Markers Of Tumor Promotion Research Articles

PSCA is a critical biomarker for predicting the prognosis of KRAS/TP53 mutant pancreatic cancer patients

Background: Partly due to the limited effect of chemotherapy or other therapeutic strategies, which may be due to the insufficient knowledge of the tumor promotion markers and targets, pancreatic cancer (PC) holds the position of one of the most malignant tumors. This study aims to find a diagnosis/therapeutic molecule that can predict the prognosis of PC with different gene background. Methods: The Cancer Genome Atlas (TCGA) pancreatic duct adenocarcinoma (PAAD)–based single nucleotide polymorphisms and gene expression data were used to find the differentially expressed genes (DEGs) between KRAS/TP53 mutant samples and no gene mutation samples. Gene Set Enrichment Analysis (GSEA)-based Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis and R-based gene oncology (GO) or immune cell invasion assay were used to explore the above DEGs involved pathways. The single-center PC cohort accompanied with next-generation sequence testing was used to verify the TCGA PAAD–based bioinformatic results. Results: First, we found PC patients who harbored KRAS and/or TP53 gene mutation have poor overall survival. Besides, the enrichment analysis showed that mutant KRAS/TP53 was correlated with PC tumor-promotion–related pathways and immune microenvironment. Next, we detected that prostate stem cell antigen (PSCA) was one of the most differential genes in KRAS/TP53 mutant PC tissues. Indeed, the bioinformatic analysis and our clinical data showed that PSCA was a biomarker of poor prognosis in PC. Conclusion: PSCA is a critical biomarker for predicting the prognosis of KRAS/TP53 mutant PC patients.

Is Glyceryl Trinitrate, a Nitric Oxide Donor Responsible for Ameliorating the Chemical-Induced Tissue Injury In Vivo?

Oxidative stress induced by well-known toxins including ferric nitrilotriacetate (Fe-NTA), carbon tetrachloride (CCl4) and thioacetamide (TAA) has been attributed to causing tissue injury in the liver and kidney. In this study, the effect of glyceryl trinitrate (GTN), a donor of nitric oxide and NG-nitroarginine methyl ester (l-NAME), a nitric oxide inhibitor on TAA-induced hepatic oxidative stress, GSH and GSH-dependent enzymes, serum transaminases and tumor promotion markers such as ornithine decarboxylase (ODC) activity and [3H]-thymidine incorporation in rats were examined. The animals were divided into seven groups consisting of six healthy rats per group. The six rats were injected intraperitoneally with TAA to evaluate its toxic effect, improvement in its toxic effect if any, or worsening in its toxic effect if any, when given in combination with GTN or l-NAME. The single necrogenic dose of TAA administration caused a significant change in the levels of both hepatic and serum enzymes such as glutathione S-transferase (GST), glutathione reductase (GR), glutathione peroxidase (GPx), γ-glutamyl transpeptidase (GGT), glucose 6-phosphate dehydrogenase (G6PD), alanine aminotransferase (AST) and aspartate aminotransferase (ALT). In addition, treatment with TAA also augmented malondialdehyde (MDA), ornithine decarboxylase (ODC) activity and [3H]-thymidine incorporation in rats liver. Concomitantly, TAA treatment depleted the levels of GSH. However, most of these changes were alleviated by the treatment of animals with GTN dose-dependently. The protective effect of GTN against TAA was also confirmed histopathologically. The present data confirmed our earlier findings with other oxidants including Fe-NTA and CCl4. The GTN showed no change whatsoever when administered alone, however when it was given along with TAA then it showed protection thereby contributing towards defending the role against oxidants-induced organ toxicity. Overall, GTN may contribute to protection against TAA-induced oxidative stress, toxicity, and proliferative response in the liver, according to our findings.

Deoxynivalenol induced mouse skin cell proliferation and inflammation via MAPK pathway

