melphalan and 3-methyladenine

Matrine has potent antitumor activity against a broad variety of cancer cells and our previous study showed that both autophagy and apoptosis were activated during matrine-induced gastric cancer cell death. The aim of the present study was to determine the significance of autophagy in antineoplastic effects of matrine and the molecular mechanism by which matrine induces autophagy in gastric cancer cells. Western blot analysis showed that exposure of gastric cancer cells to matrine resulted in the extent of autophagy increasing in a dose- and time-dependent manner by detecting micro-tubule-associated protein 1 light chain 3 (LC3). This induction was due to activation of autophagic flux, as supported using the lysosome inhibitor, bafilomycin A1, which produced an accumulation of LC3-II. Propidium iodide staining demonstrated that matrine induced cell death in a dose-dependent manner and the autophagy inhibitor 3-methyladenine (3-MA) or bafilomycin A1 enhanced lethality of matrine against gastric cancer cells. Moreover, after pretreatment with 3-MA, some of the gastric cancer cells treated with matrine exhibited prototypical characteristics of apoptosis by transmission electron microscopy. The ability of 3-MA to increase matrine-induced apoptosis was further confirmed by Annexin V-FITC/PI staining. Also, the combination of matrine and 3-MA was more potent than matrine alone in inhibiting the proliferation of SGC-7901 cells assessed by sulphorhodamine B assay. Furthermore, administration of the pan-caspase inhibitor zVAD-fmk or autophagy inducer rapamycin decreased the matrine-induced cell death. In addition, matrine treatment did not inhibit the phosphorylation of Akt and its downstream effectors mammalian target of rapamycin (mTOR) as well as p70 ribosomal protein S6 kinase (p70S6K), although the levels of the total Akt and mTOR were decreased. These results suggest that autophagy was activated as a protective mechanism against matrine-induced apoptosis and inhibition of autophagy may be an attractive strategy for enhancing the antitumor potential of matrine in gastric cancer.

Topics: Adenine; Alkaloids; Apoptosis; Autophagy; Cell Line, Tumor; Cell Nucleus; Cell Shape; Cell Survival; Drug Synergism; Humans; Immunoglobulin G; Macrolides; Matrines; Melphalan; Microtubule-Associated Proteins; Phosphorylation; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; Quinolizines; Stomach Neoplasms

Although autophagy occurs in most tumor cells following DNA damage, it is still a mystery how this DNA-damaging event turns on the autophagy machinery in multiple myeloma (MM) and how the functional status of autophagy impacts on its susceptibility to death in response to DNA-damaging chemotherapy.. We investigate the effects of DNA damage on autophagy in MM cells and elucidate its underlying molecular mechanism. Then, we examined the impacts of pharmacologic or genetic inhibition of autophagy on DNA damage-induced apoptosis. Furthermore, the antimyeloma activity of autophagy inhibitor in combination with DNA-damaging agents was evaluated in MM xenograft models.. We showed that DNA-damaging drugs, doxorubicin and melphalan, induce caspase-dependent apoptosis and concurrently trigger Beclin 1-regulated autophagy in human MM cell lines H929 and RPMI 8226. Mechanistically, association of autophagy execution proteins Beclin 1 with class III phosphoinositide 3-kinase, which is inhibited by Bcl-2 recruitment, contributes directly to the autophagic process. Importantly, targeting suppression of autophagy by minimally toxic concentrations of pharmacologic inhibitors (hydroxychloroquine and 3-methyladenine) or short hairpin RNAs against autophagy genes, Beclin 1 and Atg5, dramatically augments proapoptotic activity of DNA-damaging chemotherapy both in vitro using MM cell lines or purified patient MM cells and in vivo in a human plasmacytoma xenograft mouse model.. These data can help unravel the underlying molecular mechanism of autophagy in DNA-damaged MM cells and also provide a rationale for clinical evaluation of autophagy inhibitors in combination with DNA-damaging chemotherapy in MM.

Topics: Adenine; Animals; Antineoplastic Combined Chemotherapy Protocols; Autophagy; Cell Line, Tumor; DNA Damage; Doxorubicin; Drug Synergism; Humans; Melphalan; Mice; Mice, Inbred C57BL; Mice, SCID; Molecular Targeted Therapy; Multiple Myeloma; Xenograft Model Antitumor Assays