Rani Kunjithapatham

3-Bromopyruvate: A New Targeted Antiglycolytic Agent and a Promise for Cancer Therapy

The pyruvate analog, 3-bromopyruvate, is an alkylating agent and a potent inhibitor of glycolysis. This antiglycolytic property of 3-bromopyruvate has recently been exploited to target cancer cells, as most tumors depend on glycolysis for their energy requirements. The anticancer effect of 3-bromopyruvate is achieved by depleting intracellular energy (ATP) resulting in tumor cell death. In this review, we will discuss the principal mechanism of action and primary targets of 3-bromopyruvate, and report the impressive antitumor effects of 3-bromopyruvate in multiple animal tumor models. We describe that the primary mechanism of 3-bromopyruvate is via preferential alkylation of GAPDH and that 3-bromopyruvate mediated cell death is linked to generation of free radicals. Research in our laboratory also revealed that 3-bromopyruvate induces endoplasmic reticulum stress, inhibits global protein synthesis further contributing to cancer cell death. Therefore, these and other studies reveal the tremendous potential of 3-bromopyruvate as an anticancer agent.

Human Hepatocellular Carcinoma in a Mouse Model: Assessment of Tumor Response to Percutaneous Ablation by Using Glyceraldehyde-3-Phosphate Dehydrogenase Antagonists

PURPOSE To characterize tumor response to percutaneous injection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antagonists in a mouse model of human hepatocellular carcinoma (HCC). MATERIALS AND METHODS Animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee. Luciferase (luc) gene-expressing Hep3B tumor-bearing athymic nude mice were randomly divided into four groups of six mice each. Tumor-specific GAPDH inhibition was achieved by using percutaneous injection of GAPDH antagonists-3-bromopyruvate (3-BrPA) or GAPDH-specific short hairpin RNA (shRNA). Tumor response to treatment was assessed by using bioluminescence imaging and analysis of GAPDH function and apoptotic markers (caspase-3, caspase-9, and positive staining for terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling). HCC samples from 34 patients were obtained from the Johns Hopkins tumor bank, as approved by the Institutional Review Board, for GAPDH expression analysis. Statistical analysis was performed by using a two-sample t test or Spearman rank correlation coefficient. RESULTS In vitro, 3-BrPA affected Hep3B cell viability (half maximal inhibitory concentration = 0.15 mmol/L), and GAPDH shRNA suppressed (45.5%) colony formation. In vivo, percutaneous injection of GAPDH antagonists into luc-Hep3B tumors decreased bioluminescence imaging signal and viability (3-BrPA, P < .0001; GAPDH shRNA, P = .03). The 3-BrPA treatment primarily inhibited GAPDH activity (74.5%) compared with its expression (34.3%), whereas GAPDH shRNA inhibited both activity (60.6%) and expression (44.4%). Targeted inhibition of GAPDH by using 3-BrPA or shRNA induced apoptosis. HCC samples from patients demonstrated a strong correlation between GAPDH upregulation and the proto-oncogene c-jun expression (r = 0.543, P = .003). CONCLUSION Percutaneous injection of GAPDH antagonists induces apoptosis and blocks Hep3B tumor progression, which demonstrates the therapeutic potential of targeting GAPDH in human HCC.

The Pyruvic Acid Analog 3-Bromopyruvate Interferes With the Tetrazolium Reagent MTS in the Evaluation of Cytotoxicity

3-Bromopyruvate (3BrPA) is a pyruvate analog known for its alkylating property. Recently, several reports have documented the antiglycolytic and anticancer effects of 3BrPA and its potential for therapeutic applications. 3BrPA-mediated cytotoxicity has been evaluated in vitro by various methods including tetrazolium salt (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide)-based assays such as MTT, MTS, and so on. However, growing body of evidences has shown that tetrazolium reagent may interfere with the test compounds. In this study, we investigated whether the tetrazolium reagent interferes with the assessment of 3BrPA cytotoxicity. The results of the tetrazolium-based MTS assay were compared with 3 distinct cell viability detection methods, that is, Trypan Blue staining, ATP depletion, and Annexin V staining in 2 different cell lines, Vx-2 and HepG2. The MTS assay data showed false positive results by indicating increased cell viability at 1 mM and 2 mM 3BrPA whereas the other cell viability assays demonstrated that both Vx-2 and HepG2 cells are not viable at the same treatment conditions. In order to validate the direct interaction of 3BrPA with MTS reagent, we tested cell-free media incubated with different concentrations of 3BrPA. The results of cell-free media showed an increase in absorbance in a dose-dependent manner confirming the interaction of MTS with 3BrPA. Thus, our data clearly demonstrate that 3BrPA interferes with the accuracy of MTS-based cytotoxicity evaluation. Hence, we suggest that employing multiple methods of biochemical as well as morphological cytotoxicity assays is critical to evaluate 3BrPA-mediated cell death.

