cis-Acting Elements within the Candida albicans ERG11 Promoter Mediate the Azole Response through Transcription Factor Upc2p

Brian G. Oliver; Jia L. Song; Jake H. Choiniere; Theodore C. White

doi:10.1128/ec.00331-06

cis-Acting Elements within the Candida albicans ERG11 Promoter Mediate the Azole Response through Transcription Factor Upc2p

Eukaryotic Cell ◽

10.1128/ec.00331-06 ◽

2007 ◽

Vol 6 (12) ◽

pp. 2231-2239 ◽

Cited By ~ 38

Author(s):

Brian G. Oliver ◽

Jia L. Song ◽

Jake H. Choiniere ◽

Theodore C. White

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Regulatory Elements ◽

Antifungal Drugs ◽

Start Codon ◽

Luciferase Reporter ◽

Ergosterol Biosynthesis ◽

Luciferase Reporter Gene ◽

Enhancer Element

ABSTRACT The azole antifungal drugs are used to treat infections caused by Candida albicans and other fungi. These drugs interfere with the biosynthesis of ergosterol, the major sterol in fungal cells, by inhibiting an ergosterol biosynthetic enzyme, lanosterol 14 α-demethylase, encoded by the ERG11 gene. In vitro, these drugs as well as other ergosterol biosynthesis inhibitors increase ERG11 mRNA expression by activation of the ERG11 promoter. The signal for this activation most likely is the depletion of ergosterol, the end product of the pathway. To identify cis-acting regulatory elements that mediate this activation, ERG11 promoter fragments have been fused to the luciferase reporter gene from Renilla reniformis. Promoter deletions and linker scan mutations localized the region important for azole induction to a segment from bp −224 to −251 upstream of the start codon, specifically two 7-bp sequences separated by 13 bp. These sequences form an imperfect inverted repeat. The region is recognized by the transcription factor Upc2p and functions as an enhancer of transcription, as it can be placed upstream of a heterologous promoter in either direction, resulting in the azole induction of that promoter. The promoter constructs are not azole inducible in the upc2/upc2 homozygous deletion, demonstrating that Upc2p controls the azole induction of ERG11. These results identify an azole-responsive enhancer element (ARE) in the ERG11 promoter that is controlled by the Upc2p transcription factor. No other ARE is present in the promoter. Thus, this ARE and Upc2p are necessary and sufficient for azole induction of ERG11.

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Drug-Induced Regulation of the MDR1 Promoter in Candida albicans

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.49.7.2785-2792.2005 ◽

2005 ◽

Vol 49 (7) ◽

pp. 2785-2792 ◽

Cited By ~ 42

Author(s):

Jo Beth Harry ◽

Brian G. Oliver ◽

Jia L. Song ◽

Peter M. Silver ◽

John T. Little ◽

...

Keyword(s):

Candida Albicans ◽

Inducible Promoter ◽

Antifungal Drugs ◽

Start Codon ◽

Luciferase Reporter ◽

Mrna Levels ◽

Luciferase Reporter Gene ◽

Drug Induced ◽

Drug Induction ◽

Major Facilitator

ABSTRACT Resistance of Candida albicans to azole antifungal drugs is mediated by two types of efflux pumps, encoded by the MDR1 gene and the CDR gene family. MDR1 mRNA levels in a susceptible clinical isolate are induced by benomyl (BEN) but not by other drugs previously shown to induce MDR1. To monitor MDR1 expression under several conditions, the MDR1 promoter was fused to the Renilla reniformis luciferase reporter gene (RLUC). The promoter was monitored for its responses to four oxidizing agents, five toxic hydrophobic compounds, and an alkylating agent, all shown to induce major facilitator pumps in other organisms. Deletion constructs of the MDR1 promoter were used to analyze the basal transcription of the promoter and its responses to the toxic compound BEN and the oxidizing agent tert-butyl hydrogen peroxide (T-BHP). The cis-acting elements in the MDR1 promoter responsible for induction by BEN were localized between −399 and −299 upstream of the start codon. The cis-acting elements responsible for MDR1 induction by T-BHP were localized between −601 and −500 upstream of the start codon. The T-BHP induction region contains a sequence that resembles the YAP1-responsive element (YRE) in Saccharomyces cerevisiae. This Candida YRE was placed upstream of a noninducible promoter in the luciferase construct, resulting in an inducible promoter. Inversion or mutation of the 7-bp YRE eliminated induction. Many of the drugs used in this analysis induce the MDR1 promoter at concentrations that inhibit cell growth. These analyses define cis-acting elements responsible for drug induction of the MDR1 promoter.

