As expected, all these effects were reversed by LDHA overexpression in the PCK1-overexpressing cells (Supplementary Physique 6HCK). 18F-FDG accumulation in primary ccRCC tissue. We also exhibited that PCK1 reduces the stability of LDHA through posttranslational regulation. Finally, we showed that the effects of PCK1 on glucose metabolism, cell proliferation and metastasis are mediated via the inhibition of LDHA. Conclusion Our study identified a novel molecular mechanism underlying the Warburg effect. PCK1 may serve as a candidate prognostic biomarker, and targeting the PCK1/LDHA pathway might be a new strategy to selectively inhibit tumor metabolism in human ccRCC. with or without PCK1 re-expression were subcutaneously inoculated into 4-week-old male BALB/c severe combined immunodeficiency mice (Shanghai Laboratory Animal Center, China). Mice were subjected to 18F-FDG PET scans and sacrificed 28 days after injection. All the subcutaneous tumors were removed and weighed. The tumor tissues were collected for weight measurements and IHC analysis. 18F-FDG microPET Imaging Mice were fasted for 6 h and then administered 18F-FDG (250 Ci for each mouse) by tail vein injection. Forty-five minutes later, the animals were placed on a warm pad, and a 10-min emission scan was acquired using a MicroPET (Inveon, Siemens) with 2.5% isoflurane anesthesia. The regions of interest were drawn around the tumors on scan slices, and the maximal standard uptake values (SUVmax) were calculated to assess the 18F-FDG ability of tumors. Statistical Analysis All data were statistically analyzed using GraphPad Prism 5 (GraphPad Software, San Diego, CA, USA) or SPSS 18.0 software (SPSS, Chicago, Ill, USA). Statistical significance for the association between the binary representation of PCK1 or LDHA expression with the paired tumor versus peritumor specimens, age, sex, tumor size, histological grade, status of lymph node metastasis and tumor stage was assessed using the in control cells (Physique 2A), suggesting that aerobic glycolysis might be involved in the regulation of cell proliferation by PCK1. To further explore the effects of PCK1 around the glycolytic phenotype in ccRCC, we Homocarbonyltopsentin performed 18F-FDG uptake assays in vitro. We observed that glucose uptake was apparently upregulated in the absence of PCK1 but Rabbit Polyclonal to ELL significantly downregulated when PCK1 was overexpressed in 769-P and Caki-1 cells (Physique 2B). Lactate, a dead-end product in cellular glycolytic metabolism, is usually of great physiologic significance in tumor cells because it can drive cell proliferation and migration and might even function as a potential nutrient for tumors. We found that lactate production was increased by the stable knockdown of Homocarbonyltopsentin PCK1 in both ccRCC cell lines. In addition, these findings were further supported by the results of the overexpression experiments in cancer cells (Physique 2C). To further demonstrate the significance of PCK1 in glucose metabolism, extracellular flux analysis was performed. Compared with the control ccRCC cells, the shcells exhibited an increased ECAR and decreased OCR, and these findings were supported by the results of overexpressing PCK1 (Supplementary Physique 3 and Physique 2DCF). In addition, activation of oxidative phosphorylation by PCK1 was also exhibited by analyzing the flux of [6-14C] glucose in cancer cells. 14CO2 from 6-14C-glucose can be released only by the TCA cycle, which indicates the flux of glucose metabolism in the mitochondrial pathway. The proportion of 6-14CO2 in the total CO2 released was reduced in 769-P and Caki-1 cells with stably silenced PCK1 but increased in the ccRCC cells overexpressing PCK1 (Physique 2G). Altogether, these results provide evidence that PCK1 shunts glucose from glycolysis to oxidative phosphorylation metabolism, which is the opposite of what occurs in the Warburg effect. Open in a separate window Physique 2 PCK1 regulates glycolysis in cultured ccRCC cells. Notes: (A) 769-P and Caki-1 cells with stably knocked down PCK1 or the unfavorable control were or were not treated with 2-DG (2.5 mM). The numbers of cells were counted every 24 h. The bars represent the meansSEM (n=3). (BCG) PCK1 regulates glucose Homocarbonyltopsentin uptake (B), lactate production.