Shalmoli Bhattacharyya

Autophagy in Cancer Stem Cells: A Potential Link Between Chemoresistance, Recurrence, and Metastasis

Abstract Cancer cells require an uninterrupted nutritional supply for maintaining their proliferative needs and this high demand in concurrence with inadequate supply of blood and nutrition induces stress in these cells. These cells utilize various strategies like high glycolytic flux, redox signaling, and modulation of autophagy to avoid cell death and overcome nutritional deficiency. Autophagy allows the cell to generate ATP and other essential biochemical building blocks necessary under such adverse conditions. It is emerging as a decisive process in the development and progression of pathophysiological conditions that are associated with increased cancer risk. However, the precise role of autophagy in tumorigenesis is still debatable. Autophagy is a novel cytoprotective process to augment tumor cell survival under nutrient or growth factor starvation, metabolic stress, and hypoxia. The tumor hypoxic environment may provide site for the enrichment/expansion of the cancer stem cells (CSCs) and successive rapid tumor progression. CSCs are characteristically resistant to conventional anticancer therapy, which may contribute to treatment failure and tumor relapse. CSCs have the potential to regenerate for an indefinite period, which can impel tumor metastatic invasion. From last decade, preclinical research has focused on the diversity in CSC content within tumors that could affect their chemo- or radio-sensitivity by impeding with mechanisms of DNA repair and cell cycle progression. The aim of this review is predominantly directed on the recent developments in the CSCs during cancer treatment, role of autophagy in maintenance of CSC populations and their implications in the development of promising new cancer treatment options in future.

Autophagy inhibition suppresses the tumorigenic potential of cancer stem cell enriched side population in bladder cancer.

The mechanisms that underlie tumor formation and progression have not been elucidated in detail in cancer biology. Recently, the identification of a tumor cell subset defined as cancer stem cells (CSCs), which is enriched for tumor initiating capacity, has engendered new perspectives towards selective targeting of tumors. In this study, we isolated the side population (SP) cells which share characteristics of CSCs from bladder cancer cell lines, T24 and UM-UC-3 by fluorescence activated cell sorting. The cells were cultured in serum free medium and expression profile of stem cell like markers (SOX-2, NANOG, KLF-4 and OCT-4), drug resistant genes (ABCG2 and MDR1) and spheroid forming capability were examined in SP, non-side population (NSP) and bulk T24 and UM-UC-3 cells. We observed that SP cells possessed a higher mRNA expression of SOX-2, NANOG, KLF-4, OCT-4, ABCG2, and MDR1 as well as a higher spheroid forming ability as compared to other bulk cells or NSP cells. The SP cells had low ROS levels and high GSH/GSSG ratio which may contribute to radio-resistance. The SP cells also showed substantial resistance to gemcitabine, mitomycin and cisplatin compared with the NSP counterpart. A high autophagic flux was observed in the SP cells. Both pharmacological and siRNA mediated inhibition of autophagy potentiated the chemotherapeutic effects of gemcitabine, mitomycin and cisplatin in these cells. We concluded that the ABCG2 expressing SP cells show autophagy associated cell survival and may be a potent target for developing more effective treatment in bladder carcinoma to enhance patient survival.

A potentially important excretory-secretory product of Giardia lamblia.

In this study we have reported the detailed characterization of a 58 kDa excretory-secretory product (ESP) of Giardia lamblia. The method of purification has been simplified which has improved the purification fold as well as the yield of the ESP. The binding efficacy of disialoganglioside (GD2) to the purified ESP was found to be maximum among all other gangliosides used. The N-terminal sequence of the immunoreactive 29 kDa peptide obtained from partial tryptic digest of the ESP was found to be AD-FVPQVST. The IgG against the purified ESP (IgGES) showed cross-reactivity with the binding subunit of the commercially available cholera toxin and also with two protein bands of western cottonmouth moccasin snake toxin. The ESP could accumulate fluid in the intestine of sealed adult mice and also induce morphological changes in HEp-2 cells. The crude extract of G. lamblia trophozoites preincubated with Escherichia coli revealed 8-fold augmentation in the cytopathic activity on HEp-2 cells as compared to that of crude preparation from trophozoites only.

