Krishnegowda Gowdahalli

Evidence for Ligand-Mediated Selective Modulation of Aryl Hydrocarbon Receptor Activity

The concept of selective receptor modulators has been established for the nuclear steroid hormone receptors. Such selective modulators have been used therapeutically with great success in the treatment of cancer. However, this concept has not been examined with regard to the aryl hydrocarbon receptor (AHR) because of the latent toxicity commonly associated with AHR activation. AHR-mediated toxicity is primarily derived from AHR binding to its dioxin response element (DRE) and driving expression of CYP1 family members, which have the capacity to metabolize procarcinogens to genotoxic carcinogens. Recent evidence using a non-DRE binding AHR mutant has established the DRE-independent suppression of inflammatory markers by the AHR. We wished to determine whether such DRE-independent repression with wild-type AHR could be dissociated from canonical DRE-dependent transactivation in a ligand-dependent manner and, in doing so, prove the concept of a selective AHR modulator (SAhRM). Here, we identify the selective estrogen receptor (ER) modulator Way-169916 as a dually selective modulator, binding both ER and AHR. Inflammatory gene expression associated with the cytokine-inducible acute-phase response (e.g., SAA1 and CRP) are diminished by Way-169916 in an AHR-dependent manner. Furthermore, activation of AHR by Way-169916 fails to stimulate canonical DRE-driven AHR-mediated CYP1A1 expression, thus eliminating the potential for AHR-mediated genotoxic stress. Such anti-inflammatory activity in the absence of DRE-mediated expression fulfills the major criteria of an SAhRM, which suggests that selective modulation of AHR is possible and renders the AHR a therapeutically viable drug target for the amelioration of inflammatory disease.

Synthesis and Characterization of a Novel iNOS/Akt Inhibitor Se,Se′-1,4-phenylenebis(1,2-ethanediyl)bisisoselenourea (PBISe) – against colon cancer

Our studies demonstrate that substitution of sulfur with selenium in known iNOS inhibitor increases the compounds potency by several folds in variety of different cancers cell lines tested. Hence, this approach may be used as a strategy to increase the efficacy of the anticancer agents.

Synthesis, microsome-mediated metabolism, and identification of major metabolites of environmental pollutant naphtho[8,1,2-ghi]chrysene.

Naphtho[8,1,2- ghi]chrysene, commonly known as naphtho[1,2- e]pyrene (N[1,2- e]P) is a widespread environmental pollutant, identified in coal tar extract, air borne particulate matter, marine sediment, cigarette smoke condensate, and vehicle exhaust. Herein, we determined the ability of rat liver microsomes to metabolize N[1,2- e]P and an unequivocal assignment of the metabolites by comparing them with independently synthesized standards. We developed the synthesis of both the fjord region and the K-region dihydrodiols and various phenolic derivatives for metabolite identification. The 12-OH-N[1,2- e]P, fjord region dihydrodiol 14 and diol epoxide 15 were synthesized using a Suzuki cross-coupling reaction followed by the appropriate manipulation of the functional groups. The K-region trans-4,5-dihydrodiol ( 18) was prepared by the treatment of N[1,2- e]P with OsO 4 to give cis-dihydrodiol 16, followed by pyridinium chlorochromate oxidation to quinone 17, and finally reduction with NaBH 4 to afford the dihydrodiol 18 with the desired trans stereochemistry. The 9-OH-N[1,2- e]P ( 30) and N[1,2- e]P trans-9,10-dihydrodiol ( 32) were also synthesized following a Suzuki cross-coupling approach starting from 1,2,3,6,7,8-hexahydropyrene-4-boronic acid. The metabolism of N[1,2- e]P with rat liver microsomes led to several dihydrodiol and phenolic metabolites as assessed by the HPLC trace. The 11,12-dihydrodiol and 4,5-dihydrodiol were identified as major dihydrodiol metabolites. The synthesized 9,10-dihydrodiol, on the other hand, did not match with any of the peaks in the metabolism trace. Among the phenols, only 12-OH-N[1,2- e]P was identified in the metabolism. The other phenolic derivatives synthesized, that is, the 4-/5-, 9-, 10-, and 11-hydroxy derivatives, were not detected in the metabolism trace. In summary, N[1,2- e]P trans-11,12-dihydrodiol was the major metabolite formed along with N[1,2- e]P 4,5- trans-dihydrodiol and 12-OH-N[1,2- e]P on exposure of rat liver microsomes to N[1,2- e]P. The presence of N[1,2- e]P in the environment and formation of fjord region dihydrodiol 14 as a major metabolite in in vitro metabolism studies strongly suggest the role of N[1,2- e]P as a potential health hazard.

