Roles of aldo-keto reductases 1B10 and 1C3 and ATP-binding cassette transporter in docetaxel tolerance

Docetaxel (DTX) is widely used for treatment of inveterate lung and prostate cancers, but its continuous administration elicits the hyposensitivity. Here, we established the DTX-resistant variants of human lung cancer A549 and androgen-independent prostate cancer Du145 cells and found that the resistance development provoked aberrant up-regulations of aldo-keto reductase (AKR) 1B10 and AKR1C3 in A549 and Du145 cells, respectively. In addition, the sensitivity to the DTX toxicity was significantly decreased and increased by overexpression and knockdown of the two AKR isoforms, respectively. Furthermore, the resistant cells exhibited a decreased level of reactive 4-hydroxy-2-nonenal formed during DTX treatment, and the decrease was alleviated by adding the AKR inhibitors, inferring that the two AKRs confer the chemoresistance through elevating the antioxidant properties. The development of DTX resistance was also associated with enhanced expression of an ATP-binding cassette (ABC) transporter ABCB1 among the ABC transporter isoforms. The combined treatment with inhibitors of the two AKRs and ABCB1 additively sensitized the resistant cells to DTX. Intriguingly, the AKR1B10 inhibitor also suppressed the lung cancer cross-resistance against cisplatin. The results suggest that combined treatment with AKRs (1B10 and 1C3) and ABCB1 inhibitors exerts overcoming effect against the cancer resistance to DTX and cisplatin, and can be used as the adjuvant therapy.     

Aldo-keto reductases AKR1C1, AKR1C2 and AKR1C3 may enhance progesterone metabolism in ovarian endometriosis

Endometriosis is a very common disease that is characterized by increased formation of estradiol and disturbed progesterone action. This latter is usually explained by a lack of progesterone receptor B (PR-B) expression, while the role of pre-receptor metabolism of progesterone is not yet fully understood. In normal endometrium, progesterone is metabolized by reductive 20α-hydroxysteroid dehydrogenases (20α-HSDs), 3α/β-HSDs and 5α/β-reductases. The aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are the major reductive 20α-HSDs, while the oxidative reaction is catalyzed by 17β-HSD type 2 (HSD17B2). Also, 3α-HSD and 3β-HSD activities have been associated with the AKR1C isozymes. Additionally, 5α-reductase types 1 and 2 (SRD5A1, SRD5A2) and 5β-reductase (AKR1D1) are responsible for the formation of 5α- and 5β-reduced pregnanes. In this study, we examined the expression of PR-AB and the progesterone metabolizing enzymes in 31 specimens of ovarian endometriosis and 28 specimens of normal endometrium. Real-time PCR analysis revealed significantly decreased mRNA levels of PR-AB, HSD17B2 and SRD5A2, significantly increased mRNA levels of AKR1C1, AKR1C2, AKR1C3 and SRD5A1, and negligible mRNA levels of AKR1D1. Immunohistochemistry staining of endometriotic tissue compared to control endometrium showed significantly lower PR-B levels in epithelial cells and no significant differences in stromal cells, there were no significant differences in the expression of AKR1C3 and significantly higher AKR1C2 levels were seen only in stromal cells. Our expression analysis data at the mRNA level and partially at the cellular level thus suggest enhanced metabolism of progesterone by SRD5A1 and the 20α-HSD and 3α/β-HSD activities of AKR1C1, AKR1C2 and AKR1C3.     

Murine aldo-keto reductase family 1 subfamily B: identification of AKR1B8 as an ortholog of human AKR1B10

Aldo-keto reductase family 1 member B10 (AKR1B10), over-expressed in multiple human cancers, might be implicated in cancer development and progression via detoxifying cytotoxic carbonyls and regulating fatty acid synthesis. In the present study, we investigated the ortholog of AKR1B10 in mice, an ideal modeling organism greatly contributing to human disease investigations. In the mouse, there are three aldo-keto reductase family 1 subfamily B (AKR1B) members, i.e., AKR1B3, AKR1B7, and AKR1B8. Among them, AKR1B8 has the highest similarity to human AKR1B10 in terms of amino acid sequence, computer-modeled structures, substrate spectra and specificity, and tissue distribution. More importantly, similar to human AKR1B10, mouse AKR1B8 associates with murine acetyl-CoA carboxylase-α and mediates fatty acid synthesis in colon cancer cells. Taken together, our data suggest that murine AKR1B8 is the ortholog of human AKR1B10.     

