| Backgrounds:Nasopharyngeal carcinoma(NPC)is one of the most common malignant tumors of the head and neck,which is an epithelial carcinoma originated from the inner layer of nasopharyngeal mucosa,it often occurs in the pharyngeal crypt of nasopharynx.NPC is characterized by high malignancy,easy metastasis and inconspicuous early symptoms.Early-stage nasopharyngeal carcinoma patients receiving radiation therapy alone or in combination with chemotherapy can significantly improve the clinical efficacy,however,NPC is usually detected at an advanced stage,during which it is susceptible to local recurrence,distant metastasis,and drug resistance,there are fewer treatment options and poorer clinical efficacy.Therefore,anti-tumor drugs with high activity and low toxic side effects are currently one of the main directions of tumor research.In recent years,the advantages of traditional Chinese medicine in anti-tumor have received increasing attention.As a famous herb in traditional Chinese medicine,ginseng has been used in the clinical treatment of some patients with advanced cancers due to its antitumor activities,such as immunomodulation,inhibition of tumor proliferation,induction of apoptosis,autophagy,and synergistic effects of radiotherapy,and has received better therapeutic effects.Protopanaxatriol(PPT)is a metabolite of ginseng,which is produced by hydrolysis of glucose at C-20 and C-6 positions by ginsenoside Rgl of the protopanaxatriol group,and eventually metabolized to produce the glycoside PPT.Compared with Rg1,PPT has stronger biological activity,with antitumor,anti-inflammatory,anti-diabetes,improvement of hepatic fibrosis,and other effects.As the main anti-tumor active ingredient,PPT can significantly inhibit tumor cells,increase their sensitivity to chemotherapy drugs,overcome their resistance,and improve the effectiveness of combined therapy.And although PPT has obvious antitumor effects in many kinds of tumors,PPT has not been studied in the field of antinasopharyngeal carcinoma,which we will further explore in this study.Research purpose:This study utilized TMT-labeled protein profiling assay combined with bioinformatics analysis and molecular biology experimental techniques to explore the effects of PPT on the proliferation,migration and apoptosis of nasopharyngeal carcinoma(NPC),as well as exploring the mechanisms of its action in vitro and vivo.The main research content and conclusions of this paper are as follows:1.The effect of PPT on the proliferation,metastasis and apoptosis of nasopharyngeal carcinoma cells in vitro.Anhydrous ethanol was applied to dissolve PPT and configured into different concentrations,and acted on human nasopharyngeal carcinoma cells 5-8F and 6-1 OB cells for 24 h.The survival rate of the two groups of cells treated with PPT decreased with the increase of the concentration by CCK8 assay.Next,the cloning ability of 5-8F and 6-10B cells were examined by clone formation,and it was found that PPT exerted a significant inhibitory effect on proliferating clones in a dose-dependent manner.The cell cycle progression of 5-8F and 6-10B cells treated with different concentrations of PPT was analyzed by flow cytometry.The results showed that with the increase of PPT concentration,the cells in G0/G1 phase increased significantly,while the cells in S phase or G2 phase decreased.This result suggests that PPT causes cell cycle arrest in G0/G1 phase.The migratory ability of 5-8F and 6-10B cells was examined by scratch assay and Transwell migration assay,and we found that PPT inhibited the migratory ability of tumor cells in a concentration-dependent manner,and the migratory ability of the two cell lines was significantly inhibited.We also examined the effect of PPT on apoptosis by flow cytometry.The results revealed that the apoptosis rate and the proportion of cells with early or late apoptosis induced by PPT in 5-8F and 6-10B cells increased with the increase of PPT concentration,especially the late apoptosis rate,when compared with the control group.Subsequently,the results of apoptosis detection by TUNEL showed that the number of cells with TUNEL-positive signal gradually increased with the increase of PPT concentration.The expression of apoptosis-related proteins was detected by western blot analysis.The results showed that,with the increase of PPT,the expression of apoptotic proteins Bax,Caspase 3,and Caspase 9 was significantly increased,and the expression of anti-apoptotic protein Bcl-2 expression decreased.These results indicated that PPT had a significant inhibitory effect on the viability,proliferation and migration of nasopharyngeal carcinoma cells,and significantly promoted cell apoptosis.2.Exploration of the mechanism of action of PPT against nasopharyngeal carcinoma based on proteomic assay.Through