Experimental Study And Clinical Observation Of Apatinib In The Treatment For Malignant Melanoma

Background and objectiveMelanoma is a common malignant tumor,which occurs in skin,digestive canal or respiratory tract,reproductive system,and other organs.Regional lymph nodes are normally involved in the early stage,but then disseminate to lung,liver,bones and brain through blood.Meanwhile,melanoma is a heterogeneous and ethnically diverse disease,which contains two main subtypes,mucosal and non-mucosal melanoma.There is great difference in epidemiology,clinicopathological features,gene structure and prognosis between two subtypes.Melanoma is highly invasive and malignant and the mortality rate increase sharply year by year,especially for advanced melanoma.The one-year survival rate,median survival time and the median PFS(progress free survival)is 25%,6 months and less than 2 months in advanced melanoma,respectively.However,effectiveness of traditional chemotherapy and biological therapy is very limited.Therefore,it is dramatically urgent to explore new therapeutic drugs and treatments.Mesylate apatinib as a new kind of domestic multi-targeted small molecule vascular endothelial growth factor receptor(VEGFR)tyrosine kinase inhibitors selectively inhibits the activity of VEGFR-2 tyrosine kinase and blocks the signal transduction mediated by vascular endothelial growth factor(VEGF),and further effectively inhibits tumor angiogenesis and growth.A myriad of experiments in vivo and in vitro have shown that apatinib exerts the strong antitumor activity in gastric cancer,liver cancer,breast cancer,lung cancer and other tumors.At present,apatinib has been approved by China Food and Drug Administration(CFDA)that it can be used in third-line or more than third-line treatment for advanced gastric adenocarcinoma and adenocarcinoma of stomach to esophagus.However,to date,there is no relevant study about efficacy of apatinib in the therapy of malignant melanoma.Previous data indicate that melanoma is a vigorously vasculogenic tumor.VEGFR is highly expressed in melanoma and plays a vital role in the progression of melanoma,implying that apatinib exhibits the strong inhibition of angiogenesis in treatment for malignant melanoma.Our studies mainly focus on elucidation of the role of apatinib in treatment of malignant melanoma according to the two aspects:1)The experimental study of apatinib for the treatment of malignant melanoma;2)To evaluate the safety and validity of apatinib treatment for different types malignant melanoma(mucosa and non-mucosal melanoma).Methods1 The experimental study of apatinib treatment for malignant melanoma1.1 The experimental study of Apatinib in the treatment for malignant melanoma in vitro1.1.1 Apatinib inhibited the proliferation of malignant melanoma cells1)B16 cells at a density of 2×103 cells were inoculated into 96-well plate in 100?l of DMEM(high glucose)medium containing 10%fetal bovine serum.2)The cells have all adhered to the wall at 7 hours,and the cells are divided into four groups(3 gradients/group)in the following:?.Control group:100ul fresh medium for each well;?.Dacarbazine group:The dacarbazine concentration was adjusted to 100ug/ml,50ug/ml and lOug/ml,respectively,and the final volume was adjusted to 200ul;?.Apatinib group:The apatinib concentration was adjusted to 100uM,50uM and 5uM respectively,and the final volume was adjusted to 200ul;iv.Combined treatment group:firstly add lOug/ml of dacarbazine,and then add 100uM,50uM and 5uM of apatinib,respectively,with the final volumeadjusted to 200ul.Each group was set up with 3 groups of wells.3)The inhibition rate of different drugs on B16 cell proliferation was detected by the cell proliferation assay kit(MTS method,Promega)after 72 hours:?.15ul MTS solution was added to each well and incubate at 37 0 C for 1-4 hours;?.The absorbance value(OD)of each well was measured at 490 nm;?.The calculation of the inhibition rate of cell proliferation:The OD value of each test well is subtracted from the OD value of blank well,and the OD value of each replicate hole is averaged.Cell proliferation inhibition rate%=[1-(dosing OD-blank OD)/(control OD-blank OD)]× 100%1.1.2 Cell cycle detection1)B16 cells in logarithmic growth stage were inoculated into 6 well plate with 5 × 104cells per well and cultured in 100ul DMEM medium(High sugar)containing 10%fetal bovine serum.2)After 24 hours,the cells entered the logarithmic growth phase.The cells were divided into four groups(three concentration gradients/group):?.Control group:only 2ml fresh culture medium was added;?.Dacabaxine group:the concentration of dacabaxine was adjusted to lOug/ml and the final volume was adjusted to 2ml;?.Apatinib group:the concentration of apatinib was adjusted