The In Vitro Study Of The Anti-tumor Activity And Initial Molecular Mechanisms Of Cardiac Glycosides Compound M11 On Non-small Cell Lung Cancer

Background and Objective:Asclepias curasavica. Linn, an erect, simple(or)much branched perennial herb with a somewhat woody base, belonging to the family Asclepiadaceae(milkweeds). It shows anti-inflammatory, cardiotonic, anticancer, anthelmintic activities and is used to treat piles and gonorrhea. The common garden plant also is a good source of the cardenolide cardiac glycosides. Cardiac glycosides is a kind of natural product with many kinds of biological activity. It can be extracted from a lot of plants and clinically used for increasing the cardiac contractile force. Recently, cardiac glycosides draw more attention due to their potent antitumor activities. In the primary studies, we obtained cardiac glycosides compound M11 from Asclepias curasavica. Linn. During our search for anti-tumor bioactive screening in various malignant tumors, we found that the Asclepias curasavica-derived compound M11, exerted significant inhibitory effects on the viability of various tumor cells. This research mainly include screening of M11 antitumor activity in vitro, the effects of M11 on cell cycle of tumor cells, the observation of apoptosis, the molecular mechanisms of M11 induced cell cycle arrest and apoptosis. Our study will committed to providing theoretical references for the subsequent research on this kind of agents, as well as the development of therapy of cisplatin-resistant non-small lung cancer.Methods:1. The anti-tumor effects of M11 on the viability of A549, A549/CDDP, Hep G-2,Hep G-2/ADR, MCF-7, MCF-7/ADR cells were determined by MTT assay. And then we calculated their IC50 in different times. What’s more, we detected the cell cytotoxicity of M11 on the NIH3T3, H9C2, HEK 293 and RAW246.7 cells.2. We detected the effects of M11 on cell cycle in A549、A549/CDDP cells. Using fluorescence microscopy, we observed morphological changes in the A549/CDDP cells treated with various concentrations of M11,then detected whether M11-induced cell death was related to apoptosis by flow cytometry.3. We used immunofluorescence, western blotting technique to detect the changes of autophagy related markers including autophagosome, LC3, Beclin 1 proteins expression, and then determine whether low concentrations of M11 stimulation induced autophagy in A549 / CDDP cells.4. We detected the expression levels of regulators of cell cycle arrest and apoptosis in A549/CDDP cells which including cyclins, CDKs, CDKIs, caspase family proteins, bcl-2 family proteins, PARP. SP600125, a specific inhibitor of JNK phosphorylation, we used to verify whether inhibitor of JNK could weaken the apoptosis rate.Results:1. MTT results determined that A549/CDDP cells were more sensitive to M11 than A549 cells and M11 significantly decreased the viability of A549/CDDP cells in a dose-dependent manner with an IC50 of 0.3 μM. However,M11 inhibited cellular viability on A549 cells with an IC50 value of 38.46 μM after 48 h of treatment. There is no obvious difference on cytotoxicity of M11 to R-Hep G2 cells, Hep G2 cells, MCF7 cells and MCF7/ADR cells. Meanwhile,we showed that there is no distinctly growth-inhibitory activity on several normal cell lines of various origins under the same experiment conditions which including H9C2, NIH3T3, RAW246.7 and HEK-293 cells with IC50 48.42 μM, 58.79 μM 28.07 μM and 1.86 μM.2.Cell cycle analysis showed that cell populations in G2/M phase increased compared with the cells in control group. Using fluorescence microscopy, we observed apoptosis changes in the A549/CDDP cells treated with relatively high concentrations of M11(0.5 μM and 1 μM). Furthermore, A549/CDDP cells detected by PI/Annexin V double staining assay and results revealed that M11 induced apoptosis in A549/CDDP cells. While A549 cells treated with various concentrations of M11, no marked change of apoptosis been found.3. MDC staining revealed that there are a lot of acidic vacuole in A549/CDDP cells treated with relatively low concentrations of M11(0.01 μM and 0.05 μM). Furthermore, we found that LC3 and Beclin-1 proteins were activated by M11.4. Our study demonstrates the anti-proliferative and cytotoxic activity of M11 in A549/CDDP cells. M11 induces G2/M phase cell arrest through regulation of cyclins, CDK1, CDK2, p21 and p53. M11 also triggers JNK-mediated apoptosis via the mitochondrial apoptotic pathway, which is supported by collapse of mitochondrial membrane potential, activation of caspases, alteration of bcl-2 and Bax expressions and reactive oxygen species production.Conclusions1.During our research, we found that the Asclepiascurasavica-derived compound M11, exerted strong inhibitory effects on the viability of various malignant tumors and derived multidrug resistant cell lines. What’s more, M11 exhibited much more significant cytotoxic activity against A549/CDDP cells than the parental cell line A549 cells.2. Our study showed that M11 have exerts strong growth-inhibitory and pro-apoptotic activity specifically against cisplatin-induced resistant non-small cell lung cancer(NSCLC) cells(A549/CDDP). Moreover, there is no significant cytotoxicity of M11 on several normal cells in vitro, which including NIH3T3、H9C2 and RAW246.7 cells.3. M11 induces cell autophagy in A549/CDDP cells which play a important role in protection mechanism of cell survival.4. M11 induces cell cycle arrest by regulation of cyclins、CDKs、CDKIs. Moreover, M11 mediates apoptosis by the activation of JNK in A549/CDDP cells. No significant changes of cell cycle and apoptosis had been found in A549 cells indicated that M11 can not lead to apoptosis in experiment conditions.