Thomas Primiano

Antioxidant-Inducible Genes

Publisher Summary Antioxidants inhibit the propagation of free radical reactions. Because of their widespread use as preservatives in processed foods, their biochemical effects have been investigated. Early feeding studies indicated that butylated hydroxytoluene (BHT) increased liver weight, induced proliferation of smooth endoplasmic reticulum, and elevated several hepatic microsomal mono-oxygenase activities typical of phase 1 metabolism. In addition to phase1, antioxidants also induce phase2 xenobiotic-metabolizing enzymes. Phase2 enzymes detoxify activated electrophilic metabolites of xenobiotics via conjugation of endogenous substrates such as glutathione (GSH). Antioxidants act to directly terminate the propagation of free radical reactions, and increase the activity of enzymes those readily metabolize and aid in the elimination of potential cytotoxic chemicals. Dietary administration of antioxidants induced phase 2 xenobiotic-metabolizing enzymes. Induction of phase 2 enzymes by antioxidants was found in many organs and tissues, such as liver, lung, kidney, small intestine, colon, and spleen, thereby affording protection at many anatomical sites .This chapter presents evidence for several newly identified antioxidant-inducible genes, describes the proposed mechanisms of antioxidant signal transduction leading to enhanced expression of these enzymes, and complements the information presented in related reviews concerning the mechanisms and consequences of induction of phase 2 xenobiotic-metabolizing enzymes by antioxidants. The chapter discusses the genes induced by antioxidants—cytochrome P450s, Glutathione, S-transferases, NAD(P)H: quinone reductase, UDP-glucuronosyltransferases, microsomal epoxide hydrolase, aflatoxin BI-aldehyde reductase, dihydrodiol dehydrogenases, aldehyde dehydrogenases, enzymes of glutathione and reduced nicotinamide metabolism, and other proteins and enzymes. The chapter delves into the mechanisms of gene induction by antioxidants—the antioxidant-response element (ARE), proteins binding and signal transduction through the ARE—and the consequences of antioxidant gene induction. The regulation of antioxidant-inducible genes helps understand the signals leading to cellular transformation and carcinogenesis. Antioxidants can be used to decipher the encrypted signal transduction pathways that prevent cell transformation and carcinogenesis.

Redox regulation of genes that protect against carcinogens.

Most carcinogens require activation to electrophilic metabolites or species that generate reactive oxygen in order to initiate the tumorigenic process. These reactive intermediates can, in turn, be detoxified by endogenous enzyme systems that and in the protection of cells from either toxic or mutagenic product formation. The levels of many of these enzymes are elevated by numerous compounds found in the diet, or by antioxidants. Recent evidence describes the mechanism for this induction of carcinogen detoxication enzymes to be regulated at the transcriptional level. Nuclear transcription factors bound to sites common among these carcinogen detoxication genes are activated by as yet unknown signal transduction pathways. The activity of these nuclear transcription factors are modulated by pro- and antioxidant reagents, suggesting that a redox-sensitive component governs the induction of enzymes involved in carcinogen metabolism. In this review, evidence for the redox regulation of the genes encoding carcinogen detoxication enzymes is presented. Evidence is also presented suggesting the participation of nuclear factor kappa B (NF-kappa B), mitogen-activated protein (MAP) kinase, and basic leucine zipper (bZIP) proteins and their activation pathways in this induction.

