Pyruvate carboxylase (PC) is an anaplerotic enzyme that catalyzes the carboxylation of pyruvate to oxaloacetate, which is crucial for replenishing tricarboxylic acid cycle intermediates when they are used for biosynthetic purposes. We examined the expression of PC by immunohistochemistry of paraffin-embedded breast tissue sections of 57 breast cancer patients with different stages of cancer progression. PC was expressed in the cancerous areas of breast tissue at higher levels than in the non-cancerous areas. We also found statistical association between the levels of PC expression and tumor size and tumor stage (P < 0.05). The involvement of PC with these two parameters was further studied in four breast cancer cell lines with different metastatic potentials; i.e., MCF-7, SKBR3 (low metastasis), MDA-MB-435 (moderate metastasis) and MDA-MB-231 (high metastasis). The abundance of both PC mRNA and protein in MDA-MB-231 and MDA-MB-435 cells was 2-3-fold higher than that in MCF-7 and SKBR3 cells. siRNA-mediated knockdown of PC expression in MDA-MB-231 and MDA-MB-435 cells resulted in a 50% reduction of cell proliferation, migration and in vitro invasion ability, under both glutamine-dependent and glutamine-depleted conditions. Overexpression of PC in MCF-7 cells resulted in a 2-fold increase in their proliferation rate, migration and invasion abilities. Taken together the above results suggest that anaplerosis via PC is important for breast cancer cells to support their growth and motility.

We recently showed that the anaplerotic enzyme pyruvate carboxylase (PC) is up-regulated in human breast cancer tissue and its expression is correlated with the late stages of breast cancer and tumor size [Phannasil et al., PloS One 10, e0129848, 2015]. In the current study we showed that PC enzyme activity is much higher in the highly invasive breast cancer cell line MDA-MB-231 than in less invasive breast cancer cell lines. We generated multiple stable PC knockdown cell lines from the MDA-MB-231 cell line and used mass spectrometry with 13C6-glucose and 13C5-glutamine to discern the pathways that use PC in support of cell growth. Cells with severe PC knockdown showed a marked reduction in viability and proliferation rates suggesting the perturbation of pathways that are involved in cancer invasiveness. Strong PC suppression lowered glucose incorporation into downstream metabolites of oxaloacetate, the product of the PC reaction, including malate, citrate and aspartate. Levels of pyruvate, lactate, the redox partner of pyruvate, and acetyl-CoA were also lower suggesting the impairment of mitochondrial pyruvate cycles. Serine, glycine and 5-carbon sugar levels and flux of glucose into fatty acids were decreased. ATP, ADP and NAD(H) levels were unchanged indicating that PC suppression did not significantly affect mitochondrial energy production. The data indicate that the major metabolic roles of PC in invasive breast cancer are primarily anaplerosis, pyruvate cycling and mitochondrial biosynthesis of precursors of cellular components required for breast cancer cell growth and replication.

No abstract

No abstract

No abstract

The reactivation of γ-globin chain synthesis to combine with excess free α-globin chains and form fetal hemoglobin (HbF) is an important alternative treatment for β-thalassemia. We had reported HbF induction property of natural curcuminoids, curcumin (Cur), demethoxycurcumin (DMC) and bis-demethoxycurcumin (BDMC), in erythroid progenitors. Herein, the HbF induction property of trienone analogs of the three curcuminoids in erythroleukemic K562 cell lines and primary human erythroid progenitor cells from β-thalassemia/HbE patients was examined. All three trienone analogs could induce HbF synthesis. The most potent HbF inducer in K562 cells was trienone analog of BDMC (T-BDMC) with 2.4 ± 0.2 fold increase. In addition, DNA methylation at CpG − 53, − 50 and + 6 of Gγ-globin gene promoter in K562 cells treated with the compounds including T-BDMC (9.3 ± 1.7%, 7.3 ± 1.7% and 5.3 ± 0.5%, respectively) was significantly lower than those obtained from the control cells (30.7 ± 3.8%, 25.0 ± 2.9% and 7.7 ± 0.9%, respectively P < 0.05). The trienone compounds also significantly induced HbF synthesis in β-thalassemia/HbE erythroid progenitor cells with significantly reduction in DNA methylation at CpG + 6 of Gγ-globin gene promoter. These results suggested that the curcuminoids and their three trienone analogs induced HbF synthesis by decreased DNA methylation at Gγ-globin promoter region, without effect on Aγ-globin promoter region.

Previous studies in a mouse model have indicated the anticancer potential of boiled Moringa oleifera pod (bMO)-supplemented diets; however, its molecular mechanisms are still unclear. Therefore, the present study aimed to explore the protein expression profiles responsible for the suppressive effect of bMO supplementation on azoxymethane (AOM)/dextran sodium sulfate (DSS)-induced mouse colon carcinogenesis. Analysis by gel electrophoresis and liquid chromatography-tandem mass spectrophotometry demonstrated that there were 125 proteins that were differentially expressed in mouse colon tissues between 14 experimental groups of mice. The differentially expressed proteins are involved in various biological processes, such as signal transduction, metabolism, transcription and translation. Venn diagram analysis of the differentially expressed proteins was performed in six selected mouse groups, including negative control, positive control mice induced by AOM/DSS, the AOM/DSS groups receiving preventive or therapeutic bMO diets and their bMO-supplemented control groups. This analysis identified 7 proteins; 60S acidic ribosomal protein P1 (Rplp1), fragile X mental retardation, cystatin 9, round spermatids protein, zinc finger protein 638, protein phosphatase 2C (Ppm1g) and unnamed protein product as being potentially associated with the preventive and therapeutic effects of bMO in AOM/DSS-induced mouse colon cancer. Analysis based on the search tool for interactions of chemicals (STITCH) database predicted that Rplp1 interacted with the apoptotic and inflammatory pathways, whereas Ppm1g was associated only with inflammatory networks. This proteomic analysis revealed candidate proteins that are responsible for the effects of bMO supplementation, potentially by regulating apoptotic and inflammatory signaling networks in colorectal cancer prevention and therapy.

No abstract