Abstract 4454: Genome-wide analysis of DNA methylation induced by environmental carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice

We and others have demonstrated that the administration of dibenzo[def,p]chrysene [also known as dibenzo[a,l]pyrene (DBP)], a representative example of the class of polycyclic aromatic hydrocarbon (PAH), by ip, oral gavage, or topical application onto the oral cavity induced tumors in multiple organ sites in mice; the ovary was the most susceptible tissue. We further demonstrated that the capacity of the target organs to metabolize DBP to active intermediates that can form DNA adducts may account for its tissue selective tumorigenicity. In addition, formation of DNA adducts by certain chemical carcinogens has been linked to aberrant DNA methylation, including the most extensively studied prototype PAH benzo[a]pyrene (B[a]P). The goal of this study is to examine whether alteration of DNA methylation occurs in the ovarian tissues of mice treated with DBP during the early stage of tumor development. In this study, we employed a previously established animal protocol in which the levels of DBP-DNA adducts as a function of time had been determined in the ovary following the oral administration of DBP (24 nmol, 3×/week for 5 weeks) or vehicle (DMSO) to female B6C3F1 mice (six weeks old, n = 3/group). DNA was isolated from ovary and subjected to enhanced reduced representation bisulfite sequencing (ERRBS) which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1 (Babraham Bioinformatcis, UK). The methylKit v0.9.2 R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Among 179 differentiated methylation sites (DMS) identified between DBP and vehicle-treated mice, 68 are hypermethylated and 111 are hypomethylated. About 25% of DMS are located in promoter or exon, 32% in intron and 44% in intergenic regions. DMS are located in genes including oocyte specific homeobox 2, transforming growth factor alpha, and tumor necrosis factor. Ingenuity Pathways analysis of the genes with altered methylation patterns identified top canonical pathways as growth hormone signaling, spermine biosynthesis, threonine degradation, trehalose degradation and L-serine degradation. Collectively, our previous results together with those presented here demonstrate that both genetic and epigenetic alterations may account for the carcinogenicity of DBP in the mouse ovary. Support: NIEHS R21ES020411 and NCI R01-CA173465. Citation Format: Yuan-Wan Sun, Karam El-Bayoumy, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Kun-Ming Chen. Genome-wide analysis of DNA methylation induced by environmental carcinogen dibenzo[def,p]chrysene in ovarian tissues of mice. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4454.

Abstract 2955: The effects of the environmental carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA We had previously reported the carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P) in mouse oral cavity; we also found that that diets containing black raspberry (BRB) significantly reduced the levels of DNA adducts derived from DB[a,l]P in mouse oral tissues. Epigenetic alternations including DNA methylation have been shown to contribute to carcinogenesis induced by certain carcinogens. Literature data showed that dietary BRB can modulate DNA methylation at certain gene-specific promoters; however, the genome-wide methylation alterations by DB[a,l]P and the impact of BRB are not known. Therefore, to mimic the bioassay employed in our previous carcinogenicity study, B6C3F1 female mice (n = 3/group) were fed either control diet or diet containing BRB (5%) for 2 weeks prior to topical application of DMSO containing DB[a,l]P (24 nmol, 3 times a week for 5 weeks); animals treated with DMSO and control diet were used as a control group. DNA was isolated from oral tissues and subjected to enhanced reduced representation bisulfite sequencing analysis which is a single nucleotide resolution technique used to study DNA methylation in CpG sites and the surrounding regions. Briefly, DNA was digested by MspI followed by end repair, adenylation and adapter ligation with a modification of bead size selection to capture MspI fragments of 70-320 bp size. The resulting libraries were bisulfite-converted followed by PCR amplification and read by 1×50 bp on HiSeq 2500. Base calls of bisulfite treated sequencing reads were mapped to the mm9 mouse assembly and methylation calls were performed using Bismark v0.10.1. The methylKit R package was then used to calculate the differential methylation. Differentially methylated bases with q-value 25% were extracted. Differential methylation of 30 genes was observed in DB[a,l]P compared with control; 12 genes were hypermethylated including β-catenin; hypomethylation of 18 genes was observed. Differential methylation of 960 genes was observed in mice treated with DB[a,l]P and fed BRB compared to DB[a,l]P. Ingenuity Pathways analysis was performed on each of the resulting gene sets in the three groups. Pathway analysis of the genes with altered methylation patterns identified canonical pathways for the involvement of cancer related network for genes involved in Wnt/β-catenin signaling, epithelial-mesenchymal transition (EMT), glycolysis and p53 signaling pathways in mice treated with DB[a,l]P. On the other hand, the canonical pathways identified in mice treated with DB[a,l]P and fed BRB are genes involved in glutamate receptor signaling, IGF-1 signaling, EMT, glycolysis, and protein citrullination. Using this quantitative sequencing-based approach, our work uncovers significant global DNA methylation alterations in mouse oral tissues by DB[a,l]P alone and together with BRB. Support: NCI R01-CA173465 Citation Format: Yuan-Wan Sun, Kun-Ming Chen, Yuka Imamura Kawasawa, Anna Salzberg, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The effects of the environmental carcinogen dibenzo[a,l]pyrene on genome-wide methylation and the impact of dietary black raspberry in mouse oral tissues. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2955. doi:10.1158/1538-7445.AM2015-2955

