[AKR1B10 inhibitor enhances the inhibitory effect of sorafenib on liver cancer xenograft].

Objective: To investigate the inhibitory effect of AKR1B10 inhibitor combined with sorafenib on hepatocellular carcinoma (HCC) xenograft growth. Methods: HepG2 xenograft model was established in nude mice. The mice were then randomly divided into four groups: control group, epalrestat monotherapy group, sorafenib monotherapy group and combination treatment group. Tumor volume, tumor weight, T/C ratio and the change in body weight of nude mice in each group were compared to evaluate the curative effect. Immunohistochemistry staining was used to detect the expression of Ki-67 in tumor tissues to evaluate the proliferation status of tumor cells. One-way analysis of variance was used to compare the differences between the groups. Student's t-test was used to test means of two groups and chi-square test was used for multiple samples. Results: The differences of the grafted tumor volume before and after treatment between the control group, epalrestat group, sorafenib group and combined therapy group was 238.940 ± 39.813, 124.991 ± 84.670, -26.111 ± 11.518, and -54.072 ± 17.673(mm(3)), respectively, (F = 37.048, P < 0.001). The tumor mass were 0.273 ± 0.140, 0.158 ± 0.078, 0.079 ± 0.054, 0.045 ± 0.024 (g), (F = 16.594, P < 0.001); T/C ratio were 100%, 57.9%, 28.9%, 16.5%, and Ki-67 positive rate were 23.295 ± 6.218, 13.503 ± 3.392, 7.325 ± 2.257, 4.664 ± 1.189 (%), (χ(2) = 822.203, P < 0.001) . The tumor volume (t = -3.579, P = 0.002) and Ki-67 positive rate (t = -10.003, P < 0.001) in epalrestat monotherapy group were significantly lower than control group. The tumor volume (t = 2.056, P = 0.025), tumor mass (t = 2.101, P = 0.043), and Ki-67 positive rate (t = -2.850, P = 0.005) in combination treatment group were significantly lower than sorafenib monotherapy group. Compared with the control group, the body weight of nude mice in the treatment group decreased to a certain extent, but there was no statistically significant difference between epalrestat monotherapy group and control group (t = -1.599, P = 0.262), and combined therapy and sorafenib monotherapy group (t = -0.051, P = 0.96). Conclusion: AKR1B10 inhibitor enhanced the inhibitory effect of sorafenib on hepatocellular carcinoma xenograft.

Substrate Specificity, Inhibitor Selectivity and Structure-Function Relationships of Aldo-Keto Reductase 1B15: A Novel Human Retinaldehyde Reductase.

Human aldo-keto reductase 1B15 (AKR1B15) is a newly discovered enzyme which shares 92% amino acid sequence identity with AKR1B10. While AKR1B10 is a well characterized enzyme with high retinaldehyde reductase activity, involved in the development of several cancer types, the enzymatic activity and physiological role of AKR1B15 are still poorly known. Here, the purified recombinant enzyme has been subjected to substrate specificity characterization, kinetic analysis and inhibitor screening, combined with structural modeling. AKR1B15 is active towards a variety of carbonyl substrates, including retinoids, with lower kcat and Km values than AKR1B10. In contrast to AKR1B10, which strongly prefers all-trans-retinaldehyde, AKR1B15 exhibits superior catalytic efficiency with 9-cis-retinaldehyde, the best substrate found for this enzyme. With ketone and dicarbonyl substrates, AKR1B15 also shows higher catalytic activity than AKR1B10. Several typical AKR inhibitors do not significantly affect AKR1B15 activity. Amino acid substitutions clustered in loops A and C result in a smaller, more hydrophobic and more rigid active site in AKR1B15 compared with the AKR1B10 pocket, consistent with distinct substrate specificity and narrower inhibitor selectivity for AKR1B15.

Impaired self-renewal and increased colitis and dysplastic lesions in colonic mucosa of AKR1B8-deficient mice.

