The Mechanism Of Platycodin D Inhibiting Proliferation And Inducing Apoptosis Of Melanoma A375 Cells

Objective:Malignant melanoma?MM?,characterized by its early metastasis,strong invasiveness and poor prognosis,is a highly malignant skin tumor that ranks third in skin malignancies.The incidence of melanoma has risen considerably over the past 30 years,and it has become one of the diseases that pose a major threat to human health.Early diagnosis and early treatment are the key to treating melanoma.Traditional treatments such as surgery,chemotherapy,and radiotherapy are still not ideal,so researches around the world are working to find new therapeutic drugs.In recent years,Chinese herbal medicines and traditional Chinese medicine monomers have gradually become hot spots in the field of tumor research.There is evidence that plant-derived bioactive compounds inhibit cancer through various mechanisms.Platycodin D?PD?is a main component extractsfrom Platycodon grandiflorum,a stable triterpenoid saponin,which has anti-inflammatory,immunomodulatory,and antitumor activities.Studies have shown that PD can significantly induce apoptosis in non-small cell lung cancer and liver cancer cells.However,the effacts and mechanism of PD on melanoma have not been studied.In this experiment,melanoma A375 cells were treated with PD to observe its effect on melanoma cell proliferation and apoptosis,and to study its specific molecular mechanism of PD.Methods:1. Cell culture.Melanoma A375 cells were cultured in DMEM medium containing 10%fetal calf serum,and cultured in a 37?,5%CO₂ incubator.2. CCK8 method was used to detect the effect of PD on the proliferationof melanoma A375 cells.The experiment was design with a blank control group?medium without cells and no PD?,a negative control group?medium containing cells and no PD?,and experimental groups?medium containing cells,the medium was added with 2.5,5,10,15,20?mol/L PD at different concentrations?,each set of 6 duplicate wells.After 24 h,48 h and 72 h of culture,CCK8 reagent was added to detect the effect of PD on the proliferation activity of melanoma A375 cells,and the inhibition rate and IC₅₀value of the cells under different drug concentrations were calculated.3. Observing the morphological changes of melanoma A375 cell?apoptosis induced by different concentrations of PD in an inverted microscope and a fluorescence microscope.A negative control group and experimental groups were set up.Negative control group?medium containing cells and no PD?,experimental groups?medium containing cells,the medium was added with 2.5,5,10,15,20?mol/L PD at different concentrations?.After 24 h of culture,the growth and morphological changes of the cells were observed under an inverted microscope and a fluorescent microscope after DAPI staining.4.Real-time quantitative PCR?RT-qPCR?was used to detect the m RNA expression levels of apoptosis-related genes?Bcl-2 and Bax?in melanoma A375 cells treated with different concentrations of PD.GAPDH as a control.The experimental groups were the same as the microscope observation group.After 24 h of culture,RNA was extracted for detection.5.Western blot was used to detect the apoptosis-related proteins?Bcl-2,Bax?and mitogen-activated protein kinase?MAPK?signaling pathway-related proteins?MEK,p-MEK,ERK,p-ERK,FOXO3a and p-FOXO3a?.?-actin as a control.The experimental groups were the same as the microscope observation group.After 24 h of culture,proteins were extracted for detection.6.Statistical software SPSS 25.0 was used for statistical analysis.The measurement data were expressed as?x±S.One-way ANOVA was used to compare the mean of multiple groups.SNK-q was used for pairwise comparison within groups.P<0.05 was considered statistically significant.Results:1.The results of CCK8 showed that the inhibition rates of A375 cells treated with different concentrations of PD for 24 h were?7.023±3.689?%,?17.612±4.514?%,?47.608±2.361?%,?53.583±1.354?%,?57.864±2.424?%.r=0.984.After pairwise comparison,the differences between the experimental group and the negative control group were statistically significant?P<0.05?.The inhibition rates after treatment for 48 h were?9.218±1.287?%,?19.782±1.266?%,?61.594±0.828?%,?71.463±0.721?%,?74.166±0.893?