| Cucurmosin(CUR), a new ribosome-inactivating proteins(RIP), exhibites anti-tumor and apoptosis-inducing effects both in vitro and in vivo on the human chronic myeloid leukemia K562 cell line and the murine B16 melanoma cell line. Pancreatic cancer is a devastating disease characterized by a very poor prognosis due to the low rates of early diagnosis and rapid progression, with most patients dying within 12 months after diagnosis. The goal of this study was to examine its anti-pancreatic cancer effects both in vitro and in vivo and explore the underlying molecular mechanisms with the aim of providing theoretical rationale and experimental evidence for its potential clinical application.ObjectiveTo investigate the anti-tumor effect of CUR on human pancreatic cancer PANC-1 cells in vitro and in vivo, and to identify the molecular mechanism of CUR on inducing apoptosis and chemo-sensitization.Methods1. MTT assay was used to determine the effect of CUR on the growth of PANC-1 cells in vitro, orthotopic transplantation NOD/SCID mouse model of pancreatic cancer was established to observe the anti-tumor effect of CUR in vivo.2. Electron microscopy, flow cytometric analysis and enzyme linked immunospecific assay were used to examine the apoptosis-inducing effect of CUR.3. Fluorescent staining was used to examine the change of mitochondrial membrane potential, and western blot was used to determine the protein level of caspase-3 , caspase-9, bcl-2 , bax, survivin ,smac and cytoplasmic cytochrome-c in PANC-1 cells after CUR treatment.4. MTT assay was used to detecte the cytotoxic effect of the different concentrations of CUS with GEM , and western blot was used to determine the protein level of P-170 and MDRP after CUR and GEM treatment.Results1. In vitro anti-tumor activity of CUR, PANC-1 cells were treated with 0.3125, 0.625, 2.5, 5.0, 10.0, 20.0, 40.0 and 80μg/mL CUR and cell viability was measured at 24, 48 and 72 hours using MTT assay. The inhibitory rates of the cell growth for 24 hours were 9.2, 14.4, 20.2, 32.0, 36.4, 44.1, 50.3 and 58.6% respectively. When treated for 48 hours , the inhibitory rates were 25.1, 35.6, 39.7, 46.5, 52.9, 57.0, 64.5 and 72.6% respectively. When treated for 72 hours, the inbitory rate was 31.4, 51.5, 60.1, 67.2, 75.2, 85.7, 91.0 and 93.4%.The 50% inhibitory concentrations (IC50) of CUR for 24, 48, and 72h were 40.24μg/mL, 8.24μg/mL and 1.17μg/mL respectively. CUR significantly inhibite the proliferation of PANC-1 cells in dosage- and time-dependent manner in vitro.2. In vivo anti-tumor activity of CUR, Orthotopic transplantation NOD/SCID mouse model of pancreatic cancer was established .Treatment with CUR at the dose of 0.125, 0.25 and 0.5mg/mL inhibited the growth of pancreatic cancer PANC-1 xenografs by 45.2%, 50.0% and 59.7% respectively. The anti-tumor results in vivo demonstrate that CUR was remarkably active against PANC-1 xenografs in a dose-dependent manner.3. Effects of CUR on cell cycle and apoptosis of human pancreatic cancer PANC-1 cells.①After exposure to 10ug/mL CUR for 24h, most cells presented typical morphologic changes of apoptosis such as chromatin condensation or shrunken or fragmentation nucleus by transmission electron microscopy.②Flow cytometric analysis with PI staining was used to detecet cell cycle changing after CUR intervention. Being exposed to 0, 2.5, 10.0, and 40.0ug/mL of the CUS for 72h, the percentage of G0/G1 phase cells was 46.56±5.08, 53.33±5.05,67.50±6.50 and 77.00±6.73 respectively. Being exposed to 40.0μg/mL of the CUS for 24, 48 and 72 hours, the percentage of G0/G1 phase cells was 56.60±6.65, 67.83±6.76 and 77.00±1.73%respectively, These data show that CUR induces the accumulation of PANC-1 cells in the G0/G1 phase of the cell cycle in a dose-dependent and time-dependent maner.③Flow cytometry combined with annexinv-FITC/PI staining showed that CUR induced apoptosis. When PANC-1 cells were incubated with 0 , 2.5,10,40μg/mL CUR for 72h , the rate of apoptosis were 2.50士0.13, 8.30士1.23, 23.40士2.45 and 48.50士3.65%. Being exposed to 40.0μg/mL of the CUS for 24, 48 and 72h, the rate of apoptosis was 16.51士2.97,38.51士2.38 and 48.50士3.65%. Significiant difference was found among them. Thus these data indicate that CUR induces the apoptosis of PANC-1 cells in a dose-dependent and time-dependent maner.④Enzyme linked immunospecific assay was used to detecet caspase-3 activity after CUR treatment. Being exposed to 0, 2.5, 10.0, and 40.0ug/mL of the CUS for 72h, the caspase-3 activity was 0.009,0.011,0.035 and 0.065 units. So the caspase-3 activity was enhanced by CUR in a dose-dependent maner.4 molecular mechanism of CUR inducing apoptosis. Western blot analysis indicate that treatment of PANC-1 cells with CUR.(2.5, 10, 40ug/mL) for 72h results in release of mitochondrial cytochrome C and increase expression of bax, smac, caspase-3 and caspases-9 protin level in dose-dependent maner , but survivin,bcl-2 protein level was not significiant altered by CUR .5 The 50% inhibitory concentration (IC50) of GEM decrease from 36.76 nmol/L to 12.14 nmol/Lwhen together with CUS, reversal fold is 3.02. Western blot analysis indicate that treatment of PANC-1cells with CUR(2.5,10,40ug/mL) for 72h results in a decrease of P-170 protein level in dose-dependent maner but MDRP protein level was not significiant altered by CUR .Conclusion1. CUR has superior anti-tumor efficacy on human pancreatic cancer PANC-1 cells in vitro and in vivo via cell apoptosis and G0/G1 cell cycle arrest. CUR induced the apoptosis of PANC-1 cells via the up-regulation of smac and bax gene, leading to release of mitochondrial cytochrome C and then activation of caspase-9 and caspase-3. Thus CUR induces apoptosis via the intrinsic pathway. CUR has a chemo-sensitization effect with gemcitabine on pancreatic cancer cell line PANC-1, which maybe associated with down-regulation of p-170 protein.
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