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/lp/pubmed-central/diosmetin-suppresses-human-prostate-cancer-cell-proliferation-through-1vjDQjGtvP
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- Publisher
- Pubmed Central
- Copyright
- Copyright © 2018, Spandidos Publications
- ISSN
- 1019-6439
- eISSN
- 1791-2423
- DOI
- 10.3892/ijo.2018.4407
- Publisher site
- See Article on Publisher Site
Abstract
INTERNATIONAL JOURNAL OF ONCOLOGY 53: 835-843, 2018 Diosmetin suppresses human prostate cancer cell proliferation through the induction of apoptosis and cell cycle arrest 1,2 1-3 1,2 1,2 CHRISTINE OAK , AHMAD O. KHALIFA , ILAHA ISALI , NATARAJAN BHASKARAN , 4 1,2 ETHAN WALKER and SANJEEV SHUKLA Department of Urology, Case Western Reserve University, School of Medicine; The Urology Institute, University Hospitals Cleveland Medical Center, Cleveland, OH 44106, USA; Department of Urology, Menoufia University, Shebin Al Kom, Menoufia 32519, Egypt; Department of Biomedical Engineering, Case Western Reserve University, Cleveland, OH 44106, USA Received December 8, 2017; Accepted March 30, 2018 DOI: 10.3892/ijo.2018.4407 Abstract. Diosmetin, a plant flavonoid, has been shown cell cycle regulation. The phosphorylation of specic fi kinase to exert promising effects on prostate cancer cells as an regulates Cdks and cyclin proteins (1). Additionally, c-Myc anti-proliferative and anticancer agent. In this study, using activation promotes the induction of cyclin D1 transcription western blot analysis for protein expression and o fl w cytometry through Cdk-specic fi kinase (2). Activated cyclin D1 forms for cell cycle analysis, we determined that the treatment of the complexes with Cdk4 and Cdk6, and these complexes act in LNCaP and PC-3 prostate cancer cells with diosmetin resulted the early G1 phase of the cell cycle; conversely, cyclin E forms in a marked decrease in cyclin D1, Cdk2 and Cdk4 expression complexes with Cdk2 and act in the late G1 phase and S phase levels (these proteins remain active in the G -G phases of the of the cell cycle (3). Much attention has been paid towards the 0 1 cell cycle). These changes were accompanied by a decrease in identic fi ation of novel agents with the ability to modulate the Kip1 c-Myc and Bcl-2 expression, and by an increase in Bax, p27 cell cycle by altering Cdks and acting as Cdk inhibitors in and FOXO3a protein expression, which suggests the potential human cancers (4,5). There is a need for the development of modulatory effects of diosmetin on protein transcription. The effective treatment agents with less to no toxicity. The majority treatment of prostate cancer cells with diosmetin set in motion of the anticancer agents are associated with side-effects or an apoptotic machinery by inhibiting X-linked inhibitor of drug-related toxicities, which limits their uses (6,7). Anticancer apoptosis (XIAP) and increasing cleaved PARP and cleaved strategies should ideally involve the use of an effective agent caspase-3 expression levels. On the whole, the findings of this with the ability to modulate cell cycle regulatory molecules study provide an in-depth analysis of the molecular mecha- without affecting normal cells. Diosmetin is an O-methylated nisms responsible for the regulatory effects of diosmetin on flavone (3',5,7-trihydroxy-4'-methoxyflavone) abundantly key molecules that perturb the cell cycle to inhibit cell growth, present in legumes and olive leaves, and has shown to have and suggest that diosmetin may prove to be an effective anti- potential for use as an anticancer agent (8-10). Diosmetin cancer agent for use in the treatment of prostate cancer in the selectively induces apoptosis and inhibits cancer cell growth future. without affecting normal cells (10,11). It has been suggested that diosmetin exerts growth inhibitory effects through Introduction various signal transduction pathways, which have relevance in cancer (12-14). Some studies have