| Melanoma is a malignant tumor that in most cases occurs on the skin caused by the excessive proliferation of abnormal melanocytes.It is highly invasive and lethal,and its incidence continues to increase worldwide.Studies have found that 40% of melanoma patients have BRAF gene mutation,and the 75%～ 90% mutation mode is BRAFV600 E.Vemurafenib is a small molecular biological inhibitor targeting BRAF mutations and the preferred drug for treating with the advanced melanoma in the world.Unfortunately,its effect is limited,and half of patients develop acquired resistance within a year,leading to further progression of the disease.Therefore,it is urgent to clarify the resistance mechanism and find new adjuvant therapies to reverse the resistance.Insulin receptor substrate 4（IRS4）is a substrate that can be activated by insulin receptor tyrosine kinase.Some scholars studied the drug resistance of breast cancer and found that the expression of IRS4 gradually increased during the process of breast cancer cells transforming into drug-resistant cells;After overexpression of IRS4,breast cancer cells were not sensitive to the therapeutic drugs targeting HER2 and the PI3K/Akt pathway in the cells was activated.At the same time,studies had reported that the occurrence of drug resistance in melanoma might be related to the activation of the PI3K/Akt pathway or the reactivation of Erk.In this experiment,we also found that the expression of IRS4 in drug-resistant cells was significantly higher than that in the melanoma parent cells.Therefore,we speculated that the drug resistance of melanoma may also be related to the IRS4 and PI3K/Akt pathways.Homoharringtonine（HHT）is an effective anti-cancer component in plants of the genus Harringtonine.In recent years,some researchers have found that homoharringtonine is effective in imatinib-resistant leukemia patients,which has attracted widespread attention.In addition to leukemia,homoharringtonine is also used in liver cancer,breast cancer,colorectal cancer and other cancers.Studies have reported that homoharringtonine has a strong inhibitory effect on the proliferation of the melanoma highly metastatic cell line B16,but the effect on the melanoma vemurafenib-resistant cell line A375R has not been reported.In order to clarify the effect of homoharringtonine on drug-resistant melanoma cells,and whether its mechanism is related to IRS4,this article did the following investigation.ObjectiveIn this study,melanoma A375 cell line,Vemurafenib-resistant A375 cell line（A375R）were used as the research objects in vitro.The effects of Vemurafenib alone or the combination with HHT on Vemurafenib-resistant melanoma cells were investigated to explore its role in reversing drug resistance and possible mechanisms.MethodsThe first part: 1)The cell proliferation inhibitory rate of Vemurafenib on melanoma cells A375 and A375R were detected by CCK-8 method,and the half-inhibition concentration（IC50）on two types of cells and drug resistance factor of A375R to Vemurafenib was calculated.2)Queried the expression of IRS4 in normal human cells and melanoma cells through the GEPIA database;RT-q PCR and Western blotting detected the expression of IRS4 in A375 and A375R cells;3)Western blotting detected the expression of PI3K/Akt and Erk pathway related proteins in A375 and A375R cells.The second part: 1)The effects of homoharringtonine on cell proliferation of A375R celld after treatment for 24,48 and 72 h were detected by CCK-8 assay.2)Using the clone formation experiment to detect the influence of homoharringtonine on the cell clonal formation and proliferation;3)Flow cytometry was used to detect the effects of homoharringtonine on the cell cycle of drug-resistant melanoma cells;4)Western blotting was used to detect the effects of homoharringtonine on cycle-related proteins（Cyclin E1,CDK2）and pathway-related proteins（IRS4,PI3 K,p-Akt,p-Erk）.The third part: 1)A375 and A375R cells were cultured in vitro,the cell viability of the two cells after homoharringtonine treatment were detected by CCK-8 method.A375R cells were treated with homoharringtonine of IC10 and different concentrations of Vemurafenib to detect the proliferation inhibitory effect of combination of drugs on A375R cells.According to above results,We divided into four groups: Control group,HHT group,Vem group and Vem + HHT group;2)The morphological changes of apoptosis were observed by microscope to observe the effect of Vem combined with HHT on A375R cells;3)Ed U was used to detect the effect of combination on A375R cell proliferation;4)The effect of Vemurafenib combined with HHT on apoptosis of A375R cells was detected by flow cytometry and Western blotting;5)Western blotting to detect the effect of combined drugs on IRS4,PI3K/Akt and Erk pathway related proteins;6)By