The Muriceopsis Flavida Antitumor Component Selection And Research Of Cholestane-3β,5α,6β-Triol Induced Apoptosis Mechanism In A549Cells

Background: Malignancy is one of the major diseases to cause actual harm to thehealth of people. Study on the development of new anticancer drugs and looking forrelevant target for anticancer drugs become an urgent need. Chemotherapy of cancerremains one of the primary means of treatment for malignant tumors, inducing tumorcell apoptosis is an important method in studies on anticancer drugs. Mitochondriaplay a critical role in the regulation of apoptosis. Apoptosis inducing factor (AIF)mediates caspase-independent apoptosis. In addition, c-Jun N-terminal kinases (JNK)mediate apoptosis induced by multiple extracellular stimuli. JNK are involved inexternal death receptor and intrinsic mitochondrial apoptotic pathway, and plays animportant role in the development of a variety of pathological damage, such ascancer, chronic hepatitis B, ischemia-reperfusion injury. Transcription factor AP-1(core components: the c-Jun protein) are important goals of JNK, JNKs activatingthe apoptosis signal through the increase of apoptosis gene-specific transcription orby direct phosphorylation of the transcription factor AP-1before regulatingmitochondrial apoptosis and activity of anti-apoptotic proteins, thus leading toapoptosis. Therefore, in the induction of apoptosis of tumor molecular therapy, JNKsignaling pathway plays an important role. Research shows that the gorgonians ofthe south China sea contains many unique structure and biological activities ofnatural active ingredients, Steroids are one of the representative metabolites isolatedfrom animals of gorgonians. These metabolites are reported to have growthinhibition to a wide variety of tumor cell lines.In present study, we screened the anticancer activity of19compounds on5different cancer cell lines and then a further study was made for the specificmechanisms of cholestane-3β,5α,6β-triol (LC1) inducing apoptosis of human lung carcinoma A549cells.Therefore, certain experimental theoretical was provided fordeveloping anticancer drugs.Methods and results:1. We screened the anti-cancer activity of19compounds ofgorgonian Muriceopsis flavida by MTT and CCK-8method. In the bioassay in vitro,these compounds exhibited different levels of growth inhibition activity againstA549and MG63cell lines. In particular, compound18displayed a considerableactivity, being similar as adriamycin. An futher Annexin V/PI analysis indicated thatcompound7is more potent in the induction of apoptosis.2. Annexin V-FITC/Pl double staining assay detected the apoptosis was analyzed byFCM, we found LC1significantly induced apoptosis in A549cells; Nuclearmorphology of A549cells were observed under fluorescence microscope (stained byHoechst33258), showing dramatic decrease in the amount of staining cells andnuclear chromatin pyknosis, dense thick lectin staining, fluorescence enhancement.3. The effect of LC1on A549cells cycle arrest was analyzed by FCM, We foundcells cycle arrest in the G2/M period. JC-1probe loaded, mitochondrial membranepotential (ΔΨm) was measured by fluorescence microscopy and FCM. The resultsshowed that the green fluorescent of LC1treated group increased significantly andred fluorescence decreased, LC1induces the loss of mitochondrial membranepotential, indicating that apoptosis was mediated by the mitochondrial pathway.4. A549cells subjected to caspase-3measurement using Fluorescence detection.Furthermore, the caspase inhibition assay, which uses a general caspase inhibitorZ-VAD-fmk as well as inhibitor specific to caspase-3was performed with annexinV/PI double staining analysis. However, all caspase inhibitors failed to attenuate LC1-induced apoptotic cell death. Additionally, we found LC1induces translocation ofAIF from the mitochondria to the nucleus. These results suggest that caspaseactivation is not necessary for LC1inducing A549apoptosis.5. Gene expression levels of c-Jun was analyzed by RT-PCR and Protein expressionof phosphorylation of JNK, ERK, c-Jun were detected by Western blot. We found JNK and c-Jun were activated, while P-ERK was down regulated; LC1is able toactivate the MAPK pathway and enhance the activity of the nuclear transcriptionfactor c-Jun of A549cells.6. A549cells were pretreated with JNK and ERK inhibitors respectively. Thespecificity of these inhibitors was assessed by western blotting of whole cell lysatesfor phosphorylated c-Jun as a measure of JNK activity, then apoptosis was detectedby FCM. Results showed that inhibition of JNK resulted in a significant increase incell viability after LC1treatment compared with exposure to LC1alone, whereasinhibition of ERK had no effect. These results suggest that sustained activation of theJNK–c-Jun pathway is necessary for LC1-induced apoptosis in A549cells.Conclusion:1. Gorgonian Muriceopsis flavida extract cholestane-3β,5α,6β-triolexhibited broad-spectrum growth inhibition of human carcinoma cells, and cansignificantly induced apoptosis in A549cells.2. LC1-mediated apoptosis in A549cancer cells is accompanied by DNAfragmentation, nuclear condensation, and phosphatidylserine exposure. LC1inducesmitochondrial membrane permeabilization (MMP) through loss of mitochondrialmembrane potential (ΔΨm). LC1was found to induce phosphorylation of c-JunN-terminal kinase (JNK), and apoptosis was found to be attenuated by inhibition ofJNK. Phosphorylation of JNK further activates the transcription factor AP-1protein(c-Jun), LC1may be mediated through transcription-dependent mitochondrialpathway of apoptosis in A549cells. Additionally, the final stage of apoptosis ofA549cells induced by LC1is via a caspase-independent mitochondrial pathway,which may be caused by the nuclear translocation of apoptosis-inducing factor(AIF).