Elucidation Of The Anti-tumor Effect Of PEITC In Prostate Cancer

As one of the most prevalent types of malignancy in the United States and many other deveolped countries, CaP incidence in China has increased year by year. PEITC, a well-studied member of ITCs, has shown anti-proliferative effects on PCa and preneoplastic cells. ITCs which naturally occur in cruciferous vegetables have recently received much attention as possible chemopreventive agents. PEITC can decrease the development of cancer cells by affecting the cellular signal pathway and induces cell cycle arrest. Since Microtubule make a important role in the development of cancer, the microtubules can be affected by numerous endogenous regulators and exogenous factors, and are considered as a susceptible target by numerous therapeutic drugs. Signal transducer and activator of transcription 3 (STAT3), a member of Janus kinase (JAK)/STAT signaling pathway, is a latent transcription factor which can be activated by many cytokines and growth factors. Activation of the JAK/STAT3 pathway via interleukin 6 (IL-6) has been linked to prostate cancer progression. The present study was undertaken to observe the effect of PEITC on cell microtubulin protein expression and cell proliferation in prostate cancer cells by westen bolt and FACS assay and to explore the mechanisms of PEITC treating the prostate cancer by determining the effects of PEITC on IL-6-mediated JAK-STAT3 activation in prostate cancer cells. The projects and results were as follow:1. Phenethyl isothiocyanate induces cell cycle arrest and reduction of a- and b-tubulin isotypes in human prostate cancer cellsObjectives:to study the effect of Phenethyl isothiocyanate on cell cycle arrest and reduction of a- and b-tubulin isotypes in human prostate cancer cellsMethods:Cell culture and treatments The human PCa cell lines (DU145, PC-3, LNCaP, and C4-2B) were routinely maintained in RPMI 1640 medium with 5% fetal bovine serum (FBS) and 1% penicillin/streptomycin under a humidified atmosphere of 5% CO2 at 37℃. Cells (were grown in 24- well plates for 2 to 3 days and then treated with PITC (20 or 30 mM) or PEITC (5,10, or 20 mM) for 2,8, or 24 h. For antioxidant effect experiment, cells were treated with PEITC (20 mM) in the presence or absence of NAC (5 mM). For proteasome inhibitor effect experiment, cells were treated with PEITC (20 mM) in the presence or absence of MG132 (10 or 20 mM). Analysis of cell cycle distribution was analyzed by FACS assay (Becton Dickinson, Bedford, MA). we then carried out western blot to determine the expression levels of tubulin isoforms in prostate cancer cells with PEITC treatment and in the presence or absence of MG132.To determine if the effect of PEITC on tubulins was cancer cell specific, we used a normal prostate epithelial cell line RWPE1, which was established from a histologically normal adult human prostate by immortalized with the human papillomavirus.Results:(1). Effect of PEITC on cell cycle arrestBoth of the cell lines showed a significant increase in the G 2 -M phase by PEITC (10 and 20 mM in PC-3 and 20 mM in DU145) treatment compared with no treatment. PEITC treatment also caused a significant reduction in the G0/G1-phase in both of cell lines compared with no treatment. Interestingly, PEITC treatment caused an increase of S phase cells in DU145 but a decrease of them in PC-3. On the other hand, PITC did not affect cell cycle in both cell lines.(2). PEITC reduced a- and b-tubulin expressions in prostate cancer cell linesPEITC inhibited the expression of a- and b-tubulins, while PITC had no effect on their expression in PC-3 cells. The inhibition was observed at all investigated time (2, 8, and 24 h), which indicates the suppression effect of PEITC can last for at least 24 h. However, g-tubulin was not affected by PEITC treatment at any time. The effect of PEITC on tubulin expression was further examined in other PCa cell lines including DU145, C4-2B, and LNCaP. The results showed that PEITC strongly inhibited both of tubulin expressions in DU145 and LNCaP cells,20 mM of PEITC can inhibit their expression in C4-2B cells. However, PITC had no effect on tubulin expression in all cell lines treated. Similar to cell arrest experiment, PEITC treatment neither inhibited a-tubulin expression nor b-tubulin expression in RWPE1 cells.(3). Antioxidant abrogated reduction of a- and b-tubulin expression by PEITCthe PEITC-induced reduction of both a-and b-tubulins was dramatically attenuated by treatment with NAC. In addition, PEITC induced cell morphological change, including cell rounding and subsequently floating, while PITC and NAC had no effect on PC-3, DU145, and LNCaP cells. NAC treatment could block morphological change induced by PEITC.(4).Proteasome inhibitor abrogated reduction of a- andb-tubulin expression by PEITCsuppressive effect of PEITC on tubulins was abrogated by presence of MG132.Conclusion:1. PEITC reduces a- and b-tubulin expressions can induces cell cycle arrest in prostate cancer cell lines2.PEITC-induced tubulin degradation may be mediated by proteasome pathway and ROS generation.2. Phenethyl Isothiocyanate inhibits STAT3 activation in prostate cancer cellsObjectives:To study the effect of Phenethyl Isothiocyanate on constitutive or IL-6-mediated JAK-STAT3 activation in prostate cancer cellsMethods:Cell proliferation assay and cell cycle distribution analysis The 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H tetrazolium (MTS) assay was used to detect the cell proliferation. Cells were cultured in 96-well plates at a density of 1000 cells per well with 100μl of culture medium. After incubation for 40 h, cells were treated with PEITC or DMSO as a control for 72h, then 20μl of MTS (Promega, Madison, WI) was added. After another 2 hour incubation,100μl soluble formazan containing media were used to measure the