Research On The Mechanism Of Notch Signal And Its Inhibitor In Drug-resistant Non-small Cell Lung Cancer

Background:Tumor is a kind of disequilibrium of cell division and cell death.Worldwide,lung cancer has become the leading cause of cancer death.However,in China’s urban population,the death of lung cancer patients has risen to take the first place in the cause of cancer death.Only 16% of patients with a survival of more than five years or more after diagnosis of lung cancer.By pathological classification,more than 85% of lungcancer is non-small cell lungcancer(NSCLC).At present,surgery is still the preferred treatment for non-small cell lung cancer,while chemotherapy is still the most important treatment for patients with late stage or requiring postoperative treatment.Although various drugs and treatment regimens currently used for chemotherapy can improve the prognosis of patients with advanced lung cancer to some extent,the inevitable drug toxicity and emerging drug resistance still result in treatment failure in most patients.The chemical resistance of tumor drugs has become a major cause of the failure of chemotherapy to fight cancer.Therefore,further prolonging the survival period of NSCLC patients and alleviating the clinical symptoms of tumor patients have become the main purpose of current clinical treatment.Gemcitabine and paclitaxel are both clinically effective chemotherapy drugs.Gemcitabine is a nucleoside drug that ACTS by killing tumor cells in the S phase and blocking the transformation of proliferative cells from G1 to S phase.Gemcitabine inhibits DNA synthesis to kill tumor cells.It can also improve the sensitivity of tumor cells to other drugs,and at the same time.The mechanism of action of taxol is mainly by promoting tubulin dimers of combination and prevent the depolymerization and stabilize microtubules,thus inhibiting the mitosis stage normal vital microtubules dynamic reorganization,will block the cell cycle in G2 / M phase,inhibiting tumor cell replication,cannot continue to split and cell death.At the same time,paclitaxel also induces apoptosis of cancer cells and regulates human function.However,the occurrence of drug resistance leads to the inability of treatment to achieve better clinical efficacy,resulting in increased difficulties in the treatment of lung cancer.Notch signaling pathway is involved in cell proliferation,differentiation,apoptosis,adhesion and epithelial-mesenchymal transformation,and its abnormalities may lead to tumor formation.The abnormal activation of Notch may regulate the proliferation and invasion of tumor cells by proteins downstream.Studies have shown that abnormal activation of Notch pathway is related to drug resistance of ovarian cancer.Ovarian cancer cells activated by Notch3 can develop resistance to platinum-based chemotherapy drugs.Our previous studies also indicated that Notch signaling pathway plays a role in the prognosis of non-small cell lung cancer.To solve the problem of poor clinical efficacy caused by patients’ resistance to gemcitabine and paclitaxel,we started to study from the Notch signal pathway for seeking solutions.To explore the role of this signaling pathway in drug resistance of gemcitabine and paclitaxel,and to search for new therapeutic regimens for drug resistance of non-small cell lung cancer.Objective:(1)To explore the role of the Notch signaling pathway in the treatment of drug-resistant non-small cell lung cancer: to detect the effects of paclitaxel and gemcitabine on the clonal formation of H1299 cells and A549 cells after the intervention of the Notch signaling pathway blocker GSI-IX.(2)The expression and changes of the Notch3 protein in paclitaxel and gemcitabine resistant cell lines were detected.The role of Notch signal pathway in drug resistance of lung cancer was analyzed.(3)The cross-sensitivity of paclitaxel and gemcitabine to growth inhibition of their respective drug-resistant cell lines was compared.(4)The effect of Notch signaling blocker GSI-IX on growth,proliferation and apoptosis of drug-resistant lung cancer cells was analyzed,so as to explore whether GSI-IX can induce apoptosis by activating the apoptotic pathway,so as to improve the sensitivity of chemotherapy drugs paclitaxel and gemcitabine in drug-resistant cells.Methods:(1)Cell culture: H1299 and A549 cells were cultured in this study,and corresponding drug-resistant cell lines were induced by paclitaxel and gemcitabine respectively.