Role of interleukin-6 in cancer progression and therapeutic resistance.

In the last several decades, the number of people dying from cancer-related deaths has not reduced significantly despite phenomenal advances in the technologies related to diagnosis and therapeutic modalities. The principal cause behind limitations in the curability of this disease is the reducing sensitivity of the cancer cells towards conventional anticancer therapeutic modalities, particularly in advance stages of the disease. Amongst several reasons, certain secretory factors released by the tumour cells into the microenvironment have been found to confer resistance towards chemo- and radiotherapy, besides promoting growth. Interleukin-6 (IL-6), one of the major cytokines in the tumour microenvironment, is an important factor which is found at high concentrations and known to be deregulated in cancer. Its overexpression has been reported in almost all types of tumours. The strong association between inflammation and cancer is reflected by the high IL-6 levels in the tumour microenvironment, where it promotes tumorigenesis by regulating all hallmarks of cancer and multiple signalling pathways, including apoptosis, survival, proliferation, angiogenesis, invasiveness and metastasis, and, most importantly, the metabolism. Moreover, IL-6 protects the cancer cells from therapy-induced DNA damage, oxidative stress and apoptosis by facilitating the repair and induction of countersignalling (antioxidant and anti-apoptotic/pro-survival) pathways. Therefore, blocking IL-6 or inhibiting its associated signalling independently or in combination with conventional anticancer therapies could be a potential therapeutic strategy for the treatment of cancers with IL-6-dominated signalling.

Per a 10 protease activity modulates CD40 expression on dendritic cell surface by nuclear factor-kappaB pathway.

Serine protease activity of Per a 10 from Periplaneta americana modulates dendritic cell (DC) functions by a mechanism(s) that remains unclear. In the present study, Per a 10 protease activity on CD40 expression and downstream signalling was evaluated in DCs. Monocyte-derived DCs from cockroach-allergic patients were treated with proteolytically active/heat-inactivated Per a 10. Stimulation with active Per a 10 demonstrated low CD40 expression on DCs surface (P < 0·05), while enhanced soluble CD40 level in the culture supernatant (P < 0·05) compared to the heat-inactivated Per a 10, suggesting cleavage of CD40. Per a 10 activity reduced the interleukin (IL)-12 and interferon (IFN)-γ secretion by DCs (P < 0·05) compared to heat-inactivated Per a 10, indicating that low CD40 expression is associated with low levels of IL-12 secretion. Active Per a 10 stimulation caused low nuclear factor-kappa B (NF-κB) activation in DCs compared to heat-inactivated Per a 10. Inhibition of the NF-κB pathway suppressed the CD40 expression and IL-12 secretion by DCs, further indicating that NF-κB is required for CD40 up-regulation. CD40 expression activated the tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6), thereby suggesting its involvement in NF-κB activation. Protease activity of Per a 10 induced p38 mitogen-activated protein kinase (MAPK) activation that showed no significant effect on CD40 expression by DCs. However, inhibiting p38 MAPK or NF-κB suppressed the secretion of IL-12, IFN-γ, IL-6 and TNF-α by DCs. Such DCs further reduced the secretion of IL-4, IL-6, IL-12 and TNF-α by CD4(+) T cells. In conclusion, protease activity of Per a 10 reduces CD40 expression on DCs. CD40 down-regulation leads to low NF-κB levels, thereby modulating DC-mediated immune responses.

Differences in DNA condensation and release by lysine and arginine homopeptides govern their DNA delivery efficiencies.

Designing of nanocarriers that can efficiently deliver therapeutic DNA payload and allow its smooth intracellular release for transgene expression is still a major constraint. The optimization of DNA nanocarriers requires thorough understanding of the chemical and structural characteristics of the vector-nucleic acid complexes and its correlation with the cellular entry, intracellular state and transfection efficiency. L-lysine and L-arginine based cationic peptides alone or in conjugation with other vectors are known to be putative DNA delivery agents. Here we have used L-lysine and L-arginine homopeptides of three different lengths and probed their DNA condensation and release properties by using a multitude of biophysical techniques including fluorescence spectroscopy, gel electrophoresis and atomic force microscopy. Our results clearly showed that although both lysine and arginine based homopeptides condense DNA via electrostatic interactions, they follow different pattern of DNA condensation and release in vitro. While lysine homopeptides condense DNA to form both monomolecular and multimolecular complexes and show differential release of DNA in vitro depending on the peptide length, arginine homopeptides predominantly form multimolecular complexes and show complete DNA release for all peptide lengths. The cellular uptake of the complexes and their intracellular state (as observed through flow cytometry and fluorescence microscopy) seem to be controlled by the peptide chemistry. The difference in the transfection efficiency of lysine and arginine homopeptides has been rationalized in light of these observations.

