| Objective: Malignant melanoma is the most malignant tumor in human skin cancer,which has high metastasis rate and high mortality but poor prognosis.Therefore,looking for some safe and effective drugs has become a research hotspot at home and abroad.Scholars have been committed to the development of highly effic ient and sensitive anti-cancer drugs.At present,Chinese herbal medic ine is a new breakthrough in the field of cancer research.Solanine is one of the main steroidal glycoalkaloid in potato.In recent years,It has been found that solanine has antimicrobial and antitumor effects,to which many scholars have paid attention.At present,Studies have shown that solanine possesses an anti-tumor property on many cells,but there are very few study on melanoma cells.Through the treatment of different solanine concentrations on melanoma A375 cells,this research mainly focuses on the effect of solanine on A375 cell proliferation,apoptosis and apoptosis related proteins,in order to investigate the antitumor mechanism of solanine,and to provide experimental and theoretical basis for its further clinical application.Metnods:1 Cell culture.The melanoma A375 cells were cultured in DMEM medium containing 10% fetal bovine serum,placed on the incubator of 37℃and 5% CO2.2 Inhibitory effect of solanine on the proliferation of melanoma A375 cells detected by CCK8.The experiment was divided into blank control group(only medium without cell without drug),negative control group(culture medium containing cell without drug)and experimental groups(culture medium containing cell with drug of the following concentrations: 4.6 μmol/L,9.2 μmol/L,13.8 μmol/L,18.4 μmol/L,23.0 μmol/L),each group of 6 holes.The proliferation of the A375 cells were tested by CCK8 after different concentrations of drug effected 24 h,48h and 72 h.Inhibition rates of the corresponding concentration and IC50 were calculated after the A375 cells groomed 24 h,48h and 72 h with different concentrations of solanine.3 The changes in apoptosis morphology of A375 cells with different concentrations of solanine were observed under an inverted optical microscope and fluorescence microscope.The experiment was divided into negative control group(solanine was 0 μmol/L)and experimental groups(9.2 μmol/L,13.8 μmol/L,18.4 μmol/L of solanine).4 The expression of apoptosis-related genes(Caspase-3,Bcl-2 and Bax)in mRNA were detected by RT-q PCR.5 The expression of apoptosis-related proteins(Caspase-3,Bcl-2 and Bax)were investigated by Western blot.6 The data was presented as mean ±SD.Statistical significance was evaluated by One-way ANOVA among groups with SPSS16.0 software.P <0.05 was considered statistically significant.Results:1 Cells without solanine grew in diamond shape with uniform s ize,regular morphology,well adherence and strong proliferation(Figure 2,3);2 CCK8 showed that different concentrations(4.6 μmol/L,9.2 μmol/L,13.8 μmol/L,18.4 μmol/L,23.0 μmol/L)of solanine in A375 cells for 24 hours,the cell inhibition rates were respectively(2±2.4)%,(11.4±1.4)%,(36.6±1.8)%,(63.7±0.3)% and(68.4 ±1.4)%.There was no significant difference between the negative control group and the experimental group with 4.6 μmol/L,and there was significant difference among the rest(P<0.05).For 48 hours,the cell inhibition rates were respectively(7.6 ±3.0)%,(15.3 ±2.8)%,(60.9 ±3.4)%,(76.6 ±2.9)% and(78.6 ±3.8)%.There was no significant difference between the concentration of 18.4μmol/L and 23μmol/L,and there was significant difference among the rest(P<0.05).For 72 hours,the cell inhibition rates were respectively(16.3±1.4)%,(28.7±3.7)%,(64.8±4.4)%,(95.8±1.2)%,(101.1±4.5)%.There was no significant difference between the concentration of 18.4 μmol/L and 23.0 μmol/L,and there was significant difference among the rest(P<0.05)(Figure 4,5,table 1).Different concentrations of solanine in A375 cells for 24 h,48h and 72 h,the IC50 were 16.2μmol/L,12.9μmol/L,11.8μmol/ L.Therefore,we selected 9.2 μmol/L,13.8 μmol/L,18.4 μmol/L of solanine in A375 cells for 24 hours for subsequent experiments;3 Under the inverted microscope,the cells in the control group had regular and clear morphology,uniform size,full cytoplasm,strong shading,well adherence and strong proliferation.In the experimental group(9.2 μmol/L,13.8 μmol/L,18.4 μmol/L)cells morphology changed,with smaller size,irregular contour,cell shrinkage,losing and malapposition.Susp ension cells increased(Figure 6,7).DAPI staining results showed the control group cells with the same size and uniform distribution of nuclei,while the experimental group presented nuclear pyknosis,nuclear edge set and apoptotic bodies formation(Figure 8);4 RT-qPCR showed different concentrations of solanine in A375 cells for 24 hours,the expression amount of gene Bax were respectively 1.370±0.335,1.721±0.711,2.371±0.494,the negative control group was 1.064±0.516,there was no significant difference between the 9.2 μmol/L,13.8 μmol/L and 18.4 groups(P>0.05),and there was significant difference among the rest(P<0.05)(Fig.9,Table 2);the expression amount of gene Caspase-3 were respectively 1.346±0.289,1.619±0.299,2.338±0.033,the negative control group was 1.018±0.267,there was no significant difference between the the negative control group,9.2 μmol/L and 13.8 μmol/L groups(P>0.05),and there was significant difference among the rest(P<0.05)(Fig.11,Table 2);the expression amount of gene Bcl-2 were respectively 0.975±0.109,0.705±0.228,0.360±0.110,the negative control group was 1.007±0.166,there was significant difference between 18.4 μmol/L and the negative control groups,9.2 μmol/L and 18.4 μmol/L groups(P<0.05),and there was no difference among the rest(P>0.05)(Fig.10,Table 2);5 Western blot showed different concentrations of solanine in A375 cells for 24 hours,the expression amount of protein Bax were respectively 0.543±0.049,0.824±0.059,0.857±0.046,the negative control group was 0.440±0.039,there was no significant difference between the 13.8 μmol/L and 18.4 groups(P>0.05),and there was significant difference among the rest(P<0.05)(Fig.12,13,Table 3);the expression amount of protein Caspase-3 were respectively 0.409±0.050,0.526±0.029,0.613±0.050,the negative control group was 0.403 ±0.037,there was no significant difference between the the negative control group and 9.2 μmol/L(P>0.05),and there was significant difference among the rest(P<0.05)(Fig.12,15,Table 3);the expression amount of protein Bc l-2 were respectively 0.275±0.026,0.145±0.018,0.045±0.018,the negative control group was 0.254±0.021,there was no significant difference between the the negative control group and 9.2 μmol/L(P>0.05),and there was significant difference among the rest(P<0.05)(Fig.12,14,Table 3).Conclusions:1 In a certain range,solanine has significantly inhibitory effect on melanoma A375 cells proliferation in a time and dose dependent manner.2 In a certain range,solanine can induce the apoptos is of melanoma A375 cells.The higher the concentration is,the more significant inducing apoptosis effects it has.3 Solanine induced to the apoptosis of melanoma cells A375 may relate to mitochondrial apoptosis pathways.It activates caspase-3 by down-regulating the ratio of Bcl-2 to Bax. |
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