Abstract 230: The inhibitory effects of an anthocyanin enriched fraction of black raspberry (BRB), protocatechuic acid and ferulic acid on DB[a,l]P-induced DNA adduct formation in mouse oral tissues

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck region; in the USA, over 45,000 cases and about 11,000 deaths from the disease occur annually. Progress in the prevention and control of OSCC has been hampered by the lack of appropriate animal models that would reflect human exposure. Tobacco smoking is considered a major etiological factor in the development of oral cancer. Previous studies have demonstrated the ability of diets containing 5-10% freeze-dried black raspberry (BRB) powder to inhibit the development of chemically-induced cancers in multiple organ sites in rodents including 7,12-dimethylbenz(a)anthracene (DMBA) induced squamous cell carcinomas (SCC) in the hamster cheek pouch. However, DMBA is not present in the environment and the hamster cheek pouch model may not be applicable to humans. We had previously reported the carcinogenicity of dibenzo[a,l]pyrene (DB[a,l]P), an environmental pollutant and a tobacco smoke component, in mouse oral cavity. Recently, we demonstrated that administration of BRB at 5% in the diet can significantly reduce DNA adducts resulted from the administration of DB[a,l]P. It has also been shown that anthocyanins are the most abundant compounds in BRBs that can account for much of their antioxidant activity. In the current study, under identical conditions we compare the inhibitory effects of an anthocyanin enriched fraction extracted from black raspberry (BRBE), protocatechuic acid (PA, a major metabolite of anthocyanins) or ferulic acid (FA, a major phenolic acid in raspberries shown to reduce oral carcinogenesis) with BRB on DNA adducts induced by DB[a,l]P in mouse oral cavity. Levels of DB[a,l]PDE-DNA adducts were quantified by a LC-MS/MS method. We demonstrated that the administration of BRBE (1.6 %), protocatechuic acid (0.2%) or ferulic acid (0.05%) in the diet, starting 2 weeks before DB[a,l]P (24 nmol, 3 times a week for 5 weeks) significantly resulted in 29.6%, 23.0% or 25.3% reduction of the level of (-)-anti-trans-DB[a,l]PDE-dA in murine oral tissues, respectively, compared to a 19.2% reduction of BRB. Our results clearly showed that the anthocyanin components of BRB and its metabolite, PA, are responsible for the inhibitory effects of BRB on the DB[a,l]P- induced DNA adducts formation; in addition, BRB and its related components can inhibit the metabolic activation of DB[a,l]P, and may prevent the subsequent mutagenesis and carcinogenesis resulting from exposure to DB[a,l]P. Supported by NIH grant #CA173465. Citation Format: Kun-Ming Chen, Shangmin Zhang, Yuan-Wan Sun, Cesar Aliaga, Krishnegowda Gowdahalli, Shantu Amin, Gary Stoner, Karam El-Bayoumy. The inhibitory effects of an anthocyanin enriched fraction of black raspberry (BRB), protocatechuic acid and ferulic acid on DB[a,l]P-induced DNA adduct formation in mouse oral tissues. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 230. doi:10.1158/1538-7445.AM2014-230

Abstract 252: Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by the tobacco carcinogen, dibenzo(a,l)pyrene and its metabolites in cultured rat oral fibroblasts