PURPOSE: Ulcerative colitis and colitis-associated colorectal cancer (CAC) is a serious health issue, but etiopathological factors remain unclear. Aldo-keto reductase 1B10 (AKR1B10) is specifically expressed in the colonic epithelium, but downregulated in colorectal cancer. This study was aimed to investigate the etiopathogenic role of AKR1B10 in ulcerative colitis and CAC. EXPERIMENTAL DESIGN: Ulcerative colitis and CAC biopsies (paraffin-embedded sections) and frozen tissues were collected to examine AKR1B10 expression. Aldo-keto reductase 1B8 (the ortholog of human AKR1B10) knockout (AKR1B8(-/-)) mice were produced to estimate its role in the susceptibility and severity of chronic colitis and associated dysplastic lesions, induced by dextran sulfate sodium (DSS) at a low dose (2%). Genome-wide exome sequencing was used to profile DNA damage in DSS-induced colitis and tumors. RESULTS: AKR1B10 expression was markedly diminished in over 90% of ulcerative colitis and CAC tissues. AKR1B8 deficiency led to reduced lipid synthesis from butyrate and diminished proliferation of colonic epithelial cells. The DSS-treated AKR1B8(-/-) mice demonstrated impaired injury repair of colonic epithelium and more severe bleeding, inflammation, and ulceration. These AKR1B8(-/-) mice had more severe oxidative stress and DNA damage, and dysplasias were more frequent and at a higher grade in the AKR1B8(-/-) mice than in wild-type mice. Palpable masses were seen in the AKR1B8(-/-) mice only, not in wild-type. CONCLUSIONS: AKR1B8 is a critical protein in the proliferation and injury repair of the colonic epithelium and in the pathogenesis of ulcerative colitis and CAC, being a new etiopathogenic factor of these diseases.

Inflammatory mediators accelerate metabolism of benzo[a]pyrene in rat alveolar type II cells: the role of enhanced cytochrome P450 1B1 expression.

Long-term deregulated inflammation represents one of the key factors contributing to lung cancer etiology. Previously, we have observed that tumor necrosis factor-α (TNF-α), a major pro-inflammatory cytokine, enhances genotoxicity of benzo[a]pyrene (B[a]P), a highly carcinogenic polycyclic aromatic hydrocarbon, in rat lung epithelial RLE-6TN cells, a model of alveolar type II cells. Therefore, we analyzed B[a]P metabolism in RLE-6TN cells under inflammatory conditions, simulated using either recombinant TNF-α, or a mixture of inflammatory mediators derived from activated alveolar macrophage cell line. Inflammatory conditions significantly accelerated BaP metabolism, as evidenced by decreased levels of both parent B[a]P and its metabolites. TNF-α altered production of the metabolites associated with dihydrodiol-epoxide and radical cation pathways of B[a]P metabolism, especially B[a]P-dihydrodiols, and B[a]P-diones. We then evaluated the role of cytochrome P450 1B1 (CYP1B1), which is strongly up-regulated in cells treated with B[a]P under inflammatory conditions, in the observed effects. The siRNA-mediated CYP1B1 knock-down increased levels of B[a]P and reduced formation of stable DNA adducts, thus confirming the essential role of CYP1B1 in B[a]P metabolism under inflammatory conditions. TNF-α also reduced expression of aldo-keto reductase 1C14, which may compete with CYP1B1 for B[a]P-7,8-dihydrodiol and divert it from the formation of ultimate B[a]P dihydrodiol epoxide. Together, the present data suggests that the CYP1B1-catalyzed metabolism of polycyclic aromatic hydrocarbons might contribute to their enhanced bioactivation and genotoxic effects under inflammatory conditions.

Production of non-fucosylated antibodies by co-expression of heterologous GDP-6-deoxy-D-lyxo-4-hexulose reductase.

All IgG-type antibodies are N-glycosylated in their Fc part at Asn-297. Typically, a fucose residue is attached to the first N-acetylglucosamine of these complex-type N-glycans. Antibodies lacking core fucosylation show a significantly enhanced antibody-dependent cell-mediated cytotoxicity (ADCC) and an increased efficacy of anti-tumor activity. In cases where the clinical efficacy of an antibody is to some extent mediated by its ADCC effector function, afucosylated N-glycans could help to reduce dose requirement and save manufacturing costs. Using Chinese hamster ovary (CHO) cells as a model, we demonstrate here that heterologous expression of the prokaryotic enzyme GDP-6-deoxy-d-lyxo-4-hexulose reductase within the cytosol can efficiently deflect the fucose de novo pathway. Antibody-producing CHO cells that were modified in this way secrete antibodies lacking core fucose as demonstrated by MALDI-TOF mass spectrometry and HPAEC-PAD monosaccharide analysis. Engineering of the fucose de novo pathway has led to the construction of IgGs with a strongly enhanced ADCC effector function. The method described here should have broad practical applicability for the development of next-generation therapeutic antibodies.