Induction of Leukotriene B4 Metabolism by Cancer Chemopreventive Agents

Dithiolethiones and other cancer chemopreventive agents inhibit the production of experimentally produced tumors by elevating the expression of several genes that encode for known cytoprotective enzymes1. Complementary DNA clones representing dithiolethione-inducible gene-1 (DIG-1*) were isolated from rat liver via differential hybridization screening2. The deduced amino acid sequence of DIG-1 was found to have 80% identity with the human liver enzyme leukotriene B4 (LTB4)-12-hydroxydehydrogenase (LTB4 DH)3,4. DIG-1, purified >400-fold from the liver of rats dosed with l,2-dithiole-3-dithiolethione (D3T), possessed an NADP+-dependent activity to convert LTB4 to 12-oxo-LTB4 similar to LTB4DH 3. The formation of 12-oxo- LTB4 by LTB4 DH is the first step in the catabolism of LTB4 4,5. Subsequent conversion of 12-oxo- LTB4 to 10,11-dihydro-12-oxo- LTB4 (Met I) and 10,11,14,15-tetrahyro- 12-oxo- LTB4 (Met II) by as yet unidentified reductases has been described in liver and kidney cytosolic preparations as shown in Fig. 16. These metabolic products of LTB4 have diminished pro-inflammatory capacities, such as enhancing the mobilization of intracellular calcium, putatively via antagonism of the LTB4 receptor7. Suppression of the pro-inflammatory actions of LTB4, such as stimulation of neutrophil chemotaxis and Superoxide anion generation, suggests that the catabolism of LTB4 is a cytoprotective process.

Abstract 287: Specific delivery of immunostimulatory RNA via nanoparticles blocks growth of primary and disseminated ovarian tumors

One important mechanism of immune evasion is the accumulation of tumor-infiltrating regulatory T cells creating an immunosuppressive microenvironment in situ. Many new therapies target immune checkpoint receptors, CTLA-4 and PD-1, on immunosuppressive T-cells to reverse tumor immune evasion. Small interfering RNAs (siRNAs) are double stranded, sequence-specific inhibitors of gene expression. The paradigm for using siRNA to block cancer is to target mRNA sequences of oncogenes. Several limitations have suppressed the use of siRNA in human cancer therapy, such as off-target effects and lack of tumor-specific delivery. Additionally, certain siRNA sequence motifs activate toll-like receptors that induce the systemic innate immune response, including induction of interferon and interleukin-6 release that is traditionally considered as an undesirable clinical outcome. The hypothesis proposed here is to control the innate immune response by directing it only to tumors and not other organs by delivering an immunostimulatory siRNA (isRNA) directly to tumors. An isRNA designed to silence no human genes was synthesized as an unmodified triphosphate 19-mer double-stranded RNA with a GUGA motif on the 5′ antisense end. This isRNA was delivered into human ovarian tumors grown from SK-OV-3 cells surgically implanted into the ovaries of immunosuppressed mice by an L1CAM-targeted elastin-like polypeptide (ELP-L) nanoparticle. The isRNA/ELP-L nanocomplex was dosed into ten mice by i.p. injection 14 times at 10 mg/kg with respect to the isRNA over 28 days. The isRNA was delivered to the tumors, not to liver, spleen, heart, or lungs. The total tumor mass and number of disseminated nodules both decreased by 50% with respect to vehicle control. Six ovaries harboring tumors were resected from isRNA/ELP-L nanocomplex-dosed mice and each displayed elevated levels of interferon alpha and interleukin 6 mRNA as determined by RT-PCR. Mean AST, ALT and BUN serum biomarkers of liver and kidney function were elevated by 68%, 128% and 14%, respectively, compared to vehicle dosed mice; none of which were outside average normal levels. These results indicate that specifically targeting isRNA to tumors using a targeted nanoparticle may provide an effective therapy for ovarian cancer, either alone or in combination with agents that affect immune check point pathways in tumors. Citation Format: Thomas Primiano, Bey-ih Chang. Specific delivery of immunostimulatory RNA via nanoparticles blocks growth of primary and disseminated ovarian tumors. [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 287. doi:10.1158/1538-7445.AM2015-287

Abstract LB-103: L1CAM-targeted delivery of siRNA using elastin-like polypeptide (ELP) nanoparticles inhibits the growth of human tumors implanted in mice