The Role of Notch, Hedgehog, and Wnt Signaling Pathways in the Resistance of Tumors to Anticancer Therapies.

Resistance to therapy is the major hurdle in the current cancer management. Cancer cells often rewire their cellular process to alternate mechanisms to resist the deleterious effect mounted by different therapeutic approaches. The major signaling pathways involved in the developmental process, such as Notch, Hedgehog, and Wnt, play a vital role in development, tumorigenesis, and also in the resistance to the various anticancer therapies. Understanding how cancer utilizes these developmental pathways in acquiring the resistance to the multi-therapeutic approach cancer can give rise to a new insight of the anti-therapy resistance mechanisms, which can be explored for the development of a novel therapeutic approach. We present a brief overview of Notch, Hedgehog, and Wnt signaling pathways in cancer and its role in providing resistance to various cancer treatment modalities such as chemotherapy, radiotherapy, molecular targeted therapy, and immunotherapy. Understanding the importance of these molecular networks will provide a rational basis for novel and safer combined anticancer therapeutic approaches for the improvement of cancer treatment by overcoming drug resistance.

Cancer biomarkers - current perspectives.

In the recent years, knowledge about cancer biomarkers has increased tremendously providing great opportunities for improving the management of cancer patients by enhancing the efficiency of detection and efficacy of treatment. Recent technological advancement has enabled the examination of many potential biomarkers and renewed interest in developing new biomarkers. Biomarkers of cancer could include a broad range of biochemical entities, such as nucleic acids, proteins, sugars, lipids, and small metabolites, cytogenetic and cytokinetic parameters as well as whole tumour cells found in the body fluid. A comprehensive understanding of the relevance of each biomarker will be very important not only for diagnosing the disease reliably, but also help in the choice of multiple therapeutic alternatives currently available that is likely to benefit the patients. This review provides a brief account on various biomarkers for diagnosis, prognosis and therapeutic purposes, which include markers already in clinical practice as well as various upcoming biomarkers.

Endogenous and induced oxidative stress in multi-cellular tumour spheroids: implications for improving tumour therapy.

The endogenous oxidative stress in tumours is determined by the status of mitochondrial, metabolic, oxygen (hypoxia) and inherent enzymatic as well as non-enzymatic antioxidant defense systems, which influence tumour growth and respond to anticancer therapeutics. Induced oxidative stress is one of the important determinants of the outcome of treatment with certain chemotherapeutic drugs and ionizing radiation. The mild to moderate levels of reactive oxygen species (ROS) have often been found to trigger prosurvival responses, thereby contributing to the resistance against therapy. The higher levels of ROS stimulate multiple death pathways viz. typical and atypical apoptosis, necrosis etc, thereby enhancing the therapeutic efficiency. Therefore, approaches employing therapeutic agents that generate ROS efficiently in the tumour cells and enhance the antioxidant defense system in the normal cells could significantly enhance the therapeutic gain. Multi-cellular tumour spheroids (MCTS) offer an excellent in vitro system that mimics endogenous oxidative stress often observed in tumours, arising due to a number of factors (gradients of oxygen and nutrients, altered intercellular interaction and tumour necrosis factor), besides antioxidant defense systems similar to tumours in vivo. More importantly, MCTS resemble tumours in vivo with reference to the induced oxidative stress related responses, particularly following combinations of certain chemotherapeutic drugs and metabolic inhibitors and differs significantly from the responses in monolayer cultures. Therefore, MCTS appear to be excellent in vitro models, ideally suited for developing novel therapies that are based on the generation of oxidative stress in tumours. The present review provides a modest account on the utility of MCTS in understanding the role of oxidative stress in treatment-induced responses of tumours for designing therapies and therapeutics.

Radiation induces EIF2AK3/PERK and ERN1/IRE1 mediated pro-survival autophagy.