Several toxicological manifestations of deoxynivalenol (DON), a mycotoxin, are well documented; however, dermal toxicity is not yet explored. The effect of topical application of DON to mice was studied using markers of skin proliferation, inflammation and tumor promotion. Single topical application of DON (84–672nmol/mouse) significantly enhanced dermal hyperplasia and skin edema. DON (336 and 672nmol) caused significant enhancement in [3H]-thymidine uptake in DNA along with increased myeloperoxidase and ornithine decarboxylase activities, suggesting tissue inflammation and cell proliferation. Furthermore, DON (168nmol) caused enhanced expression of RAS, and phosphorylation of PI3K/Akt, ERK, JNK and p38 MAPKs. DON exposure also showed activation of transcription factors, c-fos, c-jun and NF-κB along with phosphorylation of IkBα. Enhanced phosphorylation of NF-κB by DON caused over expression of target proteins, COX-2, cyclin D1 and iNOS in skin. Though a single topical application of DMBA followed by twice weekly application of DON (84 and 168nmol) showed no tumorigenesis after 24weeks, however, histopathological studies suggested hyperplasia of the epidermis and hypertrophy of hair follicles. Interestingly, intestine was also found to be affected as enlarged Peyer's patches were observed, suggesting inflammatory effects which were supported by elevation of inflammatory cytokines after 24weeks of topical application of DON. These results suggest that DON induced cell proliferation in mouse skin is through the activation of MAPK signaling pathway involving transcription factors NFκB and AP-1, further leading to transcriptional activation of downstream target proteins c-fos, c-jun, cyclin D1, iNOS and COX-2 which might be responsible for its inflammatory potential.

Novel metabolites in cyanobacterium Cylindrospermopsis raciborskii with potencies to inhibit gap junctional intercellular communication

Despite intensive research into toxic bloom-forming cyanobacteria, the majority of their metabolites remain unknown. The present study explored in detail a novel bioactivity identified in cyanobacteria, i.e. inhibition of gap junctional intercellular communication (GJIC), a marker of tumor promotion. The extracellular mixture (exudate) of the cyanobacterial strain Cylindrospermopsis raciborskii (SAG 1.97) was fractionated by semi-preparative reversed phase HPLC, and the fractions assessed for their potencies to inhibit GJIC. Two non-polar fractions that significantly inhibited GJIC were further fractionated, tested and analyzed using multiple mass spectrometric methods. Investigations led to the identification of a putative chemical compound (molecular formula C18H34O3, m/z 299.2581 for the [M+H]+ ion) responsible for observed bioactivities. Specific inhibitors of signaling pathways were used to screen for biochemical mechanisms beyond GJIC inhibition, and the results indicate the involvement of ERK1/2 kinases via a mechanism related to the action of epidermal growth factor EGF but clearly distinct from other anthropogenic tumor promoters like polychlorinated biphenyls or polycyclic aromatic hydrocarbons. The chemical and in vitro toxicological characterizations of the newly described metabolite provide important insights into the still poorly understood health impacts of complex toxic cyanobacterial blooms and indicate that currently applied monitoring practices may underestimate actual risks.

Chemopreventive effects of aloin against 1,2-dimethylhydrazine-induced preneoplastic lesions in the colon of Wistar rats

Chemoprevention opens new window in the prevention of all types of cancers including colon cancer. Aloin, an anthracycline in plant pigment, can be utilized as a protective agent in cancer induction. In the present study, we have evaluated the chemopreventive efficacy of aloin against 1,2-dimethylhydrazine (DMH)-induced preneoplastic lesions in the colon of Wistar rats. DMH-induced aberrant crypt foci (ACF) and mucin-depleted foci (MDF) have been used as biomarkers of colon cancer. Efficacy of aloin against the colon toxicity was evaluated in terms of biochemical estimation of antioxidant enzyme activities, lipid peroxidation, ACF, MDF, histopathological changes, and expression levels of molecular markers of inflammation and tumor promotion. Aloin pretreatment ameliorates the damaging effects induced by DMH through a protective mechanism that involved reduction in increased oxidative stress enzymes (p < 0.001), ACF, MDF, cyclooxygenase-2, inducible nitric oxide synthase, interleukin-6, proliferating cell nuclear antigen protein expression, and tumor necrosis factor-α (p < 0.001) release. From the results, it could be concluded that aloin clearly protects against chemically induced colon toxicity and acts reasonably by inducing antioxidant level, anti-inflammatory and antiproliferative markers.

The effects of dissociated glucocorticoids RU24858 and RU24782 on TPA-induced skin tumor promotion biomarkers in SENCAR mice

