Swatilekha Ghosh

Inhibition of Epithelial to Mesenchymal Transition by E-cadherin Up-regulation via Repression of Slug Transcription and Inhibition of E-cadherin Degradation DUAL ROLE OF SCAFFOLD/MATRIX ATTACHMENT REGION-BINDING PROTEIN 1 (SMAR1) IN BREAST CANCER CELLS

Background: Epithelial-mesenchymal transition (EMT) is an important program in tumor metastasis. Results: SMAR1 inhibits EMT by up-regulating E-cadherin in a dual manner via repression of Slug transcription and inhibition of E-cadherin degradation. Conclusion: SMAR1 functions as a critical protein in regulating EMT. Significance: This study provides a potential mechanism for the contribution of SMAR1 in inhibiting breast cancer metastasis. The evolution of the cancer cell into a metastatic entity is the major cause of death in patients with cancer. It has been acknowledged that aberrant activation of a latent embryonic program, known as the epithelial-mesenchymal transition (EMT), can endow cancer cells with the migratory and invasive capabilities associated with metastatic competence for which E-cadherin switch is a well-established hallmark. Discerning the molecular mechanisms that regulate E-cadherin expression is therefore critical for understanding tumor invasiveness and metastasis. Here we report that SMAR1 overexpression inhibits EMT and decelerates the migratory potential of breast cancer cells by up-regulating E-cadherin in a bidirectional manner. While SMAR1-dependent transcriptional repression of Slug by direct recruitment of SMAR1/HDAC1 complex to the matrix attachment region site present in the Slug promoter restores E-cadherin expression, SMAR1 also hinders E-cadherin-MDM2 interaction thereby reducing ubiquitination and degradation of E-cadherin protein. Consistently, siRNA knockdown of SMAR1 expression in these breast cancer cells results in a coordinative action of Slug-mediated repression of E-cadherin transcription, as well as degradation of E-cadherin protein through MDM2, up-regulating breast cancer cell migration. These results indicate a crucial role for SMAR1 in restraining breast cancer cell migration and suggest the candidature of this scaffold matrix-associated region-binding protein as a tumor suppressor.

PEGylated-thymoquinone-nanoparticle mediated retardation of breast cancer cell migration by deregulation of cytoskeletal actin polymerization through miR-34a

Thymoquinone (TQ), a major active constituent of black seeds of Nigella sativa, has potential medical applications including spectrum of therapeutic properties against different cancers. However, little is known about their effect on breast cancer cell migration, which is the cause of over 90% of deaths worldwide. Herein, we have synthesized TQ-encapsulated nanoparticles using biodegradable, hydrophilic polymers like polyvinylpyrrolidone (PVP) and polyethyleneglycol (PEG) to overcome TQs poor aqueous solubility, thermal and light sensitivity as well as consequently, minimal systemic bioavailability which can greatly improve the cancer treatment efficiency. Sizes of synthesized TQ-Nps were found to be below 50 nm and they were mostly spherical in shape with smooth surface texture. Estimation of the zeta potential also revealed that all the three TQ-Nps were negatively charged which also facilitated their cellular uptake. In the present investigation, we provide direct evidence that TQ-Nps showed more efficiency in killing cancer cells as well as proved to be less toxic to normal cells at a significantly lower dose than TQ. Interestingly, evaluation of the anti-migratory effect of the TQ-Nps, revealed that PEG4000-TQ-Nps showed much potent anti-migratory properties than the other types. Further studies indicated that PEG4000-TQ-Nps could significantly increase the expression of miR-34a through p53. Moreover, NPs mediated miR-34a up-regulation directly down-regulated Rac1 expression followed by actin depolymerisation thereby disrupting the actin cytoskeleton which leads to significant reduction in the lamellipodia and filopodia formation on cell surfaces thus retarding cell migration. Considering the biodegradability, non-toxicity and effectivity of PEG4000-TQ-Nps against cancer cell migration, TQ-Nps may provide new insights into specific therapeutic approach for cancer treatment.