Cellular effects of ionizing radiation include oxidative damage to macromolecules, unfolded protein response (UPR) and metabolic imbalances. Oxidative stress and UPR have been shown to induce macroautophagy/autophagy in a context-dependent manner and are crucial factors in determining the fate of irradiated cells. However, an in-depth analysis of the relationship between radiation-induced damage and autophagy has not been explored. In the present study, we investigated the relationship between radiation-induced oxidative stress, UPR and autophagy in murine macrophage cells. A close association was observed between radiation-induced oxidative burst, UPR and induction of autophagy, with the possible involvement of EIF2AK3/PERK (eukaryotic translation initiation factor 2 alpha kinase 3) and ERN1/IRE1 (endoplasmic reticulum [ER] to nucleus signaling 1). Inhibitors of either UPR or autophagy reduced the cell survival indicating the importance of these processes after radiation exposure. Moreover, modulation of autophagy affected lethality in the whole body irradiated C57BL/6 mouse. These findings indicate that radiation-induced autophagy is a pro-survival response initiated by oxidative stress and mediated by EIF2AK3 and ERN1. Abbreviations: ACTB: actin, beta; ATF6: activating transcription factor 6; ATG: autophagy-related; BafA1: bafilomycin A1; CQ: chloroquine; DBSA: 3,5-dibromosalicylaldehyde; EIF2AK3: eukaryotic translation initiation factor 2 alpha kinase 3; ERN1: endoplasmic reticulum (ER) to nucleus signaling 1; IR: ionizing radiation; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; 3-MA: 3-methyladenine; MTOR: mechanistic target of rapamycin kinase; NAC: N-acetyl-L-cysteine; PARP1: poly (ADP-ribose) polymerase family, member 1; 4-PBA: 4-phenylbutyrate; Rap: rapamycin; ROS: reactive oxygen species; UPR: unfolded protein response; XBP1: x-box binding protein 1.

Targeting protein acetylation for improving cancer therapy.

Acetylation is one of the most important post-translational modification of proteins determining the structure, function and intracellular localization that plays an important role in the signal transduction pathways related to diverse cell functions, both during unstimulated and stress conditions. Protein acetylation in cells is regulated by a co-ordinated action of histone acetyl transferases (HAT) and histone deacetylases(HDAC) that ensures the maintenance of homeostasis and execution of activities related to damage response viz. DNA repair, cell cycle delay, apoptosis and senescence. Since inhibition of histone deacetylation, stalls the progress of many nuclear events including proliferation and damage response events on the one hand and the levels of deacetylases are elevated in many tumours on the other. Histone deacetylase has been among the targets for the development of anticancer drugs and adjuvant. The recent observation showing acetylation of proteins by calreticulin (an endoplasmic reticulum resident protein) with a high efficiency when polyphenolic acetates are the acetyl group donating molecules and acetyl CoA as weak substrate extends the realm of protein acetylation beyond HAT/HDAC combination. Elucidation of the relative roles of HAT/HDAC mediated acetylation viz. a calreticulin mediated acetylation in cell function under a variety of stress conditions would hold key to the design of drugs targeting protein acetylation system.

Mitochondrial biogenesis and metabolic hyperactivation limits the application of MTT assay in the estimation of radiation induced growth inhibition.

Metabolic viability based high throughput assays like MTT and MTS are widely used in assessing the cell viability. However, alteration in both mitochondrial content and metabolism can influence the metabolic viability of cells and radiation is a potential mitochondrial biogenesis inducer. Therefore, we tested if MTT assay is a true measure of radiation induced cell death in widely used cell lines. Radiation induced cellular growth inhibition was performed by enumerating cell numbers and metabolic viability using MTT assay at 24 and 48 hours (hrs) after exposure. The extent of radiation induced reduction in cell number was found to be larger than the decrease in MTT reduction in all the cell lines tested. We demonstrated that radiation induces PGC-1α and TFAM to stimulate mitochondrial biogenesis leading to increased levels of SDH-A and enhanced metabolic viability. Radiation induced disturbance in calcium (Ca2+) homeostasis also plays a crucial role by making the mitochondria hyperactive. These findings suggest that radiation induces mitochondrial biogenesis and hyperactivation leading to increased metabolic viability and MTT reduction. Therefore, conclusions drawn on radiation induced growth inhibition based on metabolic viability assays are likely to be erroneous as it may not correlate with growth inhibition and/or loss of clonogenic survival.

Calcium channel antagonist (nifedipine) attenuates Plasmodium berghei-specific T cell immune responses in Balb/C mice.

