A Pilot Study Of The Molecular Mechanism Of EphB4-induced Cisplatin Resistance Of Skin Melanoma

BackgroundAs a highly malignant tumor originated from melanocytes of neural crest, melanoma takes up 1~3% of all malignant tumors. It is often seen on the skin, being the most commonly seen skin cancer besides skin squamous cell carcinoma and basal cell carinoma. Melanoma has a trend of early metastasis which, especially to distant organs, can cause a mortality of 80%. The incidence of melanoma in China has increased during the recent years, with a number of newly diagnosed cases about 20,000 per year, where a familial aggregation is also observed. The main problems with treating melanoma lie in that 1) the tumor is hardly symptomatic at early stages while metastasis usually occurs soon after the detection of primary lesion, which makes early diagnosis and treatment often impossible; and 2) melanoma is not sensible to most chemotherapy drugs and drug resistance is commonly seen, which often makes chemotherapy ineffective.Nowadays, multiple therapeutic methods including targeted therapy and immune therapy are showing some effectiveness in the treatment of melanoma. However, cytotoxic, i.e. chemotherapy drugs are still preserved for late-staged, metastasized melanoma where none of the methods above is actually effective.Until dacarbazine was widely used as a “gold standard” for the treatment of melanoma, alkylating agent analogues such as cisplatin(Cis) had been used as a somehow effective therapy alone or in combination with other drugs to treat many solid malignancies including melanoma. However, compared with other solid malignancies, melanoma shows low response and rapidly developed resistance to Cis, limiting the use of this drug in its therapy. For a long time, people have been working on the Cis resistance mechanisms with the hope of lengthening and expanding the clinical use of Cis. From a present point of view, the mechanisms are very complicated, including declined apoptosis, enhanced DNA damage repair, recombinant activation, malabsorption of drug, dysfunction of cell membrane, etc., of which failure of induction of apoptosis by Cis is considered the most important mechanism. It is helpful to dig in the Cis resistance mechanisms of melanoma so that people can get better control of late-staged melanoma and expand the clinical use of Cis and its analogues. The present study, composed of four parts, aims to explain from new angles the Cis resistance mechanisms of skin melanoma, and hopefully to have some positive impact on the treatment of melanoma.Part I Nude Mouse Model of Cis-resistant A375 MelanomaOjective: This part of study aims to establish nude mouse models of A375 melanoma resistant to Cis(Cis-resistant models) for the subsequent experimental steps and researches in the future.Methods: Human skin A375 melanoma cells which have a relatively strong invasiveness were recovered and cultured and inoculated into 100 BALB/c female nude mice at 4×106 cells per mouse. Tumors were seen about 30 days later. The mice were divided into 10 groups(10 in each group) and treated with intraperitoneal injection(IP) of Cis 2mg/Kg, twice a week for 40 weeks. After that, the Cis-resistant models were assessed according to the percentage of weight change of the mice, the volume and weight of melanoma in the mice.Results: A375 cells were routinely recovered, cultured and inoculated into the nude mice whose tumor bears were good 30 days later, where the volumes and weights of tumors all met the requirement of the experiment, indicating the successful establishment of nude mouse models of A375 melanoma. After induction by Cis, Groups 3 and 6 were found to have acquired resistance to Cis, where all these mice developed tumors significantly larger and heavier than that of the mice in other groups(Cis-sensible models). Worsened conditions, obvious weight losses and acute or late toxic reactions were not observed in the Cis-resistant mice compared to the Cis-sensible mice, indicating the successful establishment of Cis-resistant models.Conclusion: Based on the successful establishment of nude mouse models of A375 melanoma, the Cis-resistant models were also established through a long-term Cis induction, which laid the foundation for the subsequent experimental steps and researches in the future.Part II EphB4-induced Cis resistance of MelanomaObjective: To explore the gene that induces Cis resistance of melanoma by gene chips and find a new therapeutic target of melanoma.Methods: The RNA of Cis-resistant and Cis-sensible melanoma cells were respectively extracted and made into gene chips, which then underwent differential gene expression analysis and were compared with multiple tumor activation-related genes that are commonly seen. High expression of EphB4 was demonstrated in Cis-resistant melanoma cells and then confirmed with immunohistochemical(IHC) staining and Western blot(WB). Cis-resistant models were then established in a large quantity and randomly divided into PBS group, Cis group, NVP-BHG712 group and Cis+ NVP-BHG712 group, respectively, with 10 mice in each group. The four groups were treated with PBS(IP, QD), cisplatin(2mg/kg, twice a week), NVP-BHG712(a specific EphB4 inhibitor, PO, QD) and Cis+ NVP-BHG712, respectively. After 21 days’ continuous treatment, each mouse’s weight change in percentage, the tumor volume and weight were measured in order to testify the validity and reliability of EphB4 as a new therapeutic target of melanoma.Results: The high expression of EphB4 was demonstrated