suggested that diosmetin The most common and slow-growing cancer in males is exerts anti-carcinogenic effects on cancer cells through the prostate cancer. The prerequisites for any type of cancer, induction of apoptosis (9,10,14). Recently in an in vivo model including prostate cancer are increased cell viability and cell system, diosmetin treatment was shown to delay acute myeloid survival, traits which are inherited through the dysregulation leukemia tumor progression (15). Various kinases and proteins of cell cycle events. Cyclin-dependent kinases (Cdks) and have been reported as the potential sites at which diosmetin their modulatory cyclin partners are the key molecules of funtions, and these include the c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and AKT signaling pathways, as well as the p53/p21 pathways (9,10,16). Diosmetin-induced cell growth arrest has been shown to be associated with observed a marked decrease in cyclin activity Correspondence to: Dr Sanjeev Shukla, Department of Urology, Cip1 and Cdk levels, with an increase in the levels of p21 /p53 Case Western Reserve University, School of Medicine, 10900 Euclid and Cdk inhibitor expressions (16). Moreover, it has been Avenue, Cleveland, OH 44106, USA suggested that diosmetin exerts anticancer effects on hepa- E-mail: sanjeev.shukla@case.edu tocellular carcinoma cells, partly through the p53 enzyme, Key words: prostate cancer cells, diosmetin, growth inhibition which regulates cytochrome P450 CYP1A (9). However, the role of diosmetin in prostate cancer has not yet been fully OAK et al: DIOSMETIN SUPPRESSES PROSTATE CANCER CELL PROLIFERATION elucidated. Thus, the present study aimed to investigate the role of diosmetin in the cell cycle machinery and the induction of the apoptosis of prostate cancer cells. Material and methods Reagents and cell lines. Diosmetin (>99% purity; chemical structure shown in Fig. 1), 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and propidium iodide (PI) were obtained from Sigma-Aldrich; Merck KGaA (Darmstadt, Germany). The prostate cancer cells (LNCaP Figure 1. Structure of diosmetin; 3',5,7-trihydroxy-4'-methoxya fl vone. and PC-3) were purchased from the American Type Culture Collection (Manassas, VA, USA). The RPMI-1640 medium used to culture the cells, phosphate-buffered saline (PBS) and trypsin were purchased from Thermo Fisher Scientific (Waltham, into the major groove of double-stranded DNA and produces MA, USA). The reagents used for western blot analysis were a highly-u fl orescent adduct that can be excited at 488 nm with purchased from Bio-Rad Laboratories (Hercules, CA, USA). broad emission centered around 600 nm (17). Cell culture and treatment. Both human prostate androgen- Western blot analysis. Total cell lysate was prepared using dependent cancer cells (LNCaP) and human prostate lysis buffer containing 10 µg/ml phenylmethylsulfonyl u fl o - androgen-independent cancer cells (PC-3) were cultured ride (PMSF), 20 µg/ml protease inhibitor cocktail (PIC), in RPMI-1640 medium with 10 and 5% fetal bovine serum, 20 µg/ml sodium u fl oride and 10 µg/ml sodium orthovanadate respectively. The cells were treated with diosmetin at various (Na VO ). The protein concentration in the cell lysate was 3 4 concentrations (5-80 µM) for various periods of time (24, 48 estimated using Lowry protein assay and 25 µg of protein and 72 h). The cells were grown at 37˚C with 5% CO /95% air was aliquoted to run a sodium dodecyl sulfate polyacrylamide and were passaged every 48 h using trypsin-ethylene-diamine- gel electrophoresis (SDS-PAGE) using a 4-20% Tri-glycine tetra-acetic acid (EDTA). gel. Data from the gel were transferred onto a nitrocel- lulose membrane and incubated with the anti-cyclin D1 Cell viability assay (MTT assay). The PC-3 or LNCaP cells (sc-450), anti-Cdk2 (sc-6248), anti-Cdk4 (sc-260), anti-p27 were seeded into 96-well a fl t-bottomed plates at 25% conu fl - (sc-393380), anti-GAPDH (sc-166545), anti-Bax (sc-493), ency. After 24 h of normal growth, the medium was carefully anti-Bcl2 (sc-7382), anti-c-Myc (sc-40), anti-cyclin E (sc-248) removed without disturbing the cells and the cells were treated were procured from Santa Cruz Biotechnology (Santa Cruz, with diosmetin at various concentrations (0, 2.5, 5, 10, 20, 40 CA, USA). Additionally anti-XIAP (#2042), anti-FOXO3a and 80 µM) and sterile dimethyl sulfoxide (DMSO) used as (#12829), anti-cleaved caspase-3 (#9661), anti-cleaved PARP a vehicle control. Each drug dosage was repeated 12 times (#5625) procured from Cell Signaling Technology (Danvers, within the 96-well plate. After the cells were treated for various MA, USA) and anti-β-actin (#A1978) were procured from periods of time (24, 48 and 72 h), the treatment was terminated. Sigma-Aldrich. Secondary antibodies used were anti-mouse MTT was dissolved in sterile DMSO at 5 mg/ml and a working (#HAF007) anti-rabbit (#HAF008) from R&D Systems solution was made of 1 ml from stock MTT:4 ml media. Since (Minneapolis, MN, USA). All antibodies were diluted to MTT is light-sensitive, the procedure was performed in the 1:1,000 dilution of stock solution of 1 mg/ml. Densitometric presence of very little light. The working solution was added analysis of the protein bands were performed using the Kodak to the cells followed by incubation at 37˚C for 3 h to form 2000R imaging system digitalized scientic fi software program. formazan crystals, which are purple-colored. Subsequently, the MTT solution was removed and the cells/crystals were Statistical analysis. Each experiment was performed at least solubilized with DMSO and shaken for 10 min. The results in triplicate. The results are expressed as the means ± standard were read at 540 nm using a microplate reader with a built-in deviation. Statistical analysis was performed using analysis of UV/VIS spectrophotometer (FLUOstar Omega Microplate variance (ANOVA) with a post hoc Fisher's LSD test between Reader, BMG Labtech, Cary, NC, USA). IC values were the treated and the control groups. As there were multiple determined to the concentration of diosmetin to reduce by 50% groups for the time points and diosmetin concentrations, we the growth of the treated cells in 24 h. Data were analyzed to therefore also applied Kruskal-Wallis tests and the normality determine the percentage of cells unaffected by treatment and test to confirm that there was no difference. A P-value <0.05 the standard deviation between the 12 different trials. was considered to indicate a statistically signic fi ant difference. Cell cycle analysis (flow cytometry). The untreated and Results treated LNCaP and PC-3 cells were fixed by using cold 100% methanol and stored at -20˚C indefinitely. The cells were then Prostate cancer cell growth inhibition by diosmetin. Treatment removed from the methanol and added to RNAse solution that of the prostate cancer cells, LNCaP (androgen-responsive) included sodium azide, EDTA and PBS. This was followed and PC-3 cells (androgen-refractory), with diosmetin exerted by incubation at 37˚C for 15-30 min and chilling at 4˚C for diosmetin dose-dependent (0-80 µM) inhibitory effects on 10 min. PI was then used to stain the cells, since it intercalates cell growth, compared to vehicle (DMSO-treated) controls INTERNATIONAL JOURNAL OF ONCOLOGY 53: 835-843, 2018 Figure 2. Human prostate cancer cell growth inhibition by diosmetin. Asynchronous (A) LNCaP and (B) PC-3 cells were treated with diosmetin at increasing concentrations and exposure times and later subjected to MTT assay. Values are represented as the means ± SD from 3 independent experiments. P<0.05 for time points vs. vehicle (DMSO) controls. Figure 3. DNA content cell cycle analysis profiling of (A) LNCaP and (B) PC-3 cells following diosmetin treatment. These cells were initially treated with the vehicle (DMSO-treated) or with increasing doses of diosmetin for 24 h, stained with dye (propidium iodide) and analyzed by o fl w cytometry. Cell percent - ages were calculated using cell t fi computer software in G -G , S and G -M phases and values were plotted as averages from 3 different sets of experiments 0 1 2 performed in duplicate. P<0.05 for G -G phase vs. control. 