Nifedipine and verapamil (Martin et al. Science 1987;235:899-901) are a class of calcium channel blockers involved in the reversal of chloroquine (CQ) drug resistance in CQ-sensitive Plasmodium spp. Nifedipine alters calcium-dependent functions of macrophages and neutrophils during Plasmodium berghei malaria. However, knowledge of nifedipine-induced immunomodulation of T cell functions during P. berghei malaria is still limited. We investigated the effect of nifedipine on the immune status of splenic T cells during P. berghei malaria. The intracellular calcium levels were determined in the FURA-2A/M loaded T cells by spectrofluorometry. Splenic T cell proliferation, phosphatidylserine (PS) externalization, Fas expression and Bcl2/Bax expression were determined by flow cytometry. We report a significant increase in mean percent parasitemia in nifedipine-treated and P. berghei-infected mice. Although nifedipine treatment alone did not affect the resting state free calcium levels in splenic T cells, the rise in intracellular calcium levels of T cells following P. berghei infection was significantly less in nifedipine-treated mice compared to untreated groups at various parasitemia levels. Antigen-specific splenic T cell proliferation and apoptosis was ablated in nifedipine-treated and untreated groups at various parasitemia levels. The study unequivocally reflects the suppression of P. berghei-specific T cell immune responses by nifedipine.

Differential mechanisms of radiosensitization by 2-deoxy-D-glucose in the monolayers and multicellular spheroids of a human glioma cell line.

In vitro studies using monolayer cultures of human tumor cell lines have shown that 2-DG selectively inhibits energy-dependent DNA repair and cellular recovery processes in cancer cells. However, monolayer cultures differ greatly from the complex environmental conditions generated in solid tumors that develop inhomogeneous hypoxic and necrotic regions. In contrast, multicellular spheroids mimic heterogeneous cellular behavior and the consequent functional characteristics of in vivo solid tumors, and serve as important in vitro model to investigate tumor biology and responses to potential therapeutic agents. The present study compares the radiomodification by 2-DG in monolayer cultures and spheroids of a human glioma cell line (BMG-1) to gain insight into the effects in solid tumors. In spheroids, the glucose consumption (2.1 p mole/cell/h) and lactate production (3.67 p mole/cell/h) was nearly 2-3 fold higher than in monolayer cells (0.83 and 1.43 p mole/cell/h respectively). Presence of 2-DG (5 mM) for 2-4 h inhibited the glucose usage and lactate production by 70% in spheroids, while a 35% reduction was observed in monolayer cells. Under these conditions, 2-DG drastically enhanced the radiation-induced cell death of spheroids (by 2-3 folds); while a 40% increase was observed in monolayer cells. Radiosensitization by 2-DG in monolayer cells was primarily due to an increase in mitotic death (23%) linked to cytogenetic damage (micronuclei), whereas a profound induction of apoptosis (40%) accounted for the sensitization in spheroids. Although the Bcl-2 and Bax levels were significantly higher in spheroids, Bcl-2/Bax ratio was similar in monolayers and spheroids. Comet assay revealed a late onset of DNA breaks in the presence of 2- DG following irradiation only in spheroids, which corroborated well with the late onset of oxidative stress. 2-DG did not induce a significant cell cycle delay in monolayers, while a transient G(2) delay was apparent in spheroids.

Establishment and characterization of multicellular spheroids from a human glioma cell line; Implications for tumor therapy.