Tailored-CuO-nanowire decorated with folic acid mediated coupling of the mitochondrial-ROS generation and miR425-PTEN axis in furnishing potent anti-cancer activity in human triple negative breast carcinoma cells.

Metal oxide nanoparticles are the forthcoming anti-tumor therapeutics and provide a versatile platform in the development of therapeutic approaches for drug-resistant cancers such as triple negative breast cancer (TNBC). Copper oxide nanoparticles have been characterized as anti-cancer agents but its toxicity has been a matter of concern. Herein, we have developed a targeted CuO Nanowire fabricated with Folic acid (CuO-Nw-FA) that enables enhanced cellular uptake in TNBC cells without imparting significant toxicity in normal cellular system. In the present study, we enumerated that CuO-Nw-FA caused mitochondrial-dependent apoptosis in MDAMB-231 cells. Furthermore, CuO-Nw-FA mediated cytosolic retardation of NF-κB favoured inactivation of miR-425 and henceforth activated PTEN to induce apoptosis in TNBC cells. Simultaneously, CuO-Nw-FA also restricted the in-vitro cell migration through the miR-425/PTEN axis via pFAK. Studies extended to ex-ovo and in-vivo mice models further validated the efficacy of CuO-Nw-FA. Additionally, the accumulations of nanoparticles in tumor as well as different organs in mice were examined by in-vivo biodistribution and ex-vivo optical imaging studies. Thus our results cumulatively propose that CuO-Nw-FA cross-talks two distinct signalling pathways to induce apoptosis and retard migration in TNBC cells and raises the possibility for the use of CuO-Nw-FA as a potent anti-tumor agent.

Nifetepimine, a Dihydropyrimidone, Ensures CD4+ T Cell Survival in a Tumor Microenvironment by Maneuvering Sarco(endo)plasmic Reticulum Ca2+ ATPase (SERCA)

Background: Tumor-induced SERCA3 up-regulation is a major cause of death of CD4+T lymphocytes leading to immune suppression in cancer bearers. Results: Nifetepimine down-modulates SERCA3 expression and thereby protects the lymphocytes from tumor-induced apoptosis. Conclusion: The present finding strongly suggests nifetepimine as a potent immuno-restoring agent that protects T lymphocytes from tumor insult. Significance: The results suggest that nifetepimine may be developed into a potent immuno-restoring agent in tumor-bearers. Multiple mechanisms have been proposed by which tumors induce T cell apoptosis to circumvent tumor immune-surveillance. Although sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) have long been known to regulate intracellular Ca2+ homeostasis, few studies have examined the role of SERCA in processes of T lymphocyte survival and activation. In this context it remains largely unexplored as to how tumors jeopardize SERCA function to disable T cell-mediated anti-tumor immunity. Here, we show that human CD4+ T cells in the presence of tumor conditions manifested an up-regulation of SERCA3 expression that resulted in development of endoplasmic reticulum stress leading to CD4+ T cell apoptosis. Prostaglandin E2 produced by the tumor cell plays a critical role in up-regulating SERCA3 by enhancing the binding of its transcription factor Sp1. Gene manipulation and pharmacological approaches further established that an increase in SERCA expression also resulted in subsequent inhibition of PKCα and -θ and retention of NFκB in the cytosol; however, down-modulation of SERCA3 expression by a dihydropyrimidone derivative, ethyl-4-(3-nitro)-phenyl-6-methyl-2-oxo-1,2,3,4-tetrahydropyrimidine-5 carboxylate (nifetepimine), protected the CD4+ T cells from tumor-induced apoptosis. In fact, nifetepimine-mediated restoration of PKC activity resulted in nuclear translocation of p65NFκB, thereby ensuring its survival. Studies further undertaken in a tumor-bearing mice model revalidated the immunoprotective role of nifetepimine. Our present study thus strongly suggests that imbalance in cellular calcium homeostasis is an important factor leading to CD4+ T cell death during cancer and holds promise that nifetepimine may have the potential to be used as an immunorestoring agent in cancer bearers.

