Growing attention is being directed toward insects as a novel and sustainable source of protein for pet food. The aim of the study was to evaluate nutrient digestibility of a diet containing black soldier fly larvae as its main protein source. Moreover, the purpose of the study was to compare the traditional in vivo total collection method with the in vivo marker method and in vitro digestibility method. Two isonitrogenous and isoenergetic dry diets containing either venison meal (CTRL diet) or black soldier fly larvae meal (BSF diet) as their primary sources of proteins were fed to six adult dogs, according to a Latin square design. The digestibility of nutrients was determined using both in vivo (“total collection” and “internal marker” approaches) and in vitro methods. The two diets showed similar nutrient digestibility values for dry matter, organic matter, ether extract, ash, and phosphorus. However, a statistical trend ( p = 0.066) was observed indicating greater protein digestibility in the BSF diet compared with the CTRL diet. Calcium digestibility was higher in the BSF diet compared with the CTRL diet ( p = 0.018). On the contrary, fiber digestibility was lower in the insect-based diet compared with the venison diet ( p < 0.001). There was no difference between total collection and internal marker methods in the assessment of in vivo digestibility for any of the nutrients considered. The in vitro digestibility values for dry matter, organic matter, and crude protein, as well as the estimated in vivo digestibility of organic matter and crude protein by the means of the predictive equation, were aligned with the in vivo results, although in vitro estimations were consistently higher compared with those obtained by in vivo analysis. Digestibility analysis of a dog food containing insect meal as the sole source of protein (36.5% inclusion) showed promising results in terms of it presenting similar values as a meat-based diet, indicating its suitability as a sustainable protein source for pet food. Moreover, the study showed that both the in vivo marker method and the in vitro method could be possible alternatives to the traditional total collection method in digestibility trials.
Ipilimumab (IPI) blocks CTLA-4 immune checkpoint resulting in T cell activation and enhanced antitumor immunity. IPI improves overall survival (OS) in 22% of patients with metastatic melanoma (MM). We investigated the association of CTLA-4 single nucleotide variants (SNVs) with best overall response (BOR) to IPI and OS in a cohort of 173 MM patients. Patients were genotyped for six CTLA-4 SNVs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, and CT60G>A). We assessed the association between SNVs and BOR through multinomial logistic regression (MLR) and the prognostic effect of SNVs on OS through Kaplan-Meier method. Both -1577G>A and CT60G>A SNVs were found significantly associated with BOR. In particular, the proportion of responders was higher in G/G genotype while that of stable patients was higher in A/A genotype. The frequency of patients experiencing progression was similar in all genotypes. MLR evidenced a strong downward trend in the probability of responsiveness/progression, in comparison to disease stability, as a function of the allele A "dose" (0, 1, or 2) in both SNVs with reductions of about 70% (G/A vs G/G) and about 95% (A/A vs G/G). Moreover, -1577G/G and CT60G/G genotypes were associated with long-term OS, the surviving patients being at 3 years 29.8 and 30.8%, respectively, as compared to 12.9 and 14.4% of surviving patients carrying -1577G/A and CT60G/A, respectively. MM patients carrying -1577G/G or CT60G/G genotypes may benefit from IPI treatment in terms of BOR and long-term OS. These CTLA-4 SNVs may serve as potential biomarkers predictive of favorable outcome in this subset of patients.
Tetrahydrolipstatin (orlistat), an inhibitor of lipases and fatty acid synthase, is used orally for long-term treatment of obesity. Although the drug possesses striking antitumor activities in vitro against human cancer cells and in vitro and in vivo against animal tumors, it also induces precancerous lesions in rat colon. Therefore, we tested the in vitro effect of orlistat on the expression of O6-methylguanine-DNA methyltransferase (MGMT), a DNA repair enzyme that plays an essential role in the control of mutagenesis and carcinogenesis. Western blot analysis demonstrated that 2-day continuous exposure to 40 µM orlistat did not affect MGMT levels in a human melanoma cell line, but downregulated the repair protein by 30-70% in human peripheral blood mononuclear cells, in two leukemia and two colon cancer cell lines. On the other hand, orlistat did not alter noticeably MGMT mRNA expression. Differently from lomeguatrib (a false substrate, strong inhibitor of MGMT) orlistat did not reduce substantially MGMT function after 2-h exposure of target cells to the agent, suggesting that this drug is not a competitive inhibitor of the repair protein. Combined treatment with orlistat and lomeguatrib showed additive reduction of MGMT levels. More importantly, orlistat-mediated downregulation of MGMT protein expression was markedly amplified when the drug was combined with a DNA methylating agent endowed with carcinogenic properties such as temozolomide. In conclusion, even if orlistat is scarcely absorbed by oral route, it is possible that this drug could reduce local MGMT-mediated protection against DNA damage provoked by DNA methylating compounds on gastrointestinal tract epithelial cells, thus favoring chemical carcinogenesis.
Introduction Temozolomide (TMZ) an antitumor DNA methylating agent, is able to induce O6-methyl-guanine DNA adducts. Its activity is largely conditioned by a fully functional mismatch repair system (MMR). In contrast, the antineoplastic effects of the agent are suppressed by the DNA repair enzyme O6-alkylguanine-DNA-alkyl-transferase (OGAT), that removes methyl adducts. The OGAT inhibitor O6-benzyl-guanine (BG) abrogates resistance to TMZ of malignant cells expressing high OGAT levels (i.e. OGAT-dependent resistance, ODR), but not of MMR-deficient tumors, or of tumors showing other resistance mechanisms unrelated to OGAT (i.e. OGAT-independent resistance, OIR). Aim of the study To establish the onset of ODR or of OIR in melanoma cells treated in vitro with TMZ alone or associated with BG. Methods Three MMR-proficient melanoma cell clones with barely detectable OGAT levels and sensitive to TMZ, originated from 3 different parental lines, were treated with TMZ (50 μM), or TMZ (50 μM)+BG (5 μM) daily for 5 days, for 4 consecutive cycles, and tested for sensitivity to TMZ or TMZ+BG, at the end of each cycle. Results Dramatic increase of chemo-resistance to TMZ alone (i.e. ranging from more than 200% to more than 1200% increase of IC50 of TMZ) was detected in all 3 clones, after as early as 1 cycle of treatment with TMZ alone (i.e. TMZ sublines). This increase was maintained essentially constant for up to 4 treatment cycles. In all TMZ sublines OGAT expression was found to be increased, and BG partially reversed resistance to TMZ, thus suggesting that resistance was, at least in part, of ODR type. A similar phenomenon was observed when the 3 clones were treated with TMZ+BG (i.e. TMZ/BG sublines). However, in this case BG reduced TMZ resistance in only 2 of the 3 TMZ/BG sublines. Consistently with these results, OGAT levels were up-regulated in the 2 TMZ/BG sublines still sensitive to TMZ+BG, but not in the TMZ/BG subline in which the inhibitor failed to alleviate TMZ resistance. Conclusions Treatment of melanomas with TMZ, alone or with the OGAT inhibitor BG, shows an unexpectedly high capacity of inducing early anti-TMZ resistance of both ODR and OIR type. The elucidation of the molecular mechanisms underlying OIR requires further studies. However, the present results provide valuable information concerning the poor therapeutic response to triazenes observed in melanoma patients. Supported by the Italian Ministry of Health.
