Empirical Study Of Geraniol Anti-pancreatic Adenocarcinoma Effect And Its Mechanism

Part Ⅰ Inhibitory effect of Geraniol on pancreatic cancer in nude miceObjective:To investigate the inhibitory effect of geraniol on hetero transplanted tumors of human pancreatic cancer cells line BXPC-3cells in nude mice.Methods:Hetero transplanted models of human pancreatic cancer cells line BXPC-3cells in nude mice were established.They were divided at random into four groups as control group,geraniol group,Gemzar group, geraniol+Gemzar group. Animals in each group were received intraperitoneal injection of geraniol,Gemzar,geraniol plus Gemzar or0.9%sodium chloride,respectively.The mice of the four groups were sacrificed at12th day after the first injection of drug.The tumor weight of the sacrificed mice was measured. Morphological and ultrastructural examinations of the tumors were carried out by light and electron microscopy.Results:Geraniol injection showed a suppressive effect on the growth of heterotransplanted tumors in the nude mice.The tumor growth inhibition rates were51.5%,50.9%and57.6%in the geraniol group,Gemzar group,geraniol+Gemzar group,respectively.The morphological study demonstrated that tumor cells displayed a typical appearance of apoptosis.Conclusion: Geraniol can markedly inhibit the growth of heterotransplanted human pancreatic cancer in nude mice. It was due, at least in part,to the induction of apoptosis in cancer cells. Part Ⅱ Effect of geraniol on the biological behavior of human pancreatic adenocarcinoma BXPC-3cellsObjective:The purpose of this study is to investigate the apoptosis-inducing and proliferation inhibition effect of geraniol on human pancreatic adenocarcinoma BXPC-3cells, and provides the experimental basis for clinical application. To examine the effects of geraniol on invasion, metastasis and mitoehondria fuctions of human pancreatic adenocarcinoma BXPC-3cells.Method:1. Culture BXPC-3cells in vitro, screen the effective working concentration of geraniol by MTT, determine its working concentration (1/2IC50), detect the mitochondrial respiratory changes in BXPC-3cells; observe cell morphologic changes in each group by inverted microscope and TEM; detect the cell cycle and apoptosis by flow cytometry; detect cell apoptosis by TUNEL.2. Cell-base adhesion assay was used to determine the effect of geraniol on the adhesion of BXPC-3cells to fibronectin(Fn).3Wound healing assay was used to determine the effect of geraniol on the migration of BXPC-3cells.Results:1. It can be concluded that the geraniol inhibition effect on the BXPC-3cell growth offers time-effect dependence and dose-effect dependence when put geraniol with a concentration of0.0675mM,0.125mM,0.25mM,0.5mM,1mM with BXPC-3for24h,48h,72h separately.2. Inhibition concentration IC50geraniol functioning at48h is0.38mM. Make the half of IC50at48h (0.19mM) as the geraniol working concentration for the experiment later.3. The result from morphology observing geraniol function on cell growth inhibiting shows:The cells in control group grow well in the state of adherence, in the geraniol experimental group, it is the fact that part of the cells ablate, adherent cells decrease obviously, the fact of the Gemzar group is that part of the cells ablate as well, adherent cells decrease, some cells become small and round. geraniol+Gemzar group:adherent cells decrease obviously.4. Result from ultramicrostructure observing geraniol function on BXPC-3cells by TEM in ultramicrostructure:The facts that BXPC-3cells are in low differentiation is morphologic character of the typical malignant tumor cells. In geraniol group, Cell junction and microvillus have disappeared and apoptotic body has appeared in part of the cells.Changes of caryocinesia image and cell apoptosis appears in part of the cells in group Gemzar. The fact in geraniol+Gemzar group is that some mitochondria swell, microvillus disappear and apoptotic body appears.5. Result from observing by flow cytometry geraniol function one cell cycle of BXPC-3human pancreatic adenocarcinoma cell:The fact in control group is that ratio of cells at stage G1is42.6%,24.7%at stage S and the ratio is32.7%at the stage G2. The fact of geraniol group is that the cell cyele has obvious arrested at stage G1, the ratio of cells at stage G1rises from42.6%to61.5%while the ratio declines from24.7%to15.9%at stage S. the ratio at stage G2declines from32.7%to22.6%; apoptos is may happen to some cells and peak of diploid may appear. The fact in Gemzar:the ratio of cells at stage G1has increased to some extent reaching to57.6%; the