Cross-talk between endoplasmic reticulum (ER) stress and the MEK/ERK pathway potentiates apoptosis in human triple negative breast carcinoma cells: role of a dihydropyrimidone, nifetepimine.

Background: Triple negative breast cancers (TNBC) are resistant to conventional therapies and molecular targets of TNBC are not explored in detail. Results: Coupling of ER stress and the MEK/ERK pathway by nifetepimine effectively kills TNBC cells. Conclusion: Nifetepimine plays a significant role in inducing apoptosis in TNBC cells. Significance: Our findings strongly suggest that nifetepimine may act as a potent anti-cancer agent for treatment of TNBC. Triple negative breast cancers (TNBC) are among the most aggressive and therapy-resistant breast tumors and currently possess almost no molecular targets for therapeutic options in this horizon. In the present study we discerned the molecular mechanisms of potential interaction between the endoplasmic reticulum (ER) stress response and the MEK/ERK pathway in inducing apoptosis in TNBC cells. Here we observed that induction of ER stress alone was not sufficient to trigger significant apoptosis but simultaneous inhibition of the MEK/ERK pathway enhanced ER stress-induced apoptosis via a caspase-dependent mechanism. Our study also demonstrated nifetepimine, a dihydropyrimidone derivative as a potent anti-cancer agent in TNBC cells. Nifetepimine down-regulated the MEK/ERK pathway in MDAMB-231 and MDAMB-468 cells and resulted in blockage of ER stress-mediated GRP78 up-regulation. Detailed mechanistic studies also revealed that nifetepimine by down-regulating pERK expression also declined the promoter binding activity of TFII-I to the GRP78 promoter and in turn regulated GRP78 transcription. Studies further extended to in vivo Swiss albino and SCID mice models also revalidated the anti-carcinogenic property of nifetepimine. Thus our findings cumulatively suggest that nifetepimine couples two distinct signaling pathways to induce the apoptotic death cascade in TNBC cells and raises the possibility for the use of nifetepimine as a potent anti-cancer agent with strong immune-restoring properties for therapeutic intervention for this group of cancer bearers.

Regioselective N1-alkylation of 3,4-dihydropyrimidine-2(1H)-ones: Screening of their biological activities against Ca2+-ATPase

A regioselective N1-alkylation of 3,4-dihydropyrimidin-2(1H)-ones using a very efficient mild base Cs(2)CO(3) and alkyl halides at room temperature has been reported. The selectivity of this methodology is excellent and the yields of the alkylated products are very good. Furthermore inhibitory action of both the 3,4-dihydropyrimidin-2(1H)-ones and the N1-alkylated derivatives were tested on Ca(2+)-ATPase, which revealed that the parent compounds can act as Ca(2+)-ATPase inhibitors whereas the N1-alkylated derivatives are inefficient for this purpose.

Phemindole, a Synthetic Di-indole Derivative Maneuvers the Store Operated Calcium Entry (SOCE) to Induce Potent Anti-Carcinogenic Activity in Human Triple Negative Breast Cancer Cells

Triple-negative breast cancer (TNBC), is a specific subtype of epithelial breast tumors that are immuno-histochemically negative for the protein expression of the estrogen receptor (ER), the progesterone receptor (PR) and lack over expression/gene amplification of HER2. This subtype of breast cancers is highly metastatic, shows poor prognosis and hence represents an important clinical challenge to researchers worldwide. Thus alternative approaches of drug development for TNBC have gained utmost importance in the present times. Dietary indole and its derivatives have gained prominence as anti-cancer agents and new therapeutic approaches are being developed to target them against TNBC. But a major drawback with 3, 3′di Indolyl methane (DIM) is their poor bioavailability and high effective concentration against TNBC. However, the Aryl methyl ring substituted analogs of DIM display interesting anti-cancer activity in breast cancer cells. In the current study we report the synthesis of a novel synthetic aryl methyl ring substituted analog of DIM, named as Phemindole as an effective anti-tumor agent against TNBC cells. Furthermore, we enumerated that Phemindole caused reactive oxygen species mediated mitochondrial-dependent apoptosis in MDAMB-231 cells. Furthermore, Phemindole mediated Store Operated Calcium Entry (SOCE) retardation favored inactivation of STIM1 and henceforth activated ER stress to induce apoptosis in TNBC cells. Simultaneously, Phemindole was also found to restrict the in vitro cell migration through its anti mitotic property and pFAK regulation. Studies extended to ex ovo and in vivo mice models further validated the efficacy of Phemindole. Thus our results cumulatively propose Phemindole as a new chemotherapeutic regime which might be effective to target the deadly aspects of the TNBC.