0 1 by MTT assay. The magnitude of cell growth inhibition in the G -G phase to 61 and 65%, respectively (Fig. 3A). In the 0 1 was higher in the LNCaP cells compared to the PC-3 cells. PC-3 cells, we observed evident S phase arrest following dios- Diosmetin exerted clear dose-dependent inhibitory effects metin treatment. Treatment of the PC-3 cells with diosmetin at on prostate cancer cell growth. Moreover, we observed that 5 µM resulted in 24% of the cells being arrest at the S phase; diosmetin also suppressed prostate cancer cell growth in a at the concentrations of 10, 20 and 40 µM, diosmetin led to 26, time-dependent manner; the effects were more noticeable at 29 and 30% of the cells being arrested at the S phase compared 48 and 72 h of treatment than at 24 h (Fig. 2). to the vehicle-treated controls (22%) (Fig. 3B). The dose- dependent increase in the S phase cell population of the PC-3 Cell cycle arrest following diosmetin treatment. Using flow cells induced by diosmetin was associated with a concomitant cytometric analysis, we assessed whether the cell growth decrease in the number of cells in the G -G phase, whereas 0 1 inhibitory effects induced by diosmetin are mediated through opposite effects were observed in the LNCaP cells. These alterations in the cell cycle. As we observed the marked differences may be due to the different origin of the cell lines, inhibitory effects of diosmetin on cell growth, we further as the LNCaP cells are androgen-sensitive and the PC-3 cells treated the asynchronous LNCaP and PC-3 cells with various are androgen refractory. concentrations (0-40 µM) of diosmetin for 24 h to determine the DNA content at various stages of the cell cycle. The vehicle Alterations in cell cycle regulatory molecules following (DMSO-treated) control LNCaP cells represented ~50% of the diosmetin dose-response treatment. Diosmetin induced the cells that were in the G -G phase of the cell cycle. Following downregulation of cyclin D1, cyclin E, Cdk2 and Cdk4 levels. 0 1 treatment with increasing concentrations of diosmetin, we Diosmetin at concentrations of 10 and 20 µM suppressed the observed that the cell number in the G -G phase increased, growth of the LNCaP and PC-3 cells. These effective diosmetin 0 1 evidenced by an increased accumulation of cells in the concentrations were also able to decrease Cdk expression levels G -G phase. Treatment with diosmetin at 5 µM resulted in 55% in the LNCaP cells at IC ± SD 10±2 µM and whereas, in PC-3 0 1 50 of the cells being arrest in the G -G phase of the cell cycle, cells it was 18±5 µM. These results suggest that diosmetin may 0 1 whereas treatment with 10 µM diosmetin further increased the be responding more like a Cdk inhibitor; however, other mech- number of cells in the G -G phase to 58%. At the concentra- anisms may be involved. We further evaluated the diosmetin 0 1 tions of 20 and 40 µM, diosmetin increased the number of cells dose-response effect on the expression levels of cyclin D1, OAK et al: DIOSMETIN SUPPRESSES PROSTATE CANCER CELL PROLIFERATION Figure 4. Dose-response effects of diosmetin on cell cycle regulatory molecules. (A) LNCaP and PC-3 cells; both asynchronous cells were exposed to increasing concentrations (0, 5, 10 and 20 µM) of diosmetin for 24 h. Total cell lysates were prepared and using SDS-PAGE, lysates proteins were resolved Kip1 and then subjected to western blot analysis for the evaluation of cyclin D1, cyclin E, Cdk2, Cdk4 and p27 . Lanes marked with ‘0’ are the DMSO control (0.2%)-treated cells. For protein band density, densitometric analysis was performed and proteins expression levels were correlated