ratio at stage S has declined somewhat to15.2%, the ratio at stage G2is low as well, reaching27.2%; the cell cycle has been blocked to stage G1.The fact in geraniol+Gemzar group:cell cycle has obvious depression at stage G1, the ratio of cells at stage G1reaches up to64.2%, ratio at stage S declines, only reaching6.3%, the ratio at stage G2increases somewhat obviously, reaching29.5%; most cells have been blocked to stage G1, that is to say blocking effect on cells mainly takes place at stage G1and the blocking effect of geraniol+ Gemzar is stronger than the effect of single geraniol or Gemzar, tpical peak of apoptosis diploid appears.6. With the effect of the three experimental groups on BXPC-3cells for48h, the mitochondrial transmembrane potential in the three groups has obviously declined compared with control group(P<0.01); the difference in three experimental groups has no statistic significance.7. The effect of geraniol on the adhesion of BXPC-3cells to fibronectin:Atfer treatment by geraniol for24hours,the ability of adhesion was attenuated between BXPC-3cells and fibronection in a dose-dependent manner. After exposed to geraniol, Gemzar and the combination of them for24hours, the adhension inhibition rate were45.20%±1.91%;47.82%±2.05%and73.36%±1.10%.8. Wound healing assay showed that the cellular motility was evidently inhibited in dose-dependent manner.9. The results of the in vitro invasion assay displayed that geraniol was able to inhibit invasion ability of BXPC-3cells dose-dependently.Conclusion:1. Geraniol has a strong proliferation inhibitory effect on a dose-and time-dependent manner against human pancreatic adenocarcinoma BXPC-3cells.2. Geraniol can enhance proliferation inhibitory effect and the apoptosis of human pancreatic adenocarcinoma BXPC-3cells induced by Gemzar, in the same time inhibit cell adhesion, migration,and invasion in human pancreatic cancer BXPC-3cells in vitro. One of probably mechanism of inducing apoptosis is cause BXPC-3human pancreatic cancer cell mitochondrial respiratory dysfunction and decrease mitochondrial transmembrane potential, trigger the cascade activation of caspase and induce apoptosis.3Geraniol can enhance the apoptosis of human pancreatic adenocarcinoma BXPC-3cells and the blocking effect play on stage G0/G1. Part Ⅲ Study on the molecular mechanisms of geraniol against pancreatic carcinomaObjective:To identify the molecular mechanism of geraniol-induced apoptosis, cell arrest and inhibition of invasion ability.Method:1. In vitro, by means of EMSA, we observed the effect of geraniol, on the transcriptional activity change of NF-K B in BXPC-3cell line.2. After treated the human pancreatic adenocarcinoma BXPC-3cells with geraniol for48h, the expression of bcl-2、Livin、Survivin、bax Procaspase-3,8,9protein were detected by west blotting.3. After treated the human pancreatic adenocarcinoma BXPC-3cells with geraniol, for48h, the expression of MMP-2、MMP9、TIMP2and cyt-c protein were detected by west blotting.4. The expression of bcl-2, Bax, Livin and Survivin was detected by RT-PCR.Results:1The transcriptional activity of NF-K B in BXPC-3cells exposed to geraniol was inhibited significantly in contrast to the control group.2After treated with geraniol for48h, Bax proteins was up-regulated, while Livin, Survivin, Bcl-2and Procaspase-3,8,9down-regulated.3Result from observing geraniol effect on protein expression of Cyt-C in mitochondria and endochylema of BXPC-3pancreatic adenocarcinoma cells by Western Blotting:the Cyt-C enjoys a high expression in the BXPC-3cell mitochondria in the control group after48h while the expression in the geraniol group was low; The Cyt-C expression is relatively low in the BXPC-3cell cytosol in control group while the expression has significantly increased in the geraniol group.4. After treated with geraniol for48h, TIMP-2proteins was up-regulated, while MMP2, MMP9and NF-κB down-regulated.5. In Xenograft The expression of bcl-2,Livin and Survivin mRNA was down-regulated, while Bax was up-regulated.Conclusion:1. The possible molecular mechanism of apoptosis of human pancreatic adenocarcinoma BXPC-3cells induced by geraniol is related to up-regulating expression of bax, and reducing expression of bcl-2, Livin, Survivin,Procaspase-3,8,9protein, and inhibit the vitality of NF-κ B.2.Geraniol can make intramitochondrial cytochrome C release to cytolymph, trigger the cascade activation of caspase and induce apoptosis.3. Geraniol can increase the expression of TIMP-2proteins while decrease the expression of MMP2, MMP9, thus inhibit the invasion ability of BXPC-3cell.