Avishek Ganguly

Targeting Mitochondrial Cell Death Pathway to Overcome Drug Resistance with a Newly Developed Iron Chelate

Background Multi drug resistance (MDR) or cross-resistance to multiple classes of chemotherapeutic agents is a major obstacle to successful application of chemotherapy and a basic problem in cancer biology. The multidrug resistance gene, MDR1, and its gene product P-glycoprotein (P-gp) are an important determinant of MDR. Therefore, there is an urgent need for development of novel compounds that are not substrates of P-glycoprotein and are effective against drug-resistant cancer. Methodology/Principal Findings In this present study, we have synthesized a novel, redox active Fe (II) complex (chelate), iron N- (2-hydroxy acetophenone) glycinate (FeNG). The structure of the complex has been determined by spectroscopic means. To evaluate the cytotoxic effect of FeNG we used doxorubicin resistant and/or sensitive T lymphoblastic leukemia cells and show that FeNG kills both the cell types irrespective of their MDR phenotype. Moreover, FeNG induces apoptosis in doxorubicin resistance T lymphoblastic leukemia cell through mitochondrial pathway via generation reactive oxygen species (ROS). This is substantiated by the fact that the antioxidant N-acetyle-cysteine (NAC) could completely block ROS generation and, subsequently, abrogated FeNG induced apoptosis. Therefore, FeNG induces the doxorubicin resistant T lymphoblastic leukemia cells to undergo apoptosis and thus overcome MDR. Conclusion/Significance Our study provides evidence that FeNG, a redox active metal chelate may be a promising new therapeutic agent against drug resistance cancers.

A Novel Copper Chelate Modulates Tumor Associated Macrophages to Promote Anti-Tumor Response of T Cells

Background At the early stages of carcinogenesis, the induction of tumor specific T cell mediated immunity seems to block the tumor growth and give protective anti-tumor immune response. However, tumor associated macrophages (TAMs) might play an immunosuppressive role and subvert this anti tumor immunity leading to tumor progression and metastasis. Methodology/Principal Findings The Cu (II) complex, (chelate), copper N-(2-hydroxy acetophenone) glycinate (CuNG), synthesized by us, has previously been shown to have a potential usefulness in immunotherapy of multiple drug resistant cancers. The current study demonstrates that CuNG treatment of TAMs modulates their status from immunosuppressive to proimmunogenic nature. Interestingly, these activated TAMs produced high levels of IL-12 along with low levels of IL-10 that not only allowed strong Th1 response marked by generation of high levels of IFN-γ but also reduced activation induced T cell death. Similarly, CuNG treatment of peripheral blood monocytes from chemotherapy and/or radiotherapy refractory cancer patients also modulated their cytokine status. Most intriguingly, CuNG treated TAMs could influence reprogramming of TGF-β producing CD4+CD25+ T cells toward IFN-γ producing T cells. Conclusion/Significance Our results show the potential usefulness of CuNG in immunotherapy of drug-resistant cancers through reprogramming of TAMs that in turn reprogram the T cells and reeducate the T helper function to elicit proper anti-tumorogenic Th1 response leading to effective reduction in tumor growth.

The role of a Schiff base scaffold, N-(2-hydroxy acetophenone) glycinate-in overcoming multidrug resistance in cancer

Drug resistance is a problem that hinders the numerous successes of chemotherapeutic intervention of cancer and continues to be a major obstacle for cures. Till date, several attempts have been made to develop suitable multidrug resistance (MDR) reversing agents. But, throughout the clinical development of MDR reversing agents, patients repeatedly suffer from toxicities. So far, some anticancer activity of Schiff bases which are the condensation products of carbonyl compounds and primary amines and their metal complexes has been described. But, overcoming multidrug resistance, by the use of such small molecules still remain unexplored. Under this backdrop, in search of less toxic and more effective MDR reversing agents our laboratory has developed the different metal chelates of Schiff base N-(2-hydroxy acetophenone)glycinate (NG) which is structurally similar to azatyrosine [L-β-(5-hydroxy-2-pyridyl)-alanine] that inhibits tumor formation by deactivating the c-Raf-1 kinase and c-Ha-ras signalling pathway. A decade-long research proposes possible strategies to overcome MDR by exploiting the chemical nature of such metal chelates. In this review we have catalogued the success of metal chelates of NG to overcome MDR in cancer. The review depict that the problem of MDR can be circumvent by synchronized activation of immunogenic cell death pathways that utilize the components of a hosts immune system to kill cancer cells in combination with other conventional strategies. The current wealth of preclinical information promises better understanding of the cellular processes underlying MDR reversing activity of metal derivatives of NG and thus exposes several cellular targets for rational designing of new generation of Schiff base metal chelates as MDR reversing agents.

