT3 mediated this drug resistance. Targeting is considered as an oncogene and found constitutively activated STAT3 with STAT3 siRNA reduced the protection of IL-6 in many types of human malignancies (19–22). STAT3 signal- against doxorubicin-induced apoptosis, indicating that STAT3 ing is a major pathway for cancer inflammation. It induces a lot signaling contributed to the anti-apoptotic effect of IL-6. More- of genes crucial for inflammation. Environmental factors, over, we further explored if a STAT3 small molecule inhibitor including UV radiation, chemical carcinogen, infection, stress, could abolish this anti-apoptotic effect. LLL12, a STAT3 small and smoke, can activate STAT3 via cytokine receptor, toll-like molecule inhibitor, blocked IL-6-induced STAT3 phosphoryla- receptor, adrenergic receptor, or nicotinic receptor (22, 23). tion, resulting in attenuation of the anti-apoptotic activity of STAT3 is activated when tyrosine 705 (Tyr-705) is phosphory- IL-6. Finally, neutralization of endogenous IL-6 with anti-IL-6 lated. Phosphorylated STAT3 molecules dimerize and translo- antibody or blockade of STAT3 with LLL12 lowered the recov- cate into the nucleus, where they bind to specific DNA response ery in SNU-449 cells after doxorubicin treatment. Therefore, elements and induce the transcription of proliferation and anti- our results demonstrated that targeting STAT3 signaling could apoptosis associated genes (19, 24), such as BCL-2, BCL-X , interrupt the anti-apoptotic function of IL-6 in human liver can- IL-17, IL-23, MCL1, and Survivin (20, 21, 25–28). The activa- cer cells. tion of STAT3 also promotes tumor angiogenesis via HIF-1 and VEGF (11, 29–31). Therefore, STAT3 is a potential target for cancer therapy. Evidence shows that inhibiting STAT3 using Interleukin-6 is a multifunctional cytokine that was origi- dominant-negative STAT3, antisense oligonucleotides and nally characterized acting in immune and inflammatory RNA interference induces tumor cell death (22, 32). responses, and inhibits apoptosis in toxic environments during A growing numbers of evidence demonstrate that tumori- inflammation (1). Growing evidence has indicated that cancers genesis caused by STAT3 is mediated by IL-6 signaling (1, 33). are associated with chronic inflammation. Although serum Therefore, targeting IL-6/STAT3 signaling pathway should be IL-6 levels were known elevated in colon cancer patients, only considered for the treatment of patients with elevated levels of recent studies by the Karin group (2) document the role of IL-6 IL-6/STAT3 signaling. IL-6 levels in liver cancer patients are 25-fold higher than healthy adults (34). The high levels of IL-6 are associated with hepatocellular carcinoma (HCC), the most * This work was supported, in whole or in part, by National Institutes of Health common liver cancer (35). R21 Grant R21CA133652-01 (to J. L.). This work was also supported by a In this study, we explored the biological role of IL-6 in human Pancreatic Cancer Action Network-AACR, National Foundation for Cancer Research grant. liver cancer cells and found that IL-6 stimulated STAT3 phos- To whom correspondence should be addressed: Center for Childhood Can- phorylation and promoted cell survival upon doxorubicin treat- cer, Dept. of Pediatrics, College of Medicine, The Ohio State University, 700 ment. Interestingly, targeting STAT3 by STAT3 siRNA, anti- Children’s Drive, Columbus, OH 43205. Tel.: 614-722-5086; Fax: 614-722- 5895; E-mail: [email protected]. IL-6 antibody or a small molecule STAT3 inhibitor, attenuated AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27429 This is an Open Access article under the CC BY license. IL-6/STAT3 Promotes Cell Survival FIGURE 1. IL-6 induces STAT3 phosphorylation. A, human hepatocyte, Hep3B, SNU-387, SNU-398, and SNU-449 cells were cultured in Hepatocyte Medium for 24 h. Endogenous IL-6 was accessed by IL-6 ELISA assay. B, Hep3B and SNU-398 cells were cultured in serum-free medium. After 24 h of serum deprivation, cells were treated with various concentrations of IL-6 for 30 min. STAT3 phosphorylation and total STAT3 were detected by Western blot. C, Hep3B and SNU-398 cells were cultured under the same conditions and then were treated with 50 ng/ml of IL-6 for 30 min. The distribution of phosphorylated STAT3 was analyzed by immunofluorescence. IL-6-induced drug resistance, suggesting that activated STAT3 turer’s protocol. STAT3 small interfering RNA (siRNA) kit and contributed to the IL-6-induced cell survival. scrambled control siRNA were purchased from Cell Signaling Technology (Beverly, MA). EXPERIMENTAL PROCEDURES Western Blot—Whole cell extracts were prepared by lysing Cell Culture, Transfection, and RNA Interference—Human the cells in RIPA buffer containing mixture proteasome inhib- hepatoma cell lines, Hep3B, SNU-387, SNU-398, SNU-449 itor. Lysates were then centrifuged at 13,300 rpm for 10 min at 4 °C, and the supernatant was collected. Protein samples were were obtained from American Type Culture Collection (ATCC, separated by SDS-PAGE, transferred onto PVDF membrane, Manassas, VA). Hep3B cells were cultured in Minimum Essen- tial Medium, Eagle (MEM) (ATCC) supplemented with 10% and immunoblotted with the appropriate antibody. Antibodies fetal bovine serum (FBS) and 1% penicillin/streptomycin. SNU- to p-STAT3 (Y705), STAT3, p-STAT1 (Y701), STAT1, p- 398 and SNU-449 cells were cultured in RPMI 1640 medium ERK1/2 (T202/Y204), p-JAK2, JAK2, p-JAK1, JAK1, GP130, GAPDH, and HRP-conjugated secondary antibodies were from (ATCC) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human hepatocytes were pur- Cell Signaling Technology. The target protein was examined by chased from ScienCell Research Laboratories (Carlsbad, CA). chemiluminescence (Cell Signaling Technology). Human hepatocytes were cultured in Hepatocyte Medium Immunofluorescence—Cells were seeded on a glass slide and fixed with cold methanol for 15 min. After a washing in (HM, ScienCell Research Laboratories) including 5% FBS, 1% hepatocyte growth supplement, and 1% penicillin/streptomy- phosphate-buffered saline (PBS), the slide was blocked with cin. Interleukin-6 (IL-6) and interferon- (IFN-) were pur- 5% normal goat serum and 0.3% Triton X-100 in PBS for at chased from Cell Sciences (Canton, MA). Antibody to human least 1 h. Then the slide was incubated with primary anti- body. After overnight incubation, the slide was washed with IL-6, which can neutralize IL-6 bioactivity, was from R &D Sys- tems (Minneapolis, MN). The human IL-6 ELISA kit was from PBST and then incubated with anti-rabbit FITC-conjugated Pepro Tech Inc (Rocky Hill, NJ). secondary antibody (Jackson ImmunoResearch Laboratories, siRNA was transfected into Hep3B cells using Lipofectamine West Grove, PA). The cells were mounted in Vectashield Hard- 2000 (Invitrogen, Carlsbad, CA) according to the manufac- Set mounting medium with DAPI (Vector Laboratories, Burl- 27430 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival FIGURE 2. SNU-449 cells are more doxorubicin-resistant than Hep3B cells. A, SNU-449 cells and Hep3B cells were treated with various concentrations of doxorubicin. After 24 h, morphological examination was performed. B, SNU-449 cells and Hep3B cells were treated under the same conditions. After 48 h, cell viability assay was performed. The data showed the percent decrease in cell viability compared with untreated cells and represented three independent results. C, endogenous phosphorylated STAT3 and total STAT3 in Hep3B and SNU-449 cells were analyzed by Western blot. ingame, CA). Pictures were captured by Leica Microsystems Statistical Analysis—The statistical significance was calcu- lated using Student’s t test. p values of 0.05 were considered (Bannockburn, IL). Apoptosis Assay—Apoptosis was measured with caspase3/7 significant. assay kit (Promega, Madison, WI) according to the manufac- turer’s protocol. Briefly, 20,000 cells were seeded into 96-well RESULTS plates. After the treatment, 100 l of Apo-One caspase3/7 rea- IL-6 Induces STAT3 Phosphorylation—To examine the en- gent was added to each well and was incubated at 37 °C for 30 dogenous IL-6 levels in different liver cancer cells, four human min. The fluorescence was measured at an excitation wave- liver cancer cell lines (Hep3B, SNU-387, SNU-398, and length range of 485 nm and an emission wavelength range of SNU449) and human hepatocytes were cultured in the same 530 nm. medium, hepatocyte medium, for 24 h. After the incubation, Viability Assay—Viability was performed with CyQUANT the IL-6 levels in the culture medium were evaluated using IL-6 NF Assay (Invitrogen) according to the manufacturer’s pro- ELISA assay. As illustrated in Fig. 1A, the IL-6 levels were tocol. Briefly, 2,000 cells were seeded into a 96-well plate. remarkably elevated in SNU-387 and SNU-449 cell lines com- After the 48-h treatment, medium was removed from the pared with human hepatocytes, Hep3B and SNU-398. To plate, and 50 l of dye binding solution were added to each explore if IL-6 would induce STAT3 phosphorylation in cells well. The plate was covered and incubated at 37 °C for 30 with lower endogenous IL-6, Hep3B, and SNU-398 cells were min. The fluorescence was measured at an excitation wave- cultured in serum-free medium for 24 h and then were treated length range of 485 nm and an emission wavelength range of with different concentrations of IL-6 (0–50 ng/ml) for 30 min. 530 nm. As illustrated in Fig. 1B, IL-6 induced STAT3 phosphorylation Clonogenic Assay—Cells were washed with PBS twice and in a dose-dependent manner, but did not alter the expression of fixed with cold methanol at 20 °C for 10 min. After 10 min, total STAT3. To confirm if IL-6-induced phosphorylated cells were stained with 1% crystal violet (25% methanol) at room STAT3 would translocate to the nucleus, the localization of temperature for 10 min. After the staining, the plates were phosphorylated STAT3 was determined by staining with anti- washed with distill water and dried. phosphorylated STAT3 primary antibody and FITC-conju- AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27431 IL-6/STAT3 Promotes Cell Survival FIGURE 3. IL-6 protects cells against doxorubicin-induced