absorbance at 490 nanometers (nm). The control without cells was used to blank the background. To test the effect of ITCs on the cell cycle, DU145 cells were plated in a 10 cm-diameter dishes treated with PEITC or PITC or vehicle (DMSO) for 20 h. the cells were analyzed on a FACScan flow cytometer.Western blot analysis Cells were seeded in 24 well plates at 2.5×104 cells/well and cultured for 2 days. Cultured cells were then treated with PEITC or PITC or DMSO (for control) at indicated concentration. For the IL-6 stimulation experiment, cells were starved with serum free medium for 2-3 h in order to remove constitutive IL-6 generation and then add 25 ng/ml IL-6 combine with PEITC or PITC or DMSO (for control) at indicated concentration. samples were subjected to Western blot analysis.Cell transfection and luciferase reporter assays Transfections were performed using the Lipofectamine reagent (Invitrogen, Camarillo, CA) for DU145 cells and genefactor (VennNova, LLC., Pompano Beach, FL) for LNCaP cells. a luciferase reporter construct containing STAT3-binding sites was transfected into DU145 and LNCaP cells. After transfection, cells were treated with or without PEITC at various concentrations in the presence or absence of IL-6. Then luciferase activity was measured by using a luciferase assay system (Promega) and Turners Design Luminometer TD 20/20we tested the effects of PEITC on IL-6-associated activation of AR-signaling. PSA-6kb and hK2-3ARE luciferase reporter gene expression vectors, representing two commonly used AR activated gene promoters, were transfected into LNCaP cells. Transfected cells were then treated with or without IL-6 in the presence or absence PEITC at concentration of 5,10,20μM or PITC at 30μM (only in Fig. 5B) for 6 h followed by luciferase analysis.ResultsEffect of PEITC on cell growth and and G2-M phase cell cycle arrest in PCa cellsPEITC inhibited cell proliferation. The proliferation of DU145 cell was reduced by 25% after treatment with PEITC at 5μM, and was further reduced by 68% at 20μM. In contrast, when cells were treated with PITC, no significant cell growth inhibition was observed.The cell-cycle study showed that G2-M population was significantly increased after treatment with 10μM of PEITC in DU145 cells for 24 h. In contrast, little effect was observed when the cells were treated with PITC.Effect of PEITC on constitutive and IL-6-induced STAT3 activation in PCa cells Total and phosphorylated STAT3 expression levels were detected by western blot analysis. An activation of STAT3 via phosphorylation at tyrosine site 705 was detected in DU145 cells. After treatment with PEITC at 5,10,20μM, the phosphorylated STAT3 protein level in cells were decreased in a dose-depended manner. In contrast, the total STAT3 showed no change.The results from western blot showed that IL-6 can induce STAT3 activation which is consistent with the previous report. The important finding is PEITC inhibited the IL-6-induced STAT3 activation in a dose-depended manner. STAT3 phosphorylation was totally blocked by PEITC at a concentration of 20μM. In order to test how fast PEITC can inhibit IL-6-induced STAT3 phosphorylation, DU145 cells were treated with IL-6 with or without PEITC for 5,15,25,40 min. PEITC inhibited IL-6 activated STAT3 phosphorylation as early as 5 min after treatment. Similarly, IL-6-induced STAT3 phosphorylation and inhibition by PEITC were also found in LNCaP cells. To further address the effects of PEITC on STAT3 activation in PCa cells, a luciferase reporter construct containing STAT3-binding sites was transfected into DU145 and LNCaP cells. After transfection, cells were treated with or without PEITC at various concentrations in the presence or absence of IL-6. Again, a constitutive STAT3 transactivation was detected in DU145 cells. A significant stimulatory effect of exogenous IL-6 on STAT3 transcriptional activity was observed in both DU145 and LNCaP cells. PEITC inhibited the constitutive and IL-6-induced STAT3 reporter activities in DU145 cells and LNCaP cells.PEITC inhibits IL-6-induced JAK2 phosphorylationTyrosine phosphorylation of JAK was assessed by Western analysis in both cell lines after treatment with IL-6 and PEITC. IL-6-induced phosphorylation of JAK2 was totally blocked by PEITC in DU145 cells and partially blocked in LNCaP cells, further supporting that PEITC inhibits IL-6-induced activation of the JAK/STAT3 pathway in PCa cells.PEITC inhibits IL-6-induced AR transcriptional activity in LNCaP cellsThe results showed that luciferase activity in both PSA-6kb and hK2-3ARE induced by IL-6 in PCa cells was significantly inhibited by PEITC.N-acetyl-L-cysteine (NAC) can affect PEITC actionWe found that NAC at a dose of 5 mM completely reversed the inhibitory effect of PEITC on DU145 cell growth. Interestingly, this reversal effect of NAC was only observed at 0 h and 0.5 h after its administration, after 1 h incubation with PEITC, NAC can not reverse the inhibitory effect of PEITC, suggesting that ROS or some kind of redox reactions occurred at the early phase of PEITC action on DU145 cells. Consistently, addition of NAC dramatically attenuated PEITC-repressed constitutive and IL-6-induced STAT3 protein phosphorylation and STAT3 transcriptional activity. Furthermore, inhibitory effect of PEITC on IL-6-stimulated AR transcriptional activity in LNCaP cell was also reversed by NAC administration.Conclusion:(1). PEITC can repress human PCa cell growth and this inhibitory effect maybe associated with G2-M cell cycle arrest.(2). IL-6 induces STAT3 activation by phosphorylation. PEITC suppresses IL-6-mediated JAK/STAT3 activation. (3). PEITC down-regulates IL-6-induced AR transcriptional activity on hK2-3ARE and PSA promoter activity.(4). ROS generation or some kind of redox reactions may, at least in part, account for the effect of PEITC on STAT3 activation.