%.r=0.982.After pairwise comparison,the differences between the experimental group and the negative control group were statistically significant?P<0.05?.The inhibition rates after treatment for 72 h were?15.902±0.287?%,?41.312±0.477?%,?67.711±1.300?%,?78.373±0.445?%,?88.085±0.547?%.r=0.996.After pairwise comparison,the differences between the experimental group and the negative control group were statistically significant?P<0.05?.The half inhibitory concentrations(IC₅₀)of A375 cells treated with PD for 24 h,48 h,and 72 h in the experimental groups were 12.05?mol/L,8.63?mol/L and 6.52?mol/L.Therefore,we selected PD concentrations of 5?mol/L,10?mol/L and 15?mol/L for 24 h for subsequent experiments.2.Observed under an inverted microscope,the negative control group were consistent in morphology and size,with clear outline,good cell adherence and less dead cells.the experimental group had smaller cell sizes,cells shrunk and shed,and had poor adherence,dead cells increased.After DAPI staining,fluorescence microscope observation showed that the nuclei in the negative control group were uniform in size and distributed uniformly.The nucleus of the experimental group showed nuclear division,nuclear shrinkage,and apoptotic body formation.With the increasing of PD concentration,the morphology change of apoptosis become more conspicuous.3.The results of RT-qPCR showed that the expression of Bcl-2 m RNA was 0.740±0.025,0.554±0.025,0.295±0.020,and 1.000±0.019 in the negative control group.GAPDH as a control.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?,the expression levels of Bax m RNA were1.448±0.085,1.867±0.042,2.534±0.054,and 1.002±0.063 in the negative control group.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?.4. Western blot results showed that melanoma A375 cells were treatedwith PD at different concentrations for 24 h,?-actin as a control,the levels of Bcl-2 protein were 0.303±0.022,0.097±0.012,0.082±0.009,and 0.417±0.002 in the negative control group.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?.the levels of Bax protein were 0.473±0.086,0.593±0.025,0.653±0.044,and 0.318±0.051 in the negative control group.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?.the levels of MEK protein were 0.650±0.045,0.675±0.031,0.681±0.051,and 0.696±0.063 in the negative control group.After pairwise comparison,there was no significant difference between the experimental group and the negative control group?P>0.05?.the levels of p-MEK protein were 0.431±0.059,0.389±0.054,0.245±0.048,and 0.684±0.038 in the negative control group.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?.the levels of ERK protein were 0.752±0.048,0.724±0.052,0.711±0.055,and 0.767±0.054 in the negative control group.After pairwise comparison,there was no significant difference between the experimental group and the negative control group?P>0.05?.the levels of p-ERK protein were 0.633±0.021,0.519±0.043,0.246±0.054,and 0.738±0.032 in the negative control group.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?,the levels of FOXO3a protein were 0.434±0.041,0.501±0.064,0.559±0.047,and 0.313±0.038 in the negative control group.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?,the levels of p-FOXO3a protein were 0.164±0.026,0.113±0.022,0.076±0.011,and 0.224±0.023 in the negative control group.After pairwise comparison,the difference between the experimental group and the negative control group was statistically significant?P<0.05?.the levels of FOXO3a/p-FOXO3a protein were 2.955±0.295,4.587±0.937,7.560±1.574,and 1.405±0.186 in the negative control group.After pairwise comparison,there was no significant difference between the group of 5?mol/L and the negative control?P>0.05?,the difference between the rest experimental group and the negative control group was statistically significant?P<0.05?.Conclusion:1.PD has a significant inhibitory effect on the proliferation of melanoma A375 cells in a certain time and concentration range,and it is time-and dose dependent.2.In a certain time and concentration range,PD can induce the apoptosis of melanoma A375 cells,and with the increase of PD concentration,the apoptosis effect is more obvious.3.PD-induced apoptosis of melanoma A375 cells may be related to mitochondrial apoptosis pathway and MAPK signaling pathway.By down-regulating Bcl-2 protein expression,up-regulating Bax and FOXO3a protein expression,and inhibiting MEK,ERK,FOXO3a phosphorylation.