Glucocorticoids (GCs) are very effective at preventing carcinogen- and tumor promoter-induced skin inflammation, hyperplasia, and mouse skin tumor formation. The effects of GCs are mediated by a well-known transcription factor, the glucocorticoid receptor (GR). GR acts via two different mechanisms: transcriptional regulation that requires DNA-binding (transactivation) and DNA binding-independent protein-protein interactions between GR and other transcription factors, such as nuclear factor kappa B (NF-κB) or activator protein 1 (AP-1; transrepression). We hypothesize that the transrepression activities of the GR are sufficient to suppress skin tumor promotion. We obtained two GCs (RU24858 and RU24782) that have dissociated downstream effects and induce only transrepression activities of the GR in a number of systems. These compounds bind the GR with high affinity and repress AP-1 and NF-κB activities while showing a lack of GR transactivation. RU24858, RU24782, or control full GCs desoximetasone (DES) and fluocinolone acetonide (FA) were applied to the dorsal skin of SENCAR mice prior to application of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA), two times per week for 2 weeks. DES, FA and RU24858 reversed TPA-induced epidermal hyperplasia and proliferation, while RU24782 treatment had no effect on these markers of skin tumor promotion. All tested compounds decreased TPA-induced c-jun mRNA levels in skin. DES, FA, and RU24858, but not RU24782, were also able to reverse TPA-induced increases in the mRNA levels of COX-2 and iNOS. These findings show that RU24858 but not RU24782 reduced TPA-induced epidermal hyperplasia, proliferation, and inflammation, while both compounds reversed c-jun mRNA increases in the skin.

Diosmin abrogates chemically induced hepatocarcinogenesis via alleviation of oxidative stress, hyperproliferative and inflammatory markers in murine model

Hepatocellular carcinoma (HCC) is a global health problem and is fourth leading cause of cancer related deaths. Now-a-days new strategies have been accounted for the chemoprevention of liver cancer due to ineffective traditional treatments against HCC. In the present study, we have shown that diosmin attenuates 2-AAF induced hepatic toxicity and early tumor promotion markers (ODC, PCNA and Ki67), its chemopreventive efficacy against DEN initiated and 2-AAF promoted hyper-proliferation and hepatocarcinogenesis in Wistar rats. Hepatocarcinogenesis has been characterized by the presence of apparent hepatic nodules, hepatic proliferation, elevation in the levels of proliferation markers (PCNA and Ki67), and inflammatory markers (COX-2 and iNOS) in DEN and 2-AAF administered rats. Protective efficacy of diosmin has been investigated in terms of its potential in reducing the percentage of visible hepatic nodules and the restoration of early tumor markers (PCNA, Ki67 and ODC), oxidative stress biomarkers, serum cytotoxicity markers (AST, ALT and LDH), cell necrosis markers (NF-kappa B and TNF-α) and inflammatory markers (COX-2 and iNos). Our study demonstrates that the inhibition of cell proliferation and down regulation of inflammatory markers may be, at least in part, the underlying mechanisms related to the liver tumor inhibition by diosmin. The present study allows us to conclude that diosmin being a dietary supplement, could be used as chemopreventive agent to prevent hepatocarcinogenesis.

Chrysin suppresses renal carcinogenesis via amelioration of hyperproliferation, oxidative stress and inflammation: Plausible role of NF-κB

Flavonoid family is a rich source of polyphenolic compounds and hence possess strong antioxidant and anti inflammatory properties. The aim of this study was to determine the efficacy of chrysin; a bio-active flavonoid as an anticancer agent. Renal cancer was initiated by single intraperitoneal (i.p.) injection of N-nitrosodiethylamine (DEN 200mg/kg BW body weight) and promoted by twice weekly administration of ferric nitrilotriacetate (Fe-NTA) 9mg Fe/kg BW for 16wk. In the present study, we report the chemopreventive effects of chrysin against (Fe-NTA) induced renal oxidative stress, inflammation, hyperproliferative response, and two-stage renal carcinogenesis. To ascertain the molecular mechanism implicated in the antitumor promoting activity of chrysin, its effect was investigated on markers of tumor promotion and inflammation: ornithine decarboxylase (ODC) activity, proliferating cell nuclear antigen (PCNA), inducible nitric oxide synthase (iNOS) and cyclo-oxygenase-2 (COX-2) expression, and on levels of proinflammatory cytokines interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), and prostaglandin E2 (PGE2). Pretreatment of animals with chrysin at both doses (20 and 40mg/kg body weight) markedly inhibited all. Further, Fe-NTA enhances renal lipid peroxidation, with concomitant reduction in reduced glutathione content (GSH), antioxidant enzymes, and phase II metabolizing enzymes. It induces serum toxicity markers, viz., blood urea nitrogen (BUN), creatinine and lactate dehydrogenase (LDH). Prophylactic treatment of animals with chrysin before the administration of Fe-NTA was effective in modulating oxidative and renal injury markers and resulted in the diminution of Fe-NTA mediated injury. These results suggest chrysin as an effective chemopreventive agent having the capability to obstruct DEN initiated and Fe-NTA promoted renal cancer in the rat model.