proliferation,apoptosis and migration assays,it was found that 80 uM PPT treatment of 5-8F and 6-10B cells for 24 h could significantly promote apoptosis and inhibit proliferation and migration.We performed TMT-labeled liquid chromatography tandem mass spectrometry(LC-MS/MS)protein profiling on three cases of 80 uM-treated 6-10B cells and three cases of untreated 6-10B cells.A total of 6174 proteins were detected,of which significantly different proteins included 46 differentially up-regulated proteins and 328 differentially down-regulated proteins.The Wnt signaling pathway was significantly enriched in both GO and KEGG databases,indicating that PPT inhibition of NPC cells may be related to the Wnt signaling pathway.By analyzing the proteins closely related to the Wnt pathway among the significantly different proteins and using molecular docking techniques,we believe that S100A4 may be an important target protein in the anti-nasopharyngeal carcinoma effect of PPT.After the precise docking of PPT with S100A4,we found that the docking binding energy of PPT with S100A4 was very high,up to-30.138 KJ/mol.PPT had a high affinity for S100A4 and showed key molecular docking with the adjacent amino acids in the active site of S100A4,which further suggested that the antitumor effect of PPT may be related to S100A4 protein.To further investigate whether PPT affects the expression of S100A4 and its detailed distribution,we performed high-resolution dSTORM imaging.The intracellular S100A4 was significantly reduced after 80 μM PPT treatment of 6-10B cells.The number of localisations and the size of the clusters are proportional to the expression of the proteins.The results showed that PPT regulated the distribution of S100A4 clusters and down-regulated S100A4 expression.At the same time,the differential proteins were significantly enriched for mitochondrial function in both GO and KEGG databases,which indicated that the antinasopharyngeal carcinoma effect of PPT was also related to the mitochondrial function,and the multi-targeting effect shown by PPT is a major feature of natural drugs.3.Research of PPT in vitro anti-nasopharyngeal carcinoma mechanismBased on the screening of signal pathways in the previous section,validation will be conducted in vitro.qRT-PCR results showed that,compared with the control group,the expression levels of S100A4 and β-catenin mRNA were all down-regulated.This result was consistent with the dSTORM imaging data.The expression of S100A4/Wnt signaling pathway-related proteins in 5-8F and 6-10B cells after PPT action or transfection of siS100A4 was also detected by Western blot.The results showed that the expression of S100A4,Cyclin D1,β-catenin,Survivin,and C-Myc were downregulated in 5-8F and 6-10B cells treated with PPT or transfected with siS100A4.After overexpressing S100A4,The expression of β-catenin protein also increased accordingly.Meanwhile,the cell viability,proliferation and migration ability of the two cells were detected by CCK8 assay,clone formation,scratch assay and Transwell migration assay after silencing S100A4 with siRNA.The results showed that,compared with the control group,the viability,proliferation and migration ability of the two cells were significantly reduced after transfected with siS100A4.And these results indicated that PPT could inhibit the viability,proliferation and migration of both nasopharyngeal carcinoma cells by down-regulating the S100A4/Wnt signaling pathway.However,significant apoptosis was observed in 5-8F and 6-1 OB cells treated by PPT,whereas it was not observed after inhibition of S100A4/Wnt signaling.This may be due to PPT inducing cell apoptosis through other targets.According to the mitochondrial pathway apoptosis-related protein we tested,PPT induced apoptosis in 5-8F and 6-1 OB cells by initiating the mitochondrial apoptotic pathway.The bioelectron microscopy images showed that compared with the control group,siNC group and siS100A group,the mitochondria in the 5-8F and 6-10B cells of the PPT-treated group were reduced in number,swollen in morphology,with cristae broken,disorganized or even disappeared,and with local rupture of the outer membrane of the mitochondria.The results of reactive oxygen pecies(ROS)assay showed that with the increase of PPT concentration,the level of ROS in the cells increased significantly,a large amount of ROS aggregation could damage the mitochondria,thus leading to the apoptosis of cells.Subsequently,the mitochondrial membrane potential(MMP)assay was performed.The MMP in cells was significantly depolarized after exposure to high concentration of PPT,which is a hallmark change in the early stage of apoptosis.The respiratory function of mitochondria and ATP production were detected by oxygen consumption rate(OCR).The results showed