Study On Atorvastatin Reverse Multi-Drug Resistance And Its Mechanism In Colon Cancer Cells

OBJECTIVE: To determine whether atorvastatin can reverse multi-drug resistance and its mechanism in human colon cancer HCT116/L-OHP cells in vitro.METHODS: The toxic effect of atorvastatin on HCT116(sensitive strain) and HCT116/L-OHP(drug-resistant strain) was measured by CCK-8 and nontoxic concentration of ATV for HCT116/L-OHP was determined. The toxic effect of oxaliplatin to HCT116 and HCT116/L-OHP was measured by CCK-8 in vitro for resistance index. HCT116/L-OHP was treated with nontoxic concentration of atorvastatin combined with oxaliplatin to observe the reversal effect of atorvastatin and get the reversal fold. Quantitative real time PCR(q RT-PCR) was used to detect the expression of LRP1,MRP1,P-gp,BRCP m RNA in HCT116/L-OHP cells treated with or without ATV after 48 h. Effect of oxaliplatin combined with atorvastatin and oxaliplatin only treated HCT116/L-OHP cells on apoptosis when treated for 48 h was detected by Annexin V-FITC/PI staining. Western blot assay was used to determine the expression of bax,bcl-2,caspase-3 in HCT116/L-OHP cells treated with oxaliplatin alone or combined with atorvastatin for 48 h treatment time and for detection of Stat3,p-Stat3, NF-κB and NF-κB/p65 in HCT116/L-OHP cells treated with or without ATV after 48 h.RESULTS: Atorvastatin was toxic to HCT116 and HCT116/L-OHP in a dose-time dependent manner and nontoxic concentration of ATV for colon cancer cells HCT116/L-OHP is 20μmol/L. Oxaliplatin was toxic to HCT116 and HCT116/L-OHP in a dose-dependent manner and the toxic effect of oxaliplatin to HCT116 and HCT116/L-OHP was compared. Statistical significance was found(P<0.05).IC50 of oxaliplatin to HCT116 and HCT116/L-OHP were 218.197 ㎎/L and 1398.773 ㎎/L, respectively. Drug-resistant index of HCT116/L-OHP was 6.41. Atorvastatin increased cell toxic effect of oxaliplatin compared to oxaliplatin only(p<0.05). After treating with 20μmol/L atorvastatin(nontoxic concentration) combined with oxaliplatin for 48 h, IC50 of oxaliplatin declined to 399.204 ㎎/L, and the reversal fold was 3.5. q RT-PCR demonstrated a decreased level of BCRP and LRP1 but the expression of P-gp and MRP1 did not change in HCT116/L-OHP cells treated with ATV after 48 h. The apoptotic rate of control group, ATV only group, oxaliplatin only group and oxaliplatin combined with atorvastatin group were(0.58±0.14)%,(0.95±0.15)%,(6.47±0.71)% and(12.26±0.74)%, respectively. ATV only group did not have statistical significance compared with the control group natural apoptosis(P>0.05) but statistical significance was found in oxaliplatin only group and oxaliplatin combined with ATV group compared with the control group(P<0.05). Moreover, the difference between oxaliplatin only group and oxaliplatin combined with ATV group was also significant(P<0.05).Western blot assay showed that atorvastatin combined with oxaliplatin group compared with oxaliplatin only group showed an upregulation of the expression of bax and caspase-3 while there was downregulation in the expressions of bcl-2 in HCT116/L-OHP cells after 48 h treatment. The expression of p-Stat3 and NF-κB/p65 protein was decreased when HCT116/L-OHP cells were treated with ATV for 48 h. However, the expression of Stat3 and NF-κB showed no obvious difference.CONCLUSION: Atorvastatin is toxic to HCT116 and HCT116/L-OHP cells in a dose-time dependent manner and nontoxic concentration of ATV for colon cancer cells HCT116/L-OHP is 20μmol/L. 20μmol/L ATV could partly reverse drug resistance of HCT116/L-OHP to oxaliplatin in vitro(the reversal fold is 3.5).The mechanism of colon cancer drug resistance reversal by ATV is decreased expression of BCRP and LRP1. It showed no obvious relationship with P-gp and MRP1. 20μmol/L ATV cooperated with oxaliplatin in increasing apoptosis of HCT116/L-OHP cells. Oxaliplatin combined with 20μmol/L ATV compared with oxaliplatin only decreased the expressions of bcl-2 and increased the expression of bax and caspase-3. Moreover,decreased expression of p-Stat3 and NF-κB/p65 in HCT116/L-OHP cells maybe the mechanism involved in drug resistance reversal by 20μmol/L ATV.