Shanmugasundaram Ganapathy-Kanniappan

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

PURPOSEnTo characterize tumor response to percutaneous injection of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antagonists in a mouse model of human hepatocellular carcinoma (HCC).nnnMATERIALS AND METHODSnAnimal 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.nnnRESULTSnIn 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).nnnCONCLUSIONnPercutaneous injection of GAPDH antagonists induces apoptosis and blocks Hep3B tumor progression, which demonstrates the therapeutic potential of targeting GAPDH in human HCC.

Systemic Delivery of Microencapsulated 3-Bromopyruvate for the Therapy of Pancreatic Cancer

Purpose: This study characterized the therapeutic efficacy of a systemically administered formulation of 3-bromopyruvate (3-BrPA), microencapsulated in a complex with β-cyclodextrin (β-CD), using an orthotopic xenograft mouse model of pancreatic ductal adenocarcinoma (PDAC). Experimental Design: The presence of the β-CD–3-BrPA complex was confirmed using nuclear magnetic resonance spectroscopy. Monolayer as well as three-dimensional organotypic cell culture was used to determine the half-maximal inhibitory concentrations (IC50) of β-CD–3-BrPA, free 3-BrPA, β-CD (control), and gemcitabine in MiaPaCa-2 and Suit-2 cell lines, both in normoxia and hypoxia. Phase-contrast microscopy, bioluminescence imaging (BLI), as well as zymography and Matrigel assays were used to characterize the effects of the drug in vitro. An orthotopic lucMiaPaCa-2 xenograft tumor model was used to investigate the in vivo efficacy. Results: β-CD–3-BrPA and free 3-BrPA demonstrated an almost identical IC50 profile in both PDAC cell lines with higher sensitivity in hypoxia. Using the Matrigel invasion assay as well as zymography, 3-BrPA showed anti-invasive effects in sublethal drug concentrations. In vivo, animals treated with β-CD–3-BrPA demonstrated minimal or no tumor progression as evident by the BLI signal as opposed to animals treated with gemcitabine or the β-CD (60-fold and 140-fold signal increase, respectively). In contrast to animals treated with free 3-BrPA, no lethal toxicity was observed for β-CD–3-BrPA. Conclusion: The microencapsulation of 3-BrPA represents a promising step towards achieving the goal of systemically deliverable antiglycolytic tumor therapy. The strong anticancer effects of β-CD–3-BrPA combined with its favorable toxicity profile suggest that clinical trials, particularly in patients with PDAC, should be considered. Clin Cancer Res; 20(24); 6406–17. ©2014 AACR.

Ultrasound-guided direct delivery of 3-bromopyruvate blocks tumor progression in an orthotopic mouse model of human pancreatic cancer

Studies in animal models of cancer have demonstrated that targeting tumor metabolism can be an effective anticancer strategy. Previously, we showed that inhibition of glucose metabolism by the pyruvate analog, 3-bromopyruvate (3-BrPA), induces anticancer effects both in vitro and in vivo. We have also documented that intratumoral delivery of 3-BrPA affects tumor growth in a subcutaneous tumor model of human liver cancer. However, the efficacy of such an approach in a clinically relevant orthotopic tumor model has not been reported. Here, we investigated the feasibility of ultrasound (US) image-guided delivery of 3-BrPA in an orthotopic mouse model of human pancreatic cancer and evaluated its therapeutic efficacy. In vitro, treatment of Panc-1 cells with 3-BrPA resulted in a dose-dependent decrease in cell viability. The loss of viability correlated with a dose-dependent decrease in the intracellular ATP level and lactate production confirming that disruption of energy metabolism underlies these 3-BrPA-mediated effects. In vivo, US-guided delivery of 3-BrPA was feasible and effective as demonstrated by a marked decrease in tumor size on imaging. Further, the antitumor effect was confirmed by (1) a decrease in the proliferative potential by Ki-67 immunohistochemical staining and (2) the induction of apoptosis by terminal deoxynucleotidyl transferase-mediated deoxyuridine 5-triphospate nick end labeling staining. We therefore demonstrate the technical feasibility of US-guided intratumoral injection of 3-BrPA in a mouse model of human pancreatic cancer as well as its therapeutic efficacy. Our data suggest that this new therapeutic approach consisting of a direct intratumoral injection of antiglycolytic agents may represent an exciting opportunity to treat patients with pancreas cancer.

Preclinical Benefit of Hypoxia-Activated Intraarterial Therapy with Evofosfamide in Liver Cancer

Purpose: To evaluate safety and characterize anticancer efficacy of hepatic hypoxia-activated intra-arterial therapy (HAIAT) with evofosfamide in a rabbit model. Experimental Design: VX2-tumor-bearing rabbits were assigned to 4 intra-arterial therapy (IAT) groups (n = 7/group): (i) saline (control); (ii) evofosfamide (Evo); (iii) doxorubicin–lipiodol emulsion followed by embolization with 100–300 μm beads (conventional, cTACE); or (iv) cTACE and evofosfamide (cTACE + Evo). Blood samples were collected pre-IAT and 1, 2, 7, and 14 days post-IAT. A semiquantitative scoring system assessed hepatocellular damage. Tumor volumes were segmented on multidetector CT (baseline, 7/14 days post-IAT). Pathologic tumor necrosis was quantified using manual segmentation on whole-slide images. Hypoxic fraction (HF) and compartment (HC) were determined by pimonidazole staining. Tumor DNA damage, apoptosis, cell proliferation, endogenous hypoxia, and metabolism were quantified (γ-H2AX, Annexin V, caspase-3, Ki-67, HIF1α, VEGF, GAPDH, MCT4, and LDH). Results: cTACE + Evo showed a similar profile of liver enzymes elevation and pathologic scores compared with cTACE. Neither hematologic nor renal toxicity were observed. Animals treated with cTACE + Evo demonstrated smaller tumor volumes, lower tumor growth rates, and higher necrotic fractions compared with cTACE. cTACE + Evo resulted in a marked reduction in the HF and HC. Correlation was observed between decreases in HF or HC and tumor necrosis. cTACE + Evo promoted antitumor effects as evidenced by increased expression of γ-H2AX, apoptotic biomarkers, and decreased cell proliferation. Increased HIF1α/VEGF expression and tumor glycolysis supported HAIAT. Conclusions: HAIAT achieved a promising step towards the locoregional targeting of tumor hypoxia. The favorable toxicity profile and enhanced anticancer effects of evofosfamide in combination with cTACE pave the way towards clinical trials in patients with liver cancer. Clin Cancer Res; 23(2); 536–48. ©2016 AACR.