adding the inhibitor of IRS4 upstream protein IGF-1R/Ins R BMS-754807 and Ins R activator MLR1023 to explore the role of IRS4 in melanoma drug-resistant cells.ResultsThe first part1.Detection by CCK-8 assay showed that the half-inhibition concentration（IC50）of Vemurafenib on melanoma cells A375 and A375R were 0.94 μmol/L and 49.09 μmol/L respectively,and the resistance factor was about 52.22 times.2.The results of GEPIA,RT-q PCR and Western blotting showed that: IRS4 is almost not expressed in normal human cells and parental melanoma cells,but highly expressed in drug-resistant cells.3.Western blotting showed that compared with A375 cells,the expression of PI3K/Akt and Erk pathway related proteins was increased in A375R cells.The second part1.After the A375R cells was treated with homoharringtonine for 24,48 and 72 hours,IC50 for homoharringtonine were 18.57 ng/m L,9.88 ng/m L and 9.23 ng/m L.It indicated that homoharringtonine could effectively inhibit the proliferation of A375R cells in a time and concentration-dependent manner.The follow-up study was conducted with 5,10,20 ng/m L of homoharringtonine for 24 h.2.Clone formation experiment:Compared with the control group,given 5,10,and 20 ng/m L homoharringtonine for 24 hours,the clone formation rates of A375R cells were 52.18%,29.72%,and 2.36%,respectively.It shows that homoharringtonine can effectively inhibit the colony formation of A375R cells in a dose-dependent manner.3.The results of cell cycle detection by FCM showed that after different concentrations of homoharringtonine acted on A375R cells,the ratio of A375R cells in G0/G1 phase increased,while the ratio of S phase and G2 phase decreased,indicating that homoharringtonine can retard A375R cells in G0/G1 phase.4.Western blotting results showed that homoharringtonine can significantly down-regulate the expression of cycle-related proteins Cyclin E1 and CDK2,and retard the cell cycle in G0/G1 phase.In addition,compared with the control group,homoharringtonine had no effect on the protein expression of Akt and Erk,but it could significantly down-regulate the protein levels of IRS4,PI3 K,p-Akt and p-Erk.The third part1.The CCK8 test results showed that homoharringtonine could effectively inhibite the proliferation of A375 and A375R cells,and the non-cytotoxic dose IC10 was 2.5 ng/m L.After treating A375R with a non-cytotoxic concentration of HHT,the IC50 of Vemurafenib on A375R cells decreased from 49.09 μmol/L to 18.90 μmol/L,and the reversal factor RF was 2.60,showing that HHT could reverse the resistance of A375R cells and enhance the sensitivity of A375R cells to vemurafenib.2.The observation results under the cell microscope showed that compared with the control group,the combination of drugs resulted in a significant decrease in the number of cells,irregular cell morphology,and growth in groups.3.Ed U results showed that: the combination of drugs could reduce the green fluorescence intensity of cells,and at the same time,the cells have obvious nuclear shrinkage and nuclear fragmentation.4.FCM showed that the apoptosis rate was 7.40 ± 0.03% in the control group,9.75 ± 0.10% in HHT group,18.18 ± 0.04% in Vem group and 35.12 ± 0.13% in Vem combined with HHT group.And Western blotting results showed that the combination group could significantly increase the expression of Bax ang reduce the expression of Bcl-2.5.Western blotting results showed that the combination group could significantly reduce the expression of IRS4,PI3 K,p-Akt and p-Erk.Both the HHT group and the inhibitor group could reduce the expression of IRS4,PI3 K,p-Akt and p-Erk,and the activator could increase their expression.ConclusionThe first part1.The A375R cell was indeed a drug-resistant cell and could be used for follow-up experiments.2.The expression of IRS4 in drug-resistant melanoma cells was significantly increased,and the PI3K/Akt and Erk pathways both were activated.The second part1.Different concentrations of homoharringtonine can effectively inhibit the proliferation and the ability of cell clone formation of A375R melanoma drug-resistant cells,and retard the cell cycle in G0/G1 phase.2.Homoharringtonine may inhibit the activation of PI3K/Akt and Erk pathways by down-regulating the expression of IRS4 protein,and retard the cell cycle in G0/G1 phase,thereby inhibiting the proliferation of drug-resistant melanoma cells.The third part1.Lower concentration of homoharringtonine could enhance the sensitivity of melanoma drug-resistant cells to vemurafenib,and the combination of homoharringtonine and vemurafenib could inhibit the proliferation and induce apoptosis of A375R cells.2.Homoharringtonine may play its role in reversing drug resistance by inhibiting IRS4/PI3K/Akt and Erk pathways. |
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