Anthraquinone Derivative C2 Reverses The Drug Resistance Of Colon Cancer Cells HCT116/L-OHP And Its Mechanism

Inhibition and destruction of cancer cells through chemotherapy is one of the main clinical treatment of colon cancer.However,in the treatment of patients with advanced colon cancer,due to the long-term exposure to the stimulation and pressure of chemical drugs,it will often make the patient’s sensitivity to the drug become worse,produce multidrug resistance,and eventually lead to the failure of chemotherapy.Therefore,there is an urgent to develop new safe and efficient drug resistance reversal agents,improve the efficacy of chemotherapy,and has become a major research direction of anticancer drugs research.Studies have found that the autophagy pathway is involved in the development of tumor multidrug resistance,so the problem of tumor multidrug resistance can be reversed by targeted regulation of autophagy.Studies have found that many anthraquinone compounds can induce autophagy and regulate drug resistance-related proteins,thereby increasing the sensitivity of tumor resistant cells to anticancer drugs.Because natural anthraquinone compounds have problems such as difficult extraction,high cost,and poor solubility,people often obtain them through chemical synthesis.The compound 1-nitro-2 acyl anthraquinone-leucine(C2)was obtained by a series of chemical reactions in the early stage of the research group.Through MTT experiments,it was proved to have anti-tumor effect little toxicity to normal cells.It has the potential to develop drug resistance reversal agent.This article focuses on the effect and molecular mechanism of C2 reversing the multidrug resistance of colon cancer cells HCT116/L-OHP.The main research results include the following aspects:1.C2 reverses the drug resistance of colon cancer HCT116/L-OHP cells.Firstly,the inhibitory effects of three anticancer drugs oxaliplatin,cisplatin and 5-fluorouracil on colon cancer sensitive cells HCT116 and drug-resistant cells HCT116/L-OHP were tested.The results show that the sensitivity of HCT116 cells to the three anticancer drugs is much greater than that of HCT116/L-OHP cells,which proves that colon cancer HCT116/L-OHP cells have better drug tolerance.Then the inhibitory effect of C2 on the proliferation of HCT116/L-OHP cells was determined by MTT experiments,and the concentration of inhibition rate less than 20% was taken as the reverse drug concentration.The drug resistance reversal effect of C2 was determined by the combination of nontoxic dose and oxaliplatin.At the same time,the observation of cell morphology showed that with the increase of the concentration of C2,the cell density gradually decreased and the morphology shrank into a spindle shape.Subsequently,cell cloning,flow cytometry to detect drug efflux ability,Western blot and q PCR to detect drug resistance protein expression.The results showed that C2 can significantly inhibit the ability of cell clone formation,inhibit cell efflux,and reverse the drug tolerance of HCT116/L-OHP cells by down-regulating the expression of P-gp and BCRP.2.C2 induces autophagy and apoptosis in HCT116/L-OHP cells.MDC experimental observations found that the green fluorescent dots increased with the increase of C2 concentration.Flow cytometry detected cell apoptosis,and the results showed that the number of cell apoptosis gradually increased and was concentration-dependent.Through Western blot and q PCR,it was found that as the concentration of C2 increased,the ratio of LC3-II/I,the expression of Beclin 1 and Cleaved caspase-3 gradually increased,and the expression of Bcl-2 gradually decreased.Then the cells were pretreated with the autophagy inhibitor 3-MA,and then combined with the drug to find that the expression of Cleaved caspase-3 was greater than that of the drug-treated group,that is,C2-induced autophagy can also activate cell apoptosis.3.C2 reverses the drug resistance of HCT116/L-OHP cells by mediating p53 and PI3K/AKT/mTOR signaling pathway.The results of Western blot and q PCR experiments showed that with the increase of C2 concentration,the expression of PI3 K,phosphorylated AKT,and mTOR were down-regulated at the protein and m RNA levels,and the expression of p53 at the protein and m RNA levels was up-regulated.It indicates that C2 may induce autophagy in HCT116/L-OHP cells by mediating p53 and PI3K/AKT/mTOR signaling pathway.Furthermore,by adding 3-MA to inhibit autophagy,it was found that the expression of P-gp and BCRP was up-regulated at the protein and m RNA levels,and the survival rate of HCT116/L-OHP cells increased slightly.These results indicate that C2 induces autophagy in cells by mediating p53 and PI3K/AKT/mTOR signaling pathways,thereby promoting cell apoptosis,thereby enhancing the sensitivity of HCT116/L-OHP cells to oxaliplatin.In conclusion,C2 reverses the drug resistance of colon cancer drug-resistant cells HCT116/L-OHP by regulating autophagy and apoptosis,providing a theoretical basis for the development of anthraquinone derivatives in tumor resistance reversal agents.