BITS Faculty Publications

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Author: search.filters.author.Chowdhury, ShibasishStart date: 2020

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    Cisplatin-induced oxidative stress regulates YAP to modulate epigenome promoting survival of osteosarcoma cells
    (2025-08) Chowdhury, Rajdeep; Chowdhury, Shibasish; Mukherjee, Sudeshna
    The widely used chemotherapeutic drug cisplatin (CDDP) is an integral part of the pre-operative chemotherapy protocol for high-grade osteosarcoma (OS). However, despite an aggressive treatment regimen, drug refractoriness is a major hindrance to successful therapy. We previously identified key transcriptomic alterations essential for the survival of OS cells following CDDP exposure. In the present study, we further demonstrate that CDDP treatment resulted in a ROS-dependent enrichment of the repressive histone mark H3K27me3 at the upstream promoter regions of growth-promoting genes such as CCNA2, and on the promoter of the negative regulator of Yes-Associated Protein (YAP)-LATS1, thereby contributing to their transcriptional repression. This was associated with a growth arrest, and quenching of ROS with N-acetyl cysteine (NAC) reversed it. Importantly, repression of LATS1 led to an increased nuclear localization of YAP, while pharmacological or genetic ablation of YAP reduced CDDP-mediated induction of repressive marks. YAP was further found to co-localize and co-immunoprecipitate with the Polycomb Repressive Complex 2 (PRC2) catalytic member-the histone methyl transferase-EZH2, indicating its putative role in mediating transcriptional repression. In lieu of the above, inhibition of YAP or reversal of the repressive chromatin state using a histone deacetylase (HDAC) inhibitor sensitized OS cells to a low-dose CDDP treatment as well. Overall, the present study demonstrates an interplay between oxidative stress, epigenetics, and YAP in modulating OS cell fate post CDDP exposure.
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    A switch from histone methyltransferase-EZH2 to demethylase KDM6A activity marks reinitiation of proliferation in cisplatin treated colorectal cancer cells
    (Elsevier, 2026-01) Chowdhury, Shibasish; Mukherjee, Sudeshna; Chowdhury, Rajdeep
    Colorectal cancer (CRC) is one of the deadliest cancers, ranking third in cancer incidence worldwide. These tumor cells often adopt unique strategies under chemotherapeutic stress to attain a reversible drug-tolerant state and evade cell death. However, the molecular adaptations associated with this transitory emergence of the drug-tolerant state remain elusive. Herein, epigenetic alterations often dictate such reversible dynamic changes, and this study aims to characterize the role of specific epigenetic modifiers governing CRC cell survival under cisplatin exposure and their subsequent relapse. We observed that under cisplatin-stress there is a drastic increase in the histone-repressive mark-H3K27me3, linked to an enhanced expression of EZH2, driving transcriptional inhibition of cell proliferation-associated genes and a proliferative arrest. Interestingly, cisplatin-induced oxidative stress increased the expression of P65 protein, which was found to interact with and regulate EZH2 expression. Quenching of ROS, cisplatin-rescue, or P65 inactivation compromised EZH2 activity, concurrent with a re-initiation of cell proliferation. Interestingly, this reversal to proliferative state was associated with an elevated activity of the histone lysine demethylase-KDM6A. The promoter elements of the proliferative genes were now occupied by KDM6A instead of EZH2. Accordingly, a genetic knockdown or pharmacological inhibition of KDM6A in vitro not only resulted in increased cell death but also prevented emergence of the re-proliferative CRC cells. Furthermore, KDM6A inhibition in combination with cisplatin, resulted in an increased tumor regression in vivo. Our study thus highlights the importance of KDM6A as a therapeutic target in preventing CRC growth and relapse which can have future therapeutic implications.
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    DRP1-mediated mitochondrial dynamics orchestrate EMT in glioblastoma cells
    (2025-12) Chowdhury, Shibasish; Chowdhury, Rajdeep; Mukherjee, Sudeshna
    Epithelial to mesenchymal transition (EMT), a differentiation process, frequently imparts invasive properties in Glioblastoma Multiforme (GBM), which leads to a poor prognosis. Cells lose apical-basal polarity, cell-cell connections, and/or chemo-resistance during EMT, which can result in the spread of cancer and the acquisition of additional stem cell-like traits. It is unclear how organelle dynamics influence EMT in this respect. The interaction between cytoskeletal and mitochondrial regulators governing GBM cell EMT is explored in this article.
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    S-nitrosylation of EZH2 alters PRC2 assembly, methyltransferase activity, and EZH2 stability to maintain endothelial homeostasis
    (Springer Nature, 2025-04) Sundriyal, Sandeep; Chowdhury, Shibasish; Majumder, Syamantak
    Nitric oxide (NO), a versatile bio-active molecule modulates cellular functions through diverse mechanisms including S-nitrosylation of proteins. Herein, we report S-nitrosylation of selected cysteine residues of EZH2 in endothelial cells, which interplays with its stability and functions. We detect a significant reduction in H3K27me3 upon S-nitrosylation of EZH2 as contributed by the early dissociation of SUZ12 from the PRC2. Moreover, S-nitrosylation of EZH2 causes its cytosolic translocation, ubiquitination, and degradation. Further analysis reveal S-nitrosylation of cysteine 329 induces EZH2 instability, whereas S-nitrosylation of cysteine 700 abrogates its catalytic activity. We further show that S-nitrosylation-dependent regulation of EZH2 maintains endothelial homeostasis in both physiological and pathological settings. Molecular dynamics simulation reveals the inability of SUZ12 to efficiently bind to the SAL domain of EZH2 upon S-nitrosylation. Taken together, our study reports S-nitrosylation-dependent regulation of EZH2 and its associated PRC2 complex, thereby influencing the epigenetics of endothelial homeostasis.
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    Repurposing of CNS accumulating drugs Gemfibrozil and Doxylamine for enhanced sensitization of glioblastoma cells through modulation of autophagy
    (Springer Nature, 2025-07) Mukherjee, Sudeshna; Chowdhury, Rajdeep; Majumder, Syamantak; Chowdhury, Shibasish; Roy, Aniruddha
    GBM is one of the most aggressive malignancies, having the greatest fatality rate and average life years lost. The current standard medicine, temozolomide (TMZ), is ineffective, requiring the development of new treatments. However, identifying and introducing a novel medicine takes time and money. In this context, repurposing FDA-approved drugs can be a novel yet efficient alternative method. Here, we, therefore, investigated the differential expression signatures of genes of patients suffering from GBM from publicly available GEO datasets and constructed a connectivity map. Functional annotation and KEGG pathway analysis showed dysregulated molecular activities and pathways. Based on their gene ontologies, putative key genes and hub genes linked with the disease were identified, and the C-MAP database was scanned for FDA-approved medicinal compounds that could alter hub gene expression or associated pathways. Our in-silico investigation showed that Gemfibrozil (Gem) and Doxylamine (Doxy) might reverse GBM disease patterns by deregulating GBM-related genes. Evaluation of the GBM inhibitory potential of these drugs through in-vitro and three-dimensional spheroid assay showed promising results. These drugs were more cytotoxic than TMZ; however, they synergised with TMZ as well. Interestingly, the cellular homeostatic process autophagy which has been implicated significantly in GBM pathogenesis and therapy resistance, was found to be inhibited by the drugs Gemfibrozil and Doxylamine, signifying their prospective potential. Therefore, in this study, we, for the first time, identify drugs with the ability to cross the blood brain barrier (BBB), with potential cytotoxic effects beyond TMZ, and with autophagy inhibitory potential, which can be further explored for repurposing against GBM.
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    Chloroquine attenuates hypoxia-mediated autophagy to curb thrombosis- an ex vivo and in vivo study
    (2024-04) Mukherjee, Sudeshna; Majumder, Syamantak; Chowdhury, Shibasish; Chowdhury, Rajdeep
    Hypoxia can trigger the activation of blood platelets, leading to thrombosis. If not addressed clinically, it can cause severe complications and fatal consequences as well. The current treatment regime for thrombosis is often palliative and includes long-term administration of anticoagulants, which has the risk of over-bleeding in injury and other secondary effects as well. This demands a deeper understanding of the process and exploration of an alternative therapeutic avenue. Interestingly, recent studies demonstrate that platelets though atypical and enucleated, possess components of autophagy machinery. This cellular homeostatic process though well-studied in non-platelet cells, is under-explored in platelets.
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    LncRNA HULC augments high glucose-associated pancreatic cancer progression and drug resistance by enhancing YAP activity and autophagy
    (Wiley, 2024-07) Mukherjee, Sudeshna; Chowdhury, Shibasish; Chowdhury, Rajdeep
    Background Information: One of the confounding factors in pancreatic cancer (PC) pathogenesis is hyperglycemia. The molecular mechanism by which high glucose (HG) influences PC severity is poorly understood. Our investigation delved into the impact of lncRNA highly upregulated in liver cancer (HULC) and its interaction with yes-associated protein (YAP) in regulating the fate of pancreatic ductal adenocarcinoma cells (PDAC) under HG-induced conditions. PDAC cells were cultured under normal or HG conditions. We thereafter measured the effect of HG on the viability of PDAC cells, their migration potential and drug resistance properties. The lncRNAs putatively dysregulated in PC and diabetes were shortlisted by bioinformatics analysis followed by wet lab validation of function. Results: HG led to enhanced proliferation and drug refractoriness in PDAC cells. HULC was identified as one of the major deregulated lncRNAs following bioinformatics analysis. HULC was found to regulate the expression of the potent transcriptional regulator – YAP through selective histone modifications at the YAP promoter. siRNA-mediated ablation of HULC resulted in a concurrent decrease in YAP transcriptional activity. Importantly, HULC and YAP were found to co-operatively regulate the cellular homeostatic process autophagy, thus inculcating drug resistance and proliferative potential in PDAC cells. Moreover, inhibition of autophagy or YAP led to a decrease in HULC levels, suggesting the existence of an inter-regulatory feedback loop. Conclusions: We observed that HG triggers aggressive properties in PDAC cells. Mechanistically, up-regulation of lncRNA HULC resulted in activation of YAP and differential regulation of autophagy coupled to increased proliferation of PDAC cells. Significance: Inhibition of HULC and YAP may represent a novel therapeutic strategy for PDAC. Furthermore, this study portrays the intricate molecular interplay between HULC, YAP and autophagy in PDAC pathogenesis
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    Inhibition of autophagy in platelets as a therapeutic strategy preventing hypoxia induced thrombosis
    (Springer Nature, 2025-02) Mukherjee, Sudeshna; Majumder, Syamantak; Chowdhury, Shibasish; Chowdhury, Rajdeep
    Hypoxia triggers activation of platelets, leading to thrombosis. If not addressed clinically, it can cause severe complications and fatal consequences. The current treatment regime for thrombosis is often palliative and include long-term administration of anticoagulants, causing over-bleeding risk and other secondary effects as well. This demands a molecular understanding of the process and exploration of an alternative therapeutic avenue. Interestingly, recent studies demonstrate that platelets exhibit functional autophagy. This cellular homeostatic process though well-studied in non-platelet cells, is under-explored in platelets. Herein, we report autophagy activation under physiologically relevant hypoxic condition (10% O2; associated with high altitude) in ex-vivo platelets and in vivo as well. We show that autophagy inhibition using chloroquine (CQ), a repurposed FDA-approved drug, can significantly reduce platelet activation, both in ex-vivo and in-vivo settings. Further, surgical ligation of inferior vena cava (IVC) was performed to induce thrombus formation. Interestingly, CQ pre-treated rats showed reduced clotting ability in surgical animals as well. Importantly, thrombosis inhibitory dose of CQ was considerably lower than the currently used drug-acetazolamide; CQ was also found to be non-toxic to the tissues. Hence, we propose that repurposing of CQ can attenuate hypoxia-induced thrombosis through inhibition of autophagy and can be explored as an effective therapeutic alternative.
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    S-nitrosylation of EZH2 at C329 and C700 interplay with PRC2 complex assembly, methyltransferase activity, and EZH2 stability to regulate endothelial functions
    (2024) Sundriyal, Sandeep; Chowdhury, Shibasish; Majumder, Syamantak
    Nitric oxide (NO), a versatile bio-active molecule modulates cellular function through diverse mechanisms including S-nitrosylation of proteins. However, the role of this post-translational modification in regulating epigenetic pathways was very limitedly explored. Herein, we report that NO causes S-nitrosylation of selected cysteine residues of EZH2 in endothelial cells (EC) resulting in SUZ12 dissociation from EZH2 bound PRC2 complex, reduced methyltransferase activity, and diminished nuclear localization eventually hampering its stability. We detected a significant reduction in H3K27me3 upon exposure to NO as contributed by the early dissociation of SUZ12 from the PRC2 complex. Longer exposure to NO donors caused EZH2 cytosolic translocation, its ubiquitination, and further degradation primarily through the autophagosome-lysosome pathway. Through in silico S-nitrosylation prediction analysis and site-directed mutagenesis assay, we identified three cysteine residues namely at locations 260, 329, and 700 in EZH2 and further determined that S-nitrosylation of cysteine 329 induced EZH2 instability while S-nitrosylation of cysteine 700 abrogated EZH2’s catalytic activity. A double mutant of EZH2 containing mutations at Cysteine 329 and 700 remained undeterred to NO exposure. Furthermore, reinforcing H3K27me3 in NO exposed EC through the use of an inhibitor of H3K27me3 demethylase, we confirmed a significant contribution of the EZH2-H3K27me3 axis in defining NO-mediated regulation of endothelial gene expression and migration. Molecular dynamics simulation study revealed SUZ12’s inability in efficiently binding to the SAL domain of EZH2 upon S-nitrosylation of C329 and C700. Taken together, our study for the first-time reports that S-nitrosylation dependent regulation of EZH2 and its associated PRC2 complex influences endothelial homeostasis.
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    Characterization of a Zeolite-Y-Encapsulated Zn(II)Salmphen Complex with Targeted Anticancer Property
    (ACS, 2023-11) Chowdhury, Shibasish; Ray, Saumi
    Resistance and severe side effects of classical chemotherapeutic drugs are major challenges to cancer therapy. New therapeutic agents and combination therapy are considered potential solutions that enhance the efficacy of the drug as well as reduce drug resistance. The success of a platinum-based anticancer drug, cisplatin, has paved the way to explore metal-centered anticancer therapeutic agents. Herein, the zeolite-Y-encapsulated Zn(II)Salmphen complex is synthesized using a flexible ligand approach. The Zn(II)Salmphen complex and its encapsulation within the supercage of zeolite-Y were characterized by elemental analysis, Fourier transform infrared (FTIR) spectroscopy, UV–vis, fluorescence, powder X-ray diffraction (PXRD), scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), NMR, and high-resolution mass spectrometry (HRMS) techniques. Elemental analysis, PXRD, and SEM, all together confirm the integrity of the zeolite framework after the encapsulation of Zn(II)Salmphen complex in it, and elemental analysis provides the Si/Al ratio and Zn content present. FTIR and XPS studies indicate the successful encapsulation of the complex. NMR and HRMS studies confirm that the Zn(II)Salmphen complex is dimer; however, within the supercage of zeolite-Y, it is expected to exist as a monomer. The extent of structural modification of the encapsulated Zn(II)Salmphen complex is intimated by electronic spectroscopic studies. The free-state Zn(II)Salmphen is a fluorescent complex, and even the encapsulated Zn(II)Salmphen complex, when taken in dimethyl sulfoxide (DMSO), shows fluorescence. In comparison to cisplatin, encapsulated Zn(II)Salmphen complex displays comparable cytotoxicity (IC50 = 2.0 ± 0.5 μg/mL at 48 h) toward breast cancer cell line, whereas free Zn(II)Salmphen has better cytotoxicity (IC50 = 1.5 ± 0.5 μg/mL at 48 h). Importantly, elemental analysis has revealed that the IC50 value, if calculated only in terms of Zn(II)Salmphen within Zn(II)Salmphen-Y, is as low as 54.59 ng/mL, indicating a very high efficacy of the drug. Interestingly, a 48 h treatment with the encapsulated Zn(II)Salmphen complex shows no toxicity toward immortal noncancerous keratinocyte cells (HaCaT), whereas cisplatin has an IC50 value of 1.75 ± 0.5 μg/mL. Internalization studies indicate that zeolite-Y targets cancer cells better than it does noncancerous ones. Hence, cellular uptake of the zeolite-encapsulated Zn(II)Salmphen complex in cancer cells is more than that in HaCaT cells, resulting in the generation of more reactive oxygen species and cell death. Significant upregulation of DNA damage response protein indicates that DNA-damage-induced cellular apoptosis could be the mechanism of drug action. Overall, the zeolite-encapsulated Zn(II)Salmphen complex could be a better alternative to the traditional drug cisplatin with minimal effect on noncancerous HaCaT cells and can also be utilized as a fluorescent probe in exploring the mechanistic pathway of its activity against cancer cells.