Genistein and metformin regulate glycerol kinase and the enzymes of glycerol 3-phosphate shuttle in a differential manner in myocytes, hepatocytes and adipocytes.

Glycerol kinase (GK) and glycerol 3-phosphate dehydrogenase (GPDH) are critical in glucose homeostasis. The role of genistein and metformin on these enzymes and glucose production was investigated in C2C12, HepG2, and 3T3-L1 cells. Enzyme kinetics, Real-Time PCR and western blots were performed to determine enzyme activities and expressions of mRNAs and proteins. Glucose production and uptake were also measured in these cells. siRNAs were used to assess their impact on the enzymes and glucose production. Ki values for the compounds were determined using purified GK and GPDH. Genistein decreased GK activity by ∼45 %, while metformin reduced cGPDH and mGPDH activities by ∼32 % and âˆ¼43 %, respectively. Insignificant changes in expressions (mRNAs and proteins) of the enzymes were observed. The compounds showed dose-dependent alterations in glucose production and uptake in these cells. Genistein non-competitively inhibited His-GK activity (Ki 19.12 µM), while metformin non-competitively inhibited His-cGPDH (Ki 75.52 µM) and mGPDH (Ki 54.70 µM) activities. siRNAs transfection showed ∼50 % and âˆ¼35 % decrease in activities of GK and mGPDH and a decrease in glucose production (0.38-fold and 0.42-fold) in 3T3-L1 cells. Considering the differential effects of the compounds, this study may provide insights into the potential therapeutic strategies for type II diabetes mellitus.

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.

Context Dependent Regulation of Human Phosphoenolpyruvate Carboxykinase Isoforms by DNA Promoter Methylation and RNA Stability.

Cytoplasmic and mitochondrial isoforms of phosphoenolpyruvate carboxykinase (PEPCK-C and PEPCK-M) regulate hepatic gluconeogenesis to control systemic glucose homeostasis. Transcriptional and post-transcriptional mechanisms may govern synthesis, maintenance and cooperative function of compartmentalized PEPCK enzymes. In a comparative analysis, we show that tumor cells consistently transcribe and translate higher levels of enzymatically active PEPCK-C than PEPCK-M and both the isoforms were present at lower levels in normal fibroblasts. Unlike in PEPCK-M, absence of glucose reduced the PEPCK-C mRNA and protein levels only in HepG2 cells. Interestingly, isoflavone genistein significantly increased PEPCK-C mRNA and protein levels in normal fibroblasts indicating cell type specific control mechanisms. Genistein also significantly affected RNA stability of PEPCK-C but not PEPCK-M in HepG2 cells. This was due to the conserved and functional mRNA destabilizing AU rich sequences at the 3'-UTR region of PEPCK-C gene and was confirmed by luciferase reporter assays suggesting that glucose deprivation and genistein targets these sequences for mRNA degradation in HepG2 cells but not in fibroblasts. Analysis of promoter methylation by luciferase reporter assays and bisulfite DNA sequencing suggested that PEPCK-C but not PEPCK-M promoter was activated by 5-aza-2-deoxycytidine by inducing cytosine demethylation at the specific CpG dinucleotides of 5'-UTR region. Taken together, our data suggests stable PEPCK-M activity and identifies intricate relationship between (1) mRNA stability and (2) promoter DNA methylation as two mechanisms of gene expression that distinguishes PEPCK-C and PEPCK-M enzyme activities in a context and cell type dependent manner during gluconeogenesis. J. Cell. Biochem. 117: 2506-2520, 2016. © 2016 Wiley Periodicals, Inc.

Phytochemicals from Flemingia vestita (Fabaceae) and Stephania glabra (Menispermeaceae) alter cGMP concentration in the cestode Raillietina echinobothrida.

Cyclic GMP (cGMP) mediates various physiological functions of nitric oxide (NO) synthesized by nitric oxide synthase (NOS). A crude peel extract and purified fraction of Flemingia vestita, as well as a crude rhizome extract of Stephania glabra and fractions were tested with respect to the activity of NOS, NO efflux and cGMP concentration in the cestode Raillietina echinobothrida in order to find out the possible mode of anthelmintic action of these plant-derived components. For comparison purposes, the parasites were also treated with pure genistein, sodium nitroprusside (SNP-a known NO donor), and the reference drug, praziquantel (PZQ). At the time of onset of paralysis in the parasites, a significant increase (32%-87%) in the NOS activity and a two to three fold increase of NO efflux into the incubation medium were observed in the treated parasites in comparison to their respective controls. The cGMP concentration in the treated parasites' tissue was also increased by 44%-103%. However, in the presence of NG-nitro-L-arginine methyl ester, a potent inhibitor of NOS, there was no increase in the cGMP concentration in the parasite tissue. This study indicates that the phytochemicals, in particular genistein and tetrahydropalmatine, from F. vestita and S. glabra, respectively, disturb the downstream signalling pathway of NO, as indicated by the change in cGMP concentration in the parasite tissue.