in Cis-resistant melanoma through differential gene expression analysis comparing Cis-sensible and Cis-resistant tumors, which was then further confirmed with IHC staining and WB. Randomization experiment illustrated that Cis+ NVP-BHG712 treatment had a significant inhibitive effect on Cis resistance of melanoma.Conclusions: Cis-resistant melanoma has a high expression of EphB4 which can be maximally inhibited by the combined use of Cis and NVP-BHG712.Part III A Pilot Study of the Molecular Mechanism of EphB4-induced Cisplatin Resistance of Skin MelanomaObjective: This part of study aims to explore the mechanism of EphB4-induced Cis resistance of melanoma by comparing the changes of key molecules in important cell signal transduction pathways and the impact of activated signal pathways on downstream biological functions such as apoptosis, so as to reveal the intact biosignal pathways through which the high expression of EphB4 impacts the drug resistance of melanoma. Additionally, the impacts of Cis and NVP-BHG712 either alone use or in combination on the activation the signal pathways were also tested. The 2nd and 3rd generations of Cis-resistant models were established and compared with the first generation to explore whether or not the drug resistance phenotype acquired is eternalMethods: WB method was used to test the activation of PI3 K and MAPK pathways in Cis-sensible and Cis-resistant melanomas, respectively. After use of Cis and NVP-BHG712, WB was used to test the inhibition of the two pathways. The apoptosis of Cis-sensible and Cis-resistance melanoma cells were tested by TUNEL staining and Annexin V/PI double staining. Finally, we established the 2nd and 3rd generations of Cis-resistant models and analyzed the expressions of EphB4, PI3 K and MAPK in all 3 generations of Cis-resistant models and proved that the drug resistance phenotype acquired was eternal.Results: Cis-resistant melanoma had higher expressions of p-AKT and p-ERK than Cis-sensible melanoma did. The use of Cis+NVP-BHG712 could maximally inhibit the activation of p-AKT and p-ERK. The expressions of p-ERK and EphB4 were both declined in the NVP-BHG712 group, implying cisplatin-induced drug resistance other than pre-existing drug-resistant cell subsets. TUNEL and Annexin V/PI staining methods both confirmed significantly declined apoptosis of Cis-resistant melanoma cells which, however, could be enhanced by the combined use of Cis and NVP-BHG712 but not by the single use of either of them. Finally, the 2nd and 3rd generations of Cis-resistant models were confirmed to have similar expressions of EphB4, PI3 K and MAPK and improved growing abilities compared with the 1st generation.Conclusion: The activation of p-AKT and p-ERK could have been the signal pathways through which EphB4 induces cisplatin resistance of melanoma. The use of Cis+NVP-BHG712 can maximally inhibit such activation while enhancing the apoptosis of melanoma cells. Additionally, the drug-resistant phenotype acquired by Cis-resistant melanoma cells is confirmed to be eternal.Part IV Role of Autophagy in EphB4-induced Cis ResistanceObjective: This part of study aims to explore the role in EphB4-induced Cis resistance played by autophagy as a basic cell function and its impact, if any, on the downstream apoptotic biological functions as whether a promotive or an inhibitive factor. The answers to these questions may hopefully help us know better about the development of Cis resistance of melanoma and provide us with new ideas of treating this cancer.Methods: The autophagy of Cis-resistant and Cis-sensible melanoma cells were observed by MDC method and the expressions of autophagy-related proteins were tested by WB method. The autophagy of PBS group, Cis group, NVP-BHG712 group and Cis+NVP-BHG712 group were all observed by MDC. The nude mice were then divided into PBS group, 3-MA group and Cis+3-MA group and underwent 21 days’ treatment using PBS(IP QD), 3-MA(a specific inhibitor of autophagy, 15mg/kg IP QD) and Cis+3-MA, respectively. The change in tumor volumes, the induced occurrence of apoptosis and the expressions of apoptosis-related proteins in each group were measured right after treatment.Results: It was revealed in our experiment that Cis-resistant melanoma cells had stronger autophagy and higher expressions of autophagy-related proteins. After drug intervention, Cis+NVP-BHG712 group had a more significantly inhibited autophagy. The use of 3-MA resulted in significant inhibition of Cis-resistant melanoma, which was further enhanced by the combined use of Cis. The combination of Cis and 3-MA also promoted the expressions of apoptosis-inducing and apoptosis-related proteins in melanoma cells.Conclusions: Autophagy plays a role in the development of Cis resistance of melanoma and is positively correlated with drug resistance. The use of Cis+3-MA can maximally inhibit Cis-resistant melanoma and induce its apoptosis.Final Conclusion1. A nude mouse model of A375 melanoma resistant to Cis was successfully established and the drug resistance acquired is an eternal phenotype.2. The Cis-resistant melanoma can be maximally inhibited through inhibition of EphB4 which is highly expressed in Cis-resistant melanoma cells, providing a new therapeutic target of drug-resistant melanoma.3. The activation of downstream PI3 K and MAPK pathways which finally cause enhanced autophagy and declined apoptosis may be the main mechanism of EphB4-induced drug resistance, which provides an important theoretic foundation of how EphB4 induces Cis resistance of melanoma.