1,2,3-Triazole tethered 1,2,4­trioxane trimer induces apoptosis in metastatic cancer cells and inhibits their proliferation, migration and invasion.

Artemisinin (ART) has been in use against different cancer cells and its derivatives and conjugates are more cytotoxic to iron-rich cancer cells. It is desirable to develop easily achievable synthetic 1,2,4-trioxanes having the same pharmacophore as that of ART. To explore more efficient compounds, a 1,2,3-triazole tethered 1,2,4­trioxane trimer (4T) was synthesized and the anti-cancer effects of ART and 4T on MDA-MB-435 and MDA-MB-231 cells were investigated concerning regulation of osteopontin (OPN) expression, which is associated with cancer progression and malignancy. 1H NMR and 13C NMR, oxidative stress analysis, flow cytometry, western blot, Real-Time PCR, transfections, luciferase assay, cell viability, proliferation, migration and chemotactic invasion assays were used in this study. It was observed that the 4T induced apoptosis by inhibiting Bcl-2 (~0.6-fold) and cleavage of caspase-3 (intrinsic pathway) in these metastatic cancer cells, and also reduced colony formation, migration and invasion of these cancer cells. The treatment of 4T decreased the reduced glutathione level and increased the activities of glucose-6-phosphate dehydrogenase and glutathione reductase in the 4T treated cancer cells as compared to their respective controls. Further, the expression of OPN was diminished (~0.5-fold) by the 4T in these cell lines. It was also observed that the key mitogen-activated protein kinase pathway proteins, mitogen-activated protein kinase kinase1/2 (~1.8-fold) and extracellular signal-regulated kinase1/2 (~16-fold), were also activated following the treatment of the 4T. However, the phosphorylated c-Jun level, a component of activator protein-1, was significantly reduced in these cancer cells upon 4T treatment. Taken together, we hypothesize that 4T may be useful for controlling cancer progression and malignancy.

MAPK pathway and SIRT1 are involved in the down-regulation of secreted osteopontin expression by genistein in metastatic cancer cells.

AIM: The regulation of secreted osteopontin (OPN) expression by genistein and its functional sequel in the metastatic cancer cells (MDA-MB-435 and MDA-MB-231) was ascertained. MAIN METHODS: Western blot and Real-Time PCR were used to analyse the proteins and mRNA transcripts, respectively. Possible transcriptional regulation of secreted OPN was analyzed by chromatin immunoprecipitation assay, bioinformatics analysis, transfection and luciferase reporter assay. The specific siRNAs and constitutive p-ERKs were used to evaluate the role of the MAPK pathway. The functional sequel of genistein in these cells was analyzed by colony formation-, migration- and invasion- assay. KEY FINDINGS: Secreted OPN expression was inhibited (up to ~0.7-fold) by genistein in these cells. Genistein (50 µM) displayed a reduction in the aggressiveness of these cells concerning colony formation rate, migration, and invasion. The p-ERK½ was increased by ~2.5-fold and ~1.5-fold upon 50 µM genistein and 15 µM resveratrol treatments at 24 h, respectively. Knockdown of ERK½ and PD98059, the inhibitor of MEK, promoted secreted OPN expression in vitro in these cells; while, the transfection of the constitutive active ERK2 (L73P and S151D) decreased the secreted OPN expression. Further, silent mating type information regulation 2 homolog 1 (SIRT1) expression in the cells was increased (~1.6-fold) upon genistein treatment (50 µM) likewise with resveratrol (~1.5-fold), an activator for SIRT1. Knockdown of SIRT1 increased OPN mRNA transcripts expression level and secreted OPN protein level in these cells. SIGNIFICANCE: MAPK pathway and SIRT1 activation are involved in the regulation of secreted OPN by genistein in these cells.

1,2,3-Triazole tethered 1,2,4-trioxanes: Studies on their synthesis and effect on osteopontin expression in MDA-MB-435 breast cancer cells.

Artemisinin and its analogs have shown potent anticancer activity in primary cancer cultures and cell lines by inhibiting cancer proliferation, metastasis, and angiogenesis. Despite its apparent compatibility to normal cells and low IC50 values in comparison to the commonly used anticancer drugs, the underlying mechanisms behind their cytotoxic effects are not yet fully understood. Surprisingly, the efficacy of synthetic 1,2,4-trioxanes against cancer has not been explored yet. Given the high antitumor activity of artemisinin dimers in comparison to their monomers, we report here the synthesis of simple 1,2,3-triazole conjugated 1,2,4-trioxanes and their potential antitumor activity by studying their inhibitory effect on osteopontin (OPN) expression in MDA-MB-435 breast cancer cells. It may be noted that despite being a strong marker to identify human tumor metastasis, no study on effect of artemisinin and its synthetic and semisynthetic derivatives on OPN expression has ever been studied. Although our initial studies did not notice any straight-line relationship between the number of trioxane units in a molecule to the extent of inhibition of OPN protein expression, we could observe better results in some cases in comparison to artemisinin. We have observed that artemisinin did not show appreciable OPN downregulation in MDA-MB-435 cancer cells, but dihydroartemisinin (DHA) and some synthetic 1,2,4-trioxane monomers and dimers showed downregulation of OPN expression. Therefore, these compounds may act as an anti-metastatic agent in controlling breast cancer cells metastasis.

Genistein represses PEPCK-C expression in an insulin-independent manner in HepG2 cells and in alloxan-induced diabetic mice.

Genistein has been reported to exert beneficial effects on type 2 diabetes mellitus (T2DM); however, the underlying molecular mechanisms involved therein have not been clearly elucidated. To address this question, the effect of genistein on the expression of phosphoenolpyruvate carboxykinase (PEPCK), and glucose production in HepG2 cells and in alloxan-induced diabetic mice was investigated. HepG2 cells were exposed to different concentration of genistein in presence or absence of modulators, and the expression of cytosolic PEPCK (PEPCK-C) and the signaling pathways was studied. Further, the biological relevance of the in vitro study was tested in alloxan-induced diabetic mice. Genistein lowered PEPCK-C expression and glucose production in HepG2 cells accompanied with increased in phosphorylation states of AMPK, MEK½, ERK½, and CRTC2. Treatment with the AMPK inhibitor (compound C) enhanced genistein-induced MEK½ and ERK½ activity indicating a potential cross-talk between the two signaling pathways. In vivo, genistein also reduced fasting glucose levels accompanied with reduced PEPCK-C expression and increased in AMPK and ERK½ phosphorylation states in the liver of genistein-treated alloxan-induced diabetic mice. Genistein fulfills the criteria of a suitable anti-diabetic agent by reducing glucose production and inhibiting PEPCK-C expression in HepG2 cells and also in alloxan-induced diabetic mice. These results indicate that genistein is an effective candidate for preventing T2DM through the modulation of AMPK-CRTC2 and MEK/ERK signaling pathways, which may allow a novel approach to modulate dysfunction in hepatic gluconeogenesis in T2DM.