Ann R. Kennedy

Protease inhibitors suppress radiation-induced malignant transformation in vitro.

LITTLE is known about the mechanisms of carcinogenesis. The fact that most carcinogens are mutagenic has led to speculation that the primary step in cancer induction may be mutational1,2; there is evidence from both in vivo2 and in vitro3 studies that a strong correlation exists between the mutagenicity and carcinogenicity of an agent. Mutagenic and carcinogenic agents, both physical and chemical, also produce similar kinds of DNA damage and repair4. Radiation-induced mutagenesis in some bacterial cells requires an error-prone DNA repair system5,6, and there is now some evidence that error-prone DNA repair may be involved in the malignant transformation of cells by radiation7,8. Protease inhibitors have been shown to suppress specifically both error-prone repair and mutagenesis in bacterial cells9,10, as well as to inhibit carcinogenesis in vivo11,12. We report here that the protease inhibitors antipain and leupeptin will suppress radiation-induced transformation in vitro as well as inhibit two-stage transformation in vitro using radiation and the promoting agent, 12-O-tetradecanoyl-phorbol-13-acetate (TPA).

Protease inhibitors as cancer chemopreventive agents

Protease inhibitors (PIs), which are widely distributed in plants and animals and have a variety of functions, interfere with cancer development in a number of ways, including suppression of oxygen radicals, oncogenes, and metastases. Epidemiologic evidence supports their prevention of major human c

Evidence that a second event in x-ray-induced oncogenic transformation in vitro occurs during cellular proliferation

It has previously been hypothesized that radiation transformation in vitro is a two-step process; the first step is a frequent alteration occurring among a large fraction of the irradiated cells, while the second step, malignant transformation, is a rare event occurring with an approximate frequency of 10(-6) among the progeny of the irradiated cells. Data are reported here on the distributions of transformed-cell clone sizes in irradiated cultures reseeded at various times post-treatment. The results suggest that the second event in transformation occurs randomly during the growth of irradiated cultures of C3H 10T1/2 cells to confluence. When the same number of irradiated C3H 10T1/2 cells were seeded into petri dishes of different sizes [35 mm (8 cm2), 60 mm (21 cm2), 100 mm (55 cm2), 150 mm (145 cm2)], the number of foci which arose per dish was dependent on the final cell numbers at confluence in the various dish sizes, such that the number of foci/cm2 was constant. When irradiated cells and parental C3H 10T1/2 cells were mixed in different proportions at low density, the number of foci which ultimately arose was a function of the number of progeny of irradiated cells present in the culture at confluence. The results presented here confirm previous studies and give further evidence that the radiation-induced malignant transformation of cells occurs in an indirect, multistage fashion.

Adaptation of the Dichlorofluorescein Assay for Detection of Radiation-Induced Oxidative Stress in Cultured Cells

Abstract Wan, X. S., Zhou, Z. and Kennedy, A. R. Adaptation of the Dichlorofluorescein Assay for Detection of Radiation-Induced Oxidative Stress in Cultured Cells. Radiat. Res. 160, 622–630, (2003). The oxidation of 2′7′-dichlorofluorescin (DCFH) to 2′7′-dichlorofluorescein (DCF), a fluorescent DCFH oxidation product, is a highly sensitive indicator that is used to measure oxidative stress in cells. In the present study, a DCF assay has been adapted to quantify oxidative stress in human breast epithelial cell cultures after exposure to γ rays. The results demonstrate that the sensitivity and specificity of the DCF assay is strongly influenced by the timing of DCFH diacetate (DCFH-DA) substrate loading in relation to radiation exposure and by the matrix in which the cells were loaded with DCFH-DA substrate. Under the conditions optimized in this study, the DCF assay is capable of detecting increased DCFH oxidation in cell cultures irradiated with γ rays at a dose as low as 1.5 cGy. The increase in fluorescence was directly proportional to the radiation dose, which ranged from 0 to 2 Gy, and a minimal level of fluorescence was observed in sham-irradiated cells. These results indicate that the DCF assay optimized in this study is highly sensitive, linear and specific for measuring oxidative stress in irradiated cells.

Protease Activity in a Hapten-Induced Model of Ulcerative Colitis in Rats

Inflammatory bowel disease (IBD) is a painfuland debilitating condition affecting the mucosal liningof the colon and other areas of the gastrointestinaltract. IBD generally falls into two major categories: ulcerative colitis (UC) and Crohns disease. Wehave utilized dinitrobenzenesulfonic acid (DNBS) toinduce experimental UC in rats. Histopathologic analysisindicates that DNBS induces a condition in animals similar to human UC. Biochemical resultsrevealed 6- to 10-fold elevated levels of serineprotease activity in colon tissue from animals with UCas compared with matched controls. We also observedelevated levels of protease activity in tissue samplesobtained from human patients with UC. Hence, our resultsdemonstrate that protease activity is increased inrodent and human UC. These proteases may play a significant role in destruction of colonictissue in IBD. Protease inhibitors that target serineproteases may be useful pharmacological agents to limittissue destruction in IBD.

Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation

Abstract Wambi, C., Sanzari, J., Wan, X. S., Nuth, M., Davis, J., Ko, Y. H., Sayers, C. M., Baran, M., Ware, J. H. and Kennedy, A. R. Dietary Antioxidants Protect Hematopoietic Cells and Improve Animal Survival after Total-Body Irradiation. Radiat. Res. 169, 384–396 (2008). The purpose of this study was to determine whether a dietary supplement consisting of l-selenomethionine, vitamin C, vitamin E succinate, α-lipoic acid and N-acetyl cysteine could improve the survival of mice after total-body irradiation. Antioxidants significantly increased the 30-day survival of mice after exposure to a potentially lethal dose of X rays when given prior to or after animal irradiation. Pretreatment of animals with antioxidants resulted in significantly higher total white blood cell and neutrophil counts in peripheral blood at 4 and 24 h after 1 Gy and 8 Gy. Antioxidants were effective in preventing peripheral lymphopenia only after low-dose irradiation. Antioxidant supplementation was also associated with increased bone marrow cell counts after irradiation. Supplementation with antioxidants was associated with increased Bcl2 and decreased Bax, caspase 9 and TGF-β1 mRNA expression in the bone marrow after irradiation. Maintenance of the antioxidant diet was associated with improved recovery of the bone marrow after sublethal or potentially lethal irradiation. Taken together, oral supplementation with antioxidants appears to be an effective approach for radioprotection of hematopoietic cells and improvement of animal survival, and modulation of apoptosis is implicated as a mechanism for the radioprotection of the hematopoietic system by antioxidants.

Bowman-Birk inhibitor concentrate reduces colon inflammation in mice with dextran sulfate sodium-induced ulcerative colitis

Bowman-Birk inhibitor concentrate (BBIC) is asoybean extract enriched in the BowmanBirk inhibitor, aprotein protease inhibitor. The Bowman-Birk inhibitorcan inhibit proteases released from inflammation mediating cells and suppress superoxide anionradical secretion from immunocytes. This studyinvestigates the ability of Bowman-Birk inhibitorconcentrate to inhibit colon inflammation in the dextransulfate sodium model of ulcerative colitis, aninflammatory bowel disease. When compared to mice on astandard diet, mice given food supplemented with 0.5%BBIC during and after dextran sulfate sodium treatment showed suppression of three of four scoredhistopathological inflammation criteria (P < 0.01),total histopathological score (P < 0.01), a 15% lowermortality rate (P < 0.01), and a delayed onset of mortality. We conclude that dietaryBowman-Birk inhibitor concentrate can beneficiallyaffect dextran sulfate sodium-treated mice and may beuseful in the treatment of human inflammatory boweldiseases, particularly ulcerative colitis.

Effects of dietary supplements on the space radiation-induced reduction in total antioxidant status in CBA mice.

Abstract Guan, J., Stewart, J., Ware, J. H., Zhou, Z., Donahue, J. J. and Kennedy, A. R. Effects of Dietary Supplements on the Space Radiation-Induced Reduction in Total Antioxidant Status in CBA Mice. Radiat. Res. 165, 373–378 (2006). In the present study, the total antioxidant status was used as a biomarker to evaluate oxidative stress induced by proton, HZE-particle and γ radiation in CBA mice. The results demonstrated that the plasma level of TAS was significantly decreased (P < 0.05) in CBA mice after exposure to a 50-cGy dose of radiation from HZE particles or a 3-Gy dose of radiation from protons or γ rays. Diet supplementation with Bowman-Birk Inhibitor Concentrate (BBIC), l-selenomethionine (l-SeM), or a combination of N-acetyl cysteine, sodium ascorbate, co-enzyme Q10 (CoQ10), α-lipoic acid, l-SeM and vitamin E succinate could partially or completely prevent the reduction in the plasma level of TAS in CBA mice exposed to proton or HZE-particle radiation. The selected antioxidant combination with or without CoQ10 has a comparable protective effect on the γ-radiation-induced drop in TAS in CBA mice. These results indicate that BBIC, l-SeM and the selected antioxidant combinations may serve as countermeasures for space radiation-induced adverse biological effects.

Effects of Bowman-Birk inhibitor on rat colon carcinogenesis.

The present study was undertaken to determine whether the Bowman-Birk inhibitor (BBI) could prevent colon carcinogenesis in rats treated with dimethylhydrazine (DMH) and whether there were adverse side effects associated with treatment with BBI for cancer prevention. BBI was evaluated in the forms of purified BBI (PBBI) or an extract of soybeans enriched in BBI, termed BBI concentrate (BBIC). The results demonstrate that PBBI and BBIC reduced the incidence and frequency of tumors in DMH-treated rats compared with animals treated with DMH alone. Autoclaved BBIC, in which the protease inhibitor activity of BBI was destroyed, had a weak and statistically insignificant, suppressive effect on DMH-induced colon carcinogenesis in rats, suggesting that the protease inhibitor activity of BBI is likely to be responsible for the anticarcinogenic activity of BBIC. Soy molasses, which contains soy isoflavones, did not have an effect on colon cancer carcinogenesis in DMH-treated rats. Similar to results from previous studies (Nauss et al. JNCI 73, 915-924, 1984), the most aggressive, malignant colon adenocarcinomas developed within or in association with gut-associated lymphoid tissue aggregates. No adverse side effects on the pancreas or animal growth were observed in rats treated with PBBI or BBIC. These results demonstrate that PBBI and BBIC may be used to prevent colon cancer without significant adverse side effects.

Suppression of X-ray induced transformation by vitamin E in mouse C3H/10T12 cells

Vitamin E (d-alpha-tocopherol) was shown to decrease X-ray induced transformation in mouse C3H/10T1/2 cells. The d-alpha-tocopherol was active in the form of succinate diluted in ethanol, but was inactive at the highest nontoxic concentration of the pure substance dissolved in oil and diluted in acetone. Vitamin E succinate was effective when present throughout the entire assay period or when treatments began after confluence was reached at day 12 post-irradiation. It was ineffective if present only for the early portion of the radiation transformation assay period, indicating that its effect may be reversible. Vitamin E did not suppress the growth and expression of transformed C3H/10T1/2 cells as foci when the transformed cells were surrounded by a monolayer of normal cells.