to 50 uM and the final volume was adjusted to 2 ml;iv.Combination group:10 ug/ml dacarbazide was added firstly,then 50 uM apatinib was added,and the final volume was adjusted to 2 ml.3)The effects of different drugs on the cell cycle of B16 cells were detected by cell cycle detection kit at 24 hours:?.According to the number of samples,the required working fluid volume was calculated.Each sample was incubated with 500?l working fluid.Firstly Rnase A and PI working fluid were prepared into dyed working fluid according to 1:9 volume;?.The collected cells were digested by trypsin without EDTA.The cell concentration was adjusted to 1×106/ml,and obtained 1 ml single cell suspension;?.After the supernatant was removed by centrifugation of single cell suspension,the cells were fixed at-20? for 2 hours with 70%500ul precooled ethanol;?.Before dyeing,ethanol was washed off with PBS,and added the prepared dyed working solution of PI/RNase A and kept it under room temperature avoiding light for 30-60 min;v.The red fluorescence at 488nm was recorded by flow cytometry.1.1.3 Apoptosis rate detection1)B16 cells in logarithmic growth stage were inoculated into 6 well plate with 5 X 104cells per well and cultured in 100ul DMEM medium(High sugar)containing 10%fetal bovine serum;2)After 24 hours,the cells had entered the logarithmic growth period and were divided the cells into four groups(3 gradients/group),and the groups were set up with 2.2.2;3)The effects of different drugs on the apoptosis of B16 cells were detected by Annexin V-FITC/PI double staining kit in 24 hours:?.The cells were digested by trypsin without EDTA and 5×105 cells were collected;?.500ul Binding Buffer heavy suspension cells were added;?.5ul Annexin V-FITC and 5ul PI were added,and then shaked and mixed;?.Kept it under room temperature without light for 10-20 min;?.Tested by flow cytometry.1.2 The experimental study of Apatiinib in the treatment for malignant melanoma in vivo(mouse model)1)The subcutaneous transplantation tumor model of C57BL/6 murine melanoma B16 was established and was divided into six groups randomly.2)The first group C57BL/6 mouse were accepted dacarbazine(80mg/kg);The second group,third group,and fourth group were accepted apatinib(50mg/kg),apatinib(100 mg/kg),apatinib(200mg/kg)in turn;The fifth group were accepted dacarbazine(80mg/kg)combined with apatinib(100mg/kg)and sixth group as control group were accepted physiological saline(0.1ml/10g).The rates of tumor growth inhibition were counted and the effect between dacarbazine and apatinib was compared.The optimal therapeutic dosage of apatinib for single therapy was established.3)Animal weights and tumor volumes were all expressed in terms of mean + SD.Statistical analysis was performed by SPSS 10.0 software.Univariate analysis was used in multiple groups.All P values reported were two sided and were calculated at a significance level of 0.05.2 Explicited differences of curative effects and adverse reactions of apatinib between mucosa and mucosal melanoma through clinical observation2.1 Safety and effectiveness of apatinib in advanced melanoma through clinical research2.1.1 This study collected clinical data of 15 advanced refractory melanoma patients in Henan Tumor Hospital who received apatinib after multi-line salvage treatments failure.2.1.2 Therapeutic schedule:15 patients received apatinib 500mg/day as initial dose,28 days per cycle and received apatinib 250mg/day when there were adverse events over patient's intolerance.2.1.3 Recorded OS,PFS,ORR,DCR and adverse events of patients.2.1.4 The research compared efficacy and safety of apatinib for mucous melanoma and cutaneous melanoma.2.1.5 All statistical procedures were carried out using SPSS(version 21.0;SPSS Company,Chicago,IL).Kaplan-Meier method and Log rank test were used for univariate analysis.Cox proportional risk regression model was used for multivariable analysis.All P values reported were two sided and were calculated at a significance level of 0.05.2.2 Differences between mucous melanoma and cutaneous melanoma in clinilopathological features and prognosis.2.2.1 This study was retrospective and consisted of 162 patients diagnosed as advanced melanoma(III or IV period)who were admitted in Henan Tumor Hospital between January 1st 2010 and December 31st 2016.2.2.2 Comparison of clinilopathological features and prognosis between mucous melanoma and cutaneous melanoma.2.2.3 Fisher's exact test and chi square test were used to compare demographic characteristics of patients in different groups.Kaplan-Meier and Log rank test were used for survival analysis.All statistical procedures were carried out using SPSS(version 21.0;SPSS Company,Chicago,IL).All P values reported were two sided and were calculated