that compared with the control group,the basal respiration,maximal respiration,and ATP production of the PPT treated group were significantly decreased,indicating that PPT induced damage to the respiratory function of mitochondria,leading to a decrease in ATP production.Since cellular energy is supplied by glycolysis in addition to oxidative phosphorylation of mitochondria,so finally we detected the cellular glycolysis by the extracellular acidification rate(ECAR).The results showed that compared with the control group,the glycolysis ability of 5-8F and 6-10B cells treated with PPT was significantly decreased,while cells transfected with siRNA did not show similar results.These results indicated that PPT could cause mitochondrial damage,leading to dysfunction,oxidative phosphorylation,and reduce glycolytic capacity,leading to reduced ATP production,disrupting the energy metabolism balance and activating the mitochondrial apoptosis pathway of nasopharyngeal carcinoma cells,thereby promoting cell apoptosis.4.Study of the anti-nasopharyngeal carcinoma effect of PPT in vivoFirstly,a nude mouse nasopharyngeal carcinoma transplant tumor model was established,with a control group,a PPT low-dose group,a PPT high-dose group,and a positive control group(cisplatin group).During the treatment process,the weight and tumor diameter of the mice were monitored,and the growth curves and tumor growth curves of the mice were plotted.It was found that mice in the PPT high-dose treatment group and the cisplatin group had slow tumor growth compared with the control group.The mice in the cisplatin group showed a decrease in body weight,and the mice in the two different doses of PPT treatment groups did not show significant weight loss.Compared with the control group,the weight of transplanted tumors in mice treated with high-dose PPT and cisplatin significantly decreased.The blood routine and liver and kidney function indexes of the mice in each group were examined,and the results showed that all the indexes of the mice in the different doses of PPT treatment groups were within the normal range,and no obvious pathological changes were found after HE staining of the organs of the mice in each group.The HE staining results showed that the tumor cells in the high-dose PPT group were sparser than those in the control group,and a small amount of necrosis was observed locally.After immunohistochemistry(IHC)staining,it was found that the expression of proliferationrelated protein Ki67 decreased,the expression of apoptosis proteins Caspase 3 and Bax increased,and the expression of anti-apoptosis protein Bcl-2 increased in the tumors of mice in the high-dose PPT group.TUNEL staining showed a significant increase in tumor cell apoptosis in the high-dose PPT group.The above results indicated that PPT could inhibit the proliferation of tumor cells and promote apoptosis in vivo,while also having good safety.Finally,the expression of S100A4 and β-catenin were in tumor tissues were detected by IHC,Western blot.The results showed that the expression of S100A4 and β-catenin were decreased in the high-dose PPT group.These results indicated that PPT also exerted anti-tumor effects in vivo by initiating mitochondriaassociated apoptosis and down-regulating the S100A4/Wnt signaling pathway,which was consistent with the results of in vitro cellular experiments.Conclusions:1.PPT not only significantly inhibited the vitality,proliferation,and migration ability of nasopharyngeal carcinoma cells in vitro,but also induced cell cycle arrest and apoptosis of nasopharyngeal carcinoma cells.2.Through protein mass spectrometry,bioinformatics analysis,molecular docking techniques,and dSTORM high-resolution imaging techniques,S100A4/Wnt signaling pathway and mitochondrial function have been preliminarily identified as important pathways and targets for the anti-nasopharyngeal carcinoma effects of PPT.The multitarget effects displayed by PPT is a significant characteristic of natural drugs.3.In vitro experiments have confirmed that PPT could inhibit the proliferation and migration of nasopharyngeal carcinoma cells by targeting the S100A4/Wnt signaling pathway,and induce apoptosis of nasopharyngeal carcinoma cells by damaging mitochondria,initiating the mitochondrial apoptosis pathway,and suppressing glycolysis.4.PPT demonstrated good efficacy and safety in the treatment of nasopharyngeal carcinoma in a murine xenograft model.PPT not only could inhibit tumor growth but also promote tumor cell apoptosis,indicating that the antitumor effects of PPT were achieved by activating the mitochondrial apoptosis pathway and downregulating the expression of S100A4/Wnt signaling pathway.This further validates the results of the in vitro experiments. |
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