to express relative con- trols (Β and C). For loading control blots were stripped and reprobed with GAPDH. P<0.05 for diosmetin concentration vs. vehicle controls. cyclin E, Cdk2 and Cdk4, operating in the G and S phase of the modulatory molecules, cyclin D1, cyclin E, Cdk2, Cdk4 and Kip1 cell cycle machinery by protein western blot analysis (Fig. 4A). p27 (Fig. 5A). Diosmetin time-response treatment (20 µM) A decreased cyclin D1 expression was observed in the LNCaP of the LNCaP cells led to a decrease in cyclin D1 expression cells following treatment with diosmetin (37.61% at 5 µM compared to the controls (6 h, 55.33%; 12 h, 72.88%; and 24 h, and 37.35% at 10 µM). In the PC-3 cells, cyclin E levels also 44.16%); in the PC-3 cells, a decrease was also observed in decreased following treatment with diosmetin (26.34% at the cyclin E levels (57.92% at 24 h). Both the Cdk2 and Cdk4 5 µM, 19.07% at 10 µM and 13.95% at 20 µM). We further levels were also markedly decreased in these cells treated with observed that the expression levels of Cdk2 and Cdk4 signifi - diosmetin. In the LNCaP cells, diosmetin treatment resulted in cantly decreased following diosmetin treatment of the prostate a decreased Cdk2 expression (53.72% at 6 h, 64.52% at 12 h cancer cells. In the LNCaP cells, diosmetin treatment resulted and 56.93% at 24 h). Similarly in the PC-3 cells, a decrease in a decreased Cdk2 expression (81.74% at 10 µM and 54.49% was observed in Cdk2 expression (63.39% at 6 h and 29.76% at 20 µM). Similarly in the PC-3 cells, Cdk2 expression also at 12 h). Moreover in the LNCaP cells treated with diosmetin, decreased following treatment with diosmetin (50.88% at Cdk4 expression decreased (25.18% at 6 h, 8.77% at 12 h 5 µM, 45.56% at 10 µM and 34.61% at 20 µM). Moreover, in the and 51.57% at 24 h). In the PC-3 cells, a decrease was also LNCaP cells following diosmetin treatment, Cdk4 expression observed in Cdk4 expression (40.54% at 12 h and 77.97% at decreased (63.96% at 5 µM, 14.79% at 10 µM and 40.07% at 24 h) (Fig. 5B and C). Similar to the diosmetin dose-response 20 µM); a decrease in Cdk4 expression was also observed in decrease in the levels of cyclins, diosmetin time-response the PC-3 cells (89.28% at 5 µM, 75.15% at 10 µM and 50% at treatment also decreased the expression levels of Cdk2 and 20 µM) (Fig. 4B and C). The Cdk2 and Cdk4 expression levels Cdk4 compared to the controls. Kip1 were decreased compared with the controls. Additionally p27 expression in the LNCaP cells increased K ip1 Additionally p27 expression in the LNCaP cells signic fi antly following diosmetin 20 µM time-response treat- increased following diosmetin dose-response treatment ment, by ~4-fold at 6 h, >3-fold at 12 h and ~9-fold at 24 h. Kip1 (67.33% at 5 µM, 143.33% at 10 µM and 109.77% at 20 µM). Similarly in the PC-3 Cells, p27 expression increased K ip1 Similarly in the PC-3 cells, p27 expression increased (83.33% at 6 h, >8-fold at 12 h and 5-fold at 24 h) compared to (143.54% at 5 µM, 171.77% at 10 µM and 153.22% at 20 µM) the vehicle-treated controls. compared to the vehicle-treated controls (Fig. 4B and C). Diosmetin induces the apoptotic response in prostate Modulation of cell cycle regulatory molecules following cancer cells. Treatment of the LNCaP and PC-3 cells with diosmetin time-response treatment. We then examined the increasing concentrations of diosmetin altered the levels of diosmetin time-dependent treatment effects on the cell cycle the apoptosis-related molecules, Bax, Bcl-2, cleaved caspase-3 INTERNATIONAL JOURNAL OF ONCOLOGY 53: 835-843, 2018 Figure 5. Time-response effects of diosmetin on cell cycle regulatory molecules. (A) LNCaP and PC-3 cells; both asynchronous cells were exposed to increasing times (0, 6, 12 and 24 h) of diosmetin (20 µM). Total cell lysates were prepared and using SDS-PAGE, lysates proteins were resolved and then Kip1 subjected to western blot analysis for the evaluation of cyclin