The Study Of P4HB Role In Predicting Efficacy Of TCM Nourishing Qi And Eliminating Phlegm Treatment On Lung Cancer

ObjectiveNourishing Qi and Eliminating Phlegm Treatment(NQEPT)" is the basic method of traditional chinese medicine(TCM) treating lung cancer in oncology department of the1st affiliated hospital of Guangzhou University of Chinese Medicine. The decoction named "YQCTF" have a major role in the treatment. It include Codonopsis, Poria, Pinellia etc. In the study of National Tenth Five Year Plan and National Eleven Five Support Program we have verified that the therapy of this method could extend the median survival period of lung cancer patients with stage Ⅲ and Ⅳ. Proly4-hydroxylase beta polypeptide(P4HB) is an endoplasmic reticulum molecular chaperone protein which play a key role in the tumor progress. The expression of P4HB is decreased in the lewis lung cancer tissue after YQCTF treatment in the earlier proteomic research of the tutor research group. This study was to research P4HB role in predicting efficacy of NQEPT on lung cancer and its related mechanism.MethodsAt first, the correlation between the expression of P4HB detected by immunohistochemical assay and clinical factors were statistically analyzed using x2test to explore the mechanism of P4HB promoting the development of lung cancer. Clinical factors include pathological type, degree of differentiation, staging, the expression of vimentin, gender, TCM syndrome type etc.Secondly, the relationship between the efficacy of NQEPT on lung cancer and the expression of P4HB was analyzed by Logistic regression statistical analysis. This is to explore the role of P4HB in predicting efficacy of this treatment on lung cancer. At last, the mechanism of P4HB role in predicting efficacy of YQCTF on lung cancer was explored. The intervention of YQCTF on cytological events which P4HB participate in to promote the progress of lung cancer was detected to validated whether P4HB is the molecular target of YQCTF in treatment of lung cancer.MTT assay was used to detect the viability of A549cells before and after medication. The microenvironment of hypoxia was simulated using CoCl2to induce epithelial mesenchymal transition of A549cells. Then phase contrast microscopy was applied to observe the morphological change of A549before and after medication. Immunohistochemical assay was used to detect the microvessel density of lewis lung cancer before and after medication. The expression of P4HB before and after medication of YQCTF in A549cells was detected by western blot, RT-PCR and immunohistochemical assay.Results1. The role of P4HB in the progress of lung cancerP411B was high expressed in lung cancer and differentiation, stage, vimentin expression was correlated to P4HB expression, but pathology, age, gender, location, concomitant disease, syndrome type of TCM and smoking status was not significantly correlated to P4HB expression. P4HB are expressed higher in the nucleus and cytoplasm in the edge region of the mose invasive lung cancer lesions and metastatic lesions. Vascular endothelial cells also have high P4HB expression. All above prompt that P4HB promote lung cancer invasion and metastasis2. P4HB role in predicting efficacy of NQEPT on lung cancerLogistic regression analysis showed that gender, whether early treatment is the independent factors that influence DCR of NQEPT on lung cancer, while female, retreated NSCLC patients in this treatment had better effect. Patients with P41IB high expression have longer survival, but the difference is not statistically significant compared with patients with P4HB low expression.3. Mechanism research of YQCTF in treatment of lung cancer by down-regulating P4HBYQCTF can inhibit the cell viability of lung cancer cell A549and lower the expression of P4HB in a dose and time dependent manner, And there are strikingly positive correlation between cell viability inhibition and down-regulated expression of P4HB by correlation analysis. Western blot demonstrated that the expressions of GADD153were enhanced after medication. RT-PCR demonstrated that the expressions of P53、Bax were increased; and Bcl-2was decreased after medication. YQCTF can lower P4HB to activate endoplasmic reticulum stress apoptosis of lung cancer cells through GADD153and Bcl-2pathway.By phase contrast microscopy, untreated A549cells showed atypical epithelial cells, CoC12cultured A549showed obvious interstitial cell morphology, while cell of interstitial cell morphology in YQCTF serum group slightly increased in contrast with untreated group, decreased significantly in compared with hypoxia group, suggesting that YQCTF can hampered hypoxia-induced EMT. RT-PCR displayed that P4HB and vimentin mRNA expression were increased after CoC12stimulation, while the expression of P4HB and vimentin gene can be lowered by YQCTF serum.Immunochemical study displayed that YQCTF can reduce microvascular density and lowered the expression of P4HB.ConclusionP4HB is the molecular target of NQEPT on lung cancer. P4HB can promote lung cancer invasion and metastasis through protecting cell survival, promoting epithelial mesenchymal transition and angiogenesis probably. YQCTF can down-regulate the expression of P4HB and induce apoptosis of lung cancer cells, inhibition of epithelial mesenchymal transition and angiogenesis. P4HB can be used as a predictor for evaluate the efficacy of NQEPT on lung cancer.