Saroj P. Mathupala

Hexokinase II: Cancer's double-edged sword acting as both facilitator and gatekeeper of malignancy when bound to mitochondria

A key hallmark of many cancers, particularly the most aggressive, is the capacity to metabolize glucose at an elevated rate, a phenotype detected clinically using positron emission tomography (PET). This phenotype provides cancer cells, including those that participate in metastasis, a distinct competitive edge over normal cells. Specifically, after rapid entry of glucose into cancer cells on the glucose transporter, the highly glycolytic phenotype is supported by hexokinase (primarily HK II) that is overexpressed and bound to the outer mitochondrial membrane via the porin-like protein voltage-dependent anion channel (VDAC). This protein and the adenine nucleotide transporter move ATP, newly synthesized by the inner membrane located ATP synthase, to active sites on HK II. The abundant amounts of HK II bind both the ATP and the incoming glucose producing the product glucose-6-phosphate, also at an elevated rate. This critical metabolite then serves both as a biosynthetic precursor to support cell proliferation and as a precursor for lactic acid, the latter exiting cancer cells causing an unfavorable environment for normal cells. Although helping facilitate this chemical warfare, HK II via its mitochondrial location also suppresses the death of cancer cells, thus increasing their possibility for metastasis and the ultimate death of the human host. For these reasons, targeting this key enzyme is currently being investigated in several laboratories in a strategy to develop novel therapies that may turn the tide on the continuing struggle to find effective cures for cancer. One such candidate is 3-bromopyruvate that has been shown recently to eradicate advanced stage, PET positive hepatocellular carcinomas in an animal model without apparent harm to the animals.

Mitochondrial bound type II hexokinase: a key player in the growth and survival of many cancers and an ideal prospect for therapeutic intervention.

Despite more than 75 years of research by some of the greatest scientists in the world to conquer cancer, the clear winner is still cancer. This is reflected particularly by liver cancer that worldwide ranks fourth in terms of mortality with survival rates of no more than 3-5%. Significantly, one of the earliest discovered hallmarks of cancer had its roots in Bioenergetics as many tumors were found in the 1920s to exhibit a high glycolytic phenotype. Although research directed at unraveling the underlying basis and significance of this phenotype comprised the focus of cancer research for almost 50 years, these efforts declined greatly from 1970 to 1990 as research into the molecular and cell biology of this disease gained center stage. Certainly, this change was necessary as the new knowledge obtained about oncogenes, gene regulation, and programmed cell death once again placed Bioenergetics in the limelight of cancer research. Thus, we now have a much better molecular understanding of the high glycolytic phenotype of many cancers, the pivotal roles that Type II hexokinase-mitochondrial interactions play in this process to promote tumor cell growth and survival, and how this new knowledge can lead to improved therapies that may ultimately turn the tide on our losing war on cancer.

Silencing of monocarboxylate transporters via small interfering ribonucleic acid inhibits glycolysis and induces cell death in malignant glioma: An in vitro study

OBJECTIVE:Dependence on glycolysis is a hallmark of malignant tumors. As a consequence, these tumors generate more lactate, which is effluxed from cells by monocarboxylate transporters (MCTs). We hypothesized that 1) MCT expression in malignant tumors may differ from normal tissue in quantity, isoform, or both; and 2) silencing MCT expression would induce intracellular acidification, resulting in decreased proliferation and/or increased cell death. METHODS:We quantified expression of MCT isoforms in human glioblastoma multiforme and glioma-derived cells lines by Western blot analysis. MCTs that were abundant or specific to glioma then were targeted in the model U-87 MG glioma cell line via small interfering ribonucleic acid-mediated gene silencing and tested for inhibition of lactate efflux, intracellular pH changes, reduced proliferation, and/or induction of cell death. RESULTS:MCT 1 and 2 were the primary isoforms expressed in human glioblastoma multiforme and glioma-derived cell lines. In contrast, MCT 3 was the predominantly expressed isoform in normal brain. Small interfering ribonucleic acid specific for MCT 1 and 2 reduced expression of these isoforms in U-87 MG cells to barely detectable levels and reduced lactate efflux by 30% individually and 85% in combination, with a concomitant decrease of intracellular pH by 0.6 units (a fourfold increase in intracellular H+). Prolonged silencing of both MCTs reduced viability by 75% individually and 92% in combination, as measured by both phenotypic and flow cytometric analyses. CONCLUSION:MCT targeting significantly reduced the viability of U-87 MG cells mediated by both apoptosis and necrosis. This indicates that the strategy may be a useful therapeutic avenue for treatment of patients with malignant glioma.

Delivery of small-interfering RNA (siRNA) to the brain.