The alteration in signal transduction parameters induced by the excretory-secretory product from Giardia lamblia

The mechanism by which Giardia lamblia exerts its pathogenicity is likely to be multifactorial. A 58 kDa enterotoxin was purified and characterized from the excretory-secretory product (ESP) of the parasite (Kaur et al. 2001). In the present study an attempt has been made to elucidate the mechanism of action of the ESP, a potentially important enterotoxin. A 41 kDa glycoprotein was identified in the mouse enterocyte membrane fraction with which the ESP interacted in a GM1-specific manner. The GTPase activity was reduced in enterocytes stimulated with the ESP, resulting in an increase in the level of adenylate cyclase-dependent cyclic adenosine monophosphate (cAMP). The activity of protein kinase A (PKA) in the enterocytes was also upregulated after ESP treatment. Ultimately, a significant increase in intracellular Ca2+ concentration and decrease in cytosolic Cl- level were noticed in ESP-stimulated mouse enterocytes. Thus it is possible that the enterotoxic ESP could bind to the 41 kDa glycoprotein (receptor?) on the enterocytes and activate the G-protein-mediated signal transduction pathway resulting in alteration of electrolyte transport.

Status of Oxidative Stress in Patients With Renal Cell Carcinoma

PURPOSE Although oxidative stress is implicated in renal cell carcinoma pathogenesis, to our knowledge changes in oxidative stress parameters in patients who undergo surgery for renal cell carcinoma have not been studied previously. We investigated the status of oxidative stress in patients with renal cell carcinoma. MATERIALS AND METHODS Reactive oxygen species, nitric oxide and glutathione were measured in the blood of 68 patients with renal tumor and in 30 age matched normal controls. Levels were measured again 1 week, and 1 and 2 months postoperatively in patients who underwent surgery for renal cell carcinoma. Levels of superoxide dismutase, catalase and lipid peroxidation were measured in tumor tissue and in normal renal parenchyma in 51 patients with renal tumor. RESULTS Significantly increased reactive oxygen species and nitric oxide, and decreased glutathione were observed in patients with renal cell carcinoma compared to normal subjects and in patients with benign tumors. Superoxide dismutase and lipid peroxidation were increased and catalase was decreased in tumor tissue compared to normal renal tissue. Oxidative stress correlated with renal cell carcinoma grade and stage but decreased after curative resection. Patients with metastatic disease had persistently increased oxidative stress parameters. Antioxidant enzyme levels in benign tumor tissue were significantly higher than in renal cell carcinoma. CONCLUSIONS Patients with renal cell carcinoma have increased oxidative stress, which is effectively alleviated by curative resection. In patients with benign tumors antioxidant defense mechanisms maintain normal redox status.

Mode of action of a potentially important excretory--secretory product from Giardia lamblia in mice enterocytes.

Giardia, a common enteric protozoan parasite is a well-recognized cause of diarrhoeal illness. The detailed mechanism of diarrhoea due to this infection is not well understood. A 58 kDa enterotoxin (ESP) was purified from the excretory-secretory product of the parasite. The present study was designed to investigate the mode of action of this enterotoxin of G. lamblia in mice enterocytes. An increase in cyclic adenosine monophosphate level, as well as intracellular Ca2+ concentration, was observed in the ESP-triggered enterocytes. The levels of phospholipase Cgamma1 and inositol triphosphate were found to be upregulated. The activity of protein kinase C (PKC) in the enterocytes was also enhanced following stimulation with the ESP. An increase in the level of reactive oxygen species in ESP-stimulated cells correlated well with the decline in the activity of antioxidant enzymes (superoxide dismutase and catalase). The significantly high levels of nitrite and citrulline indicated the generation of reactive nitrogen intermediates in the ESP-triggered enterocytes. Thus, ESP could induce cross-talk among the different signal transduction pathways in the enterocytes, which could together bring about a common secretory response.

The voyage of stem cell toward terminal differentiation: a brief overview

Presently, worldwide attempts are being made to apply stem cells and stem cell-derived products to a wide range of clinical applications and for the development of cell-based therapies. In order to harness stem cells and manipulate them for therapeutic application, it is very important to understand the basic biology of stem cells and identify the factors that govern the dynamics of these cells in the body. Several signaling pathways have emerged as key regulators of stem cells. Some of these signaling pathways regulate the stem cells proliferative capacity and therefore act as direct regulators of the stem cell, whereas others are involved in shaping and maintaining the stem cell niche and therefore act as indirect regulators of the stem cell. It is difficult to identify which signaling pathways critically affect the stem cells behavior and which are important for maintaining the quiescent population. A stem cell receives different extrinsic signals compared with the bulk population and responds to them differently. In order to manipulate these adult cells for therapeutic approaches it is crucial to identify how signaling pathways regulate stem cells either directly by regulating proliferative status or indirectly by influencing the niche. The main challenge is to identify whether different factors provide diverse extrinsic signals to the stem cell and its daughter cell population, or whether there are intrinsic differences in stem cell and daughter cell populations that is reflected in their behavior. In this study, we will focus on the various aspects of stem cell biology and differentiation, as well as exploring the potential strategies to intervene the differentiation process in order to obtain the desired yield of cells applicable in regenerative medicine.