Reprogramming of TAM toward proimmunogenic type through regulation of MAP kinases using a redox-active copper chelate

TAMs, present in the tumor microenvironment, play an immunosuppressive role, leading to tumor progression and metastasis. Recently, numerous attempts have been made to switch immunosuppressive TAMs into an immunostimulatory type. Previously, we showed that a copper chelate, viz., copper N‐(2‐hydroxy acetophenone) glycinate [CuNG], can reprogram TAMs toward the proimmunogenic type to mount an antitumor immune response, but the underlying molecular mechanisms of skewing TAMs toward the proimmunogenic type remain elusive. Herein, we tried to explore the signaling mechanisms responsible for the reprogramming of TAMs. We observed that CuNG‐induced ROS generation triggers activation of two MAPKs, i.e., p38MAPK and ERK1/2, and also causes up‐regulation of intracellular glutathione. Furthermore, activation of p38 MAPK up‐regulated the initial IL‐12 production and the activation of ERK1/2 in tandem with GSH, found responsible for IFN‐γ production by TAMs. This IFN‐γ, in turn, prolonged IL‐12 production and down‐regulated TGF‐β production and thus, plays the decisive role in CuNG‐mediated reprogramming of regulatory cytokine production by TAMs. Our work highlights that ROS‐mediated activation of MAPKs can convert suppressive macrophages toward the proimmunogenic type. Thus, the present study opens the possibility of targeting TAMs by the use of redox‐active compounds for designing a novel, therapeutic strategy against cancer.

An in vitro and in vivo study of a novel zinc complex, zinc N-(2-hydroxyacetophenone)glycinate to overcome multidrug resistance in cancer

Multiple drug resistance (MDR) remains a major clinical challenge for cancer treatment. P-glycoprotein is the major contributor and they exceed their role in the chemotherapy resistance of most of the malignancies. Attempts in several preclinical and clinical studies to reverse the MDR phenomenon by using MDR modulators have not yet generated promising results. In the present study, a co-ordination complex of zinc viz., Zn N-(2-hydroxyacetophenone)glycinate (ZnNG) has been synthesized, characterized and its antitumour activity was tested in vitro against drug sensitive and resistant human T-lymphoblastic leukemic cell lines (CCRF/CEM and CEM/ADR5000 respectively) and in vivo against Ehrlich ascites carcinoma (EAC) implanted in female Swiss albino mice. To evaluate the cytotoxic potential of ZnNG, we used sensitive CCRF/CEM and drug resistant CEM/ADR 5000 cell lines in vitro. Moreover, ZnNG also has the potential ability to reverse the multidrug resistance phenotype in drug resistant CEM/ADR 5000 cell line and induces apoptosis in combination with vinblastine. ZnNG remarkably increases the life span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of EAC in presence and in absence of doxorubicin. In addition, intraperitoneal application of ZnNG in mice does not show any systemic toxicity in preliminary trials in normal mice. To conclude, a novel metal chelate of zinc viz., ZnNG, may be a promising therapeutic agent against sensitive as well as drug resistant cancers.

A novel manganese complex, Mn-(II) N-(2-hydroxy acetophenone) glycinate overcomes multidrug-resistance in cancer

Multidrug resistance (MDR) remains a significant problem for effective cancer chemotherapy. In spite of considerable advances in drug discovery, most of the cancer cases still stay incurable because of resistance to chemotherapy. We synthesized a novel, Mn (II) complex (chelate), viz., manganese N-(2-hydroxy acetophenone) glycinate (MnNG) that exhibits considerable efficacy to overcome drug resistant cancer. The antiproliferative activity of MnNG was studied on doxorubicin resistant and sensitive human T lymphoblastic leukemia cells (CEM/ADR 5000 and CCRF/CEM). MnNG induced apoptosis significantly in CEM/ADR 5000 cells probably through generation of reactive oxygen species. Moreover, intraperitoneal (i.p.) application of MnNG at non-toxic doses caused significant increase in the life-span of Swiss albino mice bearing sensitive and doxorubicin resistant subline of Ehrlich ascites carcinoma cells.

Targeting the mitochondrial pathway to induce apoptosis/necrosis through ROS by a newly developed Schiff's base to overcome MDR in cancer.

