Sunanda Singh

Anti-genotoxicity and Glutathione S-transferase Activity in Mice Pretreated with Caffeinated and Decaffeinated Coffee

In vivo anti-genotoxic effects of caffeinated and decaffeinated instant coffee were compared in mice after pretreatment either by gavage for 10 consecutive days or in the drinking water for 2 weeks. Changes in hepatic sulfhydryl (-SH) content and glutathione S-transferase (GST) activity were evaluated in pretreated animals. Both caffeinated and decaffeinated instant coffee induced a moderate increase in -SH content and GST activity following pretreatment (with 70, 140 and 280 mg/kg body weight) by gavage for 10 days. This enhancement was not always dose dependent. The maximum effect on GST activity was observed at a dose of 140 mg/kg body weight/day. However, such an effect was not observed after administration of drinking water containing 2% caffeinated/decaffeinated instant coffee for 2 weeks. Results of the bone marrow micronucleus test for evaluating genotoxic effects revealed that both caffeinated and decaffeinated instant coffee (140 mg/kg body weight/day) could exert significant anti-genotoxic effects against ip injected benzo[a]pyrene (BP), cyclophosphamide (CPH), 7,12-dimethylbenz[a]anthracene (DMBA), mitomycin C (MMC) and procarbazine (PCB) in animals pretreated by gavage. Anti-genotoxic effects against BP, DMBA and urethane (URE) were evaluated in animals that received drinking water containing 2% caffeinated/decaffeinated instant coffee for 2 weeks. With the exception of the anti-genotoxic effect of decaffeinated coffee against DMBA, there was no significant change in genotoxicity after the above pretreatment. From this work, there is no evidence for any significant difference in the in vivo anti-genotoxicity of caffeinated and decaffeinated instant coffee.

Postnatal effect of arecoline on chlorophyllin-modulated hepatic biotransformation system enzymes in suckling neonate and lactating mice

The present study evaluates the potential of arecoline alkaloid on chlorophyllin (CHL) modulated levels of hepatic biotransformation system enzymes in suckling neonate and lactating mice. CHL [50, 100, or 200 mg/kg body weight (b.w.)/day] induced significant increases in the hepatic levels of glutathione S-transferase (GST) and sulfhydryl (-SH) in lactating dams and suckling pups of 14 or 21 days. The depleted level of hepatic Cytochrome (Cyt.) P-450 was observed only in lactating dams given 200 mg/kg b.w. CHL. Arecoline (20 mg/kg b.w.) could depress the CHL-induced levels of hepatic GST and -SH, while Cyt. P-450 and Cyt. b5 levels remained unaltered by arecoline alone or arecoline plus CHL treatment. In lactating dams the modulated levels of hepatic biotransformation system enzymes potentially could affect the detoxication efficacy of administered chemicals besides influencing the rate and extent of passage of metabolites to suckling neonate.

Direct and translactational effect of arecoline alkaloid on the clocimum oil-modulated hepatic drug metabolizing enzymes in mice.

The present study assesses the potential of arecoline alkaloid, by direct exposure in lactating dams and translactational exposure in neonates, to modulate the efficacy of clocimum oil as a blocking agent in chemopreventive pathway. Clocimum oil (25 or 50 microl/dam/day) induced a significant increase in the hepatic levels of phase II glutathione S-transferase (GST) and acid-soluble sulfhydryl in lactating dams and suckling neonates while the elevated levels of hepatic phase I cytochrome b5 (Cyt. b5) and cytochrome P-450 (P450) were observed only in the dams. Arecoline (0.6 mg/dam/day) alone did not modulate the hepatic GST and sulfhydryl levels in either dams or pups, although significant induction was observed in the hepatic levels of Cyt. b5, P450 and malondialdehyde (MDA) in lactating dams and suckling neonates. Clocimum oil-modulated hepatic levels of phase II components were depressed whereas phase I enzymes and lipid peroxides levels were further elevated by clocimum oil-plus-arecoline treatment. The direct or translactationally augmented levels of bioactivated species of the administered compounds, via enhanced phase I oxidative catalysis and less efficient GST/GSH conjugational detoxication, may suggest the antagonistic influence of arecoline on chemopreventive efficacy of clocimum oil.

