ABSTRACT
Calotropin (M11), an active compound isolated from Asclepias curasavica L., was found to exert strong inhibitory and pro-apoptotic activity specifically against cisplatin-induced resistant non-small cell lung cancer (NSCLC) cells (A549/CDDP). Molecular mechanism study revealed that M11 induced cell cycle arrest at the G2/M phase through down-regulating cyclins, CDK1, CDK2 and up-regulating p53 and p21. Furthermore, M11 accelerated apoptosis through the mitochondrial apoptotic pathway which was accompanied by increase Bax/Bcl-2 ratio, decrease in mitochondrial membrane potential, increase in reactive oxygen species production, activations of caspases 3 and 9 as well as cleavage of poly ADP-ribose polymerase (PARP). The activation and phosphorylation of JNK was also found to be involved in M11-induced apoptosis, and SP610025 (specific JNK inhibitor) partially prevented apoptosis induced by M11. In contrast, all of the effects that M11 induce cell cycle arrest and apoptosis in A549/CDDP cells were not significant in A549 cells. Drugs with higher sensitivity against resistant tumor cells than the parent cells are rather rare. Results of this study supported the potential application of M11 on the non-small lung cancer (NSCLC) with cisplatin resistance.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Apoptosis/drug effects , Asclepias/chemistry , Cardenolides/pharmacology , Drug Resistance, Neoplasm/drug effects , G2 Phase Cell Cycle Checkpoints/drug effects , Gene Expression Regulation, Neoplastic/drug effects , A549 Cells , Antineoplastic Agents, Phytogenic/isolation & purification , Apoptosis/genetics , CDC2 Protein Kinase , Cardenolides/isolation & purification , Caspase 3/genetics , Caspase 3/metabolism , Caspase 9/genetics , Caspase 9/metabolism , Cisplatin/pharmacology , Cyclin-Dependent Kinase 2/antagonists & inhibitors , Cyclin-Dependent Kinase 2/genetics , Cyclin-Dependent Kinase 2/metabolism , Cyclin-Dependent Kinase Inhibitor p21/agonists , Cyclin-Dependent Kinase Inhibitor p21/genetics , Cyclin-Dependent Kinase Inhibitor p21/metabolism , Cyclin-Dependent Kinases/antagonists & inhibitors , Cyclin-Dependent Kinases/genetics , Cyclin-Dependent Kinases/metabolism , Drug Resistance, Neoplasm/genetics , Humans , MAP Kinase Kinase 4/genetics , MAP Kinase Kinase 4/metabolism , Membrane Potential, Mitochondrial/drug effects , Mitochondria/drug effects , Mitochondria/metabolism , Plant Extracts/chemistry , Poly(ADP-ribose) Polymerases/genetics , Poly(ADP-ribose) Polymerases/metabolism , Proteolysis , Proto-Oncogene Proteins c-bcl-2/agonists , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Reactive Oxygen Species/metabolism , Signal Transduction , Tumor Suppressor Protein p53/agonists , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , bcl-2-Associated X Protein/agonists , bcl-2-Associated X Protein/genetics , bcl-2-Associated X Protein/metabolismABSTRACT
Transforming growth factor-ß1 (TGF-ß1) present in tumor microenvironment acts in a coordinated fashion to either suppress or promote tumor development. However, the molecular mechanisms underlying the effects of TGF-ß1 on tumor microenvironment are not well understood. Our clinical data showed a positive association between TGF-ß1 expression and cancer-associated fibroblasts (CAFs) in tumor microenvironment of breast cancer patients. Thus we employed starved NIH3T3 fibroblasts in vitro and 4T1 cells mixed with NIH3T3 fibroblasts xenograft model in vivo to simulate nutritional deprivation of tumor microenvironment to explore the effects of TGF-ß1. We demonstrated that TGF-ß1 protected NIH3T3 fibroblasts from Star-induced growth inhibition, mitochondrial damage and cell apoptosis. Interestingly, TGF-ß1 induced the formation of CAFs phenotype in starvation (Star)-treated NIH3T3 fibroblasts and xenografted Balb/c mice, which promoted breast cancer tumor growth. In both models, autophagy agonist rapamycin increased TGF-ß1-induced protective effects and formation of CAFs phenotypes, while autophagy inhibitor 3-methyladenine, Atg5 knockdown or TGF-ß type I receptor kinase inhibitor LY-2157299 blocked TGF-ß1 induced these effects. Taken together, our results indicated that TGF-ß/Smad autophagy was involved in TGF-ß1-induced protective effects and formation of CAFs phenotype in tumor microenvironment, which may be used as therapy targets in breast cancer.