D1, cyclin E, Cdk2, Cdk4 and p27 . Lanes marked ‘0’ are the DMSO control (0.2%)-treated cells. For protein band density, densitometric analysis was performed and protein expression levels were correlated to express relative controls (B and C). For loading control blots were stripped and reprobed with GAPDH. P<0.05 for time points vs. vehicle controls. and cleaved PARP (Fig. 6A). Diosmetin dose-response treat- In this study, we observed that diosmetin dose-response treat- ment of the LNCaP cells increased Bax expression 1.5-fold at ment of the LNCaP and PC-3 cells significantly decreased 10 µM and ~2-fold at 20 µM. In the PC-3 cells Bax expression c-Myc protein expression. Following treatment of the LNCaP also increased (110.33% at 10 µM and 103.29% at 20 µM). cells with diosmetin, c-Myc expression decreased (~27.16% at Conversely, in the LNCaP cells, Bcl-2 expression levels 10 µM and 39.43% at 20 µM); a decrease in c-Myc expression decreased (54.92% at 10 µM and 23.11% at 20 µM) and simi- was also observed in the PC-3 cells (94.77% at 10 µM and larly, Bcl-2 expression levels were also decreased in the PC-3 74.97% at 20 µM) (Fig. 7). A previous study on cancer cell lines cells (46.21% at 10 µM and 42.96% at 20 µM). The levels of the suggested that c-Myc downregulates the FOXO3a-dependent Kip1 Kip1 apoptosis inducers, cleaved caspase-3 and cleaved PARP, were activation of the p27 promoter. On the p27 promoter, also markedly increased in the cells following diosmetin dose- a functional association was observed between FOXO3a and response treatment. In the LNCaP cells, diosmetin treatment c-Myc at the proximal Forkhead binding element (19). In this resulted in increased cleaved caspase-3 expression (144.37% at study, we observed that following diosmetin treatment, c-Myc 10 µM and 143.37% at 20 µM). Similarly, in the PC-3 cells, the expression decreased, while FOXO3a expression increased. Kip1 expression of cleaved caspase-3 increased (1.5-fold at 10 µM The expression of p27 , downstream of FOXO3a, increased and >6-fold at 20 µM). Furthermore, in the LNCaP cells treated signic fi antly following diosmetin treatment. In the LNCaP cells, Kip1 with diosmetin, cleaved PARP expression increased (10-fold at p27 expression increased (166.96% at 10 µM and 160.06% 10 µM and 30-fold at 20 µM), whereas in the PC-3 cells the at 20 µM), and also increased in the PC-3 cells (159.86% at expression increased, 4-fold at 10 µM and >17-fold at 20 µM. 10 µM and 139.91% at 20 µM) (Fig. 7B and C). Moreover, in X-linked inhibitor of apoptosis protein (XIAP) expression the LNCaP cells, FOXO3a expression also increased (157.82% decreased following diosmetin dose-response treatment in at 10 µM and 191.69% at 20 µM). Similarly in the PC-3 cells, both the LNCaP and PC-3 cells. The diosmetin-treated LNCaP FOXO3a expression increased (133.27% at 10 µM and 124.68% cells exhibited a decreased expression of XIAP (78.94% at at 20 µM). Additionally, the expression levels of cyclins and 10 µM and 97.52% at 20 µM); similarly in the PC-3 cells, we Cdks decreased following diosmetin dose-response treatment. observed a decrease in XIAP expression (79.78% at 10 µM and In the LNCaP cells following diosmetin treatment, a decreased 54.19% at 20 µM) (Fig. 6B and C). Cyclin D1 expression was observed (~54.82% at 20 µM), whereas, in the PC-3 cells cyclin E expression decreased Diosmetin treatment altered proposed mechanistic molecules. (42.99% at 20 µM). Furthermore Cdk4 expression decreased c-Myc overexpression in prostate cancer patients predicts more to 73.6% at 20 µM in the LNCaP cells, whereas in the PC-3 aggressive disease progression and biochemical recur rence (18). cells Cdk4 expression decreased to 61.09% at 20 µM. OAK et al: DIOSMETIN SUPPRESSES PROSTATE CANCER CELL PROLIFERATION Figure 6. Dose-response effects of diosmetin on apoptotic and anti-apoptotic molecules. (A) LNCaP and PC-3 cells were exposed to two concentrations of