Purpose Bladder cancer is a “Warburg-like” tumor characterized by a reliance on aerobic glycolysis and expression of pyruvate kinase M2 (PKM2). PKM2 oscillates between an active tetramer and an inactive dimer. We aim to further characterize PKM2, in particular PKM2 dimer, as a urinary biomarker of bladder cancer and a potential target for treatment. Methods HTB-9, HTB-5, and UM-UC3 bladder cancer cells were assessed for proliferation under differential glucose levels using the hexosaminidase assay. Western blot and Blue-native analysis was performed for protein expression of PKM2. Shikonin, an herb that is known to bind and inhibit PKM2, was utilized to determine if PKM2 has a role in glucose usage and cellular proliferation in bladder cancer cells by caspase activity assay. Institutional review board approval was obtained to collect healthy control and bladder cancer patient urine samples. The ScheBo M2-PK EDTA Plasma Test was performed on urine samples to assess urine Tumor M2-PK values. Results The three bladder cancer cell lines tested all demonstrate statistically significant increases in proliferation when exposed to higher level of glucose (200mg/dL). Similarly, low doses of glucose (25mg/dL) result in reduced proliferation. Increased cell growth in higher glucose concentration correlated with up-regulation of PKM2 protein expression. Shikonin, a PKM2 inhibitor, reduced cell proliferation and switched PKM2 isoforms from the dimer to tetramer. Lastly, dimer PKM2 (Tumor-M2PK) levels were assessed in the urine samples from bladder cancer (Bca) patients and healthy controls. Tumor M2-PK significantly correlated with the presence of BCa in our subjects. Conclusions Our studies demonstrate the potential of PKM2, specifically the dimer (Tumor-M2PK) as a target of drug therapy and as a urinary marker for bladder cancer.
410 Background: Bladder cancer remains the fourth most common cancer in American males with a higher risk of recurrence and progression for patients with diabetes mellitus. Urothelial bladder cancer is characterized by aerobic glycolysis with upregulation of glycolytic enzymes (known as the Warburg effect) such as phosphoglycerate mutase. Phosphoglycerate mutase 2 (PGAM2), a reversible glycolytic enzyme expressed highly in muscle, represents a target for modulation because of its differential expression from another isoform, phosphoglycerate mutase 1. PGAM2 knockdown may impact bladder cancer growth significantly via its effect on glucose metabolism at different glucose concentrations seen in patients with diabetes mellitus. Methods: UM-UC3 bladder cancer cells were assessed for PGAM2 expression at different glucose concentrations via Western blot and quantitative PCR. One native UM-UC3 line, three PGAM2 knockdown lines, and one vector control cell line were included in the western blot study. Cellular proliferation was analyzed using an enzyme based hexoseaminidase assay and was further supported with an automated cell counter. The effects of cisplatin were also investigated. Results: Increased PGAM2 expression at increased glucose concentrations in UM-UC3 was confirmed by Western blot and quantitative PCR. PGAM2 knockdown cells responded differently to changes in glucose concentration compared to the control cell lines, with a large increase in growth at a low glucose level of 25mg/dL after day 4. Cell proliferation demonstrated similar growth between the knockdown and controls at higher glucose concentrations of 100 and 200mg/dL. Proliferation data using automated cell counter demonstrate the same growth trend. Conclusions: Increased cell growth of PGAM2 knockdowns, most notably at 25mg/dL, suggests that PGAM2 may play a different role in glycolysis than expected, possibly serving as a modulator in cell growth instead of a simple reversible enzyme. We are currently investigating its differential expression compared to PGAM1, an enzyme recently characterized to have an opposite effect to PGAM2.
416 Background: Hsp90 represents one of the most promising biological targets for the treatment of cancer, including bladder cancer. A number of Hsp90 inhibitors that target the N-terminal ATP-binding pocket have demonstrated potent antiproliferative effects. However, a major drawback is that they induce a prosurvival heat shock response (HSR). We demonstrate the effects of a novel Hsp90 beta selective inhibitor on bladder cancer cells, which shows potent antiproliferative effects without inducing HSR. Methods: Cell Titer-Glo luminescent anti-proliferative assay was used to determine the IC50 numbers in UC3 cells. Trypan Blue Cytotoxicity assay was performed for 24h treatment with increasing concentrations of the inhibitor. Effects of the cmpound on Hsp90’s client protein degradation were investigated by Western Blot. Results: This new compound exhibits potent anti-proliferative in bladder cancer cells. IC50 number is determined as 0.30 µM for UC3 cancer cells. The toxicity assay was also performed over UC3 cells at 24h.1uM KU new compound has the similar effects on UC3 cells as 10 uM 17AAG: inhibit the cancer cells growth to half, but maintain over 60% viability of the cells. The western blot were also performed over UC3 cells, and some new target proteins such as FGFR3 and PKM2 were investigated. The data showed that, this new compound would not induce the heat shock response like 17AAG (Hsp27), and did cause some Hsp90β related protein degradation (CXCR4). FGFR3, PKM2, Her2, Hsf-1and B-raf all show degradation to different extent. Conclusions: A novel Hsp90 inhibitor, exhibits potent anti-proliferative and cytotoxic activity along with client protein degradation, without induction of HSR in bladder cancer cell lines. The reduction of Hsp90 beta related client protein caused by this compound suggests the potential to develop isoform specific inhibitors of Hsp90 for better antitumor therapies.
359 Background: To understand and evaluate the role of glucose metabolism in bladder cancer growth, in the identification of disease, and development of potential treatment strategies. Methods: UMUC3, T24 and 253JBV cells were grown in varying glucose concentrations (25, 100 and 200mg/dl) and cell proliferation assay with Vi-Cell was performed. Next, we used Qiagen PCR array of glucose metabolic pathway of the UMUC3 cell line under different glucose concentrations. PKM2 is a driver of glycolysis and exists in an inactive dimer or active tetramer. Dimer PKM2 also known as Tumor M2-PK was measured in urine samples of bladder cancer patients using a commercially available ELISA kit (ScheBo Biotech AG). Lastly, Shikonin, a PKM2 inhibitor was evaluated as an inhibitor of bladder cancer cell proliferation using Vi-Cell. Results: Increased glucose concentration 200mg/dl leads to increased proliferation in bladder cancer cells while decreased concentration of glucose; 25mg/dl reduces proliferation compared to control (100). PCR array demonstrates genes in the glycolytic pathway genes are upregulated in cells that are grown in 200mg/dl glucose media and the TCA cycle genes are upregulated in cells that are subjected to the 25mg/dl glucose media when compared to control (100mg/dl). The enzyme pyruvate kinase M2 (PKM2) controls the transition from the glycolytic pathway to TCA cycle. We have found that 9/10 (90%) of bladder cancer urine samples show elevated levels of tumor M2-PK (>104) compared to urine from two normal subjects (~30 units ) using a commercially available ELISA kit. Conclusions: Increased glucose concentration 200mg/dl leads to increased proliferation in bladder cancer cells while decreased concentration of glucose; 25mg/dl reduces proliferation compared to control (100).
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