BACKGROUND: Multicellular spheroids, an appropriate in vitro system for simulating 3-D tumor micro-milieu can be used for evaluating and predicting tumor response to therapeutic agents including metabolic inhibitors. However, detailed understanding of the nature, distribution and sensitivity/responses of cellular sub-populations to potential therapeutic agents/strategies is required for using this unique model with optimal precision. Spheroid characteristics may also vary considerably with the origin and type of cell line used, and thorough characterization of viable and dissociated glioma cell spheroids is not yet completely known. In order to evaluate in vivo responses of gliomas to various therapeutic strategies, especially the metabolic inhibitors capable of penetrating the blood brain barrier, we have characterized continuously growing spheroids of a human glioma cell line (BMG-1) with respect to organization, growth, viability, cell survival, cell death, metabolic and mitochondrial status, oxidative stress and radiation response using microscopy, flow cytometry and enzymatic assays. Spheroids were fed daily with fresh medium in order to maintain nutrient supply to outer cellular layers while hypoxia/necrosis developed in the innermost cells of enlarging spheroids. RESULTS: Volume of spheroids, fed daily with fresh medium, increased exponentially during 7-28 days of growth through three population doublings. Proportion of G1-phase cells was higher (approximately 60%) than exponentially growing monolayer cells (approximately 48%). A significant fraction of S-phase cells turned metabolically inactive (disengaged in DNA synthesis) with increasing age of the spheroids, unlike in quiescent monolayer cultures, where the fraction of S-phase cells was less than 5%. With increasing spheroid size, increasing sub-populations of cells became non-viable and entered apoptosis or necrosis revealed by Annexin-V-FITC/PI staining. PI positive (necrotic) cells were not confined to the centre of the spheroid, but distributed at certain discrete foci. Average glucose consumption and lactate production were 2-3 folds higher in viable spheroid cells compared to monolayer cells, implying a compensatory increase in glycolysis possibly due to hypoxic environment. HIF-1alpha was expressed only in spheroids and increased in an age-dependent manner, whereas c-Myc (known to induce apoptosis in glucose-deprived cells) levels were three times higher than monolayer cells. Mitochondrial mass and activity decreased significantly during first 14 days of growth but increased with age, and were not associated with increase in ROS levels. Bcl-2 and Bax levels were higher (approximately 2 folds) than monolayers, while the ratio (Bcl/Bax) remained unaltered. Radiation-induced oxidative stress was considerably less in spheroids as compared to monolayers, and corresponded well with increase in radioresistance demonstrated by the clonogenic assay, similar to hypoxia induced radioresistance observed in tumors. CONCLUSION: Development of S-negative cells and reduced endogenous and radiation-induced ROS coupled with higher levels of anti (Bcl2) as well as pro (Bax) apoptotic regulators observed in spheroids suggest the intricate/complex nature of endogenous as well as induced stress resistance that could exist in tumors, which contribute to the treatment resistance.

Acute toxicity and cardio-respiratory effects of 2-deoxy-D-glucose: a promising radio sensitiser.

OBJECTIVE: To evaluate the acute toxicity of 2-deoxy-D-glucose (2DG) by oral (p.o.) and intravenous (i.v.) routes, and also the cardio-respiratory effects following high doses of 2DG in animal models. METHODS: The LD50 of 2DG (in water) was determined in rats and mice by p.o. route and in mice by i.v. route. The effect of 2-DG (250 mg/kg, 500 mg/kg, and 1000 mg/kg, i.v.) was studied on various cardio-respiratory parameters viz., mean arterial blood pressure, heart rate and respiratory rate in anaesthetised rats. The effect of 2DG (500 mg/kg, 1000 mg/kg, and 2000 mg/kg, p.o.) was also studied on various respiratory parameters viz., respiratory rate and tidal volume in conscious rats and mice using a computer program. RESULTS: The p.o. LD50 of 2DG was found to be >8000 mg/kg in mice and rats, and at this dose no death was observed. The LD50 in mice by i.v. route was found to be 8000 mg/kg. At this dose 2 out of 4 mice died and the death occurred within 6 h. A significant increase in the body weight was observed after p.o. administration of 2DG in rats at 500 mg/kg, 1000 mg/kg, and 2000 mg/kg doses. There was no significant change in the body weight at 4000 mg/kg and 8000 mg/kg by the p.o. route in rats and up to 8000 mg/kg by p.o. as well as i.v. routes in mice. Intravenous administration of 2DG (250 mg/kg, 500 mg/kg, and 1000 mg/kg) in anaesthetised rats showed a time-dependent decrease in the mean arterial blood pressure. There was no change in the heart rate in any of the treatment groups. The tidal volume was not changed significantly by p.o administration in conscious rats, but a significant decrease in the respiratory frequency at 500 mg/kg and 1000 mg/kg doses was observed. In the mice also there was no change in the tidal volume after p.o, administration, but the respiratory frequency decreased significantly at 2000 mg/kg dose. CONCLUSION: 2DG is a safe compound but can cause a fall in the blood pressure and a decrease in respiratory frequency at high doses.

COX-2, aspirin and metabolism of arachidonic, eicosapentaenoic and docosahexaenoic acids and their physiological and clinical significance.