Amelioration of 1,2 Dimethylhydrazine (DMH) Induced Colon Oxidative Stress, Inflammation and Tumor Promotion Response by Tannic Acid in Wistar Rats

Colon cancer is the third most common malignant neoplasm in the world and it remains an important cause of death, especially in western countries. The toxic environmental pollutant, 1, 2-dimethylhydrazine (DMH), is also a colon-specific carcinogen. Tannic acid (TA) is reported to be effective against various types of chemically induced toxicity and also carcinogenesis. In the present study, we evaluated the chemopreventive efficacy of TA against DMH induced colon toxicity in a rat model. Efficacy of TA against the colon toxicity was evaluated in terms of biochemical estimation of antioxidant enzyme activities, lipid peroxidation, histopathological changes and expression of early molecular markers of inflammation and tumor promotion. DMH treatment induced oxidative stress enzymes (p<0.001) and an early inflammatory and tumor promotion response in the colons of Wistar rats. TA treatment prevented deteriorative effects induced by DMH through a protective mechanism that involved reduction of oxidative stress as well as COX-2, i-NOS, PCNA protein expression levels and TNF-α(p<0.001) release. It could be concluded from our results that TA markedly protects against chemically induced colon toxicity and acts plausibly by virtue of its antioxidant, anti-inflammatory and antiproliferative activities.

Abstract B59: A combination of resveratrol and ursolic acid produces synergistic inhibitory effects on markers of skin tumor promotion

Abstract Resveratrol (RES), found in grapes and Ursolic Acid (UA), found in rosemary and apples, delay the onset and reduce the number of experimentally-induced skin tumors in mice and inhibit the oncogenic Akt/mammalian target of rapamycin (Akt/mTOR) and Nuclear Factor kappa B (NF\#954;B) pathways. Based on their similar downstream effects via different proximal targets, we hypothesized that a combination treatment of RES and UA would synergize to produce a stronger inhibitory effect on skin tumor promotion. Methods: The dorsal skin of FVB mice (n=8) were shaved and treated topically twice per week for 2 weeks with sub-optimal doses of 2 μmol UA, 2 μmol RES, or a combination of both in 200 μL acetone. Each treatment was followed 30 minutes later with topical application of 3.4 nmol of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) in 200 μL acetone. Animals were sacrificed either 6 h (Western blotting) or 48 h (histology) after the final TPA treatment. The skins of five mice per group were removed and the epidermis was collected by scraping for Western blotting. For immunohistochemical (IHC) analysis of epidermal proliferation three mice per group were i.p. injected with 100 mg/kg Bromodeoxyuridine (BrdU) in PBS 30 min. prior to sacrifice. Mouse skin from treated areas was fixed in formalin, paraffin embedded, and analyzed via IHC. Results: RES and UA in combination had a stronger effect on hallmarks of skin tumor promotion than was expected based on the sum of their individual inhibitory effects, indicating synergism between RES and UA. RES and UA in combination synergistically inhibited TPA-induced epidermal thickening (80.2% inhibition vs 66.1 % expected) and epidermal proliferation (100.0% inhibition vs 90.0% expected). Western blotting revealed that components of the NF\#954;B and Akt/mTOR pathways were synergistically downregulated by the RES + UA combination in mouse epidermis. TPA-induced Akt (thr308) phosphorylation (86.0% inhibition vs 26.6% expected) and NFkB subunit p65 (ser536) phosphorylation (100.0% inhibition vs 73.3% expected) were suppressed to a much stronger degree with the combination treatment than that predicted. Similar results were seen for other pathway components, such as p70S6K and ribosomal S6. The synergistic activities of RES and UA were also manifest in the HaCaT human keratinocyte cell line. Cells were treated with sub-optimal doses of RES, UA, or RES + UA and stimulated with 10 ng/ml human tumor necrosis factor alpha (TNFα) for 30 min. and cell lysates were analyzed via Western blotting. TNFα-mediated phosphorylation of p65 was not inhibited by 25 μM RES or 0.1 μM −1 μM UA, but combinations of RES + UA resulted in strong synergistic inhibition of p65 phosphorylation (&amp;gt;66% inhibition vs 0% expected). Also, when HaCaT cells were treated with NFkB- and Akt/mTOR-inhibitory doses of RES (150 μM-200 μM) and UA (5 μM-10 μM), a strong increase (5 fold) in phosphorylation/activation of the energy sensing AMP-activated protein kinase (AMPK) was observed by Western blotting. AMPK also inhibits NF\#954;B and Akt/mTOR activities in other systems, suggesting that AMPK activation may function as a mediator of the synergistic anti-tumor promoting effects of RES and UA. Conclusions: These results indicate that UA and RES synergize to inhibit markers of tumor promotion in both in vivo and in vitro skin models. Future studies will further elucidate the synergistic mechanism of RES and UA and also determine if RES and UA have a synergistic preventive effect on skin tumor promotion during two-stage skin carcinogenesis in vivo. Supported by NIH grants CA079065, supplemental CA079065-10S1 (ARRA), CA129409 and CA037111. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr B59.

