Preclinical Models of Anal Cancer Combined-Modality Therapy.

INTRODUCTION: There have been no significant changes in anal cancer treatment options in 4 decades. In this study, we highlight two preclinical models designed to assess anal cancer treatments. MATERIALS AND METHODS: Transgenic K14E6/E7 mice were treated with 7, 12-dimethylbenz(a)anthracene until anal tumors developed. Mice were treated with localized radiation in addition to chemotherapy (combined-modality therapy [CMT]) and compared to no treatment control (NTC). K14E6/E7 mouse anal spheroids with and without Pik3ca mutations were isolated and treated with vehicle, LY3023414 (LY3) (a drug previously shown to be effective in cancer prevention), CMT, or CMT + LY3. RESULTS: In the in vivo model, there was a significant increase in survival in the CMT group compared to the NTC group (P = 0.0392). In the ex vivo model, there was a significant decrease in the mean diameter of CMT and CMT + LY3-treated spheroids compared to vehicle (P ≤ 0.0001). For LY3 alone compared to vehicle, there was a statistically significant decrease in spheroid size in the K14E6/E7 group without mutation (P = 0.0004). CONCLUSIONS: We have provided proof of concept for two preclinical anal cancer treatment models that allow for the future testing of novel therapies for anal cancer.

Genetic inhibition of autophagy in a transgenic mouse model of anal cancer.

BACKGROUND: The dynamic role of autophagy in cancer development is a topic of considerable research and debate. Previously published studies have shown that anal cancer development can be promoted or prevented with the pharmacologic inhibition or induction, respectively, of autophagy in a human papillomavirus (HPV) mouse model. However, these results are confounded by the fact that the drugs utilized are known to affect other pathways besides autophagy. It has also been shown that autophagic inhibition occurs in the setting of HPV16 oncoprotein expression (E6 and E7) and correlates with increased susceptibility to anal carcinogenesis. MATERIALS AND METHODS: In this study, we employed a conditional, genetic, autophagic (Atg7) knockout mouse model to determine conclusively that autophagy has a role in anal cancer development, in the absence or presence of E6 and E7. RESULTS: In mice lacking both HPV16 oncogenes, knockout of autophagy followed by exposure to a carcinogen resulted in a tumor incidence of 40%, compared to 0% in mice treated with a carcinogen alone with an intact autophagic pathway (P = 0.007). In mice expressing either one or both HPV16 oncoproteins, the addition of genetic knockout of autophagy to carcinogen treatment did not lead to a significant difference in tumor incidence compared to carcinogen treatment alone, consistent with the ability of HPV oncogenes to inhibit autophagy in themselves. CONCLUSIONS: These results provide the first conclusive evidence for the distinct role of autophagy in anal carcinogenesis, and suggest that autophagy is a plausible target for therapies aimed at reducing anal dysplasia and anal cancer development.

The role of pharmacologic modulation of autophagy on anal cancer development in an HPV mouse model of carcinogenesis.

Autophagy is an intracellular, catabolic process that maintains cellular health. We examined the response of pharmacologic modulation of autophagy in an HPV mouse model of anal carcinogenesis. K14E6/E7 mice were treated with the topical carcinogen DMBA weekly and assessed for tumors over 20 weeks. Concurrently, they were given either chloroquine or BEZ235, to inhibit or induce autophagy, respectively. Time to tumor onset was examined. Immunofluorescence (IF) was performed for LC3ß and p62 to examine autophagy. All DMBA treated K14E6/E7 mice developed anal cancer, contrary to zero of the no DMBA treated mice. Chloroquine plus DMBA resulted in a significant decrease in the time to tumor onset compared to K14E6/E7 treated with DMBA. Only 40% BEZ235 plus DMBA treated mice developed anal cancer. Autophagic induction with DMBA and BEZ235, and autophagic inhibition with chloroquine were confirmed via IF. Anal carcinogenesis can be inhibited or induced via pharmacologic modulation of autophagy.

Cholecystokinin expression in the ß-cell leads to increased ß-cell area in aged mice and protects from streptozotocin-induced diabetes and apoptosis.

Cholecystokinin (CCK) is a peptide hormone produced in the gut and brain with beneficial effects on digestion, satiety, and insulin secretion. CCK is also expressed in pancreatic ß-cells, but only in models of obesity and insulin resistance. Whole body deletion of CCK in obese mice leads to reduced ß-cell mass expansion and increased apoptosis. We hypothesized that islet-derived CCK is important in protection from ß-cell apoptosis. To determine the specific role of ß-cell-derived CCK in ß-cell mass dynamics, we generated a transgenic mouse that expresses CCK in the ß-cell in the lean state (MIP-CCK). Although this transgene contains the human growth hormone minigene, we saw no expression of human growth hormone protein in transgenic islets. We examined the ability of MIP-CCK mice to maintain ß-cell mass when subjected to apoptotic stress, with advanced age, and after streptozotocin treatment. Aged MIP-CCK mice have increased ß-cell area. MIP-CCK mice are resistant to streptozotocin-induced diabetes and exhibit reduced ß-cell apoptosis. Directed CCK overexpression in cultured ß-cells also protects from cytokine-induced apoptosis. We have identified an important new paracrine/autocrine effect of CCK in protection of ß-cells from apoptotic stress. Understanding the role of ß-cell CCK adds to the emerging knowledge of classic gut peptides in intraislet signaling. CCK receptor agonists are being investigated as therapeutics for obesity and diabetes. While these agonists clearly have beneficial effects on body weight and insulin sensitivity in peripheral tissues, they may also directly protect ß-cells from apoptosis.

