Study of the functional product’s protein compounds digestion features

The aim of the study was to investigate the transformation of meat product’s proteins from pig hearts and aortas during enzymatic hydrolysis in an in vitro model of the gastrointestinal tract. The model consisted of three phases simulating digestion processes: “oral cavity” phase (a-amylase, pH 7.0; 2 min), “stomach” phase (pork pepsin, pH 3.0; 120 min), “intestine” phase (pork pancreatin, pH 7.0; 130 min). The product was sequentially subjected to hydrolysis, at the end of each phase, samples were taken to determine the protein concentration (biuret method) and visualize the protein fractions (one-dimensional electrophoresis). A significant increase in protein concentration at the “stomach” phase was revealed by 3.2 times, and the absolute content by 4.6 times. At the “intestine” phase, a decrease in the number of peptide complexes with copper ions by 1.8 times, the absolute protein content by 8.5% was re‑ vealed. The noted tendency was confirmed by electrophoretic studies — at the stage, simulating digestion in the stomach, the prod‑ ucts of meat product’s proteins hydrolysis were visualized; at the “intestine” phase, a low expression of protein fractions in the range of more than 10 kDa is shown. The maximum hydrolysis of protein compounds at the “stomach” phase to poly- and oligopeptides was confirmed, continuing at the “intestine” stage with the accumulation of free amino acids. This methodology makes it possible to visualize the products of hydrolysis of proteins in a meat product at all stages of the model and to monitor changes in protein concentration in the system

Inhibition of constitutive and cxc-chemokine-induced NF-κB activity potentiates ansamycin-based HSP90-inhibitor cytotoxicity in castrate-resistant prostate cancer cells

Background: We determined how CXC-chemokine signalling and necrosis factor-B (NF-B) activity affected heat-shock protein 90 (Hsp90) inhibitor (geldanamycin (GA) and 17-allylamino-demethoxygeldanamycin (17-AAG)) cytotoxicity in castrate-resistant prostate cancer (CRPC).Methods:Geldanamycin and 17-AAG toxicity, together with the CXCR2 antagonist AZ10397767 or NF-B inhibitor BAY11-7082, was assessed by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide assay in two CRPC lines, DU145 and PC3. Flow cytometry quantified apoptotic or necrosis profiles. Necrosis factor-B activity was determined by luciferase readouts or indirectly by quantitative PCR and ELISA-based determination of CXCL8 expression.Results:Geldanamycin and 17-AAG reduced PC3 and DU145 cell viability, although PC3 cells were less sensitive. Addition of AZ10397767 increased GA (e.g., PC3 IC 20: from 1.670.4 to 0.180.2 nM) and 17-AAG (PC3 IC 20: 43.77.8 to 0.641.8 nM) potency in PC3 but not DU145 cells. Similarly, BAY11-7082 increased the potency of 17-AAG in PC3 but not in DU145 cells, correlating with the elevated constitutive NF-B activity in PC3 cells. AZ10397767 increased 17-AAG-induced apoptosis and necrosis and decreased NF-B activity/CXCL8 expression in 17-AAG-treated PC3 cells.Conclusion:Ansamycin cytotoxicity is enhanced by inhibiting NF-B activity and/or CXC-chemokine signalling in CRPC cells. Detecting and/or inhibiting NF-B activity may aid the selection and treatment response of CRPC patients to Hsp90 inhibitors.</p

In vitro hypoxia-conditioned colon cancer cell lines derived from HCT116 and HT29 exhibit altered apoptosis susceptibility and a more angiogenic profile in vivo

Hypoxia is an important selective force in the clonal evolution of tumours. Through HIF-1 and other transcription factors combined with tumour-specific genetic alterations, hypoxia is a dominant factor in the angiogenic phenotype. Cellular adaptation to hypoxia is an important requirement of tumour progression independent of angiogenesis. The adaptive changes, insofar as they alter hypoxia-induced apoptosis, are likely to determine responsiveness to antiangiogenic strategies. To investigate this adaptation of tumour cells to hypoxia, we recreated in vitro the in vivo situation of chronic intermittent exposure to low-oxygen levels. The colon carcinoma cell lines HT29 and HCT116 were subjected to 40 episodes of sublethal hypoxia (4 h) three times a week. The resulting two hypoxia-conditioned cell lines have been maintained in culture for more than 2 years. In both cell lines changes in doubling times occurred: in HT29 an increase, and in HCT116 a decrease. Cell survival in response to hypoxia and to DNA damage differed strikingly in the two cell lines. The HT29 hypoxia-conditioned cells were more resistant than the parental line to a 24 h hypoxic challenge, while those from HCT116 surprisingly were more sensitive. Sensitivity to cisplatin in vitro was also significantly different for the hypoxia-conditioned compared with the parental lines, suggesting a change in pathways leading to apoptosis following DNA damage signaling. The growth of both conditioned cell lines in vivo as xenografts in immunodeficient (SCID) mice was more rapid than their parental lines, and was accompanied in each by evidence of enhanced vascular proliferation as a consequence of the hypoxia-conditioning. Thus the changes in apoptotic susceptibility were independent of altered angiogenesis. The derivation of these lines provides a model for events within hypoxic regions of colon cancers, and for the acquisition of resistance and sensitivity characteristics that may have therapeutic implications for the use of antiangiogenesis drugs