Attenuation of oxidative stress, inflammation and early markers of tumor promotion by caffeic acid in Fe-NTA exposed kidneys of Wistar rats

Iron nitrilotriacetate (Fe-NTA), a chief environmental pollutant, is known for its extensive toxic manifestations on renal system. In the present study, caffeic acid, one of the most frequently occurring phenolic acids in fruits, grains, and dietary supplements was evaluated for its shielding effect against the Fe-NTA-induced oxidative, inflammatory, and pathological damage in kidney. Fe-NTA was administered (9 mg Fe/kg body weight) intraperitoneally to the Wistar male rats on 20th day while caffeic acid was administered orally (20 and 40 mg/kg body weight) before administration of Fe-NTA. The intraperitoneal administration of Fe-NTA-enhanced lipid peroxidation, xanthine oxidase, and hydrogen peroxide generation with reduction in renal glutathione content, antioxidant enzymes, viz., catalase, glutathione peroxidase, and glutathione reductase. A sharp elevation in the levels of myloperoxidase, blood urea nitrogen (BUN), and serum creatinine has also been observed. Tumor promotion markers viz., ornithine decarboxylase (ODC) and [(3)H] thymidine incorporation into renal DNA were also significantly increased. Treatment of rats orally with caffeic acid (20 and 40 mg/kg body weight) resulted in a significant decrease in xanthine oxidase (P < 0.001), lipid peroxidation (P < 0.001), γ-glutamyl transpeptidase (P < 0.01), and H(2)O(2) (P < 0.01). There was significant recovery of renal glutathione content (P < 0.001) and antioxidant enzymes (P < 0.001). There was also a reversal in the enhancement of renal ODC activity, DNA synthesis, BUN, and serum creatinine (P < 0.001). All these changes were supported by histological observations. The results indicate that caffeic acid may be beneficial in ameliorating the Fe-NTA-induced oxidative damage and tumor promotion in the kidney of rats.

Abstract 2481: Rapamycin is a potent inhibitor of skin tumor promotion by TPA

Abstract The activation of Akt signaling occurs early during skin tumorigenesis. Data from our lab has demonstrated that the phorbol ester TPA leads to activation of Akt as well as several downstream effectors of Akt. Of particular interest is the Akt downstream signaling molecule, mTOR, which is elevated in the epidermis of wild-type mice following treatment with TPA. These studies suggest that activation of mTOR may contribute to skin tumor promotion. To further test this hypothesis, rapamycin, which is an established mTORC1 inhibitor, was used to further explore the role of mTOR signaling in epithelial carcinogenesis and specifically during tumor promotion. In a multiple treatment regimen, rapamycin was applied topically 30 min prior to TPA at doses of 1 umol, 100 nmol and 20 nmol. Rapamycin, in a dose dependent manner inhibited TPA induced mTOR activity as well as several downstream signaling proteins including p70S6KT389 and pS6S240/244 as well as p4EBP1T37/46, the FRAP/mTOR phosphorylation site of translational repressor protein 4EBP1. In addition, the highest dose of rapamycin caused a reduction in Akt-mediated phosphorylation of Akt downstream targets GSK3B as well as PRAS40 as assessed by western blotting using phosphospecific antibodies. These data suggest that multiple treatments with rapamycin at higher doses blocked both mTORC1 and mTORC2, while lower doses primarily affected mTORC1. Multiple treatment experiments evaluating the effects of rapamycin (5-200 nmol/mouse) on epidermal hyperplasia and epidermal labeling index, two short-term markers of tumor promotion by TPA, demonstrated a significant inhibition of both parameters in a dose dependent manner. Using a two-stage chemical skin carcinogenesis model with the standard DMBA/TPA protocol, the ability of rapamycin to block skin tumor promotion by TPA was evaluated. Rapamycin exerted a powerful anti-promoting effect reducing the average number of papillomas per mouse as well as the overall incidence of papillomas. In this regard, even a 5 nmol topical dose of rapamycin given 30 min prior to each TPA treatment produced approximately 50% inhibition of papilloma formation. Moreover, topical application of rapamycin to existing papillomas induced regression and/or inhibited growth. Skin tumors from this study were collected from TPA or TPA + rapamycin treated groups and are being evaluated for expression of proteins involved in cell cycle regulation as well as proteins involved in autophagy and apoptosis. The results of these experiments will be presented. Collectively, the current results indicate that signaling through mTORC1 contributes significantly to the process of skin tumor promotion, and that blocking this pathway may be an effective strategy for prevention of epithelial carcinogenesis. Supported by NIH grant CA37111. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2481.