Polyunsaturated fatty acids (PUFAs) are vital for normal growth and development and physiological function of various tissues in humans. PUFAs have immunomodulatory actions in addition to their ability to modulate inflammation, vascular reactivity, neurotransmission and stem cell biology. PUFAs and their metabolites possess both pro- and anti-inflammatory properties that underlie their actions and involvement in several diseases. Aspirin, a non-steroidal anti-inflammatory drug (NSAID), possesses both cyclo-oxygenase (COX) and lipoxygenase (LOX) inhibitory action and enhances the production of anti-inflammatory lipoxin A4 {(called as epi-lipoxin A4, aspirin-triggered lipoxins (ATLs))}. In addition, at low doses aspirin may not interfere with the production of prostacyclin (PGI2). Both lipoxin A4 and PGI2 have vasodilator, platelet anti-aggregator and anti-inflammatory actions that may underlie the beneficial actions of aspirin. Paradoxically, other NSAIDs may not have the same actions as that of aspirin on PUFA metabolism. Similar anti-inflammatory compounds are formed from eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) by the action of aspirin termed as resolvins (from EPA and DHA) and protectins and maresins from DHA. PUFAs: arachidonic acid (AA), EPA and DHA and their various products modulate not only inflammation and immune response but also possess actions on various genes, nuclear factors, cyclic AMP and GMP, G-protein coupled receptors (GPRs), hypothalamic neurotransmitters, hormones, cytokines and enzymes, and interact with nitric oxide, carbon monoxide, and hydrogen sulfide to regulate their formation and action and to form new compounds that have several biological actions. These pleiotropic actions of PUFAs and their metabolites may explain their ability to play a role in several physiological actions and diseases. The big challenge is to harness these actions to prevent and manage clinical conditions.

The glycolytic inhibitor 2-deoxy-D-glucose enhances the efficacy of etoposide in ehrlich ascites tumor-bearing mice.

Earlier studies have shown that 2-deoxy-D-glucose (2-DG), a glucose analogue and inhibitor of glycolytic ATP production significantly enhances the cytotoxic effects of anticancer agents like topoisomerase inhibitors (etoposide and camptothecin) and a radiomimetic drug (bleomycin) in established human tumor cell lines. Therefore, combination of 2-DG and DNA damage causing cytotoxic agents could be very useful in enhancing local tumor control. The purpose of the present studies was to investigate the therapeutic effects of etoposide and 2-DG in Ehrlich ascites tumor (EAT) bearing mice, grown as solid tumor as well as in the ascites form. Cell growth, cell cycle perturbations (flow cytometry), cytogenetic damage (micronuclei assay) and apoptosis (DNA content, morphological changes) were studied as parameters of cellular response, while delay in tumor growth and cure rate (tumor free survival) were evaluated as parameters of systemic response. Body weight and general condition as well as the damage to bone marrow and spleen was monitored to evaluate normal tissue toxicity. Intraperitoneal administration of etoposide (30 mg/Kg b. wt.) resulted in significant tumor growth delay and cure (approximately 11%) only in subcutaneous tumors leading to local tumor control. When etoposide was combined with 2-DG (2 g/Kg b. wt.; i.v./i.p.; 4 h after etoposide injection), these effects were further enhanced resulting in a cure rate of approximately 22% in case of subcutaneous tumors and 20% in ascites bearing mice. Analysis of cells obtained from ascitic fluid as well as solid tumors during follow up clearly showed that etoposide induced cell death was mainly due to apoptosis, which was enhanced further by 2-DG. Although, there was a significant level of toxicity revealed by reduced animal survival, decrease in body weight and damage to sensitive organ status like spleen and bone marrow at 60 mg/Kg b. wt. of etoposide, it was not significant at 30 mg/Kg b.wt. 2-DG, however, did not enhance the etoposide toxicity at both the doses. These results indicate that the administration of 2-DG can improve the local control of tumors without increasing normal tissue toxicity, thereby enhancing the therapeutic efficacy of topoisomerase inhibitor based anticancer drugs like etoposide.

Role of topoisomerases in cytotoxicity induced by DNA ligand Hoechst-33342 and UV-C in a glioma cell line.