apoptosis. A, Hep3B cells were pretreated with 2 M doxorubicin for 2 h followed by various concentrations of IL-6 for 6 h. Morphological examination was performed after treatment. B, Hep3B cells were treated under the same conditions. After 24 h, caspase3/7 activity was analyzed. The data showed the percent decrease in csapase3/7 activity compared with control cells and represented three indepen- dent results. C, SNU-449 cells were treated with 500 ng/ml of anti-IL-6 antibody or control IgG for 24 h. After the incubation, whole cell lysates were extracted. Phosphorylated STAT3 and GAPDH were detected by Western blot. D, SNU-449 cells were pretreated with 500 ng/ml of anti-IL-6 antibody or control IgG for 24 h. After the pretreatment, cells were further treated with doxorubicin for 24 h. After the 24 h treatment, cell viability was measured. The data represented eight independent results. gated secondary antibody. The nucleus was stained with DAPI. ng/ml of IL-6 effectively displayed anti-apoptotic effect. To fur- Fig. 1C demonstrated that phosphorylated STAT3 translocated ther confirm our results, Hep3B cells were treated under the to the nucleus following IL-6 treatment. similar conditions and after 24 h, apoptotic levels were mea- SNU-449 Cells Are More Doxorubicin-resistant than Hep3B sured using caspase3/7 assay. Fig. 3B showed that 10 ng/ml of Cells—To determine if liver cancer cells with higher endoge- IL-6 treatment exhibited 70% decrease in doxorubicin-caused nous IL-6 would be more resistant to drug treatment than those apoptosis. Similar results were observed in cells treated with 25 with lower endogenous IL-6, we treated SNU-449 and Hep3B and 50 ng/ml of IL-6. cells with different concentrations of doxorubicin. After 24 h of To investigate if disruption of IL-6 autocrine would down- treatment, the characteristic morphology of cell death was regulate STAT3 phosphorylation and would interrupt the anti- observed in Hep3B cells but not in SNU-449 cells (Fig. 2A). apoptotic effect of IL-6, SNU-449 cells were treated with anti- After another 24 h, cell viability assay was performed. Cell via- IL-6 antibody and phosphorylated STAT3 was analyzed by bility did not show significant change in SNU-449 treated by 2.5 Western blot. Our results demonstrated that the treatment or 5 M doxorubicin whereas 40% decrease was observed when with anti-IL-6 antibody reduced phosphorylated STAT3 levels treated by 10 M doxorubicin (Fig. 2B). In contrast, cell viability (Fig. 3C). Then we pretreated SNU-449 cells with anti-IL-6 decreased around 60, 80, and 80% when Hep3B cells were antibody overnight to neutralize the endogenous IL-6. After the treated by 2.5, 5, and 10 M doxorubicin, respectively (Fig. 2B). pretreatment, cells were treated with doxorubicin for 24 h fol- These results demonstrated that SNU-449 cells were more lowed by cell viability assay. Fig. 3D showed that cells treated resistant to doxorubicin than Hep3B cells. To examine if liver with doxorubicin and anti-IL-6 antibody exhibited 70% cancer cells with higher IL-6 would also show enhanced endog- decrease in viability compared with cells treated with doxoru- enous STAT3, we analyzed phosphorylated STAT3 in SNU- bicin alone. The data in Fig. 3 suggested that IL-6 promoted cell 449 and Hep3B cells. The Western blot results showed that survival in response to drug treatment. phosphorylated STAT3 was elevated in SNU-449 cells com- IL-6 Causes Anti-apoptosis through Activating STAT3—To pared with Hep3B cells, whereas total STAT3 level did not explore if STAT3 signaling would contribute to the anti-apo- change (Fig. 2C). ptotic activity of IL-6, we targeted STAT3 by STAT3 siRNA. IL-6 Confers Resistance to Apoptosis Induced by Doxorubicin— siRNA was transfected into Hep3B cells with less than 50% To explore if IL-6 would protect cells upon drug treatment, transfection efficiency (data not shown). The transfected cells Hep3B cells were pretreated with 2 M doxorubicin for 2 h were cultured in serum-free medium overnight and then were followed by different doses of IL-6 (0–50 ng/ml). After6hof treated with 50 ng/ml of IL-6 for 30 min. Western blot results incubation, the characteristic morphology of cell death was demonstrated a decrease in phosphorylated STAT3 compared observed in cells treated with doxorubicin whereas the addition of IL-6 rescued doxorubicin-caused cell death (Fig. 3A). 10 with control scrambled siRNA-transfected cells (Fig. 4A). Phos- 27432 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival were cultured in serum-free me- dium for 24 h and then were pre- treated with 5 or 10 M LLL12 for 2 h followed by 50 ng/ml of IL-6 for 30 min. Fig. 5A showed that IL-6 induced STAT3 phosphorylation but had no effect on LLL12-pre- treated cells. IL-6 or LLL12 treat- ment did not alter the expression levels of total STAT3. To explore if this STAT3 small molecule inhibitor would affect STAT1 activity, we pretreated Hep3B and SNU-398 cells for 2 h followed by IFN- for 30 min. Fig. 5B showed that IFN- induced STAT1 phosphoryla- tion, whereas LLL12 pretreatment did not block IFN--induced STAT1 phosphorylation. We have shown in Fig. 1C that phosphorylated STAT3 translocated to the nucleus after IL-6 treatment. To investigate if LLL12 would block this translocation, we pretreated cells with 5 M LLL12 for2hand then treated the cells with 50 ng/ml of IL-6 for 30 min. The cells were fixed and stained by anti-phos- phorylated STAT3 primary anti- body and FITC conjugated second- ary antibody. The nucleus was stained with DAPI. As illustrated in Fig. 5C, LLL12 pretreated cells did not show phosphorylated STAT3 in the nucleus. Nonactivated STAT3 is in the cytoplasm. STAT3 translo- FIGURE 4. Inhibition of phosphorylated STAT3 blocks IL-6-induced cell survival. A, two siRNAs targeting STAT3 were transfected into Hep3B cells. After overnight incubation, the cells were cultured in serum-free cates to the nucleus when it is acti- medium for 24 h followed by IL-6 for 30 min. Phosphorylated STAT3, phosphorylated AKT, and GAPDH were vated (36). To confirm if the phos- detected by Western blot. B, STAT3 siRNA-transfected Hep3B cells were treated with 2 M doxorubicin for 2 h phorylation induced by IL-6 would followed by 50 ng/ml of IL-6 for 6 h. Morphological examination was performed after treatment. C, STAT3 siRNA-transfected Hep3B cells were pretreated with doxorubicin for 2 h followed by 50 ng/ml of IL-6 for 24 h. translocate STAT3 to the nucleus Caspase3/7 activity was assayed. The data showed the percent increase in caspase3/7 compared with scram- and if LLL12 pretreatment could bled siRNA-transfected control groups and represented three independent experiments. *, p  0.02. reverse this process, we cultured phorylated AKT and GAPDH were used as controls, which Hep3B cells in serum-free medium for 24 h. Then, the were not affected by STAT3 siRNA. starved cells were pretreated with 5 M LLL12 for 2 h fol- To investigate if knocking down STAT3 would decrease the lowed by 50 ng/ml of IL-6 for 30 min. The cells were stained anti-apoptotic effect of IL-6, STAT3 siRNA-transfected cells by anti-STAT3 primary antibody and secondary antibody. were treated under the similar conditions employed in Fig. 4A. Fig. 5D demonstrated that STAT3 was in the cytoplasm As seen in Fig. 4B, STAT3 siRNA-transfected cells exhibited when cultured in serum-free medium. IL-6 treatment trans- more characteristic morphology of apoptosis induced by doxo- located STAT3 to the nucleus whereas LLL12 pretreatment rubicin compared with scrambled control siRNA transfected blocked IL-6-induced STAT3 nuclear translocation. cells. Caspase3/7 assay further showed that knocking down LLL12 Inhibits IL-6-induced STAT3 Phosphorylation in a STAT3 increased doxorubicin-induced apoptosis when treated Dose- and Time-dependent Manner—We further examined if with1or2 M doxorubicin (Fig. 4C). Therefore, the data in Fig. the inhibitory effect of LLL12 would be dose dependent. We 4 suggested that IL-6 promoted cell survival via the enhanced pretreated cells with different concentrations of LLL12 (0–5 STAT3 signaling. M) for 2 h and then treated the cells with 50 ng/ml of IL-6 for STAT3 Small Molecule Inhibitor Blocks IL-6/STAT3 Sig- 30 min. Fig. 6A demonstrated a LLL12 dose dependence. Expo- naling—To investigate if a STAT3 inhibitor could block IL-6- sure of cells to 0.5 M LLL12 was able to suppress STAT3 induced STAT3 phosphorylation, Hep3B and SNU-398 cells phosphorylation. AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27433 IL-6/STAT3 Promotes Cell Survival FIGURE 5. A STAT3 small molecule inhibitor LLL12 blocks IL-6-induced STAT3 phosphorylation. A, Hep3B and SNU-398 cells were cultured in serum-free medium for 24 h and then were pretreated with DMSO or LLL12 for 2 h followed by IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. B, Hep3B and SNU-398 cells were cultured in serum-free medium for 24 h and then were pretreated with LLL12 for 2 h followed by IFN- for 30 min. The expression levels of phosphorylated STAT1, and total STAT1 were analyzed by Western blot. C and D, Hep3B cells were treated under the same conditions, and the distribution of phosphorylated STAT3 and total STAT3 was analyzed by immunofluorescence. To study if the inhibitory effect of LLL12 would also be time min. As seen in Fig. 6C, STAT3 phosphorylation recovered in dependent, we pretreated cells with LLL12 for different time Hep3B cells 12 h after LLL12 treatment. The results suggested points (0–2 h) followed by 50 ng/ml of IL-6 for 30 min. As that the inhibitory effect of LLL12 on STAT3 phosphorylation illustrated in Fig. 6B, IL-6-induced STAT3 phosphorylation was reversible. We further blocked protein synthesis using was suppressed by LLL12 in a time-dependent manner. cycloheximide and repeated the experiment in Fig. 6C to exam- To examine if the inhibitory effect of LLL12 would be revers- ine if IL-6 would induce STAT3 phosphorylation 12 h after ible, we pretreated cells with 5 M LLL12 for 2 h. Then LLL12- LLL12 treatment. As illustrated in Fig. 6D, STAT3 phosphory- supplemented medium was removed and fresh medium with- lation recovered in cycloheximide-treated cells, indicating that out LLL12 was added to the cells. After different incubation the recovered STAT3 phosphorylation was not due to the new time points, the cells were treated with 50 ng/ml of IL-6 for 30 synthesized STAT3 protein. 