Eugenol precludes cutaneous chemical carcinogenesis in mouse by preventing oxidative stress and inflammation and by inducing apoptosis

The present study was designed to investigate the protective efficacy of eugenol against skin cancer and probe into the mechanistic aspects. Skin tumors were initiated by applying 160 nmol DMBA and promoted by twice weekly applications of 8.5 nmol TPA for 28 wk. All mice developed tumors by 13 wk of promotion. However, in mice pretreated with 30 microL eugenol, no tumors were detected until 8 wk (following anti-initiation protocol) and until 14 wk (following antipromotion protocol) of tumor promotion. PCNA and TUNEL immunohistochemistry of tumors revealed eugenol to ameliorate cell proliferation and elevate apoptosis respectively. The effect of eugenol was assessed on specific stages of carcinogenesis. Initiation with DMBA led to a significant upregulation of p53 expression with a concomitant increase in p21(WAF1) levels in epidermal cells indicating induction of damage to the DNA. However, pretreatment with eugenol led to overexpression of these genes, which probably helped stimulate apoptosis of the initiated cells. To ascertain the molecular mechanisms implicated in the antitumor promoting activity of eugenol, its effect was investigated on markers of tumor promotion and inflammation: ODC activity and iNOS and COX-2 expression, and on levels of proinflammatory cytokines (IL-6, TNF-alpha, and PGE(2)). Eugenol markedly inhibited all. Eugenol also inhibited the upstream signaling molecule: NF-kappaB, which regulates the expression of these genes. TPA-induced depletion of cutaneous GSH and antioxidant enzymes armory was also precluded by eugenol. From these results, it could be concluded that eugenol markedly protects against chemically induced skin cancer and acts possibly by virtue of its antiproliferative, anti-inflammatory, and antioxidant activities.

Inhibition of gap-junctional intercellular communication and activation of mitogen-activated protein kinases by cyanobacterial extracts – Indications of novel tumor-promoting cyanotoxins?

Toxicity and liver tumor promotion of cyanotoxins microcystins have been extensively studied. However, recent studies document that other metabolites present in the complex cyanobacterial water blooms may also have adverse health effects. In this study we used rat liver epithelial stem-like cells (WB-F344) to examine the effects of cyanobacterial extracts on two established markers of tumor promotion, inhibition of gap-junctional intercellular communication (GJIC) and activation of mitogen-activated protein kinases (MAPKs) – ERK1/2. Extracts of cyanobacteria (laboratory cultures of Microcystis aeruginosa and Aphanizomenon flos-aquae and water blooms dominated by these species) inhibited GJIC and activated MAPKs in a dose-dependent manner (effective concentrations ranging 0.5–5 mg d.w./mL). Effects were independent of the microcystin content and the strongest responses were elicited by the extracts of Aphanizomenon sp. Neither pure microcystin-LR nor cylindrospermopsin inhibited GJIC or activated MAPKs. Modulations of GJIC and MAPKs appeared to be specific to cyanobacterial extracts since extracts from green alga Chlamydomonas reinhardtii, heterotrophic bacterium Klebsiella terrigena, and isolated bacterial lipopolysaccharides had no comparable effects. Our study provides the first evidence on the existence of unknown cyanobacterial toxic metabolites that affect in vitro biomarkers of tumor promotion, i.e. inhibition of GJIC and activation of MAPKs.