Induction of autophagy by proteasome inhibitor is associated with proliferative arrest in colon cancer cells

The ubiquitin-proteasome system (UPS) and lysosome-dependent macroautophagy (autophagy) are two major intracellular pathways for protein degradation. Blockade of UPS by proteasome inhibitors has been shown to activate autophagy. Recent evidence also suggests that proteasome inhibitors may inhibit cancer growth. In this study, the effect of a proteasome inhibitor MG-132 on the proliferation and autophagy of cultured colon cancer cells (HT-29) was elucidated. Results showed that MG-132 inhibited HT-29 cell proliferation and induced G₂/M cell cycle arrest which was associated with the formation of LC3⁺ autophagic vacuoles and the accumulation of acidic vesicular organelles. MG-132 also increased the protein expression of LC3-I and -II in a time-dependent manner. In this connection, 3-methyladenine, a Class III phosphoinositide 3-kinase inhibitor, significantly abolished the formation of LC3⁺ autophagic vacuoles and the expression of LC3-II but not LC3-I induced by MG-132. Taken together, this study demonstrates that inhibition of proteasome in colon cancer cells lowers cell proliferation and activates autophagy. This discovery may shed a new light on the novel function of proteasome in the regulation of autophagy and proliferation in colon cancer cells.     

A novel nitro-oxy substituted analogue of rofecoxib reduces human colon cancer cell growth

Rofecoxib is a specific COX-2 inhibitor able to exert antiproliferative activity against colorectal cancer cells. It was withdrawn from the market after the demonstration of an increased risk of cardiovascular complications after prolonged use. Nevertheless, it remains an interesting compound for laboratory research as an experimental COX-2 inhibitor. In this study, the antiproliferative activity of a novel dinitro-oxy-substituted analogue of rofecoxib (NO-rofe), potentially less cardiotoxic, has been investigated in vitro on human colon cancer cells and compared with the action of the parent drug. Due to the fact that COX-2 inhibition is the main characteristic of coxibs, we performed all experiments in COX-2-overexpressing (HT-29) and COX-2-negative (SW-480) human colon cancer cells, to elucidate whether the observed effects were dependent on COX-2 inhibition. Moreover, experiments were performed in order to evaluate whether COX-2 pharmacological inhibition may affect beta-catenin/E-cadherin signaling pathway. NO-rofe exerted a significant antiproliferative activity on COX-2 positive HT-29 human colon cancer cells, being less effective on the COX-2 negative SW-480 human colon cancer cell line. In particular, the rofecoxib analogue retained similar potencies with respect to COX-2 inhibition but was much more active than rofecoxib in inhibiting the growth of human colon cancer cells in vitro. In addition, this novel compound resulted in the induction of membrane β-catenin/E-cadherin expression, a feature that may significantly contribute to its antiproliferative activity.     

Nitric oxide confers cisplatin resistance in human lung cancer cells through upregulation of aldo-keto reductase 1B10 and proteasome