Suppression of Breast Cancer Cell Proliferation by Selective Single-Domain Antibody for Intracellular STAT3:

Background: The serendipitous discovery of heavy-chain antibodies devoid of light chains in camelids and the subsequent development of VHHs (variable region of camelid heavy chain) have provided a very important tool for research and possibly for therapeutics. In this study, we synthesized single-domain 15-kDa antibody SBT-100 (anti-STAT3 B VHH13) against human STAT3 (signal transducer and activator of transcription) that binds selectively to STAT3 and suppresses the function of phosphorylated STAT3 (p-STAT3). Methods: Single-chain VHH nanobodies were generated by immunizing camelid with humanized STAT3. Commercially available breast cancer cell lines including MDA-MB-231, MDA-MB-468, MDA-MB-453, MCF-7, and BT474 were used. Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The association of anti-STAT3 B VHH13 with STAT3 and p-STAT3 was determined by immunoprecipitation and Western blot analyses. The efficacy of SBT-100 on the growth of MDA-MB-231 xenografts in vivo was determined using athymic mice. Statistical significance for cell proliferation was determined using analysis of variance. If a significant difference (P < .05) was observed, then Tukey-Kramer multiple comparison test was conducted. Results: SBT-100 suppressed cell proliferation of triple-negative breast cancer cells (P < .01) as well as provided significant inhibition of tumor growth (P < .05) in a xenograft model without any toxicity. Results are presented to show that anti-STAT3 B VHH13 selectively binds to STAT3 suggesting that the effects were mediated by inhibiting STAT3. Conclusions: A very large number of human malignancies and benign diseases have constitutive STAT3 activation. Therefore, the results described here suggest that anti-STAT3 B VHH13 can be developed for therapeutic intervention for cancer cells expressing STAT3 or p-STAT3.

Abstract 4699: Single domain antibody (sdAb) localizes in cancer cells to inhibit signal transducer and activator of transcription 3 (STAT3) resulting in therapeutic inhibition of multiple cancers

STAT3 is involved in the pathogenesis of many malignancies, so we developed an anti-STAT3 VHH (variable region of the heavy chain), SBT-100, that internalizes in cancer cells and binds unphosphorylated STAT3 (U-STAT3) and phosphorylated STAT3 (P-STAT3) and results in significant inhibition of multiple cancers. ATCC cell lines for triple negative breast cancers (MDA-MB-231, MDA-MB-468, MDA-MB-453), ER+/PR+ breast cancer (MCF-7), HER2+ breast cancer (BT474), pancreatic cancers (PANC-1, BX-PC3), murine mammary cancer (4T1), STAT3 null cells (PC-3), and castrate-resistant prostate cancer (DU145) were tested. Athymic nude mice were obtained from Envigo. The IL-6 Reporter Cell Assay was obtained from Promega. VEGF inhibition ELISA was done using retinal epithelial cells (ATCC). In vitro growth inhibition was done using a MTT assay. Immunoprecipitation (IP) and western blot studies in MDA-MB-231, PANC-1, HeLa, DU145, 4T1 and PC-3 showed that SBT-100 binds U-STAT3 and P-STAT3. No STAT3 binding was seen in PC-3. Binding to P-STAT3 was seen in lysates from the 4T1 cells, which have constitutively activated STAT3. Since rodent and human STAT3 have a 99% homology, rodents are excellent models for extrapolating to human disease for over production of P-STAT3. Within 24 hrs IL-6 assay showed that SBT-100 blocked the production of IL-6 (p Citation Format: Sunanda Singh, Genoveva Murillo, Amanda Rom, Avani Singh, Samara Singh, Meenakshi Parihar, Dong Chen, Rajendra Mehta, Robert Baker, Anjali Singh, Ashutosh S. Parihar. Single domain antibody (sdAb) localizes in cancer cells to inhibit signal transducer and activator of transcription 3 (STAT3) resulting in therapeutic inhibition of multiple cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4699. doi:10.1158/1538-7445.AM2017-4699