(2)Expressions of Notch3,Bcl-2 and Bax in H1299 and A549 cells and corresponding paclitaxel and gemcitabine resistant cells were detected by Western blot.(3)The effect of GSI-IX separate intervention and synergistic paclitaxel and gemcitabine on proliferation of corresponding drug-resistant H1299 and A549 cells was detected by MTT method.(4)Flow cytometry was used to detect the effect of paclitaxel and gemcitabine on apoptosis of corresponding drug-resistant H1299 and A549 cells.Results:(1)The effects of GSI-IX,gemcitabine and paclitaxel on the clonal number of H1299 cells and A549 cells alone and in combination were analyzed and compared.The results showed that the number of clones in GSI-IX group was reduced,which was comparable to that of gemcitabine and paclitaxel alone.And GSI-IX combined with gemcitabine had the greatest effect on the reduction of cell clones.It suggested that GSI-IX could improve the chemosensitivity of gemcitabine to tumor cells.(2)The expression of Notch3 protein in gemcitabine and paclitaxel resistant cell lines was detected.The expression of Notch3 protein in drug-resistant cells was significantly increased compared with that in non-resistant cells.The expression of Notch3 protein in GSI-IX cells was significantly reduced compared with that in the isolated resistant cell lines.The Notch3 signal pathway is related to drug resistance.(3)The growth inhibition effect of gemcitabine and paclitaxel on 4 groups of drug-resistant cells was detected.The results showed that the growth inhibition effect of gemcitabine on paclitaxel was stronger than that of gemcitabine.At the same time,paclitaxel inhibited the growth of two groups of cells with drug resistance to gemcitabine compared with two groups of cells with drug resistance to paclitaxel.It indicates that there is no cross-resistance between paclitaxel and gemcitabine,but cross-resistance can improve the sensitivity of chemotherapy.(4)To examine the effect of GSI-IX on proliferation of four kinds of drug-resistant cells.The results showed that GSI-IX group and GSI-IX combined with gemcitabine or paclitaxel group significantly inhibited cell proliferation compared with the blank control group.GSI-IX enhances the growth inhibition of gemcitabine or paclitaxel on four types of drug-resistant cells compared to the single gemcitabine or paclitaxel group.Furthermore,the inhibitory effect of GSI-IX at different concentrations on drug-resistant cells was different.It showed that GSI-IX can improve drug sensitivity of gemcitabine or paclitaxel.(5)Detection of GSI-IX effect on 4 kinds of drug-resistant cell,increased cell apoptosis,while detecting the Bcl-2 and the expression of Bax levels,the results showed with the increase of concentration of GSI-IX role,cut with the Bcl-2expression Bax expression levels rise,therefore,speculated that regulate the expression of apoptosis protein GSI-IX,thus affecting gemcitabine and paclitaxel on the antitumor function of the resistant cells.Conclusion:(1)By comparing the effect of GSI-IX with taxol and gemcitabine on lung cancer cells,it can be concluded that GSI-IX has anti-tumor effect similar to that of chemotherapy drugs like gemcitabine and paclitaxel.GSI-IX combined with gemcitabine can be used as a new treatment for lung cancer chemotherapy.(2)GSI-IX can improve the expression of the Notch3 protein in drug-resistant cells,and GSI-IX acting on drug-resistant cells can enhance the growth inhibition effect of different drug-resistant cells,as well as the anti-tumor effect of gemcitabine or paclitaxel.It can be inferred that the Notch3 signaling pathway is involved in tumor resistance,and blocking the Notch3 signaling pathway can improve drug sensitivity of drug-resistant tumor cells.(3)GSI-IX can increase cell apoptosis rate in drug-resistant cell lines,and relevant apoptotic proteins bcl-2 and Bax also show corresponding changes.It can be speculated that GSI-IX can change the resistance of drug-resistant H1299 cells and A549 cells to chemotherapy drugs by regulating the apoptotic pathway.It suggests that GSI-IX may be used to treat non-small cell lung cancer by promoting apoptosis of tumor cells.