at a significance level of 0.05.Results1 The experimental study of apatinib treatment for malignant melanoma1.1 Apatinib inhibited the proliferation of malignant melanoma cells1.1.1 The results showed that when concentration of dacarbazine increased,the inhibition rate increased significantly.Inapatinib group,when the concentration of apatinib was 50uM,the inhibition rate was sharply improved compared with 5uM.However,when we further increased the concentration to 100uM,the inhibition rate was not improved obviously.In combined treatment group by lOug/ml dacarbazine combined with different concentration of apatinib,we found cell proliferation inhibition rates increased significantly compared with single dacarbazine.Compared combined treatment group with apatinib(?50uM),cell proliferation inhibition rate of the later was higher.Only when dacarbazine(5uM)combined with low concentration of apatinib(5uM),cell proliferation inhibition rate of the former was higher.1.1.2 In order to furtherly study the mechanism of the inhibitory effect of apatinib on cell proliferation,we examined the cell cycle changes after adding drugs.The cell cycle arrest effect of low concentration of dacarbamazine was not obvious.Both apatinib and apatinib combined with low concentration of dacarbazide could block the cell cycle and blocked the cells in G0/G1 phase,and evenly showed a synergistic action.Apatitininte inhibited the proliferation of B16 cell by inhibiting the synthesis of DNA.1.1.3 The results of cell proliferation inhibition test showed that apatitini could inhibit the growth of B16 cell significantly.We used flow cytometry to detect the apoptosis of cells treated with drugs.Compared with the control group,the dacarbazide group showed no obvious effect in inducing apoptosis.The apoptotic rate was 7.04%;the apatinib group significantly increased the apoptotic rate to 14.49%;the combination group had the highest apoptotic rate of 24.7%(listed in figure 4 and 5).The results showed that apatinib induced the apoptosis of melanoma cell line B16.When apatinib was combined with low concentration of dacarbazide,the effect of inducing apoptosis was inproved significantly.1.2 Clinical effects of apatinib in melanoma in vivo1.2.1 Apatinib had the obvious effect to inhibit melanoma B16 subcutaneous tumor growth and the effect was improved as the increase of concentration of apatinib.1.2.2 The antitumor activity of apatinib was stronger than that of dacarbazine.2 Clinical studies on the efficacy and safety of apatinib in advanced melanoma(clinical observation)patients who received apatinib after failure of multi-line salvage treatments.2.1 Clinical research on safety and effectiveness of apatinib in advanced melanoma2.1.1 The overall median OS was 8 months,median PFS was 3.7 months,ORR was 6.7%(1/15)and DCR was 80%(12/15).2.1.2 OS of mucosal patients and non mucosal patients reached 14.5,4.5 months(HR 0.014,95%CI 0.000-12.735,P=0.022)respectively,while the PFS of both groups were 8.0,2.8 months(HR 0.095,95%CI 0.011-0.816,P=0.011).ORR of mucous patients and non mucosal patients were 20%,0%respectively.2.1.3 During the treatment,Grade 3 adverse effects were hypertension(13.3%),hand foot syndrome(6.7%),proteinuria(6.7%)and bilirubin(6.7%).2.2 Clinical study on clinicopathological features and prognosis of advanced mucosal and cutaneous melanoma2.2.1 Median PFS of mucosal and cutaneous melanoma were 13 months and 8 months,respectively.2.2.2 Results showed that the median PFS of patients with mucosal and cutaneous melanom was 14 months and 10 months(P=0.022)respectively.The median PFS of patients with mucosal and cutaneous melanoma was 6 months and 4 months(P=0.040),respectively.Results from multivariate analysis showed that age was an independent risk factor for PFS in mucosal melanoma,but stage and treatment regimens were independent risk factors for PFS in cutaneous melanoma.2.2.3 Lung was the most common metastatic organ in mucosal and cutaneous melanoma(29.3vs28.9),followed by liver(14.6vsl2.7)and brain(9.8vs8.3).The differences between both groups were not statistically significant.Conclusion1 In vitro and in vivo experiments showed that apatinib could inhibit melanoma B16 cell growth,and the effect of inhibition displayed in a dose-dependent manner.Apatitinine inhibited the synthesis of DNA of melanoma cell line B16,and induced the apoptosis of melanoma cell line B16.2 For the failure of multiline treatment of refractory melanoma,apatinib was safe and effective.Compared with cutaneous melanoma,the prognosis of mucosal melanoma patients were better.3In China,the prognosis of the patients with advanced mucosal melanoma were better than advanced cutaneous melanoma.Both of them were different in clinicopathological features.