BRB powder and extracts have been shown to inhibit tumorigenesis in oral and other alimentary-tract sites in animal models; and have promising effects on markers of carcinogenesis in several human trials. The purpose of this study was to compare the inhibitory effects of BRB and two related compounds (protocatechuic acid and kaempferol) on mutagenesis and DNA adduct formation induced by the tobacco carcinogen dibenzo[a,l]pyrene (DBP) and its metabolites, DBP-diol and DBP-diol epoxide (DBPDE), in a rat oral epithelial cell line. Mutagenesis and DNA adduct formation induced by DBP, DBP-diol and DBPDE were monitored in rat oral epithelial and fibroblast cells, containing a lacI mutagenesis reporter gene; and the order of potency in both adduct formation and mutagenesis was DBPDE>>DBP-diol>>DBP in both cell types. In the fibroblasts for mutagenesis, the potencies (in units of mutants/10E5 pfu /uM) were: DBPDE, 16,320; DBP-diol, 25; DBP1.7, (above background); background was 2.9., Similar results were obtained for epithelial cells. Analysis of DNA adducts levels is still in progress and results thus far (in units of cis + trans isomers of anti-DBPDE-dA/10E6 A, are: DBPDE, 2096, DBP-diol ≈1.8, DBP Citation Format: Joseph B. Guttenplan, Wieslawa Kosinska, Tianzhen Han, Kun-Ming Chen, Shangmin Zhang, Krishnegowda Gowdahalli, Amin Shantu, Gary Stoner, Karam El-Bayoumy. Effects of black raspberry extract (BRB) and related compounds on mutagenesis induced by the tobacco carcinogen, dibenzo(a,l)pyrene and its metabolites in cultured rat oral fibroblasts. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 252. doi:10.1158/1538-7445.AM2014-252

Abstract 5470: Genotoxic effect and epigenetic alterations induced by the environmental carcinogen dibenzo[a, l]pyrene in oral tissues of mice

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Recently we developed a novel mouse model of oral cancer induced by DB[a, l]P; we also showed the remarkable carcinogenicity and specificity of the fjord region diol epoxide metabolite, (±)-anti-11,12-dihydroxy-13,14-epoxy-11,12,13,14-tetrahydrodibenzo[a, l]pyrene (DB[a, l]PDE). Using immunohistochemistry (IHC), we have shown over-expression of p53 protein in mice oral squamous cell carcinoma and dysplastic tissues induced by DB[a, l]P and DB[a, l]PDE. Current understanding of molecular pathogenesis of oral cancer in humans suggests that both genetic and epigenetic alterations are crucial and complement each other. Our hypothesis is that both genetic and epigenetic alterations induced by DB[a, l]P can contribute to the development of oral cancer. P53 protein overexpression detected by IHC may result from p53 gene mutation or exposure to genotoxic stress. To determine whether p53 over-expression is in part due to p53 mutations, Exons 5 to 8 of p53 from 5 tumor tissues were analyzed by polymerase chain reaction single-strand conformation polymorphisms (PCR-SSCP) and direct sequencing. G to T transversion was detected in Exon 5, leading to mutation of codon 155 Arg to Leu; A to T transversion was detected in Exon 7, resulting in mutation of codon 232 Lys to stop codon. To test the epigenetic effect of DB[a, l]P, methylation specific PCR and bisulfate sequencing were used to detect methylation alteration of p16 and RAR-β promoters. Promoter hypermethylation of both p16 and RAR-β were detected in tumors induced by DB[a, l]PDE; also in oral tissues of mice treated with DB[a, l]P (24nmol, 3 times per week, for 5 weeks, sacrificed at 48 h, 1, 2 and 4 weeks after last dose). These results suggested that genotoxic DNA adducts derived from DB[a, l]P can induce mutations in critical genes, such as tumor suppressor gene p53; this environmental carcinogen also can induce p16 and RAR-β promoter hypermethylation at early stage, which can contribute to the promotion and progression of oral carcinogenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5470. doi:1538-7445.AM2012-5470

Abstract 5469: Detection of deoxyguanosine adducts in the oral tissues of mice treated with the environmental carcinogen dibenzo[a, l]pyrene by LC-MS/MS