27434 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival FIGURE 6. The STAT3 small molecule inhibitor LLL12 blocks STAT3 phosphorylation in a dose- and time-dependent manner. A, Hep3B and SNU-398 cells were cultured in serum-free medium for 24 h and then were pretreated with various concentrations of LLL12 (0.5–5 M) for 2 h followed by IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. B, Hep3B and SNU-398 cells were cultured under the same conditions and then were treated with 5 M LLL12 for different time points (0 –2 h). After pretreatment, IL-6 was added to the cultured cells. Phosphorylated STAT3 and total STAT3 were detected by Western blot. C, Hep3B cells were cultured in serum-free medium for 24 h and then were pretreated with 5 M LLL12 for 2 h. After the 2 h pretreatment, medium was discarded, and fresh medium without LLL12 was added. After the indicated incubation (0 –24 h), the cells were treated with IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. D, Hep3B cells were cultured in serum-free medium for 24 h and then were pretreated with 5 M LLL12 for 2 h. After2hof pretreatment, the medium was discarded, and fresh medium without LLL12 was added. Cycloheximide was also added into the medium to block protein synthesis. After 12 h, the cells were treated with IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. FIGURE 7. LLL12 decreases IL-6-induced cell survival. A, Hep3B cells were pretreated with 0.5 M LLL12 for 2 h. After the pretreatment, LLL12 was removed, and fresh medium was added. The cells were treated as indicated. After overnight treatment, morphological examination was performed. B, results from A were quantified by cell viability assay. C and D, Hep3B and SNU-398 cells were pretreated with 0.5 M LLL12 for 2 h. After the pretreatment, cells were treated with IL-6 for 30 min. The levels of JAK1, JAK2, GP130, p-AKT, and p-ERK1/2 were analyzed by Western blot. LLL12 Reduces the Anti-apoptotic Activity of IL-6—To inves- overnight. Fig. 7A clearly demonstrated that LLL12-pretreated tigate if a small molecule STAT3 inhibitor would decrease anti- cells showed more cell death compared with the cells without apoptotic activity of IL-6, we chose a low dose (0.5 M) of LLL12 LLL12 pretreatment. This result was quantified by cell viability to pretreat Hep3B cells for only 2 h. After pretreatment, LLL12 assay (Fig. 7B). LLL12 pretreatment completely blocked IL-6- was removed, and cells were treated with doxorubicin and IL-6 induced doxorubicin resistance. Interestingly, the 2 h pretreat- AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27435 IL-6/STAT3 Promotes Cell Survival FIGURE 8. Blockade of IL-6 or STAT3 reduces cell recovery from doxorubicin. SNU-449 cells were treated with 2M doxorubicin, anti-IL-6 antibody, or LLL12 as indicated. After overnight treatment, live cells were counted and 5,000 cells were re-plated. Cells were treated with control IgG, anti-IL-6 antibody, or LLL12, respectively. After 7 days, cells were fixed and stained with 1% crystal violet. A, pictures were captured under microscopy at different magnification. B, original plates were scanned. Three independent experiments were performed. ment with LLL12 did not cause cell death (Fig. 7, A and B). overnight treatment, live cells were counted, and 5,000 cells These data suggested that low dose of STAT3 inhibitor alone were re-plated. The cells were cultured in doxorubicin-free might not lead to cell death but was able to attenuate IL-6- medium for 7 days. Anti-IL-6 antibody or LLL12 was supple- induced cell survival upon drug treatment. mented in medium. After 7 days, cells were fixed and stained To explore if STAT3 inhibitor LLL12 would affect other with 1% crystal violet. Fig. 8 clearly showed that many cells components of IL-6/STAT3 signaling or other IL-6 signaling, recovered from control cells, whereas fewer cells recovered we pretreated Hep3B and SNU-398 cells with 0.5 M LLL12 for from anti-IL-6 antibody or LLL12-treated cells. The data in Fig. 2 h, followed by IL-6 for 30 min. JAK1, JAK2, GP130, phosphor- 8 suggested that blockade of IL-6 or STAT3 reduced cell recov- ylated AKT, and phosphorylated ERK were analyzed by West- ery from drug treatment. ern blot. Fig. 7, C and D showed that 0.5 M LLL12 did not affect Blockade of IL-6/STAT3 Does Not Affect Cell Viability in other proteins of IL-6 signaling pathways. The data in Fig. 7 Human Primary Hepatocytes Treated with Doxorubicin—To suggested that the low dose of STAT3 small molecule inhibitor investigate if blocking IL-6 or STAT3 would reduce viability in LLL12 attenuated IL-6-induced cell survival through specifi- normal liver cells, human primary hepatocytes were treated cally inhibiting STAT3 phosphorylation. with 500 ng/ml of anti-IL-6 antibody or 0.5 M LLL12 for 2 or Blockade of IL-6 or STAT3 Reduces Cell Recovery from 24 h, respectively. After 24 h, cell viability was measured. As Doxorubicin—To investigate if blocking IL-6 or STAT3 would shown in Fig. 9A, anti-IL-6 antibody or LLL12 did not reduce reduce cell recovery from doxorubicin, SNU-449 cells were cell viability compared with untreated cells. treated with 5 M doxorubicin alone, doxorubicin plus anti- We further examined if blockade of IL-6 or STAT3 signaling IL-6 antibody, or doxorubicin plus LLL12, respectively. After in human primary normal liver cells would reduce cell viability 27436 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival cellular carcinoma (HCC) is the most common liver tumor with a 7% five-year survival rate. It mainly occurs in males due to differences in IL-6 production between males and females (35). Moreover, obesity may increase the risk of liver and other cancer due to the chronic inflam- matory response caused by en- hanced levels of IL-6/STAT3 and TNF/STAT3 signaling (46). IL-6 is a key event in tumorigene- sis, but its biological function in liver cancers is not clear. Previous studies have demonstrated that IL-6/STAT3 provides hepatopro- tection against liver damage (47). Based on these results, we hypothe- sized that IL-6 would promote sur- vival of liver cancer cells via STAT3 upon drug treatment. We found that the endogenous levels of IL-6 in SNU-387 and SNU-449 liver cancer cell lines were much higher than those in human hepatocytes. Hep3B and SNU-398 cells showed similar FIGURE 9. Blockade of IL-6 or STAT3 does not affect cell viability in human primary hepatocytes treated levels of IL-6 to human hepatocytes. with doxorubicin. A, human primary hepatocytes were treated with 500 ng/ml of anti-IL-6 antibody or 0.5 M LLL12 for 2 h and 24 h, respectively. After 24 h, cell viability was measured. Non-treated cells (NT) were used as Because IL-6 levels in liver cancer control. The data represented three independent results. B, human primary hepatocytes were pretreated with patients are 25-fold higher than 500 ng/ml of anti-IL-6 antibody or 0.5 M LLL12 for 2 h. After the pretreatment, LLL12 was discarded and fresh medium with different concentrations of doxorubicin was added. After 24 h, cell viability was measured. The healthy adults (34), we wondered data represented three independent results. how Hep3B and SNU-398 would respond upon IL-6. We found that when treated with doxorubicin. Human primary hepatocytes IL-6 activated STAT3 in these two cell lines. To study the phe- were pretreated with 500 ng/ml of anti-IL-6 antibody or 0.5 M notype of IL-6 in liver cancer cells, we induced apoptosis in Hep3B cells using doxorubicin. Interestingly, exogenous addi- LLL12 for 2 h. After the pretreatment, LLL12 was discarded, tion of IL-6 protected cells toward apoptosis induced by doxo- and fresh medium with different concentrations of doxorubicin was added. After 24 h, cell viability was measured. Our results rubicin. To explore if IL-6-promoted cell survival upon doxo- showed that when doxorubicin concentration increased from rubicin would be caused by activated STAT3, we targeted STAT3 by siRNA and observed that STAT3 knocked down 0.5 to 1 M, cell viability decreased significantly. However, the cells showed higher level of apoptosis. When SNU-449 cells treatment by anti-IL-6 antibody or LLL12 with doxorubicin did not markedly reduce viability in human primary hepatocytes with enhanced endogenous IL-6 were treated with IL-6 anti- compared with the cells treated by doxorubicin alone (Fig. 9B), body, phosphorylated STAT3 was down-regulated. As a result, IL-6 antibody treated SNU-449 cells exhibited lowered cell via- indicating that unlike liver cancer cells, liver normal cells did bility, suggesting that IL-6 induced cell survival via STAT3. In not rely on IL-6/STAT3 signaling. human cervical cancer, IL-6 causes anti-apoptosis induced by DISCUSSION doxorubicin via PI3K/AKT instead of STAT3 (10). Therefore, Growing evidence has shown that inflammatory signals from IL-6 exerts its activity through different mechanisms the surrounding microenvironment promote tumor growth depending on cell types. In prostate cancer, IL-6 also causes and progression. They are also associated with survival path- anti-apoptosis via Mcl-1 (48). In pancreatic cancer, PANC-1 ways and immunosuppression (37–39). It has been well docu- cells, however, we found that IL-6 induced STAT3 in a dose- mented that inflammatory cytokine IL-6/STAT3 signaling is dependent manner, but it did not cause anti-apoptosis (data involved in many types of human cancer (2, 6, 7, 9, 40–42). IL-6 not shown), suggesting that the anti-apoptotic effect of IL-6 is can promote cell proliferation in multiple myelomas, ovarian cell type-specific. carcinoma and prostate cancer cells in vitro (41, 43, 44) and Treatment with anti-IL-6 monoclonal antibody or with len- tumor growth in vivo in prostate, lung, and breast (37, 44). It is tivirus expressing shSTAT3 has been shown to induce apopto- also related to cell migration and adhesion (45). sis in prostate cancer and glioblastoma multiforme in vitro or in Liver cancer is a major health problem because of the poor vivo (15, 49, 50). To study