Dietary grape seed proanthocyanidins inhibit 12-O-tetradecanoyl phorbol-13-acetate-caused skin tumor promotion in 7,12-dimethylbenz[a]anthracene-initiated mouse skin, which is associated with the inhibition of inflammatory responses

Grape seed proanthocyanidins (GSPs) possess anticarcinogenic activities. Here, we assessed the effects of dietary GSPs on 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced skin tumor promotion in 7,12-dimethylbenz[a]anthracene (DMBA)-initiated mouse skin. Administration of dietary GSPs (0.2 and 0.5%, wt/wt) supplemented with control AIN76A diet resulted in significant inhibition of TPA-induced skin tumor promotion in C3H/HeN mice. The mice treated with GSPs developed a significantly lower tumor burden in terms of the percentage of mice with tumors (P < 0.05), total number of tumors per group (P < 0.01, n = 20) and total tumor volume per tumor-bearing mouse (P < 0.01-0.001) as compared with the mice that received the control diet. GSPs also delayed the malignant progression of papillomas into carcinomas. As TPA-induced inflammatory responses are used routinely as markers of skin tumor promotion, we assessed the effect of GSPs on biomarkers of TPA-induced inflammation. Immunohistochemical analysis and western blotting revealed that GSPs significantly inhibited expression of cyclooxygenase-2 (COX-2), prostaglandin E(2) (PGE(2)) and markers of proliferation (proliferating cell nuclear antigen and cyclin D1) in both the DMBA-initiated/TPA-promoted mouse skin and skin tumors. In short-term experiments in which the mouse skin was treated with acute or multiple TPA applications, we found that dietary GSPs inhibited TPA-induced edema, hyperplasia, leukocytes infiltration, myeloperoxidase, COX-2 expression and PGE(2) production in the mouse skin. The inhibitory effect of GSPs was also observed against other structurally different skin tumor promoter-induced inflammation in the skin. Together, our results show that dietary GSPs inhibit chemical carcinogenesis in mouse skin and that the inhibition of skin tumorigenesis by GSPs is associated with the inhibition of inflammatory responses caused by tumor promoters.

Guggulsterone modulates MAPK and NF-kappaB pathways and inhibits skin tumorigenesis in SENCAR mice.

Guggulsterone (GUG), a resin of the Commiphora mukul tree, has been used in ayurvedic medicine for centuries to treat a variety of ailments. Recent studies have suggested that GUG may also possess anticancer effects. In the present study, we show that GUG possesses antitumor-promoting effects in SENCAR mouse skin tumorigenesis model. We first determined the effect of topical application of GUG to mice against 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced conventional markers and other novel markers of skin tumor promotion. We found that topical application of GUG (1.6 micromol per mouse) 30 min prior to TPA (3.2 nmol per mouse) application onto the skin of mice afforded significant inhibition against TPA-mediated increase in skin edema and hyperplasia. Topical application of GUG was also found to result in substantial inhibition against TPA-induced epidermal (i) ornithine decarboxylase (ODC) activity; (ii) ODC, cyclooxygenase-2 and inducible nitric oxide synthase protein expressions; (iii) phosphorylation of extracellular signal-regulated kinase 1/2, c-jun N-terminal kinases and p38; (iv) activation of NF-kappaB/p65 and IKK alpha/beta and (v) phosphorylation and degradation of I kappaB alpha. We next assessed the effect of topically applied GUG on TPA-induced skin tumor promotion in 7,12-dimethyl benz[a]anthracene-initiated mice. Compared with non-GUG-pretreated mice, animals pretreated with GUG showed significantly reduced tumor incidence, lower tumor body burden and a significant delay in the latency period for tumor appearance from 5 to 11 weeks. These results provide the first evidence that GUG possesses anti-skin tumor-promoting effects in SENCAR mice and inhibits conventional as well as novel biomarkers of tumor promotion. In summary, GUG could be useful for delaying tumor growth in humans.

Abrogation of Thioacetamide-Induced Biochemical Events of Hepatic Tumor Promotion Stage by Tannic Acid in Wistar Rats

Tannic acid is present in almost every edible plant and is generally used as a safe food additive. In this study we investigated the antioxidative and antihyperproliferative potential of tannic acid against thioacetoamide (TAA), a potent hepatotoxic-substance-induced oxidative stress and hyperproliferation biomarker. We have shown here that the activities of hepatic antioxidant enzymes, phase II metabolizing enzymes, and the glutathione content were decreased while hepatic ornithine decarboxylase activity and DNA synthesis were induced in TAA-treated animals. Tannic acid administration at two different doses prior to the TAA injection partially recovered the depleted level of glutathione, inhibited activities of antioxidant and phase II metabolizing enzymes, and resulted in significant inhibition of oxidative stress in a dose-dependent manner. Tannic acid administration before TAA treatment also resulted in a significant decrease in ODC activity and [3H]-thymidine incorporation in rat liver, which are classical markers of inflammation and tumor promotion. Our data clearly demonstrate that tannic acid possesses antioxidant and antiproliferating activities because it inhibits early biomarkers of TAA-induced tumor promotion in an in vivo animal model.

