Effects Of A Novel PPARγ Agonist On Cell Proliferation In OS-RC-2 Cells In Vivo

The peroxisome proliferator-activated receptor γ(PPARγ) is the most extensively studied subtype of the PPARs. PPARγ binds to peroxisome proliferator response elements(PPRE) as a heterodimer with members of the retinoid X receptor, provoking target gene expression. Originally, PPARγ was identified as being essential for glucose metabolism. More recent evidence implied an important role for the nuclear hormone receptor PPARγ in getting involved in many cancer cell lines including lung, breast, colorectal, prostate, bladder and renal cell. In renal cell carcinoma(RCC), synthetic PPARγ agonists, the thiazolidinediones(TZDs), have proven to inhibit tumor growth, exhibit synergistic antitumor activity and protect against nephrotoxicity caused by chemotherapy. These findings initiated further research to assess the potential of PPARγ agonists as a promising new therapeutic alternative in the treatment of RCC.Unfortunately, despite their excellent potencies, TZDs, the PPARγ agonists in clinical use, have been found to present unwanted therapeutic profiles, marked by fluid retention, weight gain and cardiovascular risk, which prompts us to find other non-TZD PPARγ agonists with potent PPARγ agonistic activity and less side effects. By cooperated with Shanghai Institute of Materia Medica, Chinese Academy of Sciences, we synthesized a novel PPARγ agonists named DH9, using computer aided drug design(CADD). Previous studies have confirmed that DH9 significantly retarded tumor growth which is similar with that of 5-FU. In particular, the high antitumor potency of DH9 with little normal tissue damage provides potential advantages in the treatment of renal cell carcinoma.In this study, we investigate the effect of a novel non-thiazolidinedione PPARγ agonist DH9 on human renal carcinoma cell line OS-RC-2. We have also explored the molecular mechanism through which DH9 inhibit cell growth and induce cell death in OS-RC-2 Cells. Methods:1. OS-RC-2 cells were treated with DH9(0, 6.25, 12.5, 25, 50 and 100 mol/L) or rosiglitazone(0, 25, 50, 100, 150 and 200 mol/L) for 24, 48 and 72 hours, the proliferation of cells was detected by MTT. OS-RC-2 cells were treated with DH9 or rosiglitazone(0, 6.25, 12.5, 25, 50 and 100 mol/L) for 48 hours, and grow inhibition was monitored by MTT assay.2. To investigate if the mechanism of the cell proliferation inhibition was PPARγ independent, we set another group adding GW9662 in the MTT assay. To establish the independence of their antitumor effects on PPARγ, we inhibited its expression using si RNA approaches. Whole cell lysates were subjected to immunoblotting with anti-PPARγ antibodies to confirm the alterations of PPARγ expression after si RNA transfection. In separate studies, MTT assay was conducted as described above.3. The cell cycle distribution and apoptotic ratio of different concentration DH9-treated cells were investigated by Flow Cytometry (FCM). Regulatory proteins of cell cycle(P27 KIP1, Cyclin D1) and apoptosis(Bcl-2, Bax) were detected by Western blot analysis. Result:1. The proliferation of renal cell carcinoma OS-RC-2 cells was effectively inhibited by DH9 in a concentration-dependent and time-dependent manner. It was noted that the efficiency of cell growth inhibition by DH9 was much more potent than rosiglitazone(P<0.05).2. The antitumor potency quantified by IC50 s in the groups pretreated with GW9662 was the same with groups without adding it. In si RNA transfection, the results of western blot analyses showed that specific PPARγ si RNA suppressed PPARγ protein expression by. However, there is no significant difference of IC50 value between PPARγ si RNA transfected group and control group after treatment of DH9.3. Cell cycle phase analysis revealed a decreased proportion of S phase, with arrest at G0/G1 in this process(P<0.05). DH9 dose-dependently caused a marked decrease in cyclin D1 expression and a significant increase in P27 KIP1 expression.4. We found that DH9 treatment for 48 h significantly increased apoptotic cell numbers in a dose-dependent manner. To explore the molecular mechanism, we examined Bcl-2 and Bax expression following DH9 treatment for 48 h in OS-RC-2 cells. We observed that DH9 could dose-dependently decreased Bcl-2 protein expression but increased Bax protein expression.Conclusion:Our findings demonstrate that DH9 showed potent inhibitory effect on proliferation of OS-RC-2 cell line independent of PPARγ. The effects may be related to the arrest of G0/ G1 phase and induction of apoptosis.