if STAT3 small molecule inhibitor prognosis. IL-6 plays a critical role in liver cancer (34). Hepato- would show similar effect to STAT3 siRNA and IL-6 antibody AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27437 IL-6/STAT3 Promotes Cell Survival 10. Wei, L. H., Kuo, M. L., Chen, C. A., Chou, C. H., Cheng, W. F., Chang, on IL-6/STAT3 signaling in liver cancer cells, we used a STAT3 M. C., Su, J. L., and Hsieh, C. Y. (2001) Oncogene 20, 5799–5809 small molecule inhibitor, which was reported by our laboratory 11. Wei, L. H., Kuo, M. L., Chen, C. A., Chou, C. H., Lai, K. B., Lee, C. N., and (51). Our previous results show that high concentration of this Hsieh, C. Y. (2003) Oncogene 22, 1517–1527 STAT3 small molecule inhibitor may cause apoptosis in differ- 12. Lin, M. T., Juan, C. Y., Chang, K. J., Chen, W. J., and Kuo, M. L. (2001) ent types of human cancer with elevated STAT3. In this study, Carcinogenesis 22, 1947–1953 we demonstrated that this STAT3 small molecule inhibitor 13. Leu, C. M., Wong, F. H., Chang, C., Huang, S. F., and Hu, C. P. (2003) Oncogene 22, 7809–7818 blocked IL-6-induced STAT3 phosphorylation in a dose-de- 14. Meng, F., Yamagiwa, Y., Ueno, Y., and Patel, T. (2006) J. Hepatol 44, pendent manner. Low concentration of LLL12 did not cause 1055–1065 cell death whereas it attenuated IL-6-induced cell survival upon 15. Smith, P. C., and Keller, E. T. (2001) Prostate 48, 47–53 doxorubicin treatment via down-regulation of phosphorylated 16. Liu, S., Ishikawa, H., Li, F. J., Ma, Z., Otsuyama, K., Asaoku, H., Abroun, S., STAT3. Our data indicate that blockade of STAT3 signaling Zheng, X., Tsuyama, N., Obata, M., and Kawano, M. M. (2005) Cancer Res. with STAT3 small molecule inhibitor may reduce IL-6-induced 65, 2269–2276 17. Liu, S., Ma, Z., Cai, H., Li, Q., Rong, W., and Kawano, M. (2010) Eur. J. anti-apoptosis. Haematol. 84, 137–144 Although LLL12 is a STAT3 structure-based molecule, we 18. Yu, C. Y., Wang, L., Khaletskiy, A., Farrar, W. L., Larner, A., Colburn, wondered if LLL12 could affect other components of IL-6/ N. H., and Li, J. J. (2002) Oncogene 21, 3949–3960 STAT3 signaling pathway. We examined JAK1, JAK2, and 19. Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G., Zhao, Y., Pestell, R. G., GP130. JAK1 and JAK2 phosphorylate STAT3 (52, 53). GP130 Albanese, C., and Darnell, J. E., Jr. (1999) Cell 98, 295–303 20. Yu, H., Kortylewski, M., and Pardoll, D. (2007) Nat. Rev. Immunol. 7, 41–51 provides docking site for STAT3 (52). LLL12 did not change 21. Kortylewski, M., Xin, H., Kujawski, M., Lee, H., Liu, Y., Harris, T., Drake, JAK1, JAK2, or GP130. IL-6 can also activate PI3K and ERK C., Pardoll, D., and Yu, H. (2009) Cancer Cell 15, 114–123 signaling pathways (9), but LLL12 did not alter them either. In 22. Yu, H., Pardoll, D., and Jove, R. (2009) Nat. Rev. Cancer 9, 798–809 conclusion, IL-6 could protect human liver cancer cells toward 23. Choudhari, S. R., Khan, M. A., Harris, G., Picker, D., Jacob, G. S., Block, T., apoptosis via STAT3 signaling. Targeting STAT3 with STAT3 and Shailubhai, K. (2007) Mol. Cancer Ther. 6, 112–121 siRNA, anti-IL-6 antibody, or a STAT3 small molecule inhibi- 24. Real, P. J., Sierra, A., De Juan, A., Segovia, J. C., Lopez-Vega, J. M., and Fernandez-Luna, J. L. (2002) Oncogene 21, 7611–7618 tor may decrease or block IL-6-induced liver cancer cell sur- 25. Epling-Burnette, P. K., Liu, J. H., Catlett-Falcone, R., Turkson, J., Oshiro, vival upon drug treatment. M., Kothapalli, R., Li, Y., Wang, J. M., Yang-Yen, H. F., Karras, J., Jove, R., Short term and long term IL-6 exposure show paradoxical and Loughran, T. P., Jr. (2001) J. Clin. Invest. 107, 351–362 effects on liver injury and repair (54). Short term production of 26. Aoki, Y., Feldman, G. M., and Tosato, G. (2003) Blood 101, 1535–1542 IL-6 is a natural response of the body during the liver injury to 27. Amin, H. M., McDonnell, T. J., Ma, Y., Lin, Q., Fujio, Y., Kunisada, K., protect cells (35). In contrast, long term exposure to IL-6 can Leventaki, V., Das, P., Rassidakis, G. Z., Cutler, C., Medeiros, L. J., and Lai, R. (2004) Oncogene 23, 5426–5434 impair liver regeneration and even increase liver injury (54). 28. Gritsko, T., Williams, A., Turkson, J., Kaneko, S., Bowman, T., Huang, M., Our results also demonstrated that ablation of IL-6/STAT3 in Nam, S., Eweis, I., Diaz, N., Sullivan, D., Yoder, S., Enkemann, S., Eschrich, human primary hepatocytes did not affect their viability under S., Lee, J. H., Beam, C. A., Cheng, J., Minton, S., Muro-Cacho, C. A., and normal conditions and did not induce more cell death when Jove, R. (2006) Clin. Cancer Res. 12, 11–19 treated with chemotherapeutic agents. 29. Niu, G., Wright, K. L., Huang, M., Song, L., Haura, E., Turkson, J., Zhang, In conclusion, IL-6/STAT3 signaling may be not crucial in S., Wang, T., Sinibaldi, D., Coppola, D., Heller, R., Ellis, L. M., Karras, J., Bromberg, J., Pardoll, D., Jove, R., and Yu, H. (2002) Oncogene 21, human primary hepatocytes. However, this signaling can pro- 2000–2008 mote hepatocellular carcinoma survival upon drug treatment. 30. Wei, D., Le, X., Zheng, L., Wang, L., Frey, J. A., Gao, A. C., Peng, Z., Huang, Blockade of IL-6/STAT3 may be able to render hepatocellular S., Xiong, H. Q., Abbruzzese, J. L., and Xie, K. (2003) Oncogene 22, carcinoma with elevated endogenous IL-6/STAT3 more sensi- 319–329 tive to chemotherapeutic agent-induced apoptosis. 31. Xu, Q., Briggs, J., Park, S., Niu, G., Kortylewski, M., Zhang, S., Gritsko, T., Turkson, J., Kay, H., Semenza, G. L., Cheng, J. Q., Jove, R., and Yu, H. REFERENCES (2005) Oncogene 24, 5552–5560 32. Gao, L., Zhang, L., Hu, J., Li, F., Shao, Y., Zhao, D., Kalvakolanu, D. V., 1. Hodge, D. R., Hurt, E. M., and Farrar, W. L. (2005) Eur. J. Cancer 41, Kopecko, D. J., Zhao, X., and Xu, D. Q. (2005) Clin. Cancer Res. 11, 2502–2512 6333–6341 2. Grivennikov, S., Karin, E., Terzic, J., Mucida, D., Yu, G. Y., Vallabha- 33. Hodge, D. R., Li, D., Qi, S. M., and Farrar, W. L. (2002) Biochem. Biophys. purapu, S., Scheller, J., Rose-John, S., Cheroutre, H., Eckmann, L., and Res. Commun. 292, 287–291 Karin, M. (2009) Cancer Cell 15, 103–113 34. Porta, C., De Amici, M., Quaglini, S., Paglino, C., Tagliani, F., Boncimino, 3. Schafer, Z. T., and Brugge, J. S. (2007) J. Clin. Invest. 117, 3660–3663 A., Moratti, R., and Corazza, G. R. (2008) Ann. Oncol. 19, 353–358 4. Bharti, A. C., Donato, N., and Aggarwal, B. B. (2003) J. Immunol. 171, 35. Naugler, W. E., Sakurai, T., Kim, S., Maeda, S., Kim, K., Elsharkawy, A. M., 3863–3871 and Karin, M. (2007) Science 317, 121–124 5. Sansone, P., Storci, G., Tavolari, S., Guarnieri, T., Giovannini, C., Taf- 36. Darnell, J. E., Jr. (1997) Science 277, 1630–1635 furelli, M., Ceccarelli, C., Santini, D., Paterini, P., Marcu, K. B., Chieco, P., 37. Bromberg, J., and Wang, T. C. (2009) Cancer Cell 15, 79–80 and Bonafe`, M. (2007) J. Clin. Invest. 117, 3988–4002 38. Balkwill, F., and Mantovani, A. (2001) Lancet 357, 539–545 6. Gao, S. P., Mark, K. G., Leslie, K., Pao, W., Motoi, N., Gerald, W. L., Travis, 39. Grivennikov, S., and Karin, M. (2008) Cancer Cell 13, 7–9 W. D., Bornmann, W., Veach, D., Clarkson, B., and Bromberg, J. F. (2007) 40. Spiotto, M. T., and Chung, T. D. (2000) Prostate 42, 186–195 J. Clin. Invest. 117, 3846–3856 41. Rabinovich, A., Medina, L., Piura, B., Segal, S., and Huleihel, M. (2007) 7. Lou, W., Ni, Z., Dyer, K., Tweardy, D. J., and Gao, A. C. (2000) Prostate 42, Anticancer Res. 27, 267–272 239–242 42. Bollrath, J., Phesse, T. J., von Burstin, V. A., Putoczki, T., Bennecke, M., 8. Spiotto, M. T., and Chung, T. D. (2000) Prostate 42, 88–98 Bateman, T., Nebelsiek, T., Lundgren-May, T., Canli, O., Schwitalla, S., 9. Culig, Z., Steiner, H., Bartsch, G., and Hobisch, A. (2005) J. Cell. Biochem. Matthews, V., Schmid, R. M., Kirchner, T., Arkan, M. C., Ernst, M., and 95, 497–505 27438 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival Greten, F. R. (2009) Cancer Cell 15, 91–102 49. Kudo, M., Jono, H., Shinriki, S., Yano, S., Nakamura, H., Makino, K., Hide, 43. Kawano, M., Hirano, T., Matsuda, T., Taga, T., Horii, Y., Iwato, K., Asaoku, T., Muta, D., Ueda, M., Ota, K., Ando, Y., and Kuratsu, J. (2009) J. Neuro- H., Tang, B., Tanabe, O., and Tanaka, H. (1988) Nature 332, 83–85 surg 111, 219–225 44. Malinowska, K., Neuwirt, H., Cavarretta, I. T., Bektic, J., Steiner, H., Di- 50. Li, G. H., Wei, H., Chen, Z. T., Lv, S. Q., Yin, C. L., and Wang, D. L. (2009) etrich, H., Moser, P. L., Fuchs, D., Hobisch, A., and Culig, Z. (2009) Endocr J. Neurooncol. 91, 165–174 Relat Cancer 16, 155–169 51. Lin, L., Hutzen, B., Li, P. K., Ball, S., Zuo, M., DeAngelis, S., Foust, E., Sobo, 45. Santer, F. R., Malinowska, K., Culig, Z., and Cavarretta, I. T. (2010) Endocr. M., Friedman, L., Bhasin, D., Cen, L., Li, C., and Lin, J. (2010) Neoplasia 12, Relat. Cancer 17, 241–253 39–50 46. Park, E. J., Lee, J. H., Yu, G. Y., He, G., Ali, S. R., Holzer, R. G., Osterreicher, 52. Heinrich, P. C., Behrmann, I., Mu¨ller-Newen, G., Schaper, F., and Graeve, C. H., Takahashi, H., and Karin, M. (2010) Cell 140, 197–208 L. (1998) Biochem. J. 334, 297–314 47. Taub, R. (2003) J. Clin. Invest. 112, 978–980 53. Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M., Mu¨ller-Newen, 48. Cavarretta, I. T., Neuwirt, H., Untergasser, G., Moser, P. L., Zaki, M. H., G., and Schaper, F. (2003) Biochem. J. 374, 1–20 Steiner, H., Rumpold, H., Fuchs, D., Hobisch, A., Nemeth, J. A., and Culig, 54. Jin, X., Zimmers, T. A., Perez, E. A., Pierce, R. H., Zhang, Z., and Koniaris, Z. (2007) Oncogene 26, 2822–2832 L. G. (2006) Hepatology 43, 474–484 AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27439 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry American Society for Biochemistry and Molecular Biology

Inhibition of STAT3 Signaling Blocks the Anti-apoptotic Activity of IL-6 in Human Liver Cancer Cells *

Download PDF
11 pages

Loading next page...
 
/lp/american-society-for-biochemistry-and-molecular-biology/inhibition-of-stat3-signaling-blocks-the-anti-apoptotic-activity-of-il-tSkOsuxAvW

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
American Society for Biochemistry and Molecular Biology
Copyright
Copyright © 2010 Elsevier Inc.