Cellular response induced by a galactose-specific adhesin of enteroaggregative Escherichia coli in INT-407 cells

In the present study, the role of a fimbrial galactose-specific adhesin of the T7 strain of enteroaggregative Escherichia coli (EAEC-T7) in the signal transduction pathways in human small intestinal epithelial cells (INT-407) was explored. The adhesin was purified by anion exchange chromatography using a Mono Q HR5/5 column in the AKTA purifier system. The characteristic stacked brick pattern of aggregative adherence of EAEC-T7 to INT-407 cells was found to be inhibited in the presence of immunoglobulin G against the purified adhesin as well as d-galactose. The adhesin induced a significant increase in the intracellular calcium concentration [Ca(2+)](i) in INT-407 cells, which was reduced in the presence of dantrolene (inhibitor of intracellular calcium stores), verapamil, calciseptin (calcium channel blockers) as well as neomycin [inhibitor of phospholipase C (PLC)]. Further, an increased level of PLCgamma1 and inositol 1,4,5-tri phosphate as well as enhanced activity of protein kinase C (PKC) in the adhesin-stimulated cells were found to be downregulated in the presence of neomycin and U73122 (inhibitors of PLC) and H-7 (inhibitor of PKC), respectively. The adhesin could also induce interleukin-8 secretion from INT-407 cells, which was inhibited in the presence of dantrolene as well as staurosporin (inhibitor of PKC). Collectively, our results have suggested that the galactose-specific adhesin-induced signal transduction pathway might play a crucial role in the EAEC-induced pathogenesis.

Secretome Cues Modulate the Neurogenic Potential of Bone Marrow and Dental Stem Cells

Dental tissue is emerging as a promising source of stem cells especially in nerve regeneration mainly due to their neural origin and ease of harvest. We isolated dental stem cells from three sources, namely, dental pulp (DPSCs), dental follicle (DFSCs), and apical papilla (SCAP), and explored the efficacy of each towards neural differentiation in comparison to bone marrow-derived stem cells. The neural differentiation potential was assessed by expression of various neural markers and neurosphere assay. We observed that DPSCs were inherently predisposed towards neural lineage. To further delineate the paracrine cues responsible for the differences in neural differentiation potential, we harvested the conditioned secretome from each of the stem cell population and observed their effect on colony formation, neurite extension, and neural gene expression of IMR-32, a pre-neuroblastic cell line. We found that neural differentiation was significantly enhanced when IMR-32 cells were treated with secretome derived from DMSCs as compared to the same from BMSCs. Th1/Th2/Th17 cytokine array revealed DPSC secretome had higher expression of the cytokines like GCSF, IFNγ, and TGFβ that promote neural differentiation. Thus, we concluded that DPSCs may be the preferred source of cells for obtaining neural lineage among the four sources of stem cells. Our results also indicate that the DPSC-secreted factors may be responsible for their propensity towards neural differentiation. This study suggests that DPSCs and their secretomes can be a potentially lucrative source for cell-based and “cell-free” (secretome) therapy for neural disorders and injury.

Cathepsin B, H and L inhibitors as cell proliferating agents: design, synthesis, computational and pharmacological studies of some novel 2-(2-naphthoyl)-6,6-dimethyl-3-aryl-2,3,6,7-tetrahydrobenzofuran-4(5H)-ones

Elevated levels of cathepsins B, H and L in disease conditions such as inflammation and cancer accentuate the need for design, synthesis and pharmacological evaluation of new compounds, keeping in view target-specific therapy. The present work describes the one pot multicomponent synthesis of some novel 2-(2-naphthoyl)-6,6-dimethyl-3-aryl-2,3,6,7-tetrahydrobenzofuran-4(5H)-ones in good yields. The synthesized compounds were analysed by spectral and X-crystallographic studies and have been found to be potential inhibitors to cathepsins B, H and L. The extent of inhibition varied with the substitution. Among the synthesized compounds, nitro-substituted compound 1d has been evaluated as most inhibitory to cathepsin H, however fluoro-substituted compound 1f was the best inhibitor of cathepsin B and cathepsin L. The compounds have been found more selective towards cathepsin L. In vitro inhibition studies correlate well when tested using MTT assay on HepG2 cells, a hepatocellular carcinoma cell line. The results validated by in silico studies performed with iGemDock predicted that among the synthesized compounds, 1d experiences the highest affinity for cathepsin B and H sites, whereas 1f has the highest affinity to cathepsin L.