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Oral cancer is the major form of head and neck squamous cell carcinoma, which is the fifth most common cancer worldwide. Tobacco smoking is one of the leading causes of oral cancer. Dibenzo[a, l]pyrene (DB[a, l]P) is the most potent carcinogenic polycyclic aromatic hydrocarbon found in tobacco smoke. Our laboratory had developed a mouse model of oral cancer induced by DB[a, l]P. Our working hypothesis is that the stereochemical course of DB[a, l]P metabolism, the conformations of the DNA adducts formed and their removal by mammalian DNA repair enzymes, and their mutagenic properties if not removed in an error-free manner, all play critical roles during the induction of oral carcinogenesis. As an initial study to test our hypothesis, we have previously developed a liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to detect and quantify DB[a, l]PDE-N6-dA adducts in oral tissues of mice treated with DB[a, l]P. We have shown that (-)-anti-cis- and (-)-anti-trans-DB[a, l]PDE-N6-dA adducts were detected from oral tissues of mice treated with DB[a, l]P. In the present study, to further test our hypothesis, we report on the development of a LC-MS/MS method to detect DB[a, l]PDE-N2-dG adducts in vivo. (±)-anti-[15N5]-DB[a, l]PDE-N2-dG adducts were synthesized as internal standards. The stereochemistry of adducts were characterized. Following the addition of internal standards, DNA isolated from oral tissues of mice treated with DB[a, l]P or DB[a, l]PDE was enzymatically hydrolyzed to 2′-deoxyribonucleosides and partially purified by solid-phase extraction. The LC-MS/MS analysis was carried out by monitoring transitions m/z 620 [M+H]+→ m/z 504 [(M+H)+−2′-deoxyribose] for DB[a, l]PDE-N2-dG adducts and m/z 625α m/z 509 for the internal standards. We have detected two N2-dG adducts from oral tissues of mice treated with DB[a, l]P, and four N2-dG adducts from oral tissues of mice treated with (±)-anti-DB[a, l]PDE. Collectively, the in vivo detection of dA and dG adducts indicated that DB[a, l]P is predominantly metabolized to (-)-anti-DB[a, l]PDE in oral tissues of mice. This sensitive LC-MS/MS method, is capable of simultaneously detecting both dA and dG adducts derived from anti-DB[a, l]PDE. Our results indicated that levels of dA adducts are significantly higher than dG adducts in vivo, which are consistent with those reported in literature in organs other than oral tissues, demonstrating that fjord region diol epoxide of DB[a, l]P predominantly form dA adducts than dG adducts. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 5469. doi:1538-7445.AM2012-5469

Abstract 2131: Synthesis and biological evaluation of novel N-alkyl derivatives of 5, 7-dibromoisatin as potent anticancer agents

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Microtubules display an important role in a variety of cellular process, including mitosis and cell division. Various anti-mitotic agents interfering with the natural dynamics of tubulin, polymerization and depolymerization inhibit cancer cell growth. The isatin is found as an endogenous molecule in humans and other mammals, and its analogues display diverse types of biological activities including anti-cancer. Recently, it has been reported that N-alkylation of 5, 7-dibromoisatin increased its cytotoxicity against a range of human cancer cell lines. In this context, it was of interest to further investigate the cytotoxicity of N-alkylated 5,7-dibromoisatin analogues like isothiocyanates and selenocyantes. Isothiocyanates are one of the most effective naturally occurring cancer chemopreventive agents that inhibit carcinogenicity in animal models. Selenium compounds have been found capable of inhibiting and/or retarding tumorigenesis in a variety of experimental animal models. Several synthetic alkyl and aryl selenocyanates have been evaluated for anticarcinogenicity in animal models. The combined literature survey of isatin derivatives, isothiocyantes and selenocyanates have prompted us to develop a novel series of 5,7-dibromoisatin containing thiocyanate, isothiocyanate and selenocyante functionalities. The cytotoxicity of a series of new N-substituted derivatives of 5,7-dibromoisatin was evaluated against a panel of four different human cancer cell lines e.g. a colon (HT29), breast (MCF-7), lung (A549) and melanoma (UACC903) and compared with the 5,7-dibromoisatin. The results showed that the cytotoxicity of the 5,7-dibromoisatin significantly increased through N-alkylation, as reported previously for the 5,7-dibromoisatin derivatives. Introduction of isothiocyante or thiocyanate groups to alkyl chain yielded the most active compounds in this series against HT29 cells. MCF-7 cell line was susceptible to compounds with substitution of selenocyanate group in the alkyl chain, indicating that selenium is playing a role in the antiproliferative activity. These selenium substituted compounds also showed good inhibitory activity against HT29 and A549 cell lines, but poor inhibitory activity in UACC903 cells. Apoptosis assay showed that at 5μM concentration, compounds containing isothiocyanates and thiocyanates induced 99% apoptosis making them the most potent compounds in HT29 cells. The selected compounds were screened for their inhibition of tubulin polymerization in a cell-free in vitro assay. The test compounds significantly inhibited the rate of microtubule polymerization at 10 μM, as compared to vinblastine, a well known microtubule destabilizer. Examination of efficacy of isothiocyanates, thiocyanates, and selenocyanates suggests that these functional groups contribute in enhancing the anticancer activity of novel isatin analogues. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2131. doi:10.1158/1538-7445.AM2011-2131