ISSN
0021-9258
eISSN
1083-351X
DOI
10.1074/jbc.m110.142752
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 35, pp. 27429 –27439, August 27, 2010 © 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A. Inhibition of STAT3 Signaling Blocks the Anti-apoptotic Activity of IL-6 in Human Liver Cancer Cells Received for publication, May 9, 2010, and in revised form, June 8, 2010 Published, JBC Papers in Press, June 18, 2010, DOI 10.1074/jbc.M110.142752 ‡ § § ‡¶1 Yan Liu , Pui-Kai Li , Chenglong Li , and Jiayuh Lin From the Department of Pediatrics, Center for Childhood Cancer, The Research Institute at Nationwide Children’s Hospital, the § ¶ Division of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, and the Experimental Therapeutics Program, The Ohio State University Comprehensive Cancer Center, The Ohio State University, Columbus, Ohio 43205 Interleukin-6 (IL-6) is a multifunctional cytokine, which may in promoting colitis-associated cancer (CAC) tumorigenesis, block apoptosis during inflammation to protect cells under very enhancing tumor initiation cell proliferation, and preventing toxic conditions. However, IL-6 also activates STAT3 in many apoptosis of premalignant intestinal epithelial cells. High levels types of human cancer. Recent studies demonstrate that high of IL-6 have been detected in many types of human epithelial levels of IL-6 are associated with hepatocellular carcinoma, the cancers (3), and correlate with proliferation or survival of mul- most common type of liver cancer. Here we reported that IL-6 tiple myeloma, breast cancer stem cells, lung adenocarcinomas, promoted survival of human liver cancer cells through activat- prostate cancer, cervical cancer, gastric cancer, and esophageal ing STAT3 in response to doxorubicin treatment. Endogenous carcinoma (4–14). IL-6 antibody alone can induce PC-3 IL-6 levels in SNU-449 cells were higher than in Hep3B cells. xenograft regression (15). Thus, blocking IL-6-mediated signal- Meanwhile, SNU-449 cells were more resistant to doxorubicin ing cascades has a potential for treatment of these human can- than Hep3B cells. Addition of IL-6 induced STAT3 activation in cers (4, 16, 17). Hep3B cells and led to protection against doxorubicin. In con- IL-6 signals through IL-6 receptor (IL-6-R), GP130, and trast, neutralizing IL-6 with anti-IL-6 antibody decreased sur- Janus kinases (JAKs). IL-6-induced JAK family members acti- vival of SNU-449 cells in response to doxorubicin. To elucidate vate three major pathways, STAT3, MAPK, and PI3K (1, 18). the mechanism of the anti-apoptotic function of IL-6, we inves- The signal transducer and activator of transcription 3 (STAT3) tigated if STAT3 mediated this drug resistance. Targeting is considered as an oncogene and found constitutively activated STAT3 with STAT3 siRNA reduced the protection of IL-6 in many types of human malignancies (19–22). STAT3 signal- against doxorubicin-induced apoptosis, indicating that STAT3 ing is a major pathway for cancer inflammation. It induces a lot signaling contributed to the anti-apoptotic effect of IL-6. More- of genes crucial for inflammation. Environmental factors, over, we further explored if a STAT3 small molecule inhibitor including UV radiation, chemical carcinogen, infection, stress, could abolish this anti-apoptotic effect. LLL12, a STAT3 small and smoke, can activate STAT3 via cytokine receptor, toll-like molecule inhibitor, blocked IL-6-induced STAT3 phosphoryla- receptor, adrenergic receptor, or nicotinic receptor (22, 23). tion, resulting in attenuation of the anti-apoptotic activity of STAT3 is activated when tyrosine 705 (Tyr-705) is phosphory- IL-6. Finally, neutralization of endogenous IL-6 with anti-IL-6 lated. Phosphorylated STAT3 molecules dimerize and translo- antibody or blockade of STAT3 with LLL12 lowered the recov- cate into the nucleus, where they bind to specific DNA response ery in SNU-449 cells after doxorubicin treatment. Therefore, elements and induce the transcription of proliferation and anti- our results demonstrated that targeting STAT3 signaling could apoptosis associated genes (19, 24), such as BCL-2, BCL-X , interrupt the anti-apoptotic function of IL-6 in human liver can- IL-17, IL-23, MCL1, and Survivin (20, 21, 25–28). The activa- cer cells. tion of STAT3 also promotes tumor angiogenesis via HIF-1 and VEGF (11, 29–31). Therefore, STAT3 is a potential target for cancer therapy. Evidence shows that inhibiting STAT3 using Interleukin-6 is a multifunctional cytokine that was origi- dominant-negative STAT3, antisense oligonucleotides and nally characterized acting in immune and inflammatory RNA interference induces tumor cell death (22, 32). responses, and inhibits apoptosis in toxic environments during A growing numbers of evidence demonstrate that tumori- inflammation (1). Growing evidence has indicated that cancers genesis caused by STAT3 is mediated by IL-6 signaling (1, 33). are associated with chronic inflammation. Although serum Therefore, targeting IL-6/STAT3 signaling pathway should be IL-6 levels were known elevated in colon cancer patients, only considered for the treatment of patients with elevated levels of recent studies by the Karin group (2) document the role of IL-6 IL-6/STAT3 signaling. IL-6 levels in liver cancer patients are 25-fold higher than healthy adults (34). The high levels of IL-6 are associated with hepatocellular carcinoma (HCC), the most * This work was supported, in whole or in part, by National Institutes of Health common liver cancer (35). R21 Grant R21CA133652-01 (to J. L.). This work was also supported by a In this study, we explored the biological role of IL-6 in human Pancreatic Cancer Action Network-AACR, National Foundation for Cancer Research grant. liver cancer cells and found that IL-6 stimulated STAT3 phos- To whom correspondence should be addressed: Center for Childhood Can- phorylation and promoted cell survival upon doxorubicin treat- cer, Dept. of Pediatrics, College of Medicine, The Ohio State University, 700 ment. Interestingly, targeting STAT3 by STAT3 siRNA, anti- Children’s Drive, Columbus, OH 43205. Tel.: 614-722-5086; Fax: 614-722- 5895; E-mail: [email protected]. IL-6 antibody or a small molecule STAT3 inhibitor, attenuated AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27429 This is an Open Access article under the CC BY license. IL-6/STAT3 Promotes Cell Survival FIGURE 1. IL-6 induces STAT3 phosphorylation. A, human hepatocyte, Hep3B, SNU-387, SNU-398, and SNU-449 cells were cultured in Hepatocyte Medium for 24 h. Endogenous IL-6 was accessed by IL-6 ELISA assay. B, Hep3B and SNU-398 cells were cultured in serum-free medium. After 24 h of serum deprivation, cells were treated with various concentrations of IL-6 for 30 min. STAT3 phosphorylation and total STAT3 were detected by Western blot. C, Hep3B and SNU-398 cells were cultured under the same conditions and then were treated with 50 ng/ml of IL-6 for 30 min. The distribution of phosphorylated STAT3 was analyzed by immunofluorescence. IL-6-induced drug resistance, suggesting that activated STAT3 turer’s protocol. STAT3 small interfering RNA (siRNA) kit and contributed to the IL-6-induced cell survival. scrambled control siRNA were purchased from Cell Signaling Technology (Beverly, MA). EXPERIMENTAL PROCEDURES Western Blot—Whole cell extracts were prepared by lysing Cell Culture, Transfection, and RNA Interference—Human the cells in RIPA buffer containing mixture proteasome inhib- hepatoma cell lines, Hep3B, SNU-387, SNU-398, SNU-449 itor. Lysates were then centrifuged at 13,300 rpm for 10 min at 4 °C, and the supernatant was collected. Protein samples were were obtained from American Type Culture Collection (ATCC, separated by SDS-PAGE, transferred onto PVDF membrane, Manassas, VA). Hep3B cells were cultured in Minimum Essen- tial Medium, Eagle (MEM) (ATCC) supplemented with 10% and immunoblotted with the appropriate antibody. Antibodies fetal bovine serum (FBS) and 1% penicillin/streptomycin. SNU- to p-STAT3 (Y705), STAT3, p-STAT1 (Y701), STAT1, p- 398 and SNU-449 cells were cultured in RPMI 1640 medium ERK1/2 (T202/Y204), p-JAK2, JAK2, p-JAK1, JAK1, GP130, GAPDH, and HRP-conjugated secondary antibodies were from (ATCC) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Human hepatocytes were pur- Cell Signaling Technology. The target protein was examined by chased from ScienCell Research Laboratories (Carlsbad, CA). chemiluminescence (Cell Signaling Technology). Human hepatocytes were cultured in Hepatocyte Medium Immunofluorescence—Cells were seeded on a glass slide and fixed with cold methanol for 15 min. After a washing in (HM, ScienCell Research Laboratories) including 5% FBS, 1% hepatocyte growth supplement, and 1% penicillin/streptomy- phosphate-buffered saline (PBS), the slide was blocked with cin. Interleukin-6 (IL-6) and interferon- (IFN-) were pur- 5% normal goat serum and 0.3% Triton X-100 in PBS for at chased from Cell Sciences (Canton, MA). Antibody to human least 1 h. Then the slide was incubated with primary anti- body. After overnight incubation, the slide was washed with IL-6, which can neutralize IL-6 bioactivity, was from R &D Sys- tems (Minneapolis, MN). The human IL-6 ELISA kit was from PBST and then incubated with anti-rabbit FITC-conjugated Pepro Tech Inc (Rocky Hill, NJ). secondary antibody (Jackson ImmunoResearch Laboratories, siRNA was transfected into Hep3B cells using Lipofectamine West Grove, PA). The cells were mounted in Vectashield Hard- 2000 (Invitrogen, Carlsbad, CA) according to the manufac- Set mounting medium with DAPI (Vector Laboratories, Burl- 27430 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival FIGURE 2. SNU-449 cells are more doxorubicin-resistant than Hep3B cells. A, SNU-449 cells and Hep3B cells were treated with various concentrations of doxorubicin. After 24 h, morphological examination was performed. B, SNU-449 cells and Hep3B cells were treated under the same conditions. After 48 h, cell viability assay was performed. The data showed the percent decrease in cell viability compared with untreated cells and represented three independent results. C, endogenous phosphorylated STAT3 and total STAT3 in Hep3B and SNU-449 cells were analyzed by Western blot. ingame, CA). Pictures were captured by Leica Microsystems Statistical Analysis—The statistical significance was calcu- lated using Student’s t test. p values of 0.05 were considered (Bannockburn, IL). Apoptosis Assay—Apoptosis was measured with caspase3/7 significant. assay kit (Promega, Madison, WI) according to the manufac- turer’s protocol. Briefly, 20,000 cells were seeded into 96-well RESULTS plates. After the treatment, 100 l of Apo-One caspase3/7 rea- IL-6 Induces STAT3 Phosphorylation—To examine the en- gent was added to each well and was incubated at 37 °C for 30 dogenous