diosmetin (10 and 20 µM) for 24 h. Total cell lysates were prepared and using SDS-PAGE, lysates proteins were resolved and then subjected to western blot analysis for the evaluation of XIAP, Bax, Bcl-2, cleaved PARP and cleaved caspase-3. Lanes marked ‘0’ are the DMSO control (0.2%)-treated cells. For protein band density, densitometric analysis was performed and protein expressions levels were correlated to express relative controls (B and C). For loading control blots were stripped and reprobed with GAPDH. P<0.05 for diosmetin concentration vs. vehicle controls. Figure 7. Dose-response effects of diosmetin on signaling pathways in human prostate cancer cells. (A) LNCaP and PC-3 cells were exposed to two concentra- tions of diosmetin (10 and 20 µM) for 24 h. Total cell lysates were prepared and using SDS-PAGE, lysates proteins were resolved and then subjected to western Kip1 blot analysis for the evaluation of c-Myc, Cdk4, cyclin D1, FOXO3a and p27 . Lanes marked ‘0’ are the DMSO control (0.2%)-treated cells. For protein band density, densitometric analysis was performed and protein expression levels were correlated to express relative controls (B and C). For loading control blots were stripped and reprobed with GAPDH. P<0.05 for diosmetin concentration vs. vehicle controls. INTERNATIONAL JOURNAL OF ONCOLOGY 53: 835-843, 2018 Figure 8. Schematic diagram of the proposed cell growth arrest signaling pathways mediated by diosmetin in human prostate cancer cells. Overexpression of c-Myc, Cdk4 and cyclin D1 in prostate cancer cells drives cell growth. Diosmetin treatment inhibits the expression of c-Myc and its downstream molecules, Kip1 cyclin D1 and Cdk4, conversely increasing FOXO3a and p27 expression levels to arrest cell growth. Moreover, diosmetin treatment potentiates the apoptotic machinery by modulating cleaved caspase-3 and cleaved PARP in prostate cancer cells. Discussion the cell cycle transition (2). In this study, we observed that the diosmetin dose-response treatment of prostate cancer cells Various factors play a role in prostate cancer progression; decreased c-Myc, cyclin D1 and Cdk4 expression levels. This diet and obesity are one of them. Both are associated with an suggests that diosmetin has the potential to modulate upstream increased risk of high-grade tumors (20,21). A recent study target c-Myc to alter the downstream cell cycle regulatory suggested that psychosocial stress also enhances prostate cascade of molecules. There is evidence to suggest that the cancer incidence and progression (22). Moreover, epigenetic Forkhead family member, FOXO3a, negatively regulates information harbored on Y-chromosomes may play a signic fi ant c-Myc (28). Forkhead family transcription factor Fox ‘O’ role in prostate cancer progression (23). Numerous preventive regulates the cell cycle, cellular differentiation, growth, tumor approaches delay the progression of prostate cancer. The suppression pathways and metabolism (29). We hypothesized development of an effective chemopreventive agent depends that the diosmetin-mediated upregulation of FOXO3a expres- on its evaluation with available biomarkers that would predict sion could lead to cell growth inhibition and apoptosis; these the potential effets of the agent in prostate cancer. To the best events were mediated by the perturbation of the G /S phase of our knowledge, this is the first study on prostate cancer of the cell cycle (Fig. 8). Chip sequencing data suggest an cells treated with diosmetin, which exhibited growth inhibi- inverse correlation between c-Myc and FOXO3a (30). Another tory and cytotoxic effects on androgen-sensitive (LNCaP) and study suggested that the FOXO3a-induced increase in miRNA androgen refractory (PC-3) cells. The data presented herein levels alters c-Myc mRNA translation (31). It has also been imply that these effects of diosmetin are due to the induction suggested FOXO3a plays a signic fi ant role in prostate cancer