Proliferation Inhibition And The Mechanism Of Morusin On Two Kinds Of Non-small Cell Lung Cancer Cell Line

Cancer is one of the major problems that threaten human health and life.Due to population aging,air pollution,smoking and other causes,more than 1.5 million people worldwide die from lung cancer each year.However,the five-year survival rate of lung cancer is only 18.1%.Therefore,looking for chemotherapy agents with higher efficacy,lower side effects is always the pursuing goal for researchers.Sangbaipi is the dried root bark of Morus alba L.In traditional Chinese medicine(TCM),Sangbaipi has the efficacy of relieving asthma and alleviating edema.It has been commonly used for the treatment of diseases such as hypertension,hyperlipidemia diabetes,bacterial infections and cancer.In the research of chemical constituents of sangbaipi,our group obtained some prenylated flavonoids.It is well-known that prenyls substitution has a significant enhancement effect on the cytotoxicity of flavonoids.We screened the antiproliferation effects of the prenylated flavonoids obtained by using MTT method.The results showed that morusin inhibited the cell growth in two kinds of non-small cell lung cancer cell lines,A549 and NCI-H292.The content of morusin in sangbaipi is relatively high,which guarantee the amount for further bioactive researchs.In this study,we use morusin as a representative compound of prenylated flavonoids to perform the anti-proliferation assay and the mechanisms exploring.Hopefully,by doing this study we can provide some preliminary data for the research of bioactivities of prenylated compounds.In this study,we first investigated the proliferation inhibition effect of morusin in lung cancer A549 and NCI-H292 cells by using MTT assay,the IC50 of morusin was determined as 12.32 ?M and 7.92 ?M for A549 and NCI-H292 cells respectively.After morusin treatment,some of the nuclei were observed as condensed and fragmented in DAPI staining,apoptotic cells were detected by Annexin V/PI assay,and cleaved caspase-9,caspase-3 and PARP were detected by immunoblotting,which suggested the induction of apoptosis by morusin.Furthermore,JC-1 staining was used for the detection of mitochondrial membrane potential(MMP),morusin induced the decrease of MMP.Cytochrome c released from mitochondria into cytoplasm,which was revealed by immunoblotting.The expression of Bcl-2 family proteins,including anti-apoptotic proteins Bcl-2,Bcl-XL and proapoptotic protein Bax,changed simultaneously.According to above-mentioned evidences,it was suggested that the cell death induced by morusin is associated with endogenous apoptosis via the mitochondrial pathway.Some dead cells did not show the typical morphology of apoptotic cells after the treatment of morusin.Also,the apoptotic rate shown in Annexin V/PI assay was lower than inhibition rate in MTT assay.This discrepancy made us thinking about the existence of other cell death pathway than apoptosis.During the investigation,the autophagy marker LC3 changed from LC3-? to LC3-?,and the expression of p62 protein decreased in a dose-dependent manner,which suggested that autophagy may be initiated after the treatment with morusin in A549 and NCI-H292 cells.The intracellular ROS content was detected by DCFH-DA.The results showed that morusin caused a significant increase of intracellular ROS levels in both A549 and NCI-H292 cells.After cotreatment of NAC(a ROS inhibitor)with morusin,the induction of both apoptosis and autophagy was blocked,which indicated that the accumulation of intracellular ROS contribute to the apoptosis and autophagy induced by morusin.MAPK/ERK,MAPK/JNK and PI3K/AKT signaling pathways are involved in the regulation of processes including cellular stress response,cell differentiation,proliferation,metabolism and apoptosis.After treatment with morusin,the phosphorylation of ERK and JNK increased in A549 and NCI-H292 cells,which indicated the activation of MAPK signaling pathway.Meanwhile,the phosphorylation of AKT decreased,indicating the inhibition of the PI3K/AKT signaling pathway.These data suggested the involvement of MAPK and PI3K pathways in the action of morusin.The co-treatment of cells with the relevant inhibitors demonstrated that MAPK pathway activation and PI3K inhibition mediated the inhibition of cell proliferation induced by morusin,also ROS correlated with the regulation of these two pathways.Based on the results of this study,we could get the conclusions that the cell death induced by morusin in A549 and NCI-H292 cells is associated with the induction of endogenous apoptosis and autophagy.Intracellular ROS promotes apoptosis and autophagy.Meanwhile,changes in the expression of MAPK/ERK,MAPK/J-NK and PI3K/AKT signaling pathways are also associated with overproduction of ROS.ROS might be a common signaling molecule in the changes of several signaling pathways induced by morusin.Further exploring was needed in order to clarify the the mechanism of action.