IL-6 levels in different liver cancer cells, four human min. The fluorescence was measured at an excitation wave- liver cancer cell lines (Hep3B, SNU-387, SNU-398, and length range of 485 nm and an emission wavelength range of SNU449) and human hepatocytes were cultured in the same 530 nm. medium, hepatocyte medium, for 24 h. After the incubation, Viability Assay—Viability was performed with CyQUANT the IL-6 levels in the culture medium were evaluated using IL-6 NF Assay (Invitrogen) according to the manufacturer’s pro- ELISA assay. As illustrated in Fig. 1A, the IL-6 levels were tocol. Briefly, 2,000 cells were seeded into a 96-well plate. remarkably elevated in SNU-387 and SNU-449 cell lines com- After the 48-h treatment, medium was removed from the pared with human hepatocytes, Hep3B and SNU-398. To plate, and 50 l of dye binding solution were added to each explore if IL-6 would induce STAT3 phosphorylation in cells well. The plate was covered and incubated at 37 °C for 30 with lower endogenous IL-6, Hep3B, and SNU-398 cells were min. The fluorescence was measured at an excitation wave- cultured in serum-free medium for 24 h and then were treated length range of 485 nm and an emission wavelength range of with different concentrations of IL-6 (0–50 ng/ml) for 30 min. 530 nm. As illustrated in Fig. 1B, IL-6 induced STAT3 phosphorylation Clonogenic Assay—Cells were washed with PBS twice and in a dose-dependent manner, but did not alter the expression of fixed with cold methanol at 20 °C for 10 min. After 10 min, total STAT3. To confirm if IL-6-induced phosphorylated cells were stained with 1% crystal violet (25% methanol) at room STAT3 would translocate to the nucleus, the localization of temperature for 10 min. After the staining, the plates were phosphorylated STAT3 was determined by staining with anti- washed with distill water and dried. phosphorylated STAT3 primary antibody and FITC-conju- AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27431 IL-6/STAT3 Promotes Cell Survival FIGURE 3. IL-6 protects cells against doxorubicin-induced apoptosis. A, Hep3B cells were pretreated with 2 M doxorubicin for 2 h followed by various concentrations of IL-6 for 6 h. Morphological examination was performed after treatment. B, Hep3B cells were treated under the same conditions. After 24 h, caspase3/7 activity was analyzed. The data showed the percent decrease in csapase3/7 activity compared with control cells and represented three indepen- dent results. C, SNU-449 cells were treated with 500 ng/ml of anti-IL-6 antibody or control IgG for 24 h. After the incubation, whole cell lysates were extracted. Phosphorylated STAT3 and GAPDH were detected by Western blot. D, SNU-449 cells were pretreated with 500 ng/ml of anti-IL-6 antibody or control IgG for 24 h. After the pretreatment, cells were further treated with doxorubicin for 24 h. After the 24 h treatment, cell viability was measured. The data represented eight independent results. gated secondary antibody. The nucleus was stained with DAPI. ng/ml of IL-6 effectively displayed anti-apoptotic effect. To fur- Fig. 1C demonstrated that phosphorylated STAT3 translocated ther confirm our results, Hep3B cells were treated under the to the nucleus following IL-6 treatment. similar conditions and after 24 h, apoptotic levels were mea- SNU-449 Cells Are More Doxorubicin-resistant than Hep3B sured using caspase3/7 assay. Fig. 3B showed that 10 ng/ml of Cells—To determine if liver cancer cells with higher endoge- IL-6 treatment exhibited 70% decrease in doxorubicin-caused nous IL-6 would be more resistant to drug treatment than those apoptosis. Similar results were observed in cells treated with 25 with lower endogenous IL-6, we treated SNU-449 and Hep3B and 50 ng/ml of IL-6. cells with different concentrations of doxorubicin. After 24 h of To investigate if disruption of IL-6 autocrine would down- treatment, the characteristic morphology of cell death was regulate STAT3 phosphorylation and would interrupt the anti- observed in Hep3B cells but not in SNU-449 cells (Fig. 2A). apoptotic effect of IL-6, SNU-449 cells were treated with anti- After another 24 h, cell viability assay was performed. Cell via- IL-6 antibody and phosphorylated STAT3 was analyzed by bility did not show significant change in SNU-449 treated by 2.5 Western blot. Our results demonstrated that the treatment or 5 M doxorubicin whereas 40% decrease was observed when with anti-IL-6 antibody reduced phosphorylated STAT3 levels treated by 10 M doxorubicin (Fig. 2B). In contrast, cell viability (Fig. 3C). Then we pretreated SNU-449 cells with anti-IL-6 decreased around 60, 80, and 80% when Hep3B cells were antibody overnight to neutralize the endogenous IL-6. After the treated by 2.5, 5, and 10 M doxorubicin, respectively (Fig. 2B). pretreatment, cells were treated with doxorubicin for 24 h fol- These results demonstrated that SNU-449 cells were more lowed by cell viability assay. Fig. 3D showed that cells treated resistant to doxorubicin than Hep3B cells. To examine if liver with doxorubicin and anti-IL-6 antibody exhibited 70% cancer cells with higher IL-6 would also show enhanced endog- decrease in viability compared with cells treated with doxoru- enous STAT3, we analyzed phosphorylated STAT3 in SNU- bicin alone. The data in Fig. 3 suggested that IL-6 promoted cell 449 and Hep3B cells. The Western blot results showed that survival in response to drug treatment. phosphorylated STAT3 was elevated in SNU-449 cells com- IL-6 Causes Anti-apoptosis through Activating STAT3—To pared with Hep3B cells, whereas total STAT3 level did not explore if STAT3 signaling would contribute to the anti-apo- change (Fig. 2C). ptotic activity of IL-6, we targeted STAT3 by STAT3 siRNA. IL-6 Confers Resistance to Apoptosis Induced by Doxorubicin— siRNA was transfected into Hep3B cells with less than 50% To explore if IL-6 would protect cells upon drug treatment, transfection efficiency (data not shown). The transfected cells Hep3B cells were pretreated with 2 M doxorubicin for 2 h were cultured in serum-free medium overnight and then were followed by different doses of IL-6 (0–50 ng/ml). After6hof treated with 50 ng/ml of IL-6 for 30 min. Western blot results incubation, the characteristic morphology of cell death was demonstrated a decrease in phosphorylated STAT3 compared observed in cells treated with doxorubicin whereas the addition of IL-6 rescued doxorubicin-caused cell death (Fig. 3A). 10 with control scrambled siRNA-transfected cells (Fig. 4A). Phos- 27432 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival were cultured in serum-free me- dium for 24 h and then were pre- treated with 5 or 10 M LLL12 for 2 h followed by 50 ng/ml of IL-6 for 30 min. Fig. 5A showed that IL-6 induced STAT3 phosphorylation but had no effect on LLL12-pre- treated cells. IL-6 or LLL12 treat- ment did not alter the expression levels of total STAT3. To explore if this STAT3 small molecule inhibitor would affect STAT1 activity, we pretreated Hep3B and SNU-398 cells for 2 h followed by IFN- for 30 min. Fig. 5B showed that IFN- induced STAT1 phosphoryla- tion, whereas LLL12 pretreatment did not block IFN--induced STAT1 phosphorylation. We have shown in Fig. 1C that phosphorylated STAT3 translocated to the nucleus after IL-6 treatment. To investigate if LLL12 would block this translocation, we pretreated cells with 5 M LLL12 for2hand then treated the cells with 50 ng/ml of IL-6 for 30 min. The cells were fixed and stained by anti-phos- phorylated STAT3 primary anti- body and FITC conjugated second- ary antibody. The nucleus was stained with DAPI. As illustrated in Fig. 5C, LLL12 pretreated cells did not show phosphorylated STAT3 in the nucleus. Nonactivated STAT3 is in the cytoplasm. STAT3 translo- FIGURE 4. Inhibition of phosphorylated STAT3 blocks IL-6-induced cell survival. A, two siRNAs targeting STAT3 were transfected into Hep3B cells. After overnight incubation, the cells were cultured in serum-free cates to the nucleus when it is acti- medium for 24 h followed by IL-6 for 30 min. Phosphorylated STAT3, phosphorylated AKT, and GAPDH were vated (36). To confirm if the phos- detected by Western blot. B, STAT3 siRNA-transfected Hep3B cells were treated with 2 M doxorubicin for 2 h phorylation induced by IL-6 would followed by 50 ng/ml of IL-6 for 6 h. Morphological examination was performed after treatment. C, STAT3 siRNA-transfected Hep3B cells were pretreated with doxorubicin for 2 h followed by 50 ng/ml of IL-6 for 24 h. translocate STAT3 to the nucleus Caspase3/7 activity was assayed. The data showed the percent increase in caspase3/7 compared with scram- and if LLL12 pretreatment could bled siRNA-transfected control groups and represented three independent experiments. *, p  0.02. reverse this process, we cultured phorylated AKT and GAPDH were used as controls, which Hep3B cells in serum-free medium for 24 h. Then, the were not affected by STAT3 siRNA. starved cells were pretreated with 5 M LLL12 for 2 h fol- To investigate if knocking down STAT3 would decrease the lowed by 50 ng/ml of IL-6 for 30 min. The cells were stained anti-apoptotic effect of IL-6, STAT3 siRNA-transfected cells by anti-STAT3 primary antibody and secondary antibody. were treated under the similar conditions employed in Fig. 4A. Fig. 5D demonstrated that STAT3 was in the cytoplasm As seen in Fig. 4B, STAT3 siRNA-transfected cells exhibited when cultured in serum-free medium. IL-6 treatment trans- more characteristic morphology of apoptosis induced by doxo- located STAT3 to the nucleus whereas LLL12 pretreatment rubicin compared with scrambled control siRNA transfected blocked IL-6-induced STAT3 nuclear translocation. cells. Caspase3/7 assay further showed that knocking down LLL12 Inhibits IL-6-induced STAT3 Phosphorylation in a STAT3 increased doxorubicin-induced apoptosis when treated Dose- and Time-dependent Manner—We further examined if with1or2 M doxorubicin (Fig. 4C). Therefore, the data in Fig. the inhibitory effect of LLL12 would be dose dependent. We 4 suggested that IL-6 promoted cell survival via the enhanced pretreated cells with different concentrations of LLL12 (0–5 STAT3 signaling. M) for 2 h and then treated the cells with 50 ng/ml of IL-6 for STAT3 Small Molecule Inhibitor Blocks IL-6/STAT3 Sig- 30 min. Fig. 6A demonstrated a LLL12 dose dependence. Expo- naling—To investigate if a STAT3 inhibitor could block IL-6- sure of cells to 0.5 M LLL12 was able to suppress STAT3 induced STAT3 phosphorylation, Hep3B and SNU-398 cells phosphorylation. AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27433 IL-6/STAT3 Promotes Cell Survival FIGURE 5. A STAT3 small molecule inhibitor LLL12 blocks IL-6-induced STAT3 phosphorylation. A, Hep3B and SNU-398 cells were cultured in serum-free medium for 24 h and then were pretreated