Sensory Characteristics of Reduced Nitrite Bacon Manufactured by the Wisconsin Process.

Bacon with a culture of lactic acid-forming bacteria, Pediococcus acidilactici , plus 0.7% sucrose and 40 or 80 mg sodium nitrite/kg (Wisconsin Process), and control bacon with 120 mg sodium nitrite/kg but no lactic acid bacteria and sucrose, were produced at three commercial bacon plants under production conditions. The bacon was stored under refrigeration for 5 to 8 wk, then subjected to sensory analyses by an experienced sensory panel. Quantitative descriptive visual analysis was performed on uncooked as well as cooked samples, and the cooked samples were served for quantitative descriptive sensory analysis. Results indicated that the test bacon with reduced amounts of sodium nitrite was as acceptable as the control bacon with no sugar and lactics, with the 80 mg/kg nitrite-bacon being the most preferred of all. These results and the results of botulinal challenge and nitrosamine tests indicate that the test process can be a satisfactory alternative to processing bacon by the conventional procedure with 120 mg sodium nitrite/kg.

Survival of Listeria monocytogenes During the Manufacture and Storage of Nonfat Dry Milk.

The ability of Listeria monocytogenes to survive in skim milk during spray drying and to persist in nonfat dry milk during storage was examined. Concentrated (30% solids) and unconcentrated skim milks were inoculated with ca. 105 to 106 L. monocytogenes /ml and spray dried (inlet temperature, 165 ± 2°C; outlet temperature 67 ± 2°C) to a moisture content of 3.6 to 6.4%. The nonfat dry milk was packaged in moisture-resistant film and stored at 25°C for up to 16 wk. A reduction of ca. 1 to 1.5 log10 L. monocytogenes /g occurred during the spray drying process, irrespective of whether the milk was concentrated or not before spray drying. The organism progressively died during storage at 25°C, with a >4-log10 CFU/g decrease occurring within 16 wk of storage.

Evaluation of the Bacteriological Safety of Low-Salt Miso.

Studies were done to evaluate the safety of three different low-salt (2.36 to 5.79% NaCl) misos inoculated with different bacterial pathogens. Clostridium botulinum types A and B (inoculum level of ca. 120 spores/g) did not produce toxin in any of the misos within 18 wk at 25°C. Staphylococcus aureus , Salmonella typhimurium and Yersinia enterocolitica (inoculum level of ca. 103 to 104 CFU/g) progessively died in all of the misos held at either 10 or 25°C. The miso samples, which were obtained from Japan (3.75 and 5.79% NaCl) and California (2.36% NaCl), had water activities of 0.843, 0.835 and 0.875, respectively, and pH values of 5.26, 5.30 and 4.73, respectively. Results indicate that low-salt misos with these properties are not likely to be bacteriological health risks.

Evaluation of the Microbiological Safety of Tempeh Made from Unacidified Soybeans.

Studies were done to evaluate the safety of tempeh made from unacidifed soybeans and inoculated with different bacterial pathogens. Pathogens were added to either the soybeans before fermentation by Rhizopus oligosporus or the tempeh after fermentation and steaming. In the latter method, the inoculated products were incubated at several different temperatures (5, 10, 15 and 25°C). Clostridium botulinum (types A and/or B) toxin was produced in 2 d during the fermentation and within 5 d at 25°C or 4 wk at 15°C in tempeh inoculated and incubated in vacuum packages after fermentation and steaming. Staphylococcus aureus grew very well (>6-log10 CFU/g increase) in 2 d during the fermentation, and grew from ca. 103 CFU/g to 108 CFU/g in 7 d at 25°C and 21 d at 15°C in tempeh inoculated after fermentation and steaming. Staphylococcal enterotoxins were detected in some of these samples. Salmonella typhimurium also grew well during the fermentation (>6-log10 CFU/g increase in 1 d), but grew relatively slowly at 25 and 15°C in tempeh inoculated after fermentation and steaming. Yersinia enterocolitica grew very well (>6-log10 CFU/g increase) in 1 d during the fermentation, and also grew well in tempeh inoculated after fermentation and steaming, with a >6 log10 CFU/g increase in 2 d at 25 or 15°C and 5 d at 10°C. Results of these studies indicate the need for maintaining: (a) a high level of sanitary practices during production and (b) good refrigeration (≤5°C) of the product following fermentation until it is used.