Estrogen regulation of apoptosis: how can one hormone stimulate and inhibit?

The link between estrogen and the development and proliferation of breast cancer is well documented. Estrogen stimulates growth and inhibits apoptosis through estrogen receptor-mediated mechanisms in many cell types. Interestingly, there is strong evidence that estrogen induces apoptosis in breast cancer and other cell types. Forty years ago, before the development of tamoxifen, high-dose estrogen was used to induce tumor regression of hormone-dependent breast cancer in post-menopausal women. While the mechanisms by which estrogen induces apoptosis were not completely known, recent evidence from our laboratory and others demonstrates the involvement of the extrinsic (Fas/FasL) and the intrinsic (mitochondria) pathways in this process. We discuss the different apoptotic signaling pathways involved in E2 (17β-estradiol)-induced apoptosis, including the intrinsic and extrinsic apoptosis pathways, the NF-κB (nuclear factor-kappa-B)-mediated survival pathway as well as the PI3K (phosphoinositide 3-kinase)/Akt signaling pathway. Breast cancer cells can also be sensitized to estrogen-induced apoptosis through suppression of glutathione by BSO (L-buthionine sulfoximine). This finding has implications for the control of breast cancer with low-dose estrogen and other targeted therapeutic drugs

Study of the functional product’s protein compounds digestion features

The aim of the study was to investigate the transformation of meat product’s proteins from pig hearts and aortas during enzymatic hydrolysis in an in vitro model of the gastrointestinal tract. The model consisted of three phases simulating digestion processes: “oral cavity” phase (a-amylase, pH 7.0; 2 min), “stomach” phase (pork pepsin, pH 3.0; 120 min), “intestine” phase (pork pancreatin, pH 7.0; 130 min). The product was sequentially subjected to hydrolysis, at the end of each phase, samples were taken to determine the protein concentration (biuret method) and visualize the protein fractions (one-dimensional electrophoresis). A significant increase in protein concentration at the “stomach” phase was revealed by 3.2 times, and the absolute content by 4.6 times. At the “intestine” phase, a decrease in the number of peptide complexes with copper ions by 1.8 times, the absolute protein content by 8.5% was re‑ vealed. The noted tendency was confirmed by electrophoretic studies — at the stage, simulating digestion in the stomach, the prod‑ ucts of meat product’s proteins hydrolysis were visualized; at the “intestine” phase, a low expression of protein fractions in the range of more than 10 kDa is shown. The maximum hydrolysis of protein compounds at the “stomach” phase to poly- and oligopeptides was confirmed, continuing at the “intestine” stage with the accumulation of free amino acids. This methodology makes it possible to visualize the products of hydrolysis of proteins in a meat product at all stages of the model and to monitor changes in protein concentration in the system

Salt Effects on Complexes of Oppositely Charged Macromolecules Having Different Affinity to Water

International audienceThe influence of salt concentration on the size and on the thermodynamic stability of interpolymer complexes composed of oppositely charged macroions having different affinity to solvent was studied from a theoretical viewpoint. It was shown that increasing salt concentration causes changes in the structure of complex particles. At low salt concentration, the particles preserve their structure and size. At a critical salt concentration, nS cr, the particle size rises sharply to a slightly larger dimension. From this concentration, the macroions forming the interpolymer complex start to separate, and the complex is fully destroyed at a salt concentration nS*. After separation, the macroions coexist in solution and with further increase in salt concentration reduce their sizes according to the screening of polyion charges by salt ions. nS cr and nS* depend on physical parameters such as the degree of polymerization of macroions, their degree of ionization, and macroion-solvent interaction parameters. Experimental data collected in the particular cases of PLL-PLCA and PLL-PLCAI complexes with polylysine qualitatively agree with the trends indicated by the theoretical approach