Background: Two fundamental difficulties in the delivery of drugs to treat central nervous system (CNS) diseases are the systemic delivery of therapeutics across the bloodbrain-barrier (BBB), and the targeting of drugs to specific tissues or cells within the brain. With the advent and promise of RNA-based therapeutics that utilize RNA interference (RNAi) to trigger specific silencing of genes within diseased tissues, the necessity to surmount such obstacles has become even more urgent. Objective: Most pre-clinical and clinical studies on delivery of RNAi to the CNS have utilized invasive, intra-cerebral delivery of RNA to the targeted tissue. Thus, methods need to be developed to facilitate delivery of therapeutically significant quantities of RNA to the CNS via the systemic route, and to elicit clinically significant RNAi effects within the CNS tissues. Methods: Cell-penetrating-peptides (CPPs) are ‘molecular delivery vehicles’ that can traverse cell membranes and co-transport peptides or polynucleotides. The present invention examines 1) the utility of CPP-RNA duplexes for delivery of RNA to CNS tissues and, 2) cell-mediated release of the RNA payload once the CPP-RNA duplex is internalized by the CNS cells. Conclusions: The invention and embodiments listed therein outline molecular tools that can be adapted for non-invasive, systemic delivery of therapeutic RNA to the CNS in a future clinical setting.

Metabolic Remodeling of Malignant Gliomas for Enhanced Sensitization during Radiotherapy: An In Vitro Study

OBJECTIVE: To investigate a novel method to enhance radiosensitivity of gliomas via modification of metabolite flux immediately before radiotherapy. Malignant gliomas are highly glycolytic and produce copious amounts of lactic acid, which is effluxed to the tumor microenvironment via lactate transporters. We hypothesized that inhibition of lactic acid efflux would alter glioma metabolite profiles, including those that are radioprotective. 1H magnetic resonance spectroscopy (MRS) was used to quantify key metabolites, including those most effective for induction of low-dose radiation-induced cell death. METHODS: We inhibited lactate transport in U87-MG gliomas with α-cyano-4-hydroxycinnamic acid (ACCA). Flow cytometry was used to assess induction of cell death in treated cells. Cells were analyzed by MRS after ACCA treatment. Control and treated cells were subjected to low-dose irradiation, and the surviving fractions of cells were determined by clonogenic assays. RESULTS: MRS revealed changes to intracellular lactate on treatment with ACCA. Significant decreases in the metabolites taurine, glutamate, glutathione, alanine, and glycine were observed, along with inversion of the choline/phosphocholine profile. On exposure to low-dose radiation, ACCA-pretreated U-87MG cells underwent rapid morphological changes, which were followed by apoptotic cell death. CONCLUSION: Inhibition of lactate efflux in malignant gliomas results in alterations of glycolytic metabolism, including decreased levels of the antioxidants taurine and glutathione and enhanced radiosensitivity of ACCA-treated cells. Thus, in situ application of lactate transport inhibitors such as ACCA as a novel adjunctive therapeutic strategy against glial tumors may greatly enhance the level of radiation-induced cell killing during a combined radioand chemotherapeutic regimen.OBJECTIVETo investigate a novel method to enhance radiosensitivity of gliomas via modification of metabolite flux immediately before radiotherapy. Malignant gliomas are highly glycolytic and produce copious amounts of lactic acid, which is effluxed to the tumor microenvironment via lactate transporters. We hypothesized that inhibition of lactic acid efflux would alter glioma metabolite profiles, including those that are radioprotective. 1H magnetic resonance spectroscopy (MRS) was used to quantify key metabolites, including those most effective for induction of low-dose radiation-induced cell death. METHODSWe inhibited lactate transport in U87-MG gliomas with α-cyano-4-hydroxycinnamic acid (ACCA). Flow cytometry was used to assess induction of cell death in treated cells. Cells were analyzed by MRS after ACCA treatment. Control and treated cells were subjected to low-dose irradiation, and the surviving fractions of cells were determined by clonogenic assays. RESULTSMRS revealed changes to intracellular lactate on treatment with ACCA. Significant decreases in the metabolites taurine, glutamate, glutathione, alanine, and glycine were observed, along with inversion of the choline/phosphocholine profile. On exposure to low-dose radiation, ACCA-pretreated U-87MG cells underwent rapid morphological changes, which were followed by apoptotic cell death. CONCLUSIONInhibition of lactate efflux in malignant gliomas results in alterations of glycolytic metabolism, including decreased levels of the antioxidants taurine and glutathione and enhanced radiosensitivity of ACCA-treated cells. Thus, in situ application of lactate transport inhibitors such as ACCA as a novel adjunctive therapeutic strategy against glial tumors may greatly enhance the level of radiation-induced cell killing during a combined radio- and chemotherapeutic regimen.

RNAi based approaches to the treatment of malignant glioma.