Elucidation of the Involvement of p14, a Sperm Protein during Maturation, Capacitation and Acrosome Reaction of Caprine Spermatozoa

Mammalian sperm capacitation is an essential prerequisite to fertilization. Although progress is being made in understanding the physiology and biochemistry of capacitation, little has been yet explored about the potential role(s) of individual sperm cell protein during this process. Therefore elucidation of the role of different sperm proteins in the process of capacitation might be of great importance to understand the process of fertilization. The present work describes the partial characterization of a 14-kDa protein (p14) detected in goat spermatozoa using an antibody directed against the purified protein. Confocal microscopic analysis reveals that the protein is present in both the intracellular and extracellular regions of the acrosomal and postacrosomal portion of caudal sperm head. Though subcellular localization shows that p14 is mainly cytosolic, however it is also seen to be present in peripheral plasma membrane and soluble part of acrosome. Immuno-localization experiment shows change in the distribution pattern of this protein upon induction of capacitation in sperm cells. Increased immunolabeling in the anterior head region of live spermatozoa is also observed when these cells are incubated under capacitating conditions, whereas most sperm cells challenged with the calcium ionophore A23187 to acrosome react, lose their labeling almost completely. Intracellular distribution of p14 also changes significantly during acrosome reaction. Interestingly, on the other hand the antibody raised against this 14-kDa sperm protein enhances the forward motility of caprine sperm cells. Rose-Bengal staining method shows that this anti-p14 antibody also decreases the number of acrosome reacted cells if incubated with capacitated sperm cells before induction of acrosome reaction. All these results taken together clearly indicate that p14 is intimately involved and plays a critical role in the acrosomal membrane fusion event.

Mephebrindole, a synthetic indole analog coordinates the crosstalk between p38MAPK and eIF2α/ATF4/CHOP signalling pathways for induction of apoptosis in human breast carcinoma cells

The efficacy of cancer chemotherapeutics is limited by side effects resulting from narrow therapeutic windows between the anticancer activity of a drug and its cytotoxicity. Thus identification of small molecules that can selectively target cancer cells has gained major interest. Cancer cells under stress utilize the Unfolded protein response (UPR) as an effective cell adaptation mechanism. The purpose of the UPR is to balance the ER folding environment and calcium homeostasis under stress. If ER stress is prolonged, tumor cells undergo apoptosis. In the present study we demonstrated an 3,3′-(Arylmethylene)-bis-1H-indole (AMBI) derivative 3,3′-[(4-Methoxyphenyl) methylene]-bis-(5-bromo-1H-indole), named as Mephebrindole (MPB) as an effective anti-cancer agent in breast cancer cells. MPB disrupted calcium homeostasis in MCF7 cells which triggered ER stress development. Detailed evaluations revealed that mephebrindole by activating p38MAPK also regulated GRP78 and eIF2α/ATF4 downstream to promote apoptosis. Studies extended to in vivo allograft mice models revalidated its anti-carcinogenic property thus highlighting the role of MPB as an improved chemotherapeutic option.

Efficient synthesis of (6-deoxy-glycopyranosid-6-yl) sulfone derivatives and their effect on Ca2+-ATPase

Synthesis of a series of novel (6-deoxy-glycopyranosid-6-yl) sulfone derivatives has been achieved using a general synthetic strategy. Yields were excellent in every case. The synthetic compounds were evaluated for their biological potential against Ca2+-ATPase, an important enzyme involves in transporting Ca2+ across the cell membranes.