Study On The Effect And Molecular Mechanism Of Melatonin On 5-fluorouracil Resistance In Hepatocellular Carcinoma

BackgroundAccording to statistics from 2018,primary liver cancer is the sixth most common tumor type in the world and the fourth most common cause of tumor-related death.Hepatocellular carcinoma?HCC?accounts for the majority of primary liver cancer worldwide.The dramatic increase in liver cancer patients and the recurrence of clinical liver cancer patients have produced a worldwide pandemic.To date,few effective chemotherapy drugs have been used to treat this malignant tumor,with the resistance of hepatocellular carcinoma to chemotherapy and radiation therapy,hepatocellular carcinoma has brought enormous health burdens to the world.Although 5-fluorouracil?5-FU?is one of choice for the treatment of advanced hepatocellular carcinoma?HCC?,it also has a variety of acquired and intrinsic drug resistance.Melatonin?N-acetyl-5-methoxytryptamine,MLT?is an endogenous guanamine that is secreted by the pineal gland into circulatory system,and it can regulate circadian rhythm.Over the past two decades,more and more scholars have discovered the multifunctional potential of melatonin,such as anti-inflammatory,anti-cancer,antioxidant activity,and endocrine rhythm regulation.It has been reported that melatonin exhibits an anti-tumor effect through anti-proliferation and pro-apoptosis.In addition,it has been reported that melatonin can enhance chemotherapy-induced apoptosis.It has been reported that melatonin inhibits the PI3K/AKT pathway.At the same time,a large number of tumor clinical samples and studies in vitro indicate that abnormally activated PI3K/AKT pathway is associated with tumor 5-FU-resistance.And the inhibitor of AKT pathway combined with 5-FU have a synergistic effect in the treatment of hepatocellular carcinoma.These research evidences suggest that melatonin may enhance the sensitivity of hepatocellular carcinoma to 5-FU,which will bring huge benefits to patients with advanced liver cancer.Hepatocellular carcinoma is usually insensitive to anti-tumor drugs,and patients with hepatocellular carcinoma who are treated with chemotherapy drugs will rapidly develop acquired drug resistance.Multidrug resistance?MDR?refers to a phenomenon in which cancer cells exposed to one drug cross-resistance to other drugs with completely different structures and functions.One of the main mechanisms of MDR is the active pumping of tumor cells,resulting in the accumulation of intracellular chemotherapeutic drugs below the effective level.P-glycoprotein?P-gp?which belongs to the ATP-binding cassette?ABC?protein superfamily is one of the most typical efflux pump that mediate MDR.Inhibitors of P-gp can increase the concentration of intracellular drug accumulation by inhibiting drug pumping,thereby sensitizing cancer cells to chemotherapy.Despite some success in early studies,most clinical trials involving P-gp inhibitors,even clinical trials of third-generation inhibitors,have not confirmed their clinical benefit.Given that chemotherapy resistance is critical for the progression of hepatocellular carcinoma,it is important and urgent to find alternatives to P-gp inhibitors.ObjectiveBy treating BEL-7402/5-FU cells with 5-FU or melatonin or 5-FU combined with melatonin,we observed whether melatonin could reverse 5-FU resistance and study the effects of melatonin on proliferation,apoptosis and P-gp expression of human hepatocellular carcinoma cells.The mechanism of melatonin reversing 5-FU resistance in BEL-7402/5-FU hepatocellular carcinoma cells was further explored by studying PI3K/AKT signaling pathway.Methods?1?BEL-7402 cells and BEL-7402/5-FU cells were cultured with twelve groups of 5-FU from low to high concentration.MTT assay was used to detect the inhibitory rate of 5-FU.IC₅₀ values of 5-FU were calculated to identify the resistance of BEL-7402/5-FU cells to 5-FU.Western-Blot method was used to detect the expression of PI3K/AKT pathway protein and P-gp in the two cells.?2?BEL-7402/5-FU cells and human normal hepatocytes LO2 were treated with nine concentrations of melatonin.MTT assay was used to detect the inhibition rate of melatonin on this two cells,and the non-toxic dose of melatonin was screened for the follow-up study.?3?BEL-7402/5-FU cells were cultured with melatonin or 5-FU or their combination for 48 hours,IC₅₀ and reversal fold were calculated by MTT assay.Apoptotic rate was detected by flow cytometry.Cell proliferation was detected by colony formation assay.PI3K/AKT pathway proteins and P-gp expression were detected by Western-Blot assay.?4?BEL-7402/5-FU cells were treated with melatonin or 5-FU or 5-FU combined with melatonin or 5-FU combined with verapamil for 48 hours,the expression of P-gp protein was detected by immunocytochemical staining,and the efflux of Rh123 was detected by flow cytometry.?5?After treated with melatonin or 5-FU or 5-FU combined with melatonin or 5-FU,melatonin and LY294002 for 48 hours,apoptotic rates were detected by flow cytometry.And the expression of PI3K/AKT pathway proteins and P-gp were detected by Western-Blotting in BEL-7402/5-FU cells.Results?1?MTT assay showed that BEL-7402/5-FU cells were about 280-fold resistant to 5-FU in comparison with BEL-7402 cells.Western-Blot showed that the relative expression levels of P-gp,PI3K and p-AKT/AKT were increased and the PTEN was decreased significantly in BEL-7402/5-FU cells.?2?Melatonin inhibited BEL-7402/5-FU cells in a time-and dose-dependent manner.The IC₁₀ value of melatonin to L02 was 0.84 mM calculated by MTT.?3?IC₅₀ value of 5-FU in BEL-7402/5-FU cells was 1455.59 ug/ml.When combined with melatonin?0.5mM?,the value was 328.23 ug/mL and the reversal fold was 4.33.?4?Melatonin can significantly increase apoptotic rate induced by 5-FU in BEL-7402/5-FU cells and inhibit colony formation.Western-Blot showed that 5-FU could significantly increase the expression of P-gp,PI3K and p-AKT in BEL-7402/5-FU cells,while melatonin could neutralize the increase.?5?PI3K inhibitor LY294002 was used to inhibit PI3K/AKT signaling pathway.Flow cytometry results showed that the addition of PI3K inhibitor could further increase the apoptotic rate and reduce the expression levels of P-gp,PI3K and p-AKT.Conclusions?1?BEL-7402/5-FU cells are a suitable cell line for studying P-gp-mediated hepatocellular carcinoma 5-FU resistance.?2?Melatonin?0.5mM?has the effect of reversing 5-FU resistance of BEL-7402/5-FU cells.?3?Melatonin combined with 5-FU can increase the apoptosis rate,and reduce the ability of clonal formation of BEL-7402/5-FU cells.?4?By inhibiting the PI3K/AKT pathway,melatonin can down-regulate P-gp expression,thereby reversing P-gp-mediated 5-FU resistance in BEL-7402/5-FU cells.