Abstract

In this study, we show that exposure of human lung cancer A549 cells to cisplatin (cis-diamminedichloroplatinum, CDDP) promotes production of nitric oxide (NO) through generation of reactive oxygen species (ROS) and resulting upregulation of inducible NO synthase (iNOS). The incubation of the cells with a NO donor, diethylenetriamine NONOate, not only reduced the CDDP-induced cell death and apoptotic alterations (induction of CCAAT-enhancer-binding protein homologous protein and caspase-3 activation), but also elevated proteolytic activity of 26S proteasome, suggesting that the activation of proteasome function contributes to the reduction of CDDP sensitivity by NO. Monitoring expression levels of six aldo-keto reductases (AKRs) (1A1, 1B1, 1B10, 1C1, 1C2, and 1C3) during the treatment with the NO donor and subsequent CDDP sensitivity test using the specific inhibitors also proposed that upregulation of AKR1B10 by NO is a key process for acquiring the CDDP resistance in A549 cells. Treatment with CDDP and NO increased amounts of nitrotyrosine protein adducts, indicative of peroxynitrite formation, and promoted the induction of AKR1B10, inferring a relationship between peroxynitrite formation and the enzyme upregulation in the cells. The treatment with CDDP or a ROS-related lipid aldehyde, 4-hydroxy-2-nonenal, facilitated the iNOS upregulation, which was restored by increasing the AKR1B10 expression. In contrast, the facilitation of NO production by CDDP treatment was hardly observed in AKR1B10-overexpressing A549 cells and established CDDP-resistant cancer cells (A549, LoVo, and PC3). Collectively, these results suggest the NO functions as a key regulator controlling AKR1B10 expression and 26S proteasome function leading to gain of the CDDP resistance.     

抑制Hedgehog信号通路改变结肠癌细胞基因表达谱

Hedgehog（Hh）信号通路在机体发育和肿瘤发生中发挥着重要作用。在该研究中,Western blot检测三株结肠癌细胞Hedgehog信号通路组分的表达,结果表明三株结肠癌细胞中HT-29细胞Hedgehog信号通路组分较完整。采用MTT和BrdU法检测Hedgehog信号通路膜受体Smo特异性抑制剂环杷明和末端转录因子Gli1/2的特异性抑制剂GANT61对HT-29细胞的影响,提示这两种抑制剂均显著抑制HT-29细胞生存率和细胞增殖率,且GANT61比环杷明更敏感。表达谱芯片检测阻断Hedgehog信号通路后HT-29细胞基因谱的变化,结合生物信息学分析,揭示HT-29细胞经环杷明和GANT61处理后基因表达呈现抑制特征,其差异基因表达主要以下调为主,其中环杷明主要影响细胞内源刺激等,而GANT61主要影响代谢和类固醇合成,并与MAPK信号通路有关联,两者均能影响细胞免疫及凋亡相关通路。这些结果提示,Hh信号通路有可能作为结肠癌的治疗靶点。     

Quantitative Evaluation of Aldo-keto Reductase Expression in Hepatocellular Carcinoma (HCC) Cell Lines

The involvement of aldo–keto reductases (AKRs) in tumorigenesis is widely reported, but their roles in the pathological process are not generally recognized due to inconsistent measurements of their expression. To overcome this problem, we simultaneously employed real-time PCR to examine gene expression and multiple reaction monitoring (MRM) of mass spectrometry (MS) to examine the protein expression of AKRs in five different hepatic cell lines. These include one relatively normal hepatic cell line, L-02, and four hepatocellular carcinoma (HCC) cell lines, HepG2, HuH7, BEL7402 and SMMC7721. The results of real-time PCR showed that expression of genes encoding the AKR1C family members rather than AKR1A and AKR1B was associated with tumor, and most of genes encoding AKRs were highly expressed in HuH7. Similar observations were obtained through MRM. Different from HuH7, the protein abundance of AKR1A and AKR1B was relatively consistent among the other four hepatic cell lines, while protein expression of AKR1C varied significantly compared to L-02. Therefore, we conclude that the abundant distribution of AKR1C proteins is likely to be associated with liver tumorigenesis, and the AKR expression status in HuH7 is completely different from other liver cancer cell lines. This study, for the first time, provided both overall and quantitative information regarding the expression of AKRs at both mRNA and protein levels in hepatic cell lines. Our observations put the previous use of AKRs as a biomarker into question since it is only consistent with our data from HuH7. Furthermore, the data presented herein demonstrated that quantitative evaluation and comparisons within a protein family at both mRNA and protein levels were