with DMSO or LLL12 for 2 h followed by IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. B, Hep3B and SNU-398 cells were cultured in serum-free medium for 24 h and then were pretreated with LLL12 for 2 h followed by IFN- for 30 min. The expression levels of phosphorylated STAT1, and total STAT1 were analyzed by Western blot. C and D, Hep3B cells were treated under the same conditions, and the distribution of phosphorylated STAT3 and total STAT3 was analyzed by immunofluorescence. To study if the inhibitory effect of LLL12 would also be time min. As seen in Fig. 6C, STAT3 phosphorylation recovered in dependent, we pretreated cells with LLL12 for different time Hep3B cells 12 h after LLL12 treatment. The results suggested points (0–2 h) followed by 50 ng/ml of IL-6 for 30 min. As that the inhibitory effect of LLL12 on STAT3 phosphorylation illustrated in Fig. 6B, IL-6-induced STAT3 phosphorylation was reversible. We further blocked protein synthesis using was suppressed by LLL12 in a time-dependent manner. cycloheximide and repeated the experiment in Fig. 6C to exam- To examine if the inhibitory effect of LLL12 would be revers- ine if IL-6 would induce STAT3 phosphorylation 12 h after ible, we pretreated cells with 5 M LLL12 for 2 h. Then LLL12- LLL12 treatment. As illustrated in Fig. 6D, STAT3 phosphory- supplemented medium was removed and fresh medium with- lation recovered in cycloheximide-treated cells, indicating that out LLL12 was added to the cells. After different incubation the recovered STAT3 phosphorylation was not due to the new time points, the cells were treated with 50 ng/ml of IL-6 for 30 synthesized STAT3 protein. 27434 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival FIGURE 6. The STAT3 small molecule inhibitor LLL12 blocks STAT3 phosphorylation in a dose- and time-dependent manner. A, Hep3B and SNU-398 cells were cultured in serum-free medium for 24 h and then were pretreated with various concentrations of LLL12 (0.5–5 M) for 2 h followed by IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. B, Hep3B and SNU-398 cells were cultured under the same conditions and then were treated with 5 M LLL12 for different time points (0 –2 h). After pretreatment, IL-6 was added to the cultured cells. Phosphorylated STAT3 and total STAT3 were detected by Western blot. C, Hep3B cells were cultured in serum-free medium for 24 h and then were pretreated with 5 M LLL12 for 2 h. After the 2 h pretreatment, medium was discarded, and fresh medium without LLL12 was added. After the indicated incubation (0 –24 h), the cells were treated with IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. D, Hep3B cells were cultured in serum-free medium for 24 h and then were pretreated with 5 M LLL12 for 2 h. After2hof pretreatment, the medium was discarded, and fresh medium without LLL12 was added. Cycloheximide was also added into the medium to block protein synthesis. After 12 h, the cells were treated with IL-6 for 30 min. Phosphorylated STAT3 and total STAT3 were detected by Western blot. FIGURE 7. LLL12 decreases IL-6-induced cell survival. A, Hep3B cells were pretreated with 0.5 M LLL12 for 2 h. After the pretreatment, LLL12 was removed, and fresh medium was added. The cells were treated as indicated. After overnight treatment, morphological examination was performed. B, results from A were quantified by cell viability assay. C and D, Hep3B and SNU-398 cells were pretreated with 0.5 M LLL12 for 2 h. After the pretreatment, cells were treated with IL-6 for 30 min. The levels of JAK1, JAK2, GP130, p-AKT, and p-ERK1/2 were analyzed by Western blot. LLL12 Reduces the Anti-apoptotic Activity of IL-6—To inves- overnight. Fig. 7A clearly demonstrated that LLL12-pretreated tigate if a small molecule STAT3 inhibitor would decrease anti- cells showed more cell death compared with the cells without apoptotic activity of IL-6, we chose a low dose (0.5 M) of LLL12 LLL12 pretreatment. This result was quantified by cell viability to pretreat Hep3B cells for only 2 h. After pretreatment, LLL12 assay (Fig. 7B). LLL12 pretreatment completely blocked IL-6- was removed, and cells were treated with doxorubicin and IL-6 induced doxorubicin resistance. Interestingly, the 2 h pretreat- AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27435 IL-6/STAT3 Promotes Cell Survival FIGURE 8. Blockade of IL-6 or STAT3 reduces cell recovery from doxorubicin. SNU-449 cells were treated with 2M doxorubicin, anti-IL-6 antibody, or LLL12 as indicated. After overnight treatment, live cells were counted and 5,000 cells were re-plated. Cells were treated with control IgG, anti-IL-6 antibody, or LLL12, respectively. After 7 days, cells were fixed and stained with 1% crystal violet. A, pictures were captured under microscopy at different magnification. B, original plates were scanned. Three independent experiments were performed. ment with LLL12 did not cause cell death (Fig. 7, A and B). overnight treatment, live cells were counted, and 5,000 cells These data suggested that low dose of STAT3 inhibitor alone were re-plated. The cells were cultured in doxorubicin-free might not lead to cell death but was able to attenuate IL-6- medium for 7 days. Anti-IL-6 antibody or LLL12 was supple- induced cell survival upon drug treatment. mented in medium. After 7 days, cells were fixed and stained To explore if STAT3 inhibitor LLL12 would affect other with 1% crystal violet. Fig. 8 clearly showed that many cells components of IL-6/STAT3 signaling or other IL-6 signaling, recovered from control cells, whereas fewer cells recovered we pretreated Hep3B and SNU-398 cells with 0.5 M LLL12 for from anti-IL-6 antibody or LLL12-treated cells. The data in Fig. 2 h, followed by IL-6 for 30 min. JAK1, JAK2, GP130, phosphor- 8 suggested that blockade of IL-6 or STAT3 reduced cell recov- ylated AKT, and phosphorylated ERK were analyzed by West- ery from drug treatment. ern blot. Fig. 7, C and D showed that 0.5 M LLL12 did not affect Blockade of IL-6/STAT3 Does Not Affect Cell Viability in other proteins of IL-6 signaling pathways. The data in Fig. 7 Human Primary Hepatocytes Treated with Doxorubicin—To suggested that the low dose of STAT3 small molecule inhibitor investigate if blocking IL-6 or STAT3 would reduce viability in LLL12 attenuated IL-6-induced cell survival through specifi- normal liver cells, human primary hepatocytes were treated cally inhibiting STAT3 phosphorylation. with 500 ng/ml of anti-IL-6 antibody or 0.5 M LLL12 for 2 or Blockade of IL-6 or STAT3 Reduces Cell Recovery from 24 h, respectively. After 24 h, cell viability was measured. As Doxorubicin—To investigate if blocking IL-6 or STAT3 would shown in Fig. 9A, anti-IL-6 antibody or LLL12 did not reduce reduce cell recovery from doxorubicin, SNU-449 cells were cell viability compared with untreated cells. treated with 5 M doxorubicin alone, doxorubicin plus anti- We further examined if blockade of IL-6 or STAT3 signaling IL-6 antibody, or doxorubicin plus LLL12, respectively. After in human primary normal liver cells would reduce cell viability 27436 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival cellular carcinoma (HCC) is the most common liver tumor with a 7% five-year survival rate. It mainly occurs in males due to differences in IL-6 production between males and females (35). Moreover, obesity may increase the risk of liver and other cancer due to the chronic inflam- matory response caused by en- hanced levels of IL-6/STAT3 and TNF/STAT3 signaling (46). IL-6 is a key event in tumorigene- sis, but its biological function in liver cancers is not clear. Previous studies have demonstrated that IL-6/STAT3 provides hepatopro- tection against liver damage (47). Based on these results, we hypothe- sized that IL-6 would promote sur- vival of liver cancer cells via STAT3 upon drug treatment. We found that the endogenous levels of IL-6 in SNU-387 and SNU-449 liver cancer cell lines were much higher than those in human hepatocytes. Hep3B and SNU-398 cells showed similar FIGURE 9. Blockade of IL-6 or STAT3 does not affect cell viability in human primary hepatocytes treated levels of IL-6 to human hepatocytes. with doxorubicin. A, human primary hepatocytes were treated with 500 ng/ml of anti-IL-6 antibody or 0.5 M LLL12 for 2 h and 24 h, respectively. After 24 h, cell viability was measured. Non-treated cells (NT) were used as Because IL-6 levels in liver cancer control. The data represented three independent results. B, human primary hepatocytes were pretreated with patients are 25-fold higher than 500 ng/ml of anti-IL-6 antibody or 0.5 M LLL12 for 2 h. After the pretreatment, LLL12 was discarded and fresh medium with different concentrations of doxorubicin was added. After 24 h, cell viability was measured. The healthy adults (34), we wondered data represented three independent results. how Hep3B and SNU-398 would respond upon IL-6. We found that when treated with doxorubicin. Human primary hepatocytes IL-6 activated STAT3 in these two cell lines. To study the phe- were pretreated with 500 ng/ml of anti-IL-6 antibody or 0.5 M notype of IL-6 in liver cancer cells, we induced apoptosis in Hep3B cells using doxorubicin. Interestingly, exogenous addi- LLL12 for 2 h. After the pretreatment, LLL12 was discarded, tion of IL-6 protected cells toward apoptosis induced by doxo- and fresh medium with different concentrations of doxorubicin was added. After 24 h, cell viability was measured. Our results rubicin. To explore if IL-6-promoted cell survival upon doxo- showed that when doxorubicin concentration increased from rubicin would be caused by activated STAT3, we targeted STAT3 by siRNA and observed that STAT3 knocked down 0.5 to 1 M, cell viability decreased significantly. However, the cells showed higher level of apoptosis. When SNU-449 cells treatment by anti-IL-6 antibody or LLL12 with doxorubicin did not markedly reduce viability in human primary hepatocytes with enhanced endogenous IL-6 were treated with IL-6 anti- compared with the cells treated by doxorubicin alone (Fig. 9B), body, phosphorylated STAT3 was down-regulated. As a result, IL-6 antibody treated SNU-449 cells exhibited lowered cell via- indicating that unlike liver cancer cells, liver normal cells did bility, suggesting that IL-6 induced cell survival via STAT3. In not rely on IL-6/STAT3 signaling. human cervical cancer, IL-6 causes anti-apoptosis induced by DISCUSSION doxorubicin via PI3K/AKT instead of STAT3 (10). Therefore, Growing evidence has shown that inflammatory signals from IL-6 exerts its activity through different mechanisms the surrounding microenvironment promote tumor growth depending on cell types. In prostate cancer, IL-6 also causes and progression. They are also associated with survival path- anti-apoptosis via Mcl-1 (48). In pancreatic cancer, PANC-1 ways and immunosuppression (37–39). It has been well docu- cells, however, we found that IL-6 induced STAT3 in a dose- mented that inflammatory cytokine IL-6/STAT3 signaling is dependent manner, but it did not cause anti-apoptosis (data involved in many types of human cancer (2, 6, 7, 9, 40–42). IL-6 not shown), suggesting that the anti-apoptotic effect of IL-6 is can promote cell proliferation in multiple myelomas, ovarian cell type-specific. carcinoma and prostate cancer cells in vitro (41, 43, 44) and Treatment with anti-IL-6 monoclonal antibody or with len- tumor growth in vivo in prostate, lung, and breast (37, 44). It is tivirus expressing shSTAT3 has been shown to induce apopto- also related to cell migration and adhesion (45). sis in prostate cancer and glioblastoma multiforme in vitro or in Liver cancer is a major health problem because of the poor vivo (15, 49, 50). To study if STAT3 small molecule inhibitor prognosis. IL-6 plays a critical role in liver cancer (34). Hepato- would show similar effect to STAT3 siRNA and IL-6 antibody AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27437 IL-6/STAT3 Promotes Cell Survival 10. Wei, L. H., Kuo, M. L., Chen, C. A., Chou, C. H., Cheng, W. F., Chang, on IL-6/STAT3 signaling in liver cancer cells, we used a STAT3 M. C., Su, J. L., and Hsieh, C. Y. (2001) Oncogene 20, 5799–5809 small molecule inhibitor, which was reported by our laboratory 11. Wei, L. H., Kuo, M. L., Chen, C. A., Chou, C. H., Lai, K. B., Lee, C. N., and (51). Our previous results show that high concentration of this Hsieh, C. Y. (2003) Oncogene 22, 1517–1527 STAT3 small molecule inhibitor may cause apoptosis in differ- 12. Lin, M. T., Juan, C. Y., Chang, K. J., Chen, W. J., and Kuo, M. L. (2001) ent types of human cancer with elevated STAT3. In this study, Carcinogenesis 22, 1947–1953 we demonstrated that this STAT3 small molecule inhibitor 13. Leu, C. M., Wong, F. H., Chang, C., Huang, S. F., and Hu, C. P. (2003) Oncogene 22, 7809–7818 blocked IL-6-induced STAT3 phosphorylation in a dose-de- 14. Meng, F., Yamagiwa, Y., Ueno, Y., and Patel, T. (2006) J. Hepatol 44, pendent manner. Low concentration of LLL12 did not cause 1055–1065 cell death whereas it attenuated IL-6-induced cell survival upon 15. Smith, P. C., and Keller, E. T. (2001) Prostate 48, 47–53 doxorubicin treatment via down-regulation of phosphorylated 16. Liu, S., Ishikawa, H., Li, F. J., Ma, Z., Otsuyama, K., Asaoku, H., Abroun, S., STAT3. Our data indicate that blockade of STAT3 signaling Zheng, X., Tsuyama, N., Obata, M., and Kawano, M. M. (2005) Cancer Res. with STAT3 small molecule inhibitor may reduce IL-6-induced 65, 2269–2276 17. Liu, S., Ma, Z., Cai, H., Li, Q., Rong, W., and Kawano, M. (2010) Eur. J. anti-apoptosis. Haematol. 84, 137–144 Although LLL12 is a STAT3 structure-based molecule, we 18. Yu, C. Y., Wang, L., Khaletskiy, A., Farrar, W. L., Larner, A., Colburn, wondered if LLL12 could affect other components of IL-6/ N. H., and Li, J. J. (2002) Oncogene 21, 3949–3960 STAT3 signaling pathway. We examined JAK1, JAK2, and 19. Bromberg, J. F., Wrzeszczynska, M. H., Devgan, G., Zhao, Y., Pestell, R. G., GP130. JAK1 and JAK2 phosphorylate STAT3 (52, 53). GP130 Albanese, C., and Darnell, J. E., Jr. (1999) Cell 98, 295–303 20. Yu, H., Kortylewski, M., and Pardoll, D. (2007) Nat. Rev. Immunol. 7, 41–51 provides docking site for STAT3 (52). LLL12 did not change 21. Kortylewski, M., Xin, H., Kujawski, M., Lee, H., Liu, Y., Harris, T., Drake, JAK1, JAK2, or GP130. IL-6 can also activate PI3K and ERK C., Pardoll, D., and Yu, H. (2009) Cancer Cell 15, 114–123 signaling pathways (9), but LLL12 did not alter them either. In 22. Yu, H., Pardoll, D., and Jove, R. (2009) Nat. Rev. Cancer 9, 798–809 conclusion, IL-6 could protect human liver cancer cells toward 23. Choudhari, S. R., Khan, M. A., Harris, G., Picker, D., Jacob, G. S., Block, T., apoptosis via STAT3 signaling. Targeting STAT3 with STAT3 and Shailubhai, K. (2007) Mol. Cancer Ther. 6, 112–121 siRNA, anti-IL-6 antibody, or a STAT3 small molecule inhibi- 24. Real, P. J., Sierra, A., De Juan, A., Segovia, J. C., Lopez-Vega, J. M., and Fernandez-Luna, J. L. (2002) Oncogene 21, 7611–7618 tor may decrease or block IL-6-induced liver cancer cell sur- 25. Epling-Burnette, P. K., Liu, J. H., Catlett-Falcone, R., Turkson, J., Oshiro, vival upon drug treatment. M., Kothapalli, R., Li, Y., Wang, J. M., Yang-Yen, H. F., Karras, J., Jove, R., Short term and long term IL-6 exposure show paradoxical and Loughran, T. P., Jr. (2001) J. Clin. Invest. 107, 351–362 effects on liver injury and repair (54). Short term production of 26. Aoki, Y., Feldman, G. M., and Tosato, G. (2003) Blood 101, 1535–1542 IL-6 is a natural response of the body during the liver injury to 27. Amin, H. M., McDonnell, T. J., Ma, Y., Lin, Q., Fujio, Y., Kunisada, K., protect cells (35). In contrast, long term exposure to IL-6 can Leventaki, V., Das, P., Rassidakis, G. Z., Cutler, C., Medeiros, L. J., and Lai, R. (2004) Oncogene 23, 5426–5434 impair liver regeneration and even increase liver injury (54). 28. Gritsko, T., Williams, A., Turkson, J., Kaneko, S., Bowman, T., Huang, M., Our results also demonstrated that ablation of IL-6/STAT3 in Nam, S., Eweis, I., Diaz, N., Sullivan, D., Yoder, S., Enkemann, S., Eschrich, human primary hepatocytes did not affect their viability under S., Lee, J. H., Beam, C. A., Cheng, J., Minton, S., Muro-Cacho, C. A., and normal conditions and did not induce more cell death when Jove, R. (2006) Clin. Cancer Res. 12, 11–19 treated with chemotherapeutic agents. 29. Niu, G., Wright, K. L., Huang, M., Song, L., Haura, E., Turkson, J., Zhang, In conclusion, IL-6/STAT3 signaling may be not crucial in S., Wang, T., Sinibaldi, D., Coppola, D., Heller, R., Ellis, L. M., Karras, J., Bromberg, J., Pardoll, D., Jove, R., and Yu, H. (2002) Oncogene 21, human primary hepatocytes. However, this signaling can pro- 2000–2008 mote hepatocellular carcinoma survival upon drug treatment. 30. Wei, D., Le, X., Zheng, L., Wang, L., Frey, J. A., Gao, A. C., Peng, Z., Huang, Blockade of IL-6/STAT3 may be able to render hepatocellular S., Xiong, H. Q., Abbruzzese, J. L., and Xie, K. (2003) Oncogene 22, carcinoma with elevated endogenous IL-6/STAT3 more sensi- 319–329 tive to chemotherapeutic agent-induced apoptosis. 31. Xu, Q., Briggs, J., Park, S., Niu, G., Kortylewski, M., Zhang, S., Gritsko, T., Turkson, J., Kay, H., Semenza, G. L., Cheng, J. Q., Jove, R., and Yu, H. REFERENCES (2005) Oncogene 24, 5552–5560 32. Gao, L., Zhang, L., Hu, J., Li, F., Shao, Y., Zhao, D., Kalvakolanu, D. V., 1. Hodge, D. R., Hurt, E. M., and Farrar, W. L. (2005) Eur. J. Cancer 41, Kopecko, D. J., Zhao, X., and Xu, D. Q. (2005) Clin. Cancer Res. 11, 2502–2512 6333–6341 2. Grivennikov, S., Karin, E., Terzic, J., Mucida, D., Yu, G. Y., Vallabha- 33. Hodge, D. R., Li, D., Qi, S. M., and Farrar, W. L. (2002) Biochem. Biophys. purapu, S., Scheller, J., Rose-John, S., Cheroutre, H., Eckmann, L., and Res. Commun. 292, 287–291 Karin, M. (2009) Cancer Cell 15, 103–113 34. Porta, C., De Amici, M., Quaglini, S., Paglino, C., Tagliani, F., Boncimino, 3. Schafer, Z. T., and Brugge, J. S. (2007) J. Clin. Invest. 117, 3660–3663 A., Moratti, R., and Corazza, G. R. (2008) Ann. Oncol. 19, 353–358 4. Bharti, A. C., Donato, N., and Aggarwal, B. B. (2003) J. Immunol. 171, 35. Naugler, W. E., Sakurai, T., Kim, S., Maeda, S., Kim, K., Elsharkawy, A. M., 3863–3871 and Karin, M. (2007) Science 317, 121–124 5. Sansone, P., Storci, G., Tavolari, S., Guarnieri, T., Giovannini, C., Taf- 36. Darnell, J. E., Jr. (1997) Science 277, 1630–1635 furelli, M., Ceccarelli, C., Santini, D., Paterini, P., Marcu, K. B., Chieco, P., 37. Bromberg, J., and Wang, T. C. (2009) Cancer Cell 15, 79–80 and Bonafe`, M. (2007) J. Clin. Invest. 117, 3988–4002 38. Balkwill, F., and Mantovani, A. (2001) Lancet 357, 539–545 6. Gao, S. P., Mark, K. G., Leslie, K., Pao, W., Motoi, N., Gerald, W. L., Travis, 39. Grivennikov, S., and Karin, M. (2008) Cancer Cell 13, 7–9 W. D., Bornmann, W., Veach, D., Clarkson, B., and Bromberg, J. F. (2007) 40. Spiotto, M. T., and Chung, T. D. (2000) Prostate 42, 186–195 J. Clin. Invest. 117, 3846–3856 41. Rabinovich, A., Medina, L., Piura, B., Segal, S., and Huleihel, M. (2007) 7. Lou, W., Ni, Z., Dyer, K., Tweardy, D. J., and Gao, A. C. (2000) Prostate 42, Anticancer Res. 27, 267–272 239–242 42. Bollrath, J., Phesse, T. J., von Burstin, V. A., Putoczki, T., Bennecke, M., 8. Spiotto, M. T., and Chung, T. D. (2000) Prostate 42, 88–98 Bateman, T., Nebelsiek, T., Lundgren-May, T., Canli, O., Schwitalla, S., 9. Culig, Z., Steiner, H., Bartsch, G., and Hobisch, A. (2005) J. Cell. Biochem. Matthews, V., Schmid, R. M., Kirchner, T., Arkan, M. C., Ernst, M., and 95, 497–505 27438 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 285 • NUMBER 35 •AUGUST 27, 2010 IL-6/STAT3 Promotes Cell Survival Greten, F. R. (2009) Cancer Cell 15, 91–102 49. Kudo, M., Jono, H., Shinriki, S., Yano, S., Nakamura, H., Makino, K., Hide, 43. Kawano, M., Hirano, T., Matsuda, T., Taga, T., Horii, Y., Iwato, K., Asaoku, T., Muta, D., Ueda, M., Ota, K., Ando, Y., and Kuratsu, J. (2009) J. Neuro- H., Tang, B., Tanabe, O., and Tanaka, H. (1988) Nature 332, 83–85 surg 111, 219–225 44. Malinowska, K., Neuwirt, H., Cavarretta, I. T., Bektic, J., Steiner, H., Di- 50. Li, G. H., Wei, H., Chen, Z. T., Lv, S. Q., Yin, C. L., and Wang, D. L. (2009) etrich, H., Moser, P. L., Fuchs, D., Hobisch, A., and Culig, Z. (2009) Endocr J. Neurooncol. 91, 165–174 Relat Cancer 16, 155–169 51. Lin, L., Hutzen, B., Li, P. K., Ball, S., Zuo, M., DeAngelis, S., Foust, E., Sobo, 45. Santer, F. R., Malinowska, K., Culig, Z., and Cavarretta, I. T. (2010) Endocr. M., Friedman, L., Bhasin, D., Cen, L., Li, C., and Lin, J. (2010) Neoplasia 12, Relat. Cancer 17, 241–253 39–50 46. Park, E. J., Lee, J. H., Yu, G. Y., He, G., Ali, S. R., Holzer, R. G., Osterreicher, 52. Heinrich, P. C., Behrmann, I., Mu¨ller-Newen, G., Schaper, F., and Graeve, C. H., Takahashi, H., and Karin, M. (2010) Cell 140, 197–208 L. (1998) Biochem. J. 334, 297–314 47. Taub, R. (2003) J. Clin. Invest. 112, 978–980 53. Heinrich, P. C., Behrmann, I., Haan, S., Hermanns, H. M., Mu¨ller-Newen, 48. Cavarretta, I. T., Neuwirt, H., Untergasser, G., Moser, P. L., Zaki, M. H., G., and Schaper, F. (2003) Biochem. J. 374, 1–20 Steiner, H., Rumpold, H., Fuchs, D., Hobisch, A., Nemeth, J. A., and Culig, 54. Jin, X., Zimmers, T. A., Perez, E. A., Pierce, R. H., Zhang, Z., and Koniaris, Z. (2007) Oncogene 26, 2822–2832 L. G. (2006) Hepatology 43, 474–484 AUGUST 27, 2010• VOLUME 285 • NUMBER 35 JOURNAL OF BIOLOGICAL CHEMISTRY 27439

Journal

Journal of Biological Chemistry – American Society for Biochemistry and Molecular Biology

Published: Aug 27, 2010

Keywords: Apoptosis; Drug Resistance; Interleukin; Liver; STAT Transcription Factor

References