Dihydromyricetin Enhanced Sensitivity Effect On The Drug-Resistant Human Breast Cancer SKBR3 Cells Induced By Herceptin Through Inhibiting PI3K/AKT Signal Activation In Vitro And In Vivo

IntroductionPeople suffering from overexpression breast cancer of the type “human epidermal growth factor receptor 2”(HER2)accounts for about 20-25% of the total breast cancer patients.Patients with this type of disease---belonging to those whose prognosis is poor---are prone to being in the state of disease progression,thus having a relatively high mortality rate.Herceptin first approved of for the targeted treatment of HER2 positive breast cancer,is a humanized monoclonal antibody that can specifically combine with extracellular IV domain of the HER2 positive cells,blocking the transmission of downstream signals.Herceptin has significantly prolonged the disease-free survival(DFS)and overall survival(OS)of HER2 positive breast cancer patients since it came out,and clinical response rate(CRR)increased significantly.The cases of advanced patients with recurrence and metastasis are also can improved with it application.That's why It has currently been recommended for the standard treatment of HER2 positive breast cancer patients.However,there are still many problems to be solved urgently in the clinical treatment,of which the most prominent ones are the Herceptin resistance phenomenon and cardiac toxicity.There are only about less than 35% of HER2 positive breast cancer patients respond positively to Herceptin monotherapy in clinical.Most,whose treatment were initially effective,later become resistant to therapy during the treatment period of 12 months.Due to a lack of reliable molecular markers to evaluate Herceptin resistance,some patients are undergoing inefficient or even ineffective treatment,causing economic loss and delay of the disease;while others appear cardiac toxicity like myocardial damage and reduced cardiac ejection fraction during treatment,showing symptoms like palpitation,chest tightness,chest pain,dyspnea and so on,herceptin treatment having to be suspended.How to predict and overcome resistance,and how to prevent the cardiotoxicity of Herceptin,become an urgent problem needed to solved in HER2 positive breast cancer therapy.Insulin like growth factor 1 receptor(IGF1R)is a transmembrane tyrosine kinase receptor.A large number of preclinical experiments confirmed that the activated IGF1 R can promote cell proliferation and inhibit cell apoptosis.overexpression and abnormal activation of IGF1 R happen in many kinds of tumors,of which the downstream PI3K/Akt signal pathway abnormal activation are often accompanied by Herceptin resistant.The trans-infected IGF1 R gene's entry into Herceptin-sensitive SKBR3 cells can lead to significant drug resistance.Among acquired Herceptin resistant BT474/HER model,the intracellular IGF1 R expression is 3 times more than the parental BT474.Application of IGF1 R antibody alpha-IR3 or small-cell-interfering RNA,which is IGF1 R intensive or IGF1 R tyrosine kinase inhibitors to inhibit the activation of IGF1 R,can increase the cell reaction to Herceptin and restore the sensitivity of drug resistant cells to Herceptin.HER2 and IGF1 R share the same downstream PI3K/AKT signal pathway.The abnormal activation or anti oncogene PTEN inactivation of such downstream signaling proteins,as insulin receptor substrate 1(IRS1),phosphatidyl inositol 3 kinase(PI3K),and protein kinase B(Akt),can lead to cell proliferation and apoptosis.It is possible that IGF1 R affects the responsiveness of cells to Herceptin through this pathway.Up to now,the interaction between IGF1 R and HER2 and the impact of the PI3K/AKT signaling pathway on Herceptin sensitivity,are not clearly known.It is a heated topic in tumor research to find the effective anti-tumor components from natural plants.The purely natural Dihydromyricetin(DMY),classified as a flavonoid compound,is Extracted from ampelopsis grossedentata.It has been verified that DMY possesses many kinds of qualities including anti oxidation,anti thrombosis,protect the liver,anti-inflammatory etc.Researchers recently discovered that DMY also possesses the anti-tumor quality of Inhibiting proliferation,promoting apoptosis of tumor cells,enhancing the anti-tumor effect of chemotherapy drugs,such as doxorubicin,when it is applied with these chemotherapy drugs and alleviating the chemotherapy-caused dysfunction of bone marrow,liver and kidney,etc.It has not been documentarily reported that DMY has the effect of increasing Herceptin sensitivity to HER2 positive breast tumor and reducing myocardial toxicity caused by Herceptin treatment.The present research on DMY is fledgeling,with few published reports at home and abroad and a lack of sufficient experimental data to ensure security in vivo.Professor Tu of China extracted artemisinin from plants and won this year's Nobel Prize in Medicine,which brought Chinese medicine to the world stage.Ampelopsis grossedentata