Study On The Effects Of ARHI On Autophagy Of Pancreatic Cancer Cells In Vitro And Its Possible Mechanisms

Pancreatic cancer is one of the most lethal types of cancers. Despite the advancement of our knowledge in recent years regarding the pathophysiology of pancreatic cancer, we still lack effective methods to treat this cancer enough to improve the outcomes significantly. So it is an important to explore the biological behavior of pancreatic cancer cells, such as, proliferation, apoptosis, invasion, metastasis, programmed cell death and molecular mechanisms of these events.Recent research suggests a complex relationship between cancer and deregulated autophagy. Several tumor suppressor proteins can induce autophagy. Both the induction and inhibition of autophagy may have the potential role in the treatment of cancers. But it should be performed according to different types or stages of cancer.ARHI, a maternally imprinted gene that belongs to the Ras superfamily was first identified in 1999. However, unlike Ras, ARHI is a tumor suppressor gene in ovarian and breast carcinomas. Re-expression of ARHI inhibits cell growth, motility and invasion in ovarian and breast cancer cells. Over-expression of ARHI with a dual adenoviral vectors induces apoptosis in ovarian and breast cancer cells. A recent research found that stable or transient physiologic expression of ARHI induces autophagy, but not apoptosis.Previous works of our group suggested that ARHI had characteristics of a tumor suppressor gene in pancreatic cancer cells. Re-expression of ARHI in pancreatic cancer cells can inhibit proliferation, motility and invasion and induce apoptosis and cell cycle arrest.Until now, we don’t know whether the re-expression of ARHI could induce autophagy in pancreatic cancer. For this reason, we observe the influence of ARHI on autophagy of pancreatic cancer cells in vitro and discuss its possible mechanisms. I. Study on the effects of ARHI on autophagy of pancreatic cancer cell line PANC-1 in vitroAim:To investigate if the re-expresion of ARHI induces autophagy in human pancreatic cancer cell line PANC-1.Methods:Recombinant plasmid pIRES2-EGFP-ARHI has been constructed successfully by our group and the plasmid was transfected into human pancreatic cancer cell line PANC-1 by lipofectamine 2000. Transfection efficiency can be detected using standard fluorescence microscopy and flow cytometry. RT-PCR and Western blot analysis were performed to examine the expression of ARHI in transiently transferred PANC-1 cells. The cells were stained with Hoechst33258 to observe the morphological changes of apoptosis. The Apoptosis rate was measured by and flow cytometry after staining with propidium iodide (PI). The cells were stained with acridine orange and examined by fluorescence microscopy to visualize acidic vesicular organelles. Transmission electron microscopy (TEM) was used to detected scattered double membrane vacuolar structures 72 hours after induction of ARHI.Result:1) The ARHI mRNA was detectable in ARHI gene transfected PANC-1 cells 48 hour after transfection; the ARHI protein was detectable in ARHI gene transfected PANC-1 cells 48 hours and 72 hours after transfection. ARHI mRNA and protein expression were not detectable in control cells.2) Apoptotic nuclear morphological changes was found in both ARHI gene transfected group and empty vector transfected control group after staining with Hoechst33258120 hours after transfection.3) The Apoptosis rate in ARHI gene transfected group was (29.6±8.4)% 120 hours after transfection, slightly but not significantly higher than control group (23.4±6.8)% (P>0.05).4) Cells with the acidic vesicular organelles 72 hours after trancfection in ARHI gene transfected group and empty vector transfected group were 9.3% and 0.4%, respectively(P<0.05).Typical double- membrane of autophagosomes were identified with TEM in ARHI gene transfected group, but not in control group.Summary:1) Re-expression of ARHI in human pancreatic cancer cell line PANC-1 can induce apoptosis slightly; 2) Re-expression of ARHI in human pancreatic cancer cell line PANC-1 can induce autophagy.Ⅱ. Study on the possible mechanisms of ARHI re-expression induced autophagy in human pancreatic cancer cell line PANC-1Aim:To investigate the possible mechanisms of ARHI re-expression induced autophagy in human pancreatic cancer cell line PANC-1.Methods:In the first part of the study we found that re-expression of ARHI gene in human pancreatic cancer cell line PANC-1 can induce autophagy. The possible mechanism of this was explored in this part. Whole cell protein and cytoplasmic protein extracts were abtained 48 hours and 72 hours after transient transfection. Western blot analysis was used to detect target proteins, including pERK. pAKT, AKT, p53. DAPKI and ATG4B. The level of nuclear p53 protein was detected by immuno-cytochemical staining method 72 hours after transient transfection. Total RNA was pepared and quantitative real-time PCR method was used to detect the levels of mRNA of TSC1, TSC2 and beclin 148 hours after transient transfection.Results:1) There was no difference of pERK protein levels between the ARHI gene transfected group and empty vector transfected group 48 hours and 72 hours after transient transfection,2) Compared with control group. ARHI re-expression down-regulated pAKT, but the levels of AKT protein were not changed 48 hours and 72 hours after transient transfection,3) There was no difference between the levels of cytoplasmic p53 protein in ARHI gene transfected group and vector transfected group 48 hours and 72 hours after transient transfection. While nuclear p53 positive cells was significantly higher in ARHI gene transfected group than control group (18% vs.8%) 72 hours after transient transfection(P<0.05),4) Compared with control group, ATG4B and DAPK 1 proteins were up-regulated in ARHI gene transfected group 72 hours after transient transfection.5) There is no difference of mRNA levels of TSC 1, TSC2 and beclin 1 between ARHI gene transfected group and control group.Summary:Transientre-expression of ARHI induced autophagy in PANC-1 cells might through the down-regulation of pAKT and up-regulation of nuclear p53, ATG4B and DAPK1. There was no relationship between ARHI induced autophagy and Ras/Raf/ERK pathway and transcription changes of TSC1, TSC2 and beclin1 in PANC-1 cells.Conclusions:1) Re-expression of ARHI gene in human pancreatic cancer cell line PANC-1 can induce autophagy.2) Transient re-expression of ARHI induced autophagy might be through the down-regulation of pAKT and up-regulation of nuclear p53, ATG4B and DAPK1 in PANC-1 cells. There was no relationship between ARHI induced autophagy and Ras/Raf/ERK pathway and transcription changes of TSC1, TSC2 and beclinl in PANC-1 cells...