The Antitumor Effects And Mechanisms Of Pseudoginsengenin DQ On Hypopharyngeal Cancer

Hypopharyngeal cancer account for about 3%-5% of all head and neck malignant tumors,and the 5-year survival rate of the patients is only about 40%.Because the initial symptoms are not typical,patients are often at an advanced stage when diagnosed,and the prognosis is poor.Surgery combined with chemotherapy or radiotherapy is the conventional treatment for hypopharyngeal cancer.Although the treatment of hypopharyngeal cancer has made good progress over the years,due to the surgery need to remove all laryngeal structures or part of the laryngeal and hypopharyngeal tissues,the patients face the problems of damage to the basic function of the organs after surgery and a significant decrease in the quality of life.Traditional anti-cancer drugs often have many side effects,therefore,the development of anti-tumor drugs with high activity and low toxicity is one of the main directions of current tumor treatment research.Ginsenosides are the most important constituents of ginseng,which have been shown to play a therapeutic role in various cancers.The metabolites of ginsenosides can regulate signaling pathways related to tumor growth and metastasis to promote tumor cell apoptosis,induce tumor cell differentiation,and target tumor stem cells.At present,secondary ginsenosides,the degradation products of ginsenosides,have been found to have stronger pharmacological effects in vivo.Pseudoginsengenin DQ(PDQ)is an octilone-type secondary ginsenoside synthesized from protopanaxadiol(PPD).Studies have found that PDQ can be used to improve aconitine-induced arrhythmias and cisplatin-induced acute kidney injury without weakening the antitumor effect.However,the antitumor activity and mechanism of PDQ on hypopharyngeal cancer are not fully understood.The purpose of this study was to investigate the anti-tumor effects and related mechanisms of PDQ on hypopharyngeal carcinoma through in vivo and in vitro experiments,which were divided into the following three parts:(1)In order to verify the effect of PDQ on the proliferation and apoptosis of hypopharyngeal cancer cells in vitro,the effect of PDQ on human hypopharyngeal carcinoma cells(Fa Du)and mouse squamous carcinoma cells(SCC7)was first determined by CCK-8 method.The cell viability of each group of cancer SCC7 cells was found to be the lowest after 24 hours of PDQ treatment,and the IC50 value(100?mol/L)was the smallest at this time.Then,the cloning and migration abilities of Fa Du and SCC7 cells treated with different concentrations of PDQ were detected by clonogenic assay and transwell migration assay.It was found that PDQ acts in a dosedependent manner but time-dependent manner.When the concentration of PDQ is higher than 60 ?mol/L,It significant inhibited the proliferation,cloning and migration ability of the two cell lines.At the same time,cell cycle analysis found that the number of cells in the G0/G1 phase of Fa Du and SCC7 cells treated with PDQ significantly increased,indicating that PDQ triggered the arrest of the hypopharyngeal cancer cell cycle.Then,the proportion of apoptotic cells in early and late phase of Fa Du cells and SCC7 cells treated with different concentrations of PDQ was analyzed by flow cytometry,both increased with the increase of PDQ concentration.After TUNEL apoptosis detection,it was also found that with the increase of PDQ concentration,the apoptosis ratio of Fa Du and SCC7 cells after 100?mol or 140?mol/L PDQ treatment was significantly increased,which was consistent with the results of flow cytometry.In order to verify the mechanism of PDQ-induced apoptosis,in this part,the expression levels of apoptosis-related proteins Bax,Cytochrome C,Caspase 9 and Caspase 3 in Fa Du and SCC7 cells detected by western blotting after PDQ treatment were significantly increased,while the expression of the anti-apoptotic protein BCL-2decreased.At the same time,the mitochondrial membrane potential detected by JC-1also found that the mitochondrial membrane potential of the two cell lines was significantly reduced after PDQ treatment.Thus,PDQ turns on the endogenous mitochondrial apoptotic pathway to induce apoptosis.