Objective: In this study, we aimed to examine the effect of MAN on human lung cancer and reveal the underlying molecular mechanism.
Methods: MTT assay was conducted to measure cell viability. Annexin V-FITC/PI staining was used to detect cell apoptosis. Confocal microscope was performed to determine the formation of autophagosomes and autolysosomes. Flow cytometry was performed to quantify cell death. Western blotting was used to determine the related-signaling pathway.
Results: In the present study, we demonstrated for the first time that MAN inhibitd cell proliferation and induced cell apoptosis in human non-small-cell lung carcinoma (NSCLC) cells. We found that MAN treatment dysregulated mitochondrial function and led to mitochondrial apoptosis in A549 and PC9 cells. Meanwhile, MAN enhanced autophagy flux by the increase of autophagosome formation, the fusion of autophagsomes and lysosomes and lysosomal function. Moreover, mTOR signaling pathway, a classical pathway regualting autophagy, was inhibited by MAN in a time- and dose-dependent mannner, resulting in autophagy induction. Interestingly, autophagy inhibition by CQ or Atg5 knockdown attenuated cell apoptosis by MAN, indicating that autophagy serves as cell death. Furthermore, autophagy-mediated cell death by MAN can be blocked by reactive oxygen species (ROS) scavenger NAC, indicating that ROS accumulation is the inducing factor of apoptosis and autophagy. In summary, we revealed the molecular mechanism of MAN against lung cancer through apoptosis and autophagy, suggesting that MAN might be a novel therapeutic agent for NSCLC treatment.
Methods: An in vitro cell model for tamoxifen-resistant HER2 overexpressed UACC732 cells was created using the pulse method. Cells were exposed to oxidized LDL (oxLDL) and very low density lipoprotein (VLDL) separately. Effects on cell morphology was studied using phase contrast microscopic changes. Percentage of cell viability was measured using proliferation assay kit. Development of tamoxifen resistance was determined based on P-gp expression with flow cytometry. Further analysis includedcell death measurement with flow cytometry method.
Results: UACC732 cells exposed to VLDL exhibited fibroblast-like morphology. This was further supported by proliferation assay, where the percentage of cell viability achieved more than 100% with 100 μg/ml of VLDL exposure, indicating cell proliferation. Findings also showed that VLDL caused reduction in expression of Pgp in resistant cells compared to resistant cells alone (p = 0.02).
Conclusion: Results of this study suggest that VLDL may play a role in growth of drug-resistant HER2-overexpressing cells. Lower expression of P-gp in presence of VLDL need to be investigated further.