Antitumor Activity Of PPARα Agonist Fenofibrate In Human Pancreatic Cancer And Relevant Mechanisms

Background:Pancreatic cancer is the fourth most common cause of adult cancer death in the U.S. 232,306 people were expected to develop pancreatic cancer,with 227,023 anticipated deaths resulting from this disease.Pancreatic cancer is a highly aggressive malignant disease.Only a small proportion of patients(10%-15%) presented with tumors amenable to surgical resection,but even with surgery,disease recurrence will occur in the majority.Despite therapeutic advances,the prognosis of patients with pancreatic cancer is extremely poor,with 5-year survival of＜5%.Chemotherapy for pancreatic cancer is only of modest benefit,with the tumor having propensity to being chemoresistant.Gemcitabine has become the standard treatment for pancreatic cancer, since it was shown to improve clinical benefit response and survival compared with 5-fluorouracil(5-FU).Although superior to bolus 5-FU,the efficacy of gemcitabine as a single agent is modest,with a median survival of only～6 months in randomized trials and a 12-month survival of＜20%.Therefore,developing more effective treatment regimens is urgently needed.Peroxisome proliferator-activated receptorα(PPARα) is a ligand-inducible transcription factor that belongs to the nuclear-hormone-receptor family.PPARαwas shown to mediate peroxisome proliferation action.PPARαagonist fenofibrate is mainly used in the treatment of dyslipidaemia.Recently,growing evidence indicated that PPARαagonist fenofibrate had antitumor effects,probably because of its anti-proliferative and pro-apoptotic activities.Reports published so far convincingly showed that fenofibrate inhibited proliferation of hepatic cancer cell lines,colon cancer cell lines,endometrical cancer cell lines and ovarian caner cell lines.However,there is no report concerning the antitumor efficacy of fenofibrate on pancreatic cancer cells.To improve the chemotherapeutic response,we investigated the potential of fenofibrate to augment gemcitabine-based chemotherapy for pancreatic cancer.Furthermore,we examined whether fenofibrate could inhibit the metastasis of pancreatic cancer.Cancer progression is often associated with prolonged activation of signal transduction pathways,such as phosphatidylinositol 3-kinase/Akt(PI3K/Akt) pathway. This constitutive activation confers resistance to conventional therapeutics.Many papers reported that most human pancreatic tumors showed high levels of activated Akt, a serine/threonine protein kinase that mediates survival signaling.Akt phosphorylates and inactivates numerous pro-apoptotic proteins,such as BAD,caspase-9 and survivin. The progression from an in situ tumor to an invasive disseminating form requires profound changes in the tumor cell phenotype(e.g.gaining the ability to migrate,to intravasate and survive in the anchorage-independent conditions in blood vessels,and to penetrate distant organs).Based on these facts,we hypothesized that the inhibition of PI3K/Akt pathway might be involved in pro-apoptotic and antimetastatic activity of fenofibrate. Part One The Anti-proliferative Effect of Fenofibrate on Pancreatic Cancer Cells and Relevant MechanismsObjective:We attempted to evaluate whether cell growth and apoptosis are affected by PPARαagonist fenofibrate.Materials and Methods:(1) Sections of formalin-fixed and paraffin embedded material from pancreatic cancer patients were analyzed to detect PPARα-specific immunoreactivity.RT-PCR and western blot assays were done to detect PPARαexpression in pancreatic cancer cell lines Bxpc-3 and Panc-1.(2) MTT assay was performed to analyze the anti-proliferative effect of fenofibrate and combined use of fenofibrate and gemcitabine on pancreatic cancer cell lines.(3) The detection of apoptotic cells by flow cytometry was done to evaluate the pro-apoptotic effect of fenofibrate on pancreatic cancer cell lines.(4) Hoechst 33258 staining was used to observe the apoptotic characteristics of pancreatic cancer cell lines with different therapeutic regimens.(5) Nude mice were implanted with pancreatic cancer cells and randomized into the following treatment groups:(a) control(normal saline),(b) only fenofibrate,(c) only gemcitabine,(d) fenofibrate and gemcitabine.PCNA staining,TUNEL and CD31 staining were used to estimate the proliferation,apoptosis and microvessel density in tumor tissues.(6) Western blot analysis was done to detect Bcl-2,caspase-9,caspase-3 in the pancreatic cancer cells with various treatment schedules.(7) Western blotting was performed to evaluate Akt,phospho-Akt and survivin in pancreatic cancer cells treated with different regimens.Results:(1) High PPARαexpression was detected in 6 of 9 pancreatic cancers.Pancreatic cancer cell lines Bxpc-3 and Panc-1 also expressed PPARα.