Amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (PhIP), found in cooked meat, is a known food carcinogen that causes several types of cancer, including breast cancer, as PhIP metabolites produce DNA adduct and DNA strand breaks. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity. To date, no study has examined the interaction of PhIP with curcumin in breast epithelial cells. The present study demonstrates the mechanisms by which curcumin inhibits PhIP-induced cytotoxicity in normal breast epithelial cells (MCF-10A). Curcumin significantly inhibited PhIP-induced DNA adduct formation and DNA double stand breaks with a concomitant decrease in reactive oxygen species (ROS) production. The expression of Nrf2, FOXO targets; DNA repair genes BRCA-1, H2AFX and PARP-1; and tumor suppressor P16 was studied to evaluate the influence on these core signaling pathways. PhIP induced the expression of various antioxidant and DNA repair genes. However, co-treatment with curcumin inhibited this expression. PhIP suppressed the expression of the tumor suppressor P16 gene, whereas curcumin co-treatment increased its expression. Caspase-3 and -9 were slightly suppressed by curcumin with a consequent inhibition of cell death. These results suggest that curcumin appears to be an effective anti-PhIP food additive likely acting through multiple molecular targets.

Scutellaria ocmulgee (Ocmulgee skullcap) is a rare plant species with medicinal and ornamental value and requires immediate conservation. We report here the first protocol for plant regeneration and Agrobacterium-mediated transformation of S. ocmulgee using leaf and shootderived transverse thin cell layer (tTCL) explants. The effect of MS and B5 salts in combination with varying levels of growth regulators and two carbon sources on shoot proliferation and plant regeneration were studied. Among the various media treatment combinations, the best shoot induction response was observed from leaf-derived tTCL explants on B5 medium supplemented with 6-benzyl aminopurine (BAP), 1-naphthalene acetic acid (NAA), and sucrose whereas shoot-derived tTCL explants produced the maximum number of shoots also on B5 medium supplemented with either BAP or thidiazuron, NAA and maltose. Shoots obtained from both types of explants were rooted on MS medium containing 5.0 lM indole butyric acid. Agrobacterium-mediated genetic transformation protocol was optimized using leaf tTCL explants. A binary plasmid pq35SGR harboring the b glucuronidase and green fluorescent protein/neomycin phosphotransferase II fusion reporter gene was used to optimize transformation parameters. Explants were co-cultivated with Agrobacterium tumefaciens EHA105 and then transferred to shoot induction and regeneration medium to generate transgenic plants. Stable gene expression was observed in transgenic cultures and plants. The presence and integration of transgenes was confirmed by polymerase chain reaction, reverse transcriptase-polymerase chain reaction and Southern blot hybridization.

Bacopa monnieri (L.) Wettst. (Scrophulariaceae) is a highly sought after medicinal plant with therapeutic properties as cognition enhancer as well as for other brain and body functions. Research was conducted to optimize a thin cell layer explant based micropropagation system to assist mass propagation. Thin cell layers (TCL) derived from leaf and internode segments were used as explants. Murashige and Skoog medium was used to formulate shoot induction, elongation, and rooting media. Shoot induction media were prepared by supplementing three concentrations (0.1, 1.0, and 10.0 µM) of four cytokinins 6-benzylaminopurine, 2-isopentenyl-adenine, 6-3-Hydroxybenzylaminopurine, and thidiazuron to study adventitious shoot bud induction response. An optimum shoot bud induction response was observed on MS medium supplemented with 10.0 µM 6-benzylaminopurine for both leaf and stem transverse thin cell layer (tTCL) explants. The average number of shoot buds from leaf tTCL explants was 59, whereas, on an average, 33 shoot buds were regenerated from internode tTCL explants. Elongation of adventitious shoot buds was achieved best in a liquid medium using Liquid Lab Rocker ® system. Elongated shoots recorded 100% rooting in MS medium supplemented with 5 µM indole butyric acid. Bacopa micropropagation employing tTCL explants for initial shoot bud induction and using LLR ® boxes in subsequent elongation step can achieve cost effective way to regenerate high volume of plantlets and biomass required for herbal industry. Leaf and stem tTCL explants both were suitable for Agrobacterium tumefaciens (EHA105) mediated genetic transformation. Successful transformation was scored * Corresponding author. L. A. Croom et al. 1233within three days of co-cultivation with Agrobacterium suspension on the basis of Enhanced Green Fluorescent Protein (EGFP) expression as an early and non-destructible screening device. Transformation frequencies of 83% and 76% were accomplished for leaf and stem tTCL explants, respectively. Greenhouse grown Bacopa plants were analyzed as fresh and dry methanolic extracts for total polyphenol content (811.93 ± 7.98 and 814 ± 17.64 GAE mg g-1) and the Trolox Equivalent Antioxidant Capacity values were 1918.25 ± 173.12 and 3163.14 ± 403.25 µmol/g, respectively.

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PhIP is a known food carcinogen found in well done meat which causes several cancers including breast cancer. PhIP metabolites produce DNA adduct and DNA strand breaks. Curcumin, obtained from the rhizome of Curcuma longa, has potent anticancer activity. So far none of the study demonstrates the inhibition of PhIP induced cytotoxicity by the co-treatment of Curcumin in normal breast epithelial cells in vitro. Therefore, we developed a model system using the MCF 10A normal breast epithelial cells to study the PhIP cytotoxicity and if cells are co-cultured with Curcumin and PhIP how it affects. Consequently, the core signaling pathways have been explored to evaluate the efficacy of Curcumin. In this study, the effectiveness of Curcumin was investigated in MCF 10A cells along with PhIP. The cytotoxic ability was detected with MTT assay, ROS activation by DCF, the influence of the cell cycle was checked with flow cytometry, DNA damage by comet assay, DNA adduct formation by anti-PhIP DNA primary, and apoptosis by Annexin-V-FITC staining. The influence of the core signaling pathways was evaluated by RT PCR and/or Western blotting which included Nrf2 (GSR, GPX, NQO1), FOXO (Catalase, GADD45, PRDX3) targets; DNA repair genes/proteins BRCA1, H2AFX and PARP-1; and tumor suppressor P16 gene expression. PhIP cytotoxicity induced the expression of various antioxidant and DNA repair genes on MCF-10A cells but co-treatment of Curcumin retained its expression level similar to untreated groups. Additionally, Curcumin co- treatment increased the expression level of tumor suppressor expression gene P-53. Expression of antioxidants genes was induced by PhIP whereas Curcumin significantly suppress the PhIP induced ROS activation, DNA strand breaks and DNA adduct formation and consequently inhibited the cell death. In conclusion, Curcumin appears to be effective to inhibit P450 mediated ROS production and PhIP-DNA adducts which consequently reduces DNA damage. Citation Format: Ashok K. Jain, Abhilash Samykutty, Carissa L. Jackson, Muthusamy Thangaraju, Darren D. Browning. Curcumin inhibit PhIP induced cytotoxicity by inhibiting ROS production, DNA strand breaks and DNA adducts formation in MCF 10A cells. [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 4106. doi:10.1158/1538-7445.AM2014-4106