RNA interference (RNAi) is a recently discovered, powerful molecular mechanism that can be harnessed to engineer gene-specific silencing in mammalian tissues. A mechanism, where short double-stranded RNA (dsRNA) molecules, when introduced into cells elicit specific “knock-down” of gene expression via degradation of targeted messenger RNA, has lately become the technique of choice for analysis of gene function in oncology research. Thus, RNAi is currently being extensively evaluated as a potential therapeutic strategy against malignant gliomas, since surgical, radiological, and chemotherapeutic interventions during the past few decades have done little to improve the poor prognosis rate for patients with these dreaded tumors. This review summarizes the pre-clinical studies that are currently underway to test the validity of RNAi as a potential therapeutic strategy against malignant gliomas, and discusses the potential technical Hurdles that remain to be overcome before the technique can become a promising clinical therapy to combat this frequently lethal disease.

Systematic comparison of dendritic cell-based immunotherapeutic strategies for malignant gliomas: In vitro induction of cytolytic and natural killer-like T cells

OBJECTIVE: To compare the efficacy of various immunotherapeutic strategies of loading dendritic cells (DCs) with whole-glioma cell antigens and characterize the effector responses induced. METHODS: DCs were either fused with major histocompatibility complex (MHC)-matched glioma cells (Fusion) or pulsed with apoptotic tumor cells (DC/Apo), total tumor ribonucleic acid (RNA) (DC/RNA), or tumor lysate (DC/Lys). These tumor-DC preparations were then assessed for their phenotype, cytokine profile, and capacity to stimulate autologous peripheral blood mononuclear cells (PBMCs) in vitro. Phenotype and tumor-specific cytolytic activities of various effector cell populations were characterized and compared. RESULTS: The various tumor-DC preparations exhibited similar phenotype and cytokine profiles irrespective of the method of loading tumor-cell antigens. However, the fusion, DC/Apo, and DC/RNA induced superior tumor cytolytic activities in PBMCs compared with DC/Lys or DC and tumor controls. DC/Apo induced the greatest expansion of tumor-specific lymphocytes, as detected by trypan blue exclusion and thymidine incorporation assays. Flow cytometric analyses also revealed the highest relative percentages of T helper cells (CD3+CD4+), cytotoxic T lymphocytes (CTLs) (CD3+CD8+), and natural killer (NK)-like T cells (CD3+CD56+) in the DC/Apo group among all the groups studied, indicating that DC/Apo induced expansion of PBMCs bearing multiple T and NK cell markers. Interestingly, isolated NK-like T cells demonstrated significantly higher tumor cytotoxicity compared with CTLs isolated from the same groups and was also non-MHC-restricted. CONCLUSION: Apoptotic tumor cells may be an optimal source of whole-tumor-cell antigen for immunotherapy of gliomas. The study also demonstrates for the first time that both CTLs and NK-like T cells are expanded and stimulated by mature, tumor-pulsed DCs.

Dendritic cell-based active specific immunotherapy for malignant glioma.

Immunotherapy is an appealing therapeutic modality for malignant gliomas because of its potential to selectively target residual tumor cells that have invaded the normal brain. Most immunotherapeutic studies are designed to exploit the capacity of dendritic cells for inducing cell-mediated effects as well as immune memory responses for destroying residual tumor cells and preventing recurrence. Although initial clinical studies on dendritic cell-based immunotherapy resulted in very limited success, they have prompted many new studies on exploring strategies to induce a more robust antitumor immune response by using novel adjuvants for maturation and activation of dendritic cells. More studies have focused on the mechanisms of immune suppression by tumor cells and the role of regulatory T cells in tumor growth and progression. In this article, the authors review the evolution of dendritic cell-based immunotherapeutic strategies for adjuvant treatment of malignant gliomas. The authors also discuss how new knowledge on tumor-intrinsic mechanisms of tolerance induction and immunosuppression are likely to shape the future of immunotherapy for high-grade gliomas.

Voltage dependent anion channel-1 (VDAC-1) as an anti-cancer target

Commentary to: Down-regulation of voltage-dependent anion channel-1 expression by RNA interference prevents cancer cell growth in vivo Inbar Koren, Ziv Raviv and Varda Shoshan-Barmatz

An agarose-based cloning-ring anchoring method for isolation of viable cell clones.

Isolation of clonal cell populations is a crucial aspect of cell biology during engineering of specific cell strains with both genotypic and phenotypic variations. The use of cloning rings is the most established method, but requires anchoring chemicals or material that can often interfere with quantitative clonal-cell isolation and causes physical damage to the cells. Here we report a non-toxic, cell culture-compatible method that uses aga-rose for embedding the cloning rings during isolation of cell clones from monolayer cultures, with enhanced cell-viability and reproducibility during downstream applications. The method is simple and rapid, minimizing the chances for desiccation or cross-contamination during colony-lifts.