plants distribute widely and abundantly in China,thus providing a reliable support for DMY extraction and research.How to integrate the time-honored and culture-rich Traditional Chinese Medicine with modernly civilized western medicine,to make the two complement each other and to develop them hand-in-hand are a very important part in our future research.Therefore,this paper intends to establish stable subcultured Herceptin resistant SKBR3-R cell lines by inducing parent SKBR3 cells with Herceptin and set up nude mice xenograft tumor model of SKBR3-R.The expression of and interaction between IGF1 R and HER2 and the expression and activation level of PI3K/AKT signaling pathway with downstream signaling molecules IRS1,AKT,PTEN in SKBR3 and SKBR3-R cells were observed in order to explore the IGF1R/HER2 heterodimers as well as the role of PI3K/AKT signaling pathway function in the development of resistance to anti-HER2 therapy.In addition,Herceptin treatment and it combines DMY were compared in terms of their respective effects on anti-tumor efficacy,DMY's effects on sensitivity of Herceptin treatment and on cardiac and hepatic toxicity were explored,and the role of PI3K/AKT signaling pathway in the treatment were researched,and the value and significance of DMY combined targeted therapy were also discussed.Part ? Dihydromyricetin enhanced the sensitivity of Herceptin to Herceptin resistant SKBR3 cells by inhibiting IGF1R/HER2 signal pathwayObjective: 1.Exploring the interaction between Herceptin-induced drug-resistant cell line SKBR3-R's two cell-membrane receptor molecules IGF1 R and HER2 and observing influence on downstream signal activation.2.Exploring the effect of IGF1R-mediated signaling pathway on Herceptin sensitivity in SKBR3 cells.3.Exploring the effect of Dihydromyricetin on SKBR3-R cells' Herceptin sensitivity and possible mechanism in drug resistance.Methods: 1.Establishing drug-resistant SKBR3-R cell lines through Herceptin induction.2.Testing the expression of cell-membrane receptor molecules IGF1 R and HER2 and downstream signaling proteins IRS1,AKT,MAPK,PTEN,and their phosphorylation activation by western blot method.3.Testing the interaction between protein molecules of IGF1 R and HER2 by immunoprecipitation.4.Measuring cell proliferation and cell viability by MTT method.5.Measuring cell cycle and apoptosis by flow cytometry.Results: 1.With stable passage,the drug-resistant SKBR3-R cell lines,whose cell cycle distribution changed significantly,were obtained from Herceptin-induced SKBR3 cells.The total number of SKBR3-R cells was 3 times of SKBR3 cells in the cycle of S phase,and the result was reversed in the cycle of G2-M phase that the number of SKBR3 cells was 3-4 times of SKBR3-R cells.2.We applied immunoprecipitation method to lysis product of the SKBR3-R cells,detecting that the IGF1R/HER2 heterodimer existed in the precipitate and vice versa.Whereas,in SKBR3 cells,there weren't any.3.After Dihydromyricetin was applied to SKBR3 and SKBR3-R cells,their proliferation ability were inhibited and the degree of inhibition went up with the increase of Dihydromyricetin concentration.4.As application of IGF1 prolonged,the amount of IGF1R/HER2 heterodimers increased slightly,phosphorylation level of IGF1 R also increased slightly.but HER2 phosphorylation level decreased slightly.The IGF1R/HER2 heterodimers had no significant effect by IGF1 stimulation,but phosphorylation of IGF1 R was faster and the level of HER2 phosphorylation gradually increased in SKBR3-R cells.The above-mentioned two groups of cells' Intracellular signaling molecules IRS1 phosphorylation levels gradually increased,PTEN protein expression gradually decreased.AKT and MAPK phosphorylation levels increased first and then decreased in both cell lines,and the same was true of SKBR3-R cells,but more obviously.5.After ?-IR3 was applied to SKBR3-R cells,the amount of IGF1R/HER2 heterodimers significantly decreased(p < 0.05),and so did the level of IGF1 R phosphorylation(p < 0.05),but the level of HER2 phosphorylation had no significantly changed.Downstream signaling protein IRS1,AKT,MAPK phosphorylation levels gradually weakened,while the PTEN expression level remained unchanged.In addition,the inhibition of SKBR3-R cell proliferation was weak when ?