Chemomodulatory effects of Terminalia chebula against nickel chloride induced oxidative stress and tumor promotion response in male Wistar rats

Nickel, a major environmental pollutant is a known potent nephrotoxic agent. In this communication we report the chemopreventive effect of Terminalia chebula on nickel chloride (NiCl 2) induced renal oxidative stress, toxicity and cell proliferation response in male Wistar rats. Administration of NiCl 2 (250 μmoL Ni/kg body weight) to male Wistar rats resulted in an increase in the reduced renal glutathione content (GSH), glutathione-S-transferase (GST), glutathione reductase (GR), lipid peroxidation (LPO), H 2O 2 generation, blood urea nitrogen (BUN) and serum creatinine with a concomitant decrease in the activity of glutathione peroxidase ( p < 0.001 ). Nickel chloride (NiCl 2) treatment also induced tumor promotion markers, viz., ornithine decarboxylase (ODC) activity and thymidine [ 3H] incorporation into renal DNA ( p < 0.001 ). Prophylactic treatment of rats with T. chebula (25 mg/kg body weight and 50 mg/kg body weight) daily for one week resulted in the diminution of NiCl 2 mediated damage as evident from the down regulation of glutathione content, GST, GR, LPO, H 2O 2 generation, BUN, serum creatinine, DNA synthesis ( p < 0.001 ) and ODC activity ( p < 0.01 ) with concomitant restoration of GPx activity. Thus, the present investigation suggests that T. chebula extract could be used as therapeutic agent for cancer prevention as evident from this study where it blocks or suppresses the events associated with chemical carcinogenesis.

Background Gene Expression in Rat Kidney: Influence of Strain, Gender, and Diet

In order to gain better insight into factors (strain, gender, and diet) influencing background variability in kidney gene expression, we examined the transcriptomes of male and female Crl:CD(SD)IGSBR (Sprague-Dawley [SD]) and CDF(Fischer 344)/CrlBR rats maintained for 19 days on three different diets (ad libitum [AL], diet restriction-75% of AL, and casein-based phytoestrogen-free diet). Kidney RNA was analyzed using Agilent Rat oligo microarrays (approximately 20,000 genes). Principal component analysis demonstrated that strain and gender have the most impact on the variability in gene expression, while diet had a lesser effect. The majority of the affected genes differed by a magnitude of four-fold or less between strains/gender, with some previously known to be sex-hormone regulated (SLC22A7 and SLC21A1). One gene of particular interest was ornithine decarboxylase, a significant marker of cell proliferation and tumor promotion, which was expressed at an 18-fold greater level in SD rats. Further analysis revealed that the difference in expression was due to the use of an alternate polyadenylation signal resulting in the production of two different sizes of transcripts. These results demonstrate that gender and strain have significant influence on gene expression which could be a confounder when comparing results, especially when it involves predictive fingerprint/patterns.

Tamarix gallica ameliorates thioacetamide–induced hepatic oxidative stress and hyperproliferative response in Wistar rats

Tamarix gallica, a hepatic stimulant and tonic, was examined for its ability to inhibit thioacetamide (TAA)-induced hepatic oxidative stress, toxicity and early tumor promotion response in male Wistar rats. TAA (6.6 mmol/kg body wt. i.p) enhanced lipid peroxidation, hydrogen peroxide content, glutathione S-transferase and xanthine oxidase with reduction in the activities of hepatic antioxidant enzymes viz., glutathione peroxidase, superoxide dismutase and caused depletion in the level of hepatic glutathione content. A marked increase in liver damage markers was also observed. TAA treatment also enhanced tumor promotion markers, ornithine decarboxylase (ODC) activity and [3H] thymidine incorporation into hepatic DNA. Pretreatment of rats orally with Tamarix gallica extract (25 and 50 mg/kg body weight) prevented TAA-promoted oxidative stress and toxicity. Prophylaxis with Tamarix gallica significantly reduced the susceptibility of the hepatic microsomal membrane for iron-ascorbate induced lipid peroxidation, H2O2 content, glutathione S-transferase and xanthine oxidase activities. There was also reversal of the elevated levels of liver marker parameters and tumor promotion markers. Our data suggests that Tamarix gallica is a potent chemopreventive agent and may suppress TAA-mediated hepatic oxidative stress, toxicity, and tumor promotion response in rats.