Effect of isoflavone from Flemingia vestita (Fabaceae) on the Ca2+ homeostasis in Raillietina echinobothrida, the cestode of domestic fowl.

The alcoholic crude root-peel extract of Flemingia vestita and its major isoflavone, genistein, have been shown to have a vermifugal/vermicidal effect by causing a flaccid paralysis accompanied by alterations in the structural architecture of the tegumental interface and metabolic activity in Raillietina echinobothrida, the cestode of domestic fowl. In the present study, the crude root-peel extract and pure genistein were tested in vitro with respect to Ca2+ homeostasis and the occurrence of some metal ions was detected in the parasite. Live cestodes were incubated in pre-defined concentrations of the crude root-peel extract, genistein and praziquantel (as reference drug), till the paralysis time with simultaneous maintenance of respective controls. In the parasite tissue, a significant amount of Ca2+ (approximately 400 microg/g dry tissue wt) was found to be present besides magnesium, iron, zinc, lead and chromium, whilst manganese, cadmium and nickel were below the level of detection. The Ca2+ concentration was decreased significantly by 39%-49%, in the parasite tissue exposed to the test materials in comparison to the respective controls. There was also an increase in Ca2+ efflux by 91%-160% into the culture medium under similar treatments. The changes in Ca2+ homeostasis may be related to the rapid muscular contraction and consequent paralysis in the parasite due to the anthelmintic stress caused by the phytochemicals of F. vestita.

Effects of phytochemicals of Flemingia vestita (Fabaceae) on glucose 6-phosphate dehydrogenase and enzymes of gluconeogenesis in a cestode (Raillietina echinobothrida).

The crude root-peel extract of Flemingia vestita, containing genistein as the major isoflavone, has a vermifugal/vermicidal effect. It acts by causing flaccid paralysis accompanied by alterations in the activities of several tegumental enzymes and other metabolic activities in the fowl tapeworm, Raillietina echinobothrida. To elucidate the mode of action of the putative phytochemicals on energy metabolism, crude root-peel extract, pure genistein and praziquantel were tested on glucose 6-phosphate dehydrogenase (G6PDH) and enzymes of gluconeogenesis--pyruvate carboxylase (PC), phosphoenolpyruvate carboxykinase (PEPCK) and fructose 1,6-bisphosphatase (FBPase)--in R. echinobothrida. The activities of G6PDH, PEPCK and FBPase were largely restricted to the cytosolic fraction, while PC was confined to the mitochondrial fraction. Following treatments, the G6PDH activity was decreased by 23-31%, whereas the activities of PC and PEPCK were increased by 32-44% and 44-49%, respectively. There was no significant effect by any of the treatments on FBPase activity. We hypothesize that the phytochemicals from F. vestita, genistein in particular, influence the key enzymes of these pathways, which is perhaps a function of high energy demand of the parasite under anthelmintic stress.

Anthelmintic efficacy of Flemingia vestita (Fabaceae): alterations in glucose metabolism of the cestode, Raillietina echinobothrida.

The root-tuber peel of Flemingia vestita and its active component, genistein, were tested in respect of glucose metabolism in the cestode, Raillietina echinobothrida. Live R. echinobothrida, collected from the intestine of freshly slaughtered domestic fowl, were incubated at 39+/-1 degrees C in defined concentrations of the root-peel crude extract (5 mg/ml), genistein (0.2 mg/ml) and praziquantel (1 microg/ml) in phosphate buffered saline with 1% of dimethyl sulphoxide with simultaneous maintenance of controls. In the treated worms, there was a significant decrease in the glycogen concentration accompanied with the decrease of glucose by 14-32%, whereas the malate concentration increased by 49-134% as compared to controls. Both in controls and treated parasites, however, the pyruvate content was not measurable. While alanine and lactate contents showed a decline by 7-25% in the parasites exposed to all test materials, the lactate efflux into the incubation medium showed 37-71% increase in treatments indicating an overall increase of lactate production in comparison to controls. The results showing a decline in the glycogen and glucose contents and a significant rise in the malate content and lactate efflux under treatment conditions suggest that the energy demand in the parasites possibly got enhanced under stress, though it did not influence a switch over towards aerobic degradation of glucose in the parasites.