DNA ligand Hoechst-33342 significantly enhances UV induced cytotoxicity in human glioma cell lines (BMG-1 & U-87) with supra additive increase in cell death, cytogenetic damage, cell cycle delay, apoptosis and inhibition of PLDR. Cytotoxicity of Hoechst-33342 arises due to its interference in the breakage-rejoining reaction of DNA topoisomerases by stabilization of cleavable complexes. Since topoisomerases have also been implicated in the generation of potentially lethal DNA breaks by interaction with various types of DNA damage including UV induced DNA lesions, we investigated in present studies the role of functional topoisomerases in the synergistic cytotoxicity of Hoechst-33342 and UV in a human glioma cell line (BMG-1). Topoisomerase I activity analyzed by the plasmid relaxation assay, was significantly enhanced upon UV irradiation, implying a possible role of this enzyme in the processing of UV induced lesions. However, this increase in the activity was reduced by >50% in cells incubated with Hoechst-33342 for 1 hr prior to irradiation. Imunoflowcytometric analysis of the chromatin bound topoisomerases I and II levels (cleavable complex) using topoisomerases I and II anti-antibodies showed a good correlation between the induction of apoptosis by Hoechst-33342 and UV and enhancement in the level of topoisomerase II mediated cleavable complexes. Induction of apoptosis was associated with a decline in the level of Bcl2. Taken together, these studies show that supra additive cytotoxic effects of UV-C and Hoechst-33342 in BMG-1 cells are consequences of enhanced stabilization of topo II mediated cleavable complexes and alterations in specific signal transduction pathways of apoptosis, besides the inhibition of topoisomerase mediated repair processes.

Glycolytic inhibitor, 2-deoxy-D-glucose, does not enhance radiation-induced apoptosis in mouse thymocytes and splenocytes in vitro.

Earlier studies have shown that 2-deoxy-D-glucose (2-DG), a glucose analogue and inhibitor of glycolytic ATP production selectively enhances radiation-induced damage in cancer cells by inhibiting the energy (ATP) dependent postirradiation DNA and cellular repair processes. A reduction in radiation induced cytogenetic damage has been reported in normal cells viz., peripheral blood lymphocytes and bone marrow cells. Since induction of apoptosis plays a major role in determining the radiosensitivity of some most sensitive normal cells including splenocytes and thymocytes, we investigated the effects of 2-DG on radiation induced apo tosis in these cells in vitro. Thymocytes and splenocytes isolated from normal Swiss albino mouse were irradiated with Co60 gamma-rays and analyzed for apoptosis at various post-irradiation times. 2-DG added at the time of irradiation was present till the termination of cultures. A time dependent, spontaneous apoptosis was evident in both the cell systems, with nearly 40% of the cells undergoing apoptosis at 12 hr of incubation. The dose response of radiation-induced apoptosis was essentially similar in both the cell systems and was dependent on the incubation time. More than 70% of the splenocytes and 60% of the thymocytes were apoptotic by 12 hr following an absorbed dose of 2 Gy. Presence of 2-DG marginally reduced the fraction of splenocytes undergoing apoptosis at all absorbed doses, while no change was observed in thymocytes. Presence of 2-DG did not significantly alter either the level or the rate of induction of spontaneous apoptosis in both these cell systems. These results are consistent with the earlier findings on radiation-induced cytogenetic damage in human PBL in vitro and mouse bone marrow cells and lend further support to the proposition that 2-DG does not enhance radiation damage in normal cells, while radiosensitizing the tumors and hence is an ideal adjuvant in the radiotherapy of tumors.

2-deoxy-D-glucose enhances the cytotoxicity of topoisomerase inhibitors in human tumor cell lines.

Resistance of tumors due to restricted drug accumulation and reversal of DNA lesions in tumor cells as well as normal tissue toxicity limit the efficacy of topoisomerase inhibition based anticancer drugs. It has been proposed that selective inhibition of energy dependent repair processes and enhanced retention of drug in cancer cells can significantly improve the therapeutic efficacy. The purpose of these studies was to verify this proposition by investigating the effects of 2-deoxy-D-glucose (2-DG) an inhibitor of the glycolytic ATP production on the cytotoxicity of certain topoisomerase inhibitors in human tumor cell lines. Human glioma (BMG-1 and U-87) and squamous carcinoma (4197 and 4451) cell lines were investigated with topo-poisons like etoposide (topo II inhibitor), camptothecin (topo I inhibitor) and hoechst-33342 (topo I and II inhibitor). DNA damage induction (halo assay), cell survival (macro colony assay), cytogenetic damage (micronuclei) and apoptosis (morphological analysis) were investigated. Presence of 2-DG for 2 h following exposure to the topoisomerase inhibitors enhanced the cell death (macro colony assay) in a concentration dependent manner and a 2-3-fold increase was observed at 5 mM (equimolar with glucose). Halo assay revealed that 2-DG inhibited the reversal of cleavable complex leading to the accumulation of DNA strand breaks. Under these conditions 2-DG enhanced the drug-induced micronuclei formation by nearly 2 folds with etoposide and hoechst-33342 and a 4-fold increase in delayed apoptosis was observed in case of etoposide. These results clearly demonstrate that presence of 2-DG for a few hours following exposure to topo-inhibitors enhances the cytotoxicity, primarily by increasing the cytogenetic damage.