(2) Fenofibrate inhibited growth of pancreatic cancer cells in a dose- and time-dependent manner.In Bxpc-3 cells,treatment with 25μM fenofibrate for 48 hours resulted in 53.1±5.6%cell growth relative to the control.In Panc-1,treatment with 50μM fenofibrate for 48 hours resulted in 67.5±2.4%cell growth relative to the control. We subsequently evaluated the effect of fenofibrate plus gemcitabine on cell growth in vitro and found that combined use of fenofibrate and gemcitabine enhanced the anti-proliferative activity.(3) As shown by flow cytometry,fenofibrate could induce apoptosis in pancreatic cancer cells.Relative to single agents,cotreatment of fenofibrate and gemcitabine induced much more apoptosis in both pancreatic cancer cell lines(P＜0.05).(4) Hoechst 33258 staining demonstrated characteristics of apoptosis in cells receiving fenofibrate and/or gemcitabine treatment.(5) For in vivo experiments,we determined the mean tumor volume in all treated groups.Single modality treatment with either fenofibrate or gemcitabine alone in mice harboring Bxpc-3 cells caused reduction in tumor volume compared with normal saline treatment after 3 weeks.Treatment with fenofibrate plus gemcitabine simultaneously resulted in greater reduction in tumor volume compared with control tumors.We next examined the expression of the cell proliferation marker PCNA and microvessel density marker CD31 in tumor tissues from the four groups.The results showed that fenofibrate in combination with gemcitabine significantly down-regulated the expression of PCNA in tumors tissues compared with the control group(P＜0.05 versus control).The data also showed that fenofibrate suppressed the expression of CD31,but the down-regulation was not statistically different.TUNEL demonstrated that fenofibrate could enhance the induction of apoptosis by gemcitabine.Cotreatment of fenofibrate and gemcitabine induced more apoptotic cells than single agent either fenofibrate or gemcitabine alone(P＜0.05).(6) Western blotting revealed significant reduction in the expression of Bcl-2 from the fenofibrate-treated pancreatic cancer cell lines compared with those from the control group.The expression of cleaved caspase-9 and cleaved caspase-3 increased with the treatment of fenofibrate.(7) Up-regulation of TRB-3 was noticed in pancreatic cancer cell lines with fenofibrate treatment.In the next step,we observed down-regulation of phosphorylation status of Akt after incubation with fenofibrate without a change in total Akt protein level. Similar down-regulation in survivin was detected after fenofibrate treatment.Conclusions:(1) Pancreatic cancer specimens and cell lines expressed PPARα.(2) Fenofibrate inhibited pancreatic cancer cells growth in a dose- and time-dependent manner.Combined treatment of fenofibrate and gemcitabine resulted in greater inhibition of cell growth.(3) Fenofibrate induced apoptosis of pancreatic cancer cells and treatment of fenofibrate plus gemcitabine potentiated the pro-apoptotic effects.(4) Fenofibrate decreased the phosphorylation status of Akt and expression of survivin via up-regulation of TRB-3. Part Two The Inhibitory Effect of Fenofibrate on Migration of Pancreatic Cancer Cells and Relevant MechanismsObjective:We attempted to evaluate whether migration of pancreatic caner cells was affected by fenofibrate.Materials and Methods:(1) We used wound-healing assay to estimate the effect of fenofibrate on migration ability of pancreatic cancer cells.(2) Cell migration was assessed quantitatively in Transwell chambers with polycarbonate filters.(3) RT-PCR was used to determine the expression of E-cadherin and snail in pancreatic cancer cell lines incubated with different therapeutic modalities.(4) Western blotting was done to detect E-cadherin,snail and phospho-GSK3βin pancreatic cancer cells after treatment with various agents.Results:(1) Wound gap was almost closed in control group after 24 hours wound introduction.In contrast,the speed of wound closure was much slower and the wounds were still widely open in the fenofibrate-treated group.(2) Transwell chamber showed that the migration index in the presence of fenofibrate was 39.9±3.7%,significantly lower than the control group(P＜0.05). Gemcitabine did not cause significant decrease of migration index.(3) RT-PCR results demonstrated that E-cadherin was up-regulated and snail was down-regulated after the fenofibrate treatment.The expression level of E-cadherin and snail was not affected by gemcitabine. (4) Western blotting further confirmed that expression of E-cadherin increased and expression of snail decreased simultaneously in the fenofibrate-treated group.The protein levels of E-cadherin and snail remained similar after gemcitabine treatment compared with the control group.(5) Down-regulation of phosphorylation status of GSK3βwas observed from the fenofibrate-treated group.Conclusions:(1) Fenofibrate inhibited migration of pancreatic cancer cells.(2) Fenofibrate treatment led to impaired migration ability of pancreatic cancer cells via the attenuation of Akt/GSK3βmediated pathway.The decreased phosphorylation level of GSK3βrestored its kinase activity and subsequently blocked snail expression. Finally,the E-cadherin protein accumulated in pancreatic cancer cells treated with fenofibrate.