Abstract P6-12-13: Single domain antibody (SBT-100) inhibits growth of human HER2+ and triple negative breast cancers (TNBC) in xenografts by binding STAT3 and P-STAT3

SBT-100 is a single domain antibody (sdAb), developed by Singh Biotechnology, that binds unphosphorylated signal transducer and activator of transcription 3 (STAT3) and phosphorylated STAT3 (P-STAT3). SBT-100 is approximately 13 kD or less than 1/10th the size of a human IgG molecule, and is able to cross the cell membrane to bind intracellular STAT3 and P-STAT3. This in turn inhibits its effects on genes that promote malignant behavior of cancer cells. SBT-100 has a short serum half-life but a long biological half-life. Since certain types of human breast cancers express P-STAT3, we wanted to determine if SBT-100 could inhibit the growth of human breast cancers in vitro and in vivo by studying its effects on MCF-7 (ER+/PR+), BT474 (HER2+), and MDA-MB-231 (TNBC) cells. BACKGROUND: Many different types of human cancers (solid tumors, leukemias, and lymphomas) are dependent on constitutive expression of (P-STAT3) for their malignant phenotype. Growth factors, tyrosine kinase receptors, cytokines (IL-6, IL-11, IL-12, IL-23), BCR-ABL, and Src are some ways that STAT3 can be activated. In turn P-STAT3 turns on genes such as Cyclin D1 & D3, MMPs, Bcl-xL, Mcl-1, survivin, VEGF, and HIF-1 alpha. Constitutive expression of P-STAT3 has been shown to promote cancer cell proliferation, survival, angiogenesis, immune suppression, and metastasis. Additionally there is increasing evidence suggesting that unphosphorylated STAT3 contributes to malignant phenotype of cancers. STAT3 is also important for the survival of cancer stem cells as well as for some human breast cancers. METHODS: Immunoprecipitation and Western blot analyses were carried out to test whether SBT-100 binds cytoplasmic STAT3 and P-STAT3 in various malignant cell lines (e.g., MDA-MB-231, PANC-1, DU145, and HeLa). MTT assays were done to determine if SBT-100 could suppress the growth of different types of human breast cancers in vitro. Xenograft cancer models using ER+/PR+ (MCF-7), HER2+ (BT474), and TNBC (MDA-MB-231) cancer cells were used to evaluate treatment with SBT-100 1mg/kg/BID (IV and/or IP route). RESULTS: Immunoprecipitation and Western blot studies demonstrated that SBT-100 binds to both STAT3 and P-STAT3 in human cancers cells (MDA-MB-231, PANC-1, DU145, and HeLa). In a three day MTT assay, at least 90% growth suppression was achieved for all three subtypes of human breast cancer, which is highly significant. In the xenograft cancer models, SBT-100 (1mg/kg/BID) treatment for 28 days, yield growth suppression as follows: MDA-MB-231 44.8% (p CONCLUSION: Singh Biotechnology9s novel sdAb, SBT-100 suppresses growth of TNBC and HER2+ human breast cancers in vivo and suppresses growth of ER+/PR+, HER2+, and TNBC cells in vitro. The most significant anti-cancer effects of SBT-100 is observed against human TNBC. Citation Format: Singh S, Murillo G, Chen D, Singh A, Singh S, Singh A, Mehta R, Parihar A. Single domain antibody (SBT-100) inhibits growth of human HER2+ and triple negative breast cancers (TNBC) in xenografts by binding STAT3 and P-STAT3 [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P6-12-13.