Multidrug resistance (MDR) in cancer, a major obstacle to successful application of cancer chemotherapy, is often characterized by over-expression of multidrug resistance-related proteins such as MRP1, P-gp or elevated glutathione (GSH) level. Efflux of drugs by functional P-gp, MRP1 and elevated GSH level can confer resistance to apoptosis induced by a range of different stimuli. Therefore, it is necessary to develop new cell death inducers with relatively lower toxicity toward non-malignant cells that can overcome MDR by induction of apoptotic or non-apoptotic cell death pathways. Herein we report the synthesis and spectroscopic characterization of a GSH depleting, redox active Schiffs base, viz., potassium-N-(2-hydroxy-3-methoxy-benzaldehyde)-alaninate (PHMBA). Cytotoxic potential of PHMBA has been studied in doxorubicin-resistant and -sensitive T lymphoblastic leukemia cells and Ehrlich ascites carcinoma (EAC) cells. PHMBA kills both the cell types irrespective of their drug-resistance phenotype following apoptotic/necrotic pathways. Moreover, PHMBA-induced cell death is associated with oxidative stress mediated mitochondrial pathway as the H(2)O(2) inhibitor PEG-Catalase abrogated PHMBA-induced apoptosis/necrosis. PHMBA induces anti-tumor activity in both doxorubicin-sensitive and -resistant EAC-tumor-bearing Swiss albino mice. The non-toxicity of PHMBA was also confirmed through cytotoxicity studies on normal cell lines like PBMC, NIH3T3 and Chang Liver. To summarise, our data provide compelling rationale for future clinical use of this redox active Schiffs base in treatment of cancer patients irrespective of their drug-resistance status.

ROS and RNS induced apoptosis through p53 and iNOS mediated pathway by a dibasic hydroxamic acid molecule in leukemia cells

Anticancer drugs induce apoptosis to cancer cells and also exhibit undesired toxicity to normal cells. Therefore development of novel agents triggering apoptosis and have low toxicity towards normal cells is most important. Hydroxamic acids suppress tumour cell growth through apoptosis but the underlying mechanism is poorly understood. Herein, we describe the apoptotic potential of a dibasic hydroxamic acid derivative, viz., oxayl bis (N-phenyl) hydroxamic acid (OBPHA), which induces apoptosis through generation of both ROS and NO in doxorubicin resistant T-lymphoblastic leukemia, CEM/ADR5000 cells. Present study discloses that OBPHA selectively kills cancerous cells irrespective of their drug resistant phenotype. We also determined the crystal structure of OBPHA to understand the structural requirements for apoptosis; the study reveals that the presence of substituted hydroxamic acid groups (-CO-NH-OH) favours the generation of NO possibly through auto degeneration. Along with the induction of caspase 3 mediated intrinsic apoptosis; OBPHA also activates p53 dependent signalling cascade and downregulates HDAC3 expression in a time dependent manner possibly due to increased ROS and NO production and simultaneous decrease in cellular GSH level. Thus ROS and NO mediated downstream signalling are essential for the anticancer effect of OBPHA. Therefore OBPHA, having a structurally relevant pharmacophore provides important insight into the development of new ROS and RNS generating chemicals inducing p53 dependent apoptosis.

Iron N-(2-hydroxy acetophenone) glycinate (FeNG), a non-toxic glutathione depletor circumvents doxorubicin resistance in Ehrlich ascites carcinoma cells in vivo

Multidrug resistance-associated protein 1 (MRP1) reduces intracellular anticancer drug accumulation either by co transporting them with glutathione (GSH) or extruding drug-GSH conjugates outside of the cell. Thus, MRP1 confers multidrug resistance (MDR) and worsen successful chemotherapeutic treatment against cancer. Although the exact mechanism of MRP1 involved in MDR remains unknown, the elevated level of intracellular GSH is considered as a key factor responsible for MDR in cancer. Hence the quest for non-toxic molecules that are able to deplete intracellular GSH has profound importance to subdue MDR. The present preclinical study depicts the resistance reversal potentiality of an iron complex; viz. Ferrous N-(2-hydroxy acetophenone) glycinate (FeNG) developed by us in doxorubicin resistant Ehrlich ascites carcinoma (EAC/Dox) cells. FeNG potentiate cytotoxic effect of doxorubicin on EAC/Dox cells ex vivo and also increases the survivability EAC/Dox bearing Swiss albino mice in vivo as well. Moreover, in vivo administration of FeNG significantly depletes intracellular GSH with ensuant increase in doxorubicin concentration in EAC/Dox cells without alternation of MRP1 expression. In addition, intra-peritoneal (i.p.) application of FeNG in normal or EAC/Dox bearing mice does not cause any systemic toxicity in preliminary trials in mouse Ehrlich ascites carcinoma model. Therefore, the present report provides evidence that FeNG may be a promising new resistance modifying agent against drug resistant cancers.