-IR3 or Herceptin treated SKBR3-R cells respectively,but the proliferation ability of SKBR3-R cells decreased significantly when the two worked together(p < 0.05).6.IGF1 R phosphorylation levels decreased in both SKBR3 and SKBR3-R cells along with the increasing concentration of Dihydromyricetin treatment,and the decreasing effect was more obvious than the increasing effect(p < 0.05).Dihydromyricetin decreased the phosphorylation level of HER2 of SKBR3-R cells,but had no obvious effecrct on that of SKBR3 cells.There was no significant difference in the expression of IGF1 R and HER2 in the two groups of cells.Through Dihydromyricetin pretreatment first and then Herceptin treatment,the phosphorylation level of IGF1 R and HER2 significantly decreased in SKBR3-R cells compared with Herceptin-monotherapy-treated group,and especially IGF1 R phosphorylation levels decreased more significantly(p < 0.05).IRS1,AKT,MAPK protein phosphorylation levels did not change significantly,showing bad sensitivity to Herceptin,in Herceptin monotherapy group.After Dihydromyricetin pretreatment,the sensitivity of SKBR3-R cells to Herceptin was obviously enhanced,IRS1 and AKT phosphorylation decreased significantly(p < 0.05).But the level of MAPK phosphorylation was not affected by Dihydromyricetin and Herceptin treatment.In addition,Dihydromyricetin-treated cells' PTEN expression was obviously raised in treatment groups(p < 0.05).7.After Dihydromyricetin pretreatment,SKBR3 cells and SKBR3-R cells were more sensitive to Herceptin,and more apoptotic,with the latter being so more obviously(p < 0.05).Conclusion: 1.The formation of IGF1R/HER2 heterodimers in HER2 overexpression human breast cancer SKBR3-R cells,is one of the important reasons for Herceptin-resistance,inhibiting the formation of IGF1R/HER2 heterodimers can increase the Herceptin sensitivity in SKBR3-R cells.2.Dihydromyricetin can inhibit the formation and activation of IGF1R/HER2 heterodimers,blocked the IRS1/PI3K/AKT downstream signaling transmission,improve the anti-oncogene PTEN expression and significantly enhance SKBR3-R cells' sensitivity to Herceptin.Part ? Dihydromyricetin restored Herceptin sensitivity to drug resistant SKBR3-R cells transplanted nude mice in vivoObjectives: 1.To observe the effects of Dihydromyricetin on Herceptin sensitivity of nude mice into which SKBR3-R Cells Xenograft Tumors were Transplanted.2.To observe the effects of Dihydromyricetin on PI3K/AKT signaling pathway in vivo.3.To observe the effects of Dihydromyricetin on heart and liver function of nude mice in vivo.4.To observe the effect of DMY combined with Herceptin treatment on circulating tumor cells(CTC)of transplanted tumor in mice.Methods: 1.To establish SKBR3-R cell nude mice Xenograft tumor models.When the establishment was done,we randomized the mice in good state and at similar weight into 4 groups: control(NS),Herceptin(H),Dihydromyricetin(D)and Herceptin+ Dihydromyricetin(H+D).Each group contained 6 mice on average with the experimental period of 21 days.2.Every 3-4 days,The length and diameter of the tumor were measured by vernier caliper and we calculated the volume according to the formula.Body weight of the mice was measured by electronic balance.3.On the last day,the21 st day,all the experimental nude mice were killed.The serum LDH,CK,ALT and AST levels in each mice analyzed by automatic biochemical analyzer at the end of the experiment.4.The expression levels of PTEN?AKT?P-AKT?IRS1?P-IRS1 of the tumor tissue were measured by Western blot method.5.The expression levels of HER2,IGF1 R,P-AKT,P-IRS1 and PTEN in tumor tissues were detected by immunohistochemical with SP staining.6.CTC was separated and collected by negative enrichment technique,and identified by immunofluorescence technique(im FISH).Results: 1.The whole course of the experiment was 21 days,and no mouse died during the experimental period.The mice in NS group had a body weight rise trend but those in other treatment groups saw an irregular rise and fall in body weight during the experimental period,with the overall difference not significant(p < 0.05).2.The tumor volumes of all mice had an increasing rise in each group during the experimental period.The increasing rate of tumor volume in(H+D)group was significantly lower than that of(NS)group(p < 0.05).3.When the treatment was over,the serum LDH,CK,ALT and AST levels had a tendency to grow up in Herceptin group compared with control group at the end of treatment,and the rise of LDH was more obvious.The serum LDH,CK,ALT and AST levels declined significantly in Herceptin +Dihydromyricetin group compared with NS group(p< 0.05).4.There was no difference of The PTEN expression and phosphorylation degree of Insulin receptor substrate 1(p-IRS1)levels between herceptin monotherapy group and control group at the end of the experiment.The expression of PTEN in tumor tissue increased,and the level of p-IRS1?p-AKT decreased,when combined with Dihydromyricetin therapy in Herceptin group,the difference was statistically significant(p< 0.05).There were no significant differences in the expression levels of IGF1R?HER2? total AKT and IRS1 in all groups(p>0.05).5.Compared with(NS)group,(H+D)saw a more apparent decrease in CTC(p < 0.05).Conclusion: 1.Dihydromyricetin can improve nude mice SKBR3-R cell transplanted tumors' Herceptin.2.Dihydromyricetin has a certain protective impact on nude mice's heart and liver functions.3.Dihydromyricetin can inhibit abnormal activation of tumor cells'.4.Dihydromyricetin combined with Herceptin treatment can significantly reduce the amount of CTC in mice serum,and lower the risk of tumor recurrence and metastasis.