(2)Hypoxia is a common phenomenon in solid tumors.Hypoxia-inducible factor1(HIF-1?)is the central regulator of the hypoxia adaptation process of tumor cells,which can regulate the level of glycolysis in cells through its downstream protein glucose transporter 1(GLUT1).It has been demonstrated that ginsenosides can inhibit tumor growth by inhibiting the expression of HIF-1?.In this part,the expressions of HIF-1? and GLUT1 in 70 human hypopharyngeal carcinoma samples were detected by immunohistochemistry.It was found that both proteins were highly expressed in hypopharyngeal carcinoma tissues,and their expression levels were positively correlated.The results of survival analysis showed that compared with the low expression group,the overall survival time of the patients in the high expression group of HIF-1? was decreased,and the expression of HIF-1? was correlated with the tumor differentiation degree of the patients with hypopharyngeal carcinoma.The clinical stage or tumor differentiation was correlated with the expression of GLUT1.At the same time,after inhibition of HIF-1? expression by si RNA interference,the CCK-8 and transwell migration assays showed that the proliferation and migration of Fa Du or SCC7 cells reduced significantly.The above experiments jointly proved the important regulatory effect of HIF-1? on tumor growth.The first part found that PDQ can inhibit the proliferation of hypopharyngeal cancer cells and induce apoptosis.In order to verify whether the mechanism of PDQ's anti-hypopharyngeal cancer action is related to HIF-1?,this part the relationship between PDQ and HIF-1? was analyzed through molecular docking.Which proved that HIF-1? may be the potential target of PDQ.Next,through western blot and q RT-PCR experiments,it was found that both HIF-1? and GLUT1 were expressed under normoxia or hypoxia,and PDQ could inhibit the expression of both proteins under different oxygen levels.PDQ inhibited the expression of HIF-1?and GLUT1 protein and m RNA in Fa Du and SCC7 cells in a concentration-dependent manner.d STORM imaging also found that PDQ not only reduced the number of GLUT1 protein clusters on the cell membrane,but also redistributed its arrangement.The detection of glucose uptake in cells showed that the relative glucose uptake levels were significantly reduced after the treatment of higher concentrations of PDQ,while the levels of reactive oxygen species increased after PDQ treatment.(3)In the in vivo experiment,the mouse hypopharyngeal carcinoma xenograft model was established,the negative and positive control groups were set up,the weight and tumor size of the mice were monitored and the tumor growth curve was drawn.With the increase of administration times,the tumor growth in the cisplatin group and PDQ high-dose group was slow.After the end of PDQ administration,the body weight of the mice in the PDQ group did not decrease significantly,the tumor volume and weight in the cisplatin group was the smallest,followed by the PDQ high-dose group.No significant pathological changes were observed in the main organs of the PDQ highdose group after HE staining.At the same time,the levels of red blood cells,white blood cells,monocytes and lymphocytes in the blood of mice in the PDQ high-dose group,as well as serum aminotransferase,urea nitrogen and creatinine levels were also within the normal range.Immunohistochemical staining of the tumor showed that HIF-1?,GLUT1,anti-apoptotic protein BCL-2 and proliferation-related protein Ki67 were decreased in PDQ high-dose group,and the expression of apoptotic protein BAX was correspondingly increased.The TUNEL test of tumor tissue also proved that after high dose of PDQ,the apoptosis rate in tumor of mice was significantly increased.In this study,through in vitro and in vivo experiments,it was found that PDQ regulates the HIF-1?-GLUT1 signaling pathway by targeting HIF-1?,inhibiting the level of glycolysis and glucose uptake in hypopharyngeal cancer cells,and enabling cells to obtain energy,which leads to mitochondrial damage.The damaged mitochondria release reactive oxygen species into cells,which further promotes cell apoptosis.PDQ inhibited the proliferation,cloning and migration of hypopharyngeal cancer cells in a concentration-and dose-dependent manner,and at the same time induced cell apoptosis and inhibited the growth of mouse xenografts.PDQ has less side effects,and the safety of in vivo application is relatively good.HIF-1? plays an important regulatory role in this process.Hypopharyngeal carcinoma patients with high expression of HIF-1? had a faster decline in overall survival and worse tumor differentiation,while patients with high GLUT1 expression also had worse clinical stage and tumor differentiation.In this study,computer-aided techniques were used to predict HIF-1? as a potential target factor of PDQ.The nanoscale super-resolution imaging was realized by d STORM technology,which proved the anti-hypopharyngeal cancer mechanism of PDQ at the molecular level,and provided theoretical and experimental basis for PDQ's anti-hypopharyngeal cancer research.