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Small interfering RNA (siRNA) drugs provide ideal means for perturbing intracellular oncogenic targets. However, specific delivery of siRNA to tumors has proven to be difficult. An ELP was engineered with an N-terminal region that binds to L1 cell adhesion molecule (L1CAM), and a C-terminal region that binds siRNA. Upon binding of siRNA, the L1CAM targeted ELP (ELP-L) spontaneously assembled into a spherical nanocomplex with the siRNA protected within, and the CAM-binding region protruding from the surface. These nanoparticles were used to deliver siRNA to SKOV3 tumors formed following surgical implantation of the cells into the left ovary of mice. Two weeks after implantation, mice were dosed with vehicle, ELP-L-complexed control siRNA (siCON), or ELP-L-complexed siEVI1, an siRNA to the oncogenic transcription factor EVI1, that is known to enable cell proliferation and inhibits apoptosis in ovarian tumor cells. To initially assess biodistribution, a single dose of ELP-L-DY800-labeled siEVI1 (10 mg/kg, ip) localized into ovarian tumors as early as 4 h, and remained for 72 h, as measured by co-registration of near infrared fluorescence with tumor bioluminescence in live mice. The ELP-L-DY800-siEVI1 was eliminated via kidney and did not accumulate in heart, lung, spleen or liver showing specific localization to tumors. To evaluate efficacy, the mean flux of bioluminescence from ovarian tumors of ten mice receiving 14 doses of 10 mg/kg siEVI1 in ELP-L over 28 days decreased by 92% with respect to mice dosed with 5% dextrose. Immunoblot and qPCR results showed EVI1 protein and mRNA levels decreased more than 50% in mice dosed with siEVI1 compared to siCON or vehicle. There were no observable adverse effects in the mice throughout the dosing period. These results are consistent with an RNA interference mechanism and suggest that ELP-L effectively delivers siEVI1 to tumors with limited toxicity. Furthermore, EVI1 appears to be a good target for ovarian cancer therapy and silencing by siRNAs is an effective means of triggering the death of ovarian tumor cells. Citation Format: Thomas Primiano, Bey-Dih Chang, Jeremy D. Heidel. L1CAM-targeted delivery of siRNA using elastin-like polypeptide (ELP) nanoparticles inhibits the growth of human tumors implanted in mice. [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 LB-103. doi:10.1158/1538-7445.AM2014-LB-103

Abstract 1946: Silencing of EVI1 with siRNA inhibits the growth of ovarian tumor cells

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL There is currently no effective treatment for ovarian cancer. EVI1 (ecotropic viral integration site 1) is an oncogenic transcription factor that enables proliferation and inhibits apoptosis of ovarian tumor cells. Small interfering RNA (siRNA) drugs provide ideal means for perturbing intracellular oncogenic targets. Five (5) siRNA (21+21 nt) sequences were identified by in silico analysis that target the coding region of human EVI1 (siEVI1 A-E). Each of these siEVI1 species were transfected with Dharmafect4 at 25nM into SKOV3, ES-2, OVCAR3, OV-90, TOV-112D, PA-1, UWB1.289, and Coav-3 ovarian tumor cells. The non-tumorigenic cell line Hs832.Tc was used as a control. Four species (siEVI1 B-E) decreased the viability of each tumor cell line by at least 70% with none of the five siEVI1 species altered the viability of control cells, demonstrating the anti-proliferative effect of targeting EVI1 with siRNA. Both siEVI1-B and -D potently decreased the viability of ovarian tumor cells, having IC50 values of 0.8 and 0.2 nM, respectively, which compare favorably to an IC50 of 15 nM for paclitaxel. RT-PCR and immunoblot results showing dose-dependent decreases in EVI1 mRNA and protein using either siEVI1-B or-D are consistent with the mechanism of the anti-proliferative effect occurring via RNA interference. Further, dose-dependent increases in annexin staining upon siRNA treatment indicate that induction of apoptosis contributes to the decrease in cell viability, a result consistent with inhibition of EVI1 activity. These results suggest that EVI1 is a good target for ovarian cancer therapy and silencing by siRNAs is an effective means of triggering the death of ovarian tumor cells. These findings also suggest that anti-EVI1 siRNAs have the potential to be effective therapeutics for the treatment of patients with ovarian cancer. 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 1946. doi:1538-7445.AM2012-1946