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Azole Resistance by Loss of Function of the Sterol Δ5,6-Desaturase Gene (ERG3) in Candida albicans Does Not Necessarily Decrease Virulence

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.05720-11 ◽

2012 ◽

Vol 56 (4) ◽

pp. 1960-1968 ◽

Cited By ~ 53

Author(s):

L. A. Vale-Silva ◽

A. T. Coste ◽

F. Ischer ◽

J. E. Parker ◽

S. L. Kelly ◽

...

Keyword(s):

Candida Albicans ◽

Premature Stop Codon ◽

Antifungal Drugs ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Wild Type ◽

Disseminated Candidiasis ◽

Double Base

ABSTRACTThe inactivation ofERG3, a gene encoding sterol Δ5,6-desaturase (essential for ergosterol biosynthesis), is a known mechanism ofin vitroresistance to azole antifungal drugs in the human pathogenCandida albicans. ERG3inactivation typically results in loss of filamentation and attenuated virulence in animal models of disseminated candidiasis. In this work, we identified aC. albicansclinical isolate (VSY2) with high-level resistance to azole drugsin vitroand an absence of ergosterol but normal filamentation. Sequencing ofERG3in VSY2 revealed a double base deletion leading to a premature stop codon and thus a nonfunctional enzyme. The reversion of the double base deletion in the mutant allele (erg3-1) restored ergosterol biosynthesis and full fluconazole susceptibility in VSY2, confirming thatERG3inactivation was the mechanism of azole resistance. Additionally, the replacement of bothERG3alleles byerg3-1in the wild-type strain SC5314 led to the absence of ergosterol and to fluconazole resistance without affecting filamentation. In a mouse model of disseminated candidiasis, the clinicalERG3mutant VSY2 produced kidney fungal burdens and mouse survival comparable to those obtained with the wild-type control. Interestingly, while VSY2 was resistant to fluconazole bothin vitroandin vivo, theERG3-derived mutant of SC5314 was resistant onlyin vitroand was less virulent than the wild type. This suggests that VSY2 compensated for thein vivofitness defect ofERG3inactivation by a still unknown mechanism(s). Taken together, our results provide evidence that contrary to previous reports inactivation ofERG3does not necessarily affect filamentation and virulence.

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Cloning and Functional Analysis of Rat Tweety-Homolog 1 Gene Promoter

Neurochemical Research ◽

10.1007/s11064-021-03374-2 ◽

2021 ◽

Author(s):

Malgorzata Gorniak-Walas ◽

Karolina Nizinska ◽

Katarzyna Lukasiuk

Keyword(s):

Transcriptional Regulation ◽

Reporter Gene ◽

Hippocampal Neurons ◽

Gene Promoter ◽

Promoter Sequence ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Gene Assay

AbstractTweety-homolog 1 protein (Ttyh1) is abundantly expressed in neurons in the healthy brain, and its expression is induced under pathological conditions. In hippocampal neurons in vitro, Ttyh1 was implicated in the regulation of primary neuron morphology. However, the mechanisms that underlie transcriptional regulation of the Ttyh1 gene in neurons remain elusive. The present study sought to identify the promoter of the Ttyh1 gene and functionally characterize cis-regulatory elements that are potentially involved in the transcriptional regulation of Ttyh1 expression in rat dissociated hippocampal neurons in vitro. We cloned a 592 bp rat Ttyh1 promoter sequence and designed deletion constructs of the transcription factors specificity protein 1 (Sp1), E2F transcription factor 3 (E2f3), and achaete-scute homolog 1 (Ascl1) that were fused upstream of a luciferase reporter gene in pGL4.10[luc2]. The luciferase reporter gene assay showed the possible involvement of Ascl1, Sp1, and responsive cis-regulatory elements in Ttyh1 expression. These findings provide novel information about Ttyh1 gene regulation in neurons.