of prostate cancer cell arrest the at G and S phases of the suppression. The overexpression of cyclin D has been found cell cycle followed by the activation of apoptotic machinery, in various types of cancer, as well as cell lines derived from as evidenced by the increase in cleaved caspase-3 expression. tumors, which is linked with uncontrolled cell growth (32). In mammalian cells, alterations in cell cycle machinery Cyclin D overexpression in cells represents a shorter G phase result in changes in cell viability and cell growth. Studies have with a rapid transition into the S phase (33). Based on current demonstrated that an association exists between cell cycle and other studies (16), this provides evidence that diosmetin dysregulation and cancer; cell cycle inhibition is one of the has the ability to arrest cancer cell growth, and may thus be mechanisms that controls the growth of cancer (24,25). It has a potential candidate for chemopreventive and therapeutic recently been suggested that diosmetin exerts a substantial strategies in prostate cancer. Cip Kip growth inhibitory effect on prostate carcinoma cells by altering Cdk inhibitors (CKIs), such as p21 /p27 and the INK4 the G /S phase of the cell cycle. In eukaryotes, cell cycle families of proteins inhibit cyclin-Cdk complexes to modulate mechanisms are controlled by the orchestrated role of protein cell cycle progression (34). Similarly, this study demonstrated kinase complexes. Each complex contains at least a catalytic that diosmetin acts as a CKI in the G and S phases of the cell subunit, Cdks and a potential partner known as cyclins (26). cycle in the LNCaP and PC-3 cells, respectively. Moreover, Kip1 Cyclin E and cyclin D are the major players which lead to we observed a marked upregulation of p27 expression in G /S phase cell cycle arrest. Both these cyclins in association the LNCaP and PC-3 cells following diosmetin treatment, with Cdk2, Cdk4 or Cdk6 lead to Rb gene phosphorylation and which represented G and S phase cell cycle arrest. This may cell growth, whereas hyper-phosphorylated Rb further leads to be one of the molecular mechanisms through which diosmetin the release of the E2F complex. This free E2F from Rb then suppresses the viability of prostate cancer cells. activates c-Myc, which results in cell cycle progression and A potent suppressor of the apoptotic pathway is the Bcl-2 cell viabilty (27). It has been suggested that c-Myc silenced protein, which regulates apoptosis by the permeabilization cells signic fi antly exhibited reduced levels of Cdk4-cyclin D1 of the mitochondrial membrane (35). Bcl-2 is found to be and CDK6-cyclin D1 complexes during the G to S phase of overexpressed in ~50% of all human tumors (36). Moreover, 0 OAK et al: DIOSMETIN SUPPRESSES PROSTATE CANCER CELL PROLIFERATION Bcl-2 forms a heterodimer with Bax (pro-apoptotic member), Consent for publication and this interaction abolishes the pro-apoptotic functions of Bax protein. Thus, it has been proposed that the ratio between Not applicable. Bax/Bcl-2 will determine whether the cell will undergo apop- tosis or not. In both the LNCaP and PC-3 prostate cancer cells, Competing interests we observed a signic fi ant decrease in Bcl-2 protein expression following diosmetin treatment. Conversely, diosmetin treat- The authors declare that they have no competing interests. ment of these cells increased Bax protein expression. Thus, References this suggested that diosmetin treatment altered the Bax to Bcl-2 ratio in favor of apoptosis. 1. Asghar U, Witkiewicz AK, Turner NC and Knudsen ES: The In this study, we present experimental findings that history and future of targeting cyclin-dependent kinases in diosmetin induces the apoptosis of prostate cancer cells by cancer therapy. Nat Rev Drug Discov 14: 130-146, 2015. 2. 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Journal
International Journal of Oncology – Pubmed Central
Published: May 16, 2018