Experimental Study Of The Anti-tumor And Its Mechanisms Of Cerulenin Combined With Gemcitabine On Human Pancreatic Cancer Cell Line BXPC-3

Part I: Effect and mechanism of cerulenin on human pancreaticcarcinoma cell line BXPC-3Objective: To study the effect of cerulenin on the growth and apoptosis of humanpancreatic cancer cell line BXPC-3in vitro,and to explore its possible mechanisms.Methods: The growth of pancreatic cancer cell BXPC-3treated by differentconcentrations of cerulenin (0,2.5,5,10,20,40μg/ml) was evaluated by CCK8assay,and inhibition ratios were calculated in all groups and suitable IC50of cerulenin wereworked out by calculation；With using different concentrations cerulenin (0,5,10,20μg/ml)to deal with BXPC-3, the apoptosis ratioes and changes of cell cycle were detected by flowcytometry and AnnexinV/PI fluos stainging；and the expression of FAS mRNA,Bcl-2mRNA,Bax mRNA,Caspase8mRNA,Caspase9mRNA and Caspase3mRNA weredetected by RT-PCR；Bcl-2,Bax proteinum were detected by Western Blot in vitro；Caspase3,Caspase8and Caspase9activation was measured using a caspase fluorometricassay kit.Results：(1) Cerulenin could inhibit the growth of the human pancreatic cancer cellline BXPC-3, and the inhibitory effect had dose-and-time dependence (P<0.05).(2)Theearly stage apoptosis and cyclomorphosis of BXPC-3cells was detected by flow cytometryand AnnexinV/PI fluos stainging after they were incubated48hours with cerulenin.Withthe increase of Cer concentration, ratio of S phase increased from(5.18±0.25)to(25.15±1.15)and ratio of G2phase declined homologously(P<0.05),The early stage apoptosis rate ofBXPC-3cells was (11.67±0.65)%when they were incubated with5μg/ml Cer and the rate was (48.8±1.23)%with20μg/ml Cer. Appearantly, apoptosis rate was positively correlatedto Cer concentration.(3)The expression of FAS mRNA and Bcl-2mRNA decreased withconcentration-dependence after BXPC-3cells were incubated48hours with Cer, whichwas detected by RT-PCR. Otherwise, The expression of Caspase8mRNA,Caspase9mRNA,Caspase3mRNA and Bax mRNA increased with concentration-dependence,Comparisons of all Cer groups and control group have significant difference(P<0.05).(4)The expression of Bcl-2was downregulated and the expression of Bax wasupregulated significantly after BXPC-3cells were incubated48hours with Cer, which wasdetected by Western Blot. And the ratio of Bcl-2/Bax were (4.29±0.15),(1.82±0.02),(1.01±0.08),(0.38±0.05) when BXPC-3were incubated with0,5,10,20μg/ml Cer(P<0.05).(5)With the increase of Cer concentration, the activity of Caspase8,Caspase9and Caspase3was increased gradually in all Cer groups, Comparisons all Cer groups andcontrol group have significant difference (P<0.05). At the5,10,20μg/ml Cerconcentration,the caspase3activity was increased as (1.75±0.31),(3.27±0.01),(4.96±0.02)times as control group, the caspase9activity was increased as (1.40±0.4),(2.12±0.06),(3.39±0.02)times as control group, the caspase8activity was increased as(1.26±0.07),(1.86±0.11),(2.5±0.02)times as control group.Conclusion：Cerulenin could inhabit the growth of human pancreatic cancer cell lineBXPC-3in a dose-and-time dependent manner, and its mechanisms may directly inhibittummor cells to proliferate and arrest cell cycle in S stage. And it mainly induces apoptosisthrough mitochondrion pathways. Part Ⅱ: Effect and mechanism of Cer in combination with GEM onpancreatic cancer cell BXPC-3Objective: To investigate the effect and mechanism of action in pancreatic cancerBXPC-3cell treated by Cer in combination with GEM.Methods: BXPC-3cells were cultivated with10μg/ml Cer and20μmol/L GEM. OD values were detected by CCK8assay and their inhibition ratios were calculated and earlyapoptosis of them were detected by AnnexinV/PI double staining method and theexpression of Bcl-2mRNA and Bax mRNA were detected by RT-PCR, Bcl-2,Baxproteinum were detected by Western Blot.Result: Both Cer and GEM could inhibit effectivly BXPC-3cell' proliferation.Cerulenin combinated with gemcitabine had synergistic effect and their combination couldincrease both early stage apoptosis obviously and advanced stage apoptosis, the early stageapoptosis was (31.37±1.04) when BXPC-3were cultivated with10μg/ml Cer,the ratewas(38.33±0.75)with20μmmol/L GEM and the rate was (69.43±0.83)%with10μg/ml Cercombined with20μg/ml Cer. And the expression of Bcl-2mRNA,Bcl-2protein weredownregulated, and the expression of Bax mRNA, Bax protein were upregulated.Comparisons of treated groups and control group have significant difference (P<0.05) andcomparisons of single drug groups and combination group have also significant difference(P<0.05).Conclusion： Cerulenin could obviously inhibit pancreatic cancer cell BXPC-3proliferation in a dose-and-time dependent manner in coordination with Gemcitabine.Itsmajor mechanisms maybe promote apoptosis of cells.