Reversal of Anchorage-Independent Multicellular Spheroid into a Monolayer Mimics a Metastatic Model

Lack of an in vitro model of metastasis has been a major impediment in understanding the molecular regulation of metastatic processes, and identification of specific therapeutic targets. We have established an in vitro model which displayed the signatures of metastatic phenotype such as migration, invasiveness, chemoresistance and expression of cancer stem-cell markers. This in vitro model was developed by the induction of reversal of multicellular spheroids that were generated by anchorage-independent growth. In vivo data further validated the metastatic phenotype of the in vitro model. Besides delineating the molecular events of metastasis, this model could also improve the screening efficiency of antimetastatic agents.

Occurrence of a Multimeric High-Molecular-Weight Glyceraldehyde-3-phosphate Dehydrogenase in Human Serum

Cellular glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a phylogenetically conserved, ubiquitous enzyme that plays an indispensable role in energy metabolism. Although a wealth of information is available on cellular GAPDH, there is a clear paucity of data on its extracellular counterpart (i.e., the secreted or extracellular GAPDH). Here, we show that the extracellular GAPDH in human serum is a multimeric, high-molecular-weight, yet glycolytically active enzyme. The high-molecular-weight multimers of serum GAPDH were identified by immunodetection on one- and two-dimensional gel electrophoresis using multiple antibodies specific for various epitopes of GAPDH. Partial purification of serum GAPDH by DEAE Affigel affinity/ion exchange chromatography further established the multimeric composition of serum GAPDH. In vitro data demonstrated that human cell lines secrete a multimeric, high-molecular-weight enzyme similar to that of serum GAPDH. Furthermore, LC-MS/MS analysis of extracellular GAPDH from human cell lines confirmed the presence of unique peptides of GAPDH in the high-molecular-weight subunits. Furthermore, data from pulse-chase experiments established the presence of high-molecular-weight subunits in the secreted, extracellular GAPDH. Taken together, our findings demonstrate the presence of a high-molecular-weight, enzymatically active secretory GAPDH in human serum that may have a hitherto unknown function in humans.

Metabolic perturbation sensitizes human breast cancer to NK cell-mediated cytotoxicity by increasing the expression of MHC class I chain-related A/B

Cleavage or shedding of the surface antigen, MHC class I chain-related (MIC) protein (A/B) has been known to be one of the mechanisms by which tumor cells escape host immune surveillance. Thus, any strategy to augment the surface expression of MICA/B could facilitate anticancer immune response. Here, we demonstrate that metabolic perturbation by the glycolytic inhibitor, 3-bromopyruvate (3-BrPA) augments the surface expression of MICA/B in human breast cancer cell lines, MDA-MB-231 and T47D. Data from in vitro studies show that a non-toxic, low-dose of 3-BrPA is sufficient to perturb energy metabolism, as evident by the activation of p-AMPK, p-AKT and p-PI3K. Further, 3-BrPA-treatment also elevated the levels of MICA/B in human breast cancer cell lines. Significantly, 3-BrPA-dependent increase in MICA/B levels also enhanced the sensitivity of cancer cells to natural killer (NK-92MI)-mediated cytotoxicity. In vivo, 3-BrPA-pretreated cells demonstrated greater sensitivity to NK-92MI therapy than their respective controls. The antitumor effect was confirmed by a reduction in tumor size and decreased tumor viability as observed by bioluminescence imaging. Histological examination and TUNEL staining demonstrated that NK-92MI administration promoted apoptosis in 3-BrPA-pretreated cells. Taken together, our data show that targeting energy metabolism could be a novel strategy to enhance the effectiveness of anticancer immunotherapeutics.

Statins impair glucose uptake in tumor cells

Statins play a pivotal role in lowering the blood cholesterol level, which is critical for patients with hypercholesterolemia. In addition to its benefits in cardiovascular protection, statins have been found to be useful in several other clinical conditions, including cancer. In a recent report that appeared in Neoplasia, Malenda et al., have demonstrated that statins inhibit glucose uptake in cancer cells. Using multiple statins and glucose analogs (18FDG and 6-NBDG) they showed that inhibition of cholesterol synthesis underlies the blockade of glucose uptake in several cancer cell lines. Further, based on an exploratory clinical study, they also showed that diagnostic PET-CT imaging in patients treated for hypercholesterolemia was affected due to statin-mediated inhibition of glucose uptake. As the finding is based on the data from a single patient (out of four), it seems that (1) the need for a large cohort study and (2) the detailed characterization of the molecular mechanisms underlying such biological effects would be justified.