The Effect Of K-ras Mutation To Drug Sensitivity Of Anti-cancer Drugs And The Mechanism

K-ras is a member of ras gene family, whose mutant frequency is high as90%in pancreatic cancer. According to recent reports, mutant K-ras is related to induction and progression of tumors. What’s more, drug resistances to agents such as anti-EGFR antibodies and EGFR-TKIs are found in K-ras mutant tumors. However, there are few reports whether mutant K-ras would reduce the effects of gemcitabine,5-FU, cisplatin or docetaxel. The study of mechanism how K-ras mutation induces drug resistance in vitro is important to the guidance of rational use of drug in clinic.ObjectiveTo construct the recombinant plasmids containing different types of K-ras mutation and express them in human pancreatic cell line Bxpc-3. Cell growth was studied after stable transfection as well as the effect of mutant K-ras to downstream signals. Drug resistances of cells expressing mutant K-ras to anti-cancer agents such as erlotinib, gefitinib, gemcitabine,5-FU, SN-38, docetaxel and cisplatin were studied. What’s more, we explored the role of K-ras mutation on drug resistance, from both PK and PD.MethodsThe full sequence of K-ras gene carrying Bam HI and Xho I sites was inserted into pAcGFP1-C3vector to construct KRAS-pAcGFP1-C3(WT, G12V, G12R, G12D, G13D). Ligation products were transformed to E. coli DH5a, cultured and extracted. Plasmids were transfected into human pancreatic cancer cell line Bxpc-3. We picked stable expressing clones and confirmed them using western blot. Growth curve were analyzed as well as the expression of downstream signals. MTT assay was performed to study the drug resistances of transfected Bxpc-3cells to erlotinib, gefitinib, gemcitabine,5-FU, docetaxel and cisplatin.To explore the mechanism how mutant K-ras induced resistance to anti-cancer drugs, we tested the influences to cell cycle using PI as well as apoptosis using DAPI and expression of PARP. What’s more, MEK inhibitor U0126was used to find whether MEK/ERK pathway is involved in the induction of drug resistance.HPLC was used to explore the differences of different K-ras mutant cell lines in ingestion of SN-38or docetaxel. What’s more, we used Calcein-AM assay to test the expression and function of P-gp of the cells. We tested the relationship of K-ras mutation and expression of transporters to find whether it’s a potential mechanism of drugs resistance induced by K-ras mutation.ResultsFull sequences of K-ras (WT, G12V, G12R, G12D and G13D) were successfully inserted into pAcGFP1-C3vector to construct KRAS-pAcGFP1-C3. Vector and recombinant plasmids were transfected into human pancreatic cancer cell line Bxpc-3and picked the stably expressing clones. After transfection, vacuolation and shrinking occurred in some cells with higher fluorescence intensity than other transfected cells. The expression of K-Ras-GFP fusion protein was showed similar in WT and mutant K-ras transfected Bxpc-3cells using western blot.After transfection, the growth of Bxpc-3cells was slowed. The vector transfected cells grew a little faster than the KRAS-pAcGFP1-C3transfected cells. In all five picked K-Ras overexpressed clones, the KRAS(G13D)-pAcGFP1-C3transfected cells grew slower than all the other clones. In addition, the WT and other mutant clones showed comparable in vitro growth. The levels of total AKT and ERK were proved to be similar in different mutant and WT clones, but phosphorylation of AKT in G12V clone was higher than WT and the other mutant clones, while G12V, G12R and G12D showed higher expression of p-ERK.MTT assay was performed to determine the role of K-ras mutation on the sensitivities of chemotherapy agents. For the EGFR tyrosine kinase inhibitors (EGFR-TKIs), the expression of G12V and G12D mutant K-Ras was associated with obvious resistance to gefitinib, while showed less reduced sensitivity to erlotinib. For the cytotoxic agents, the mutant clones showed an increased resistance to gemcitabine,5-FU, and docetaxel when compared with the WT clone, whereas the response to cisplatin was not changed. Moreover, the expression of a specific K-Ras mutation induced a different drug sensitivity pattern. Specifically, the expression of G13D was associated with a strong resistance to gemcitabine and docetaxel, whereas the expression of G12V resulted in a remarkable reduced response to gefitinib and5-FU. As to SN-38, the active metabolite of irinotecan, G12D and G13D showed a remarkable resistance.Further, we explored how K-ras mutation induced drug resistance to docetaxel. According to the result of cell cycle determination using PI, G2/M arrest induced by docetaxel showed no differences between G12V mutant K-ras transfected cells and WT. But mutant K-ras could reduce cell apoptosis induced by docetaxel. Confirming this result, the expression of c-PARP of mutant K-ras transfected cells was less than WT group after incubating with100nM docetaxel for24h. G12V mutation cells showed the lowest expression of c-PARP, which could partly explain the resistance of docetaxel. The down regulation of K-Ras by siRNA resulted the increasing of c-PRAP, which comfirmed the relationship of K-ras and apoptpsis. What’s more, neither PI3K/AKT nor MEK/ERK pathway might be involved in apoptosis induced by docetaxel.We also studied the influence of K-ras mutation to pharmacokinetics of drugs. The results of HPLC showed that the K-ras mutant cell lines ingest less SN-38or docetaxel comparing with WT cell lines. The drug resistance induced by K-ras mutation may be related to the reduced drug accumulation, whose mechanism is still waiting to be explored.ConclusionDrug resistance of gefitinib, gemcitabine,5-FU and docetaxel may be related to K-ras mutation, which showed no effect to erlotinib and cisplatin. Mutant K-ras would not affect the cell cycle arrest, but reduced cell apoptosis caused by docetaxel, which finally resulted in drug resistance in K-ras mutant Bxpc-3cells. What’s more, this effect may not have direct connection to PI3K/AKT or MEK/ERK pathway. K-ras mutation is related to the reduced ingestion of anti-cancer drugs and expression of transporters, which may be one of the mechanisms how mutant K-ras induced drug resistance. In our study, we studied if mutant K-ras could cause drug resistances to the six anti-cancer drugs and explored the mechanism. The findings would be able to explain some results in clinical trials.