Modification of the radiation induced damage by 2-deoxy-D-glucose in organ cultures of human cerebral gliomas.

Effects of a glucose antimetabolite, 2-deoxy-D-glucose (2-DG), on the gamma ray induced radiation damage have been studied in organ cultures of human cerebral gliomas. Percentage of cells with micronuclei (M-fraction) was used to assay the radiation damage. Experimental data indicate the following results. Untreated cerebral gliomas show considerable spatial heterogeneity in M-fraction; In spite of this heterogeneity, increases in M-fraction induced by gamma rays can be clearly observed, if multiple and randomly selected explants are analyzed for each group; The radiation induced M-fraction in different gliomas varies over a wide range; Presence of 2-DG (5 mM) for 4 h after irradiation leads to an increase in the radiation induced M-fraction in the majority of tumors, while in a smaller number (congruent to 25%) a decrease is observed under similar conditions. These results can be explained on the basis of a model postulating differential effects of 2-DG on the energy linked modulations of the processes of repair and fixation of DNA damage, which competitively influence the formation of micronuclei.

Hematoporphyrin derivatives potentiate the radiosensitizing effects of 2-deoxy-D-glucose in cancer cells.

PURPOSE: Two deoxy-D-glucose (2-DG), an inhibitor of glucose transport and glycolysis, has been shown to differentially inhibit the repair of radiation damage in cancer cells by reducing the flow of metabolic energy. Since hematoporphyrin derivatives (Hpd) inhibit certain enzymes of the respiratory metabolism, resulting in an increase in the glucose usage and glycolysis, Hpd could possibly enhance the energy-linked radiosensitizing effects of 2-DG in cancer cells. The purpose of the present work was to verify this suggestion. METHODS AND MATERIALS: Two human tumor cell lines (cerebral glioma, BMG-1 and squamous cell carcinoma, 4197) and a murine tumor cell line (Ehrlich ascites tumor [EAT], F-15) in vitro were investigated. A commercially available preparation of Hpd, Photosan-3 (PS-3) was used in the present studies. Cells incubated with 0-10 microg/ml PS-3 for 0-24 h before irradiation were exposed to 2.5 Gy of Co-60 gamma rays and maintained under liquid holding conditions for 1-4 h to facilitate repair. 2-DG (0-5 mM) added at the time of irradiation was present during the liquid holding. Radiation-induced cytogenetic damage (micronuclei formation) and cell death (macrocolony assay) were analyzed as parameters of radiation response. Effects of these radiosensitizers on glucose usage and glycolysis were also studied by measuring the glucose consumption and lactate production using enzymatic assays. RESULTS: The glucose consumption and lactate production of BMG-1 cells (0.83 and 1.43 pmole/cell/h) were twofold higher than in the 4197 cells (0.38 and 0.63 pmole/cell/h). Presence of PS-3 (10 microg/ml) enhanced the rate of glycolysis (glucose consumption and lactate production) in these cells by 35% to 65%, which was reduced by 20% to 40% in the presence of 5 mM 2-DG. In exponentially growing BMG-1 and EAT cells, presence of 2-DG (5 mM; equimolar with glucose) for 4 hours after irradiation increased the radiation-induced micronuclei formation and cell death by nearly 40%, whereas no significant effects could be observed in 4197 cells. In EAT cells, radiation was also observed to induce apoptotic death, which was significantly increased in the presence of the combination (PS-3 + 2-DG). The combination (PS-3 + 2-DG) enhanced the radiation damage in all three cell systems by 60-100%. Furthermore, the radiosensitizing effects of the combination (PS-3 + 2-DG) were higher at pH 6.7 as compared to pH 7. 4. In the plateau phase, presence of 2-DG alone did not significantly influence the radiation response of either BMG-1 or of 4197 cells, whereas in combination with PS-3, 2-DG enhanced the radiation damage in both these cell lines by 40% to 50%. Furthermore, in BMG-1 cells, the effects of 2-DG were observed to be reversible to a very great extent, while that of the combination were mostly irreversible. CONCLUSION: The hematoporphyrin derivative PS-3 enhances the radiosensitizing effects of 2-DG in cancer cells, possibly by further reducing the energy supply leading to an irreversible inhibition of DNA repair, and increased cytogenetic damage and cell death. Since both these compounds have been used in clinical practice, further studies to investigate their use in improving radiotherapy of tumors are warranted.