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Role of Candida albicans Transcription Factor Upc2p in Drug Resistance and Sterol Metabolism

Eukaryotic Cell ◽

10.1128/ec.3.6.1391-1397.2004 ◽

2004 ◽

Vol 3 (6) ◽

pp. 1391-1397 ◽

Cited By ~ 131

Author(s):

Peter M. Silver ◽

Brian G. Oliver ◽

Theodore C. White

Keyword(s):

Drug Resistance ◽

Transcription Factor ◽

Candida Albicans ◽

Transmembrane Domain ◽

Homozygous Deletion ◽

Antifungal Drugs ◽

Deletion Strain ◽

Ergosterol Biosynthesis ◽

Heterozygous Deletion ◽

Nuclear Localization Signals

ABSTRACT In Candida albicans, drug resistance to clinically important antifungal drugs may be regulated through the action of transcription factors in a manner that may or may not be similar to regulation in Saccharomyces cerevisiae. A search of the C. albicans genome identified a single homolog of the S. cerevisiae transcription factor genes UPC2 (ScUPC2) and ECM22 (ScECM22) that have been associated with regulation of ergosterol biosynthesis. Sequence analysis of this C. albicans UPC2 (CaUPC2) gene identifies two domains, an anchoring transmembrane domain and a transcription factor region containing multiple nuclear localization signals and a fungal Zn(2)-Cys(6) binuclear cluster domain. Heterozygous deletion, homozygous deletion, and reconstructed strains of CaUPC2 as well as the parental strain were tested against several antifungal drugs, including ergosterol biosynthesis inhibitors. The CaUPC2 homozygous deletion strain showed marked hypersusceptibility to most drugs, compared to the parental and reconstructed strains. The deletion strains accumulate significantly less radiolabeled cholesterol, suggesting reduced ergosterol scavenging in those strains. When grown under azole drug pressure, the parental, heterozygous deletion and reconstructed strains of CaUPC2 upregulate the ERG2 and ERG11 ergosterol biosynthesis genes, while the homozygous deletion strain shows no such upregulation. Consistent with these results, CaUPC2 deletion strains show reduced ergosterol levels, which may explain the increased susceptibilities of the CaUPC2 deletion strains. Thus, it appears that CaUPC2 acts as a transcription factor involved in the regulation of ergosterol biosynthetic genes and as a regulator of sterol uptake across the plasma membrane.

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Effect of LINC00320 Regulating the Expression of PLEKHA1 through the Transcription Factor MYC in Glioma Cell Proliferation, Migration, Invasion and Apoptosis In vivo and In vitro

10.21203/rs.3.rs-306949/v1 ◽

2021 ◽

Author(s):

Jianyong Ji ◽

Pengfei Xue ◽

Juan Zheng ◽

Rongrong Li ◽

Jinyue Fu ◽

...

Keyword(s):

Transcription Factor ◽

Glioma Cells ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Luciferase Reporter Gene Assay ◽

Over Expression ◽

Gene Assay ◽

Rip Assay

Abstract Aim: This study was carried out to explore the mechanism and function of LINC00320 in the development of glioma by regulating PLEKHA1 expression through transcription factor MYC.Methods: By searching LINCDISEASE database and through difference analysis of glioma chip, glioma related lncRNAs were screened, and lncRNA-transcription factor-mRNA triplet was predicted through lncMAP database. The expressions of LINC00320 and PLEKHA1 were detected in glioma and normal controls, followed by the detection of the proliferation, invasion, migration, and apoptosis of glioma cells by using CCK-8 method, Transwell assay, and flow cytometry, respectively. In addition, the expression patterns of MMP9 and cleaved-Caspase 3 were detected with Western Blot. Furthermore, the possible mechanism of LINC00320 was predicted in gliomas by LncMAP. RIP assay was performed to verify the interaction between LINC00320 and MYC, and ChIP assay was applied to validate the binding of MYC and PLEKHA1 promoter. The existence of binding site between MYC and PLEKHA1 promoter were determined by dual luciferase reporter gene assay. Lastly, in vivo test was conducted by using nude mice as the objects of study for verification of the results obtained by in vitro tests.Results: LINC00320 was found to be significantly down-expressed in glioma, and patients with low expression levels of LINC00320 exhibited an even worse prognostic outcome. Over-expression of LINC00320 in glioma cells brought about a significant reduction in cell proliferation, migration, invasion, and promoted apoptosis. There was a significant decrease in the protein expression of MMP9 but remarkable increase in that of cleaved-Caspase 3 after LINC00320 over-expression. LncRNA-transcription factor-mRNA triplet prediction showed that LINC00320 regulated the expression of PLEKHA1 through MYC. RIP assay demonstrated that MYC could significantly enrich LINC00320, Chip assay showed that MYC bound with the PLEKHA1 promoter, and dual luciferase reporter gene assay further confirmed the presence of binding site between MYC and PLEKHA1 promoter. Cell function experiment verified that PLEKHA1 could reverse the effect of LINC00320 over-expression.Conclusion: Over-expression of LINC00320 can attenuate the binding of MYC with PLEKHA1 by recruiting MYC, and ultimately inhibit the proliferation, migration and invasion, and promote the apoptosis of glioma cells.

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Azole-resistant alleles of ERG11 in Candida glabrata trigger activation of the Pdr1 and Upc2A transcription factors.

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.02098-21 ◽

2022 ◽

Author(s):

Bao Gia Vu ◽

W. Scott Moye-Rowley

Keyword(s):

Transcription Factor ◽

Candida Albicans ◽

Candida Glabrata ◽

Double Mutant ◽

Antifungal Drugs ◽

Cross Resistance ◽

Ergosterol Biosynthesis ◽

Azole Resistance ◽

Pathogenic Yeast ◽

Cellular Effects

Azoles, the most commonly used antifungal drugs, specifically inhibit the fungal lanosterol α-14 demethylase enzyme, which is referred to as Erg11. Inhibition of Erg11 ultimately leads to a reduction in ergosterol production, an essential fungal membrane sterol. Many Candida species, such as Candida albicans , develop mutations in this enzyme which reduces the azole binding affinity and results in increased resistance. Candida glabrata is also a pathogenic yeast that has low intrinsic susceptibility to azole drugs and easily develops elevated resistance. In C. glabrata , these azole resistant mutations typically cause hyperactivity of the Pdr1 transcription factor and rarely lie within the ERG11 gene. Here, we generated C. glabrata ERG11 mutations that were analogous to azole resistance alleles from C. albicans ERG11 . Three different Erg11 forms (Y141H, S410F, and the corresponding double mutant (DM)) conferred azole resistance in C. glabrata with the DM Erg11 form causing the strongest phenotype. The DM Erg11 also induced cross-resistance to amphotericin B and caspofungin. Resistance caused by the DM allele of ERG11 imposed a fitness cost that was not observed with hyperactive PDR1 alleles. Crucially, the presence of the DM ERG11 allele was sufficient to activate the Pdr1 transcription factor in the absence of azole drugs. Our data indicate that azole resistance linked to changes in ERG11 activity can involve cellular effects beyond an alteration in this key azole target enzyme. Understanding the physiology linking ergosterol biosynthesis with Pdr1-mediated regulation of azole resistance is crucial for ensuring the continued efficacy of azole drugs against C. glabrata .

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Unusual 5'-regulatory structure and regulation of the murine Mlc1 gene: Lack of promoter-specific functional elements

Journal of Nucleic Acids Investigation ◽

10.4081/jnai.2011.2314 ◽

2011 ◽

Vol 2 (1) ◽

pp. 11

Author(s):

Darja Henseler ◽

Jonathan D. Turner ◽

Matthias Eckhardt ◽

Maaike Van der Mark ◽

Yanina Revsin ◽

...

Keyword(s):

Reporter Gene ◽

In Silico ◽

Promoter Activity ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Regulatory Structure ◽

Luciferase Reporter Gene ◽

Promoter Regions ◽

Potential Promoter

The MLC1 gene is involved in an autosomal recessive neurological disorder, megalencephalic leucoencephalopathy with subcortical cysts (MLC), which is characterized by macrocephaly during the first year of life and swollen white matter (leucoencephaly). Variants of MLC1 have also been associated with psychiatric disorders such as schizophrenia, major depression and bipolar disorder. Currently, little is known about the encoded protein (MLC1). Judging from its similarity to other known proteins, it may serve as a trans-membrane transporter. However, the function of the encoded protein and its gene regulation has not been investigated successfully so far. We investigated the 5’ region of the murine Mlc1 with respect to regulatory elements for gene expression. A promoter search and an in silico analysis were conducted. Luciferase reporter gene constructs with potential promoter regions were created to study promoter activity in vitro. We found two alternative first exons for the murine Mlc1 but were not able to detect any promoter activity for the investigated reporter gene constructs in different cell lines, thus pointing to the presence of essential cis-acting elements far outside of the region. In silico analysis indicated an uncommon promoter structure for Mlc1, with CCAAT-boxes representing the only noticeable elements.

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Histone ChIP-Seq identifies differential enhancer usage during chondrogenesis as critical for defining cell-type specificity

10.1101/727370 ◽

2019 ◽

Author(s):

Kathleen Cheung ◽

Matthew J. Barter ◽

Julia Falk ◽

Carole Proctor ◽

Louise N. Reynard ◽

...

Keyword(s):

Dna Methylation ◽

Transcription Factor ◽

Human Mesenchymal Stem Cells ◽

Cell Types ◽

Regulatory Elements ◽

Luciferase Reporter ◽

Epigenetic Mechanisms ◽

Enhancer Activity ◽

Chromatin States

AbstractEpigenetic mechanisms are known to regulate gene expression during chondrogenesis. In this study, we have characterised the epigenome during in vitro differentiation of human mesenchymal stem cells (hMSCs) into chondrocytes. Chromatin immunoprecipitation followed by next-generation sequencing (ChIP-seq) was used to assess a range of N-terminal post-transcriptional modifications (marks) to histone H3 lysines (H3K4me3, H3K4me1, H3K27ac, H3K27me3 and H3K36me3) in both hMSCs and differentiated chondrocytes. Chromatin states were characterised using histone ChIP-seq and cis-regulatory elements were identified in chondrocytes. Chondrocyte enhancers were associated with chondrogenesis related gene ontology (GO) terms. In silico analysis and integration of DNA methylation data with chondrogenesis chromatin states revealed that enhancers marked by histone marks H3K4me1 and H3K27ac were de-methylated during in vitro chondrogenesis. Similarity analysis between hMSC and chondrocyte chromatin states defined in this study with epigenomes of cell-types defined by the Roadmap Epigenomics project revealed that enhancers are more distinct between cell-types compared to other chromatin states. Motif analysis revealed that the transcription factor SOX9 is enriched in chondrocyte enhancers. Luciferase reporter assays confirmed that chondrocyte enhancers characterised in this study exhibited enhancer activity which may be modulated by inducing DNA methylation and SOX9 overexpression. Altogether, these integrated data illustrate the cross-talk between different epigenetic mechanisms during chondrocyte differentiation.SummaryHuman mesenchymal stem cells are able to differentiate into chondrocytes, the cell type found in cartilage, making them an accessible system to study gene regulation during this process. Epigenetic mechanisms such as histone modifications and DNA methylation together with transcription factor binding play a role in activating and repressing gene expression. In this study, we investigated the genome-wide histone modification changes during chondrocyte differentiation. Integration of this data with DNA methylation and SOX9 transcription factor ChIP-seq revealed epigenetic changes at gene enhancer elements. Regions of the genome that transition from non-enhancers to enhancers in chondrocytes are enriched for SOX9 transcription factor binding sites. Luciferase reporter assays revealed that enhancer activity may be modulated by manipulating DNA methylation and SOX9 expression. This study has defined important regulatory elements in chondrocytes which could serve as targets for future mechanistic studies.

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Efficacy of Novel Schiff base Derivatives as Antifungal Compounds in Combination with Approved Drugs Against Candida Albicans

Medicinal Chemistry ◽

10.2174/1573406415666181203115957 ◽

2019 ◽

Vol 15 (6) ◽

pp. 648-658 ◽

Cited By ~ 1

Author(s):

Manzoor Ahmad Malik ◽

Shabir Ahmad Lone ◽

Parveez Gull ◽

Ovas Ahmad Dar ◽

Mohmmad Younus Wani ◽

...

Keyword(s):

Schiff Base ◽

Candida Albicans ◽

Antifungal Activity ◽

Fungal Infections ◽

Total Sterol ◽

Antifungal Drugs ◽

New Drugs ◽

Ergosterol Biosynthesis ◽

Dependent Manner ◽

Schiff Base Derivatives

Background: The increasing incidence of fungal infections, especially caused by Candida albicans, and their increasing drug resistance has drastically increased in recent years. Therefore, not only new drugs but also alternative treatment strategies are promptly required. Methods: We previously reported on the synergistic interaction of some azole and non-azole compounds with fluconazole for combination antifungal therapy. In this study, we synthesized some non-azole Schiff-base derivatives and evaluated their antifungal activity profile alone and in combination with the most commonly used antifungal drugs- fluconazole (FLC) and amphotericin B (AmB) against four drug susceptible, three FLC resistant and three AmB resistant clinically isolated Candida albicans strains. To further analyze the mechanism of antifungal action of these compounds, we quantified total sterol contents in FLC-susceptible and resistant C. albicans isolates. Results: A pyrimidine ring-containing derivative SB5 showed the most potent antifungal activity against all the tested strains. After combining these compounds with FLC and AmB, 76% combinations were either synergistic or additive while as the rest of the combinations were indifferent. Interestingly, none of the combinations was antagonistic, either with FLC or AmB. Results interpreted from fractional inhibitory concentration index (FICI) and isobolograms revealed 4-10-fold reduction in MIC values for synergistic combinations. These compounds also inhibit ergosterol biosynthesis in a concentration-dependent manner, supported by the results from docking studies. Conclusion: The results of the studies conducted advocate the potential of these compounds as new antifungal drugs. However, further studies are required to understand the other mechanisms and in vivo efficacy and toxicity of these compounds.

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Benzisothiazolone Derivatives Exhibit Cytotoxicity in Hodgkin’s Lymphoma Cells through NF-κB Inhibition and are Synergistic with Doxorubicin and Etoposide

Anti-Cancer Agents in Medicinal Chemistry ◽

10.2174/1871520620666200213103513 ◽

2020 ◽

Vol 20 (6) ◽

pp. 715-723

Author(s):

Natarajan Nandakumar ◽

Pushparathinam Gopinath ◽

Jacob Gopas ◽

Kannoth M. Muraleedharan

Keyword(s):

Synergistic Effect ◽

Hodgkin’S Lymphoma ◽

A549 Cells ◽

Hodgkin's Lymphoma ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Lymphoma Cells ◽

Nuclear Extracts ◽

Gene Assay

Background: The authors investigated the NF-κB inhibitory role of three Benzisothiazolone (BIT) derivatives (1, 2 and 3) in Hodgkin’s Lymphoma cells (L428) which constitutively express activated NF-κB. All three compounds showed dose-dependent NF-κB inhibition (78.3, 70.7 and 34.6%) in the luciferase reporter gene assay and were found cytotoxic at IC50 values of 3.3μg/ml, 4.35μg/ml and 13.8μg/ml, respectively by the XTT assay. BIT 1and BIT 2 (but not BIT 3) suppressed both NF-κB subunits p50 and p65 in cytoplasmic and nuclear extracts in a concentration-dependent manner. Furthermore, BIT 1 showed a moderate synergistic effect with the standard chemotherapy drugs etoposide and doxorubicin, whereas BIT 2 and 3 showed a moderate additive effect to antagonistic effect. Cisplatin exhibited an antagonist effect on all the compounds tested under various concentrations, except in the case of 1.56μg/ml of BIT 3 with 0.156μg/ml of cisplatin. The compounds also inhibited the migration of adherent human lung adenocarcinoma cells (A549) in vitro. We conclude that especially BIT 1 and BIT 2 have in vitro anti-inflammatory and anti-cancer activities, which can be further investigated for future potential therapeutic use. Methods: Inspired by the electrophilic sulfur in Nuphar alkaloids, monomeric and dimeric benzisothiazolones were synthesized from dithiodibenzoic acid and their NF-κB inhibitory role was explored. NF-κB inhibition and cytotoxicity of the synthesized derivatives were studied using luciferase reporter gene assay and XTTassay. Immunocytochemistry studies were performed using L428 cells. Cell migration assay was conducted using the A549 cell line. L428 cells were used to conduct combination studies and the results were plotted using CompuSyn software. Results: Benzisothiazolone derivatives exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. Potent compounds showed suppression of both NF-κB subunits p50 and p65 in a concentrationdependent manner, both in cytoplasmic and nuclear extracts. Combination studies suggest that benzisothiazolone derivatives possess a synergistic effect with etoposide and doxorubicin. Furthermore, the compounds also inhibited the migration of A549 cells. Conclusion: Benzisothiazolones bearing one or two electrophilic sulfur atoms as part of the heterocyclic framework exhibited cytotoxicity in Hodgkin’s Lymphoma cells through NF-κB inhibition. In addition, these derivatives also exhibited a synergistic effect with etoposide and doxorubicin along with the ability to inhibit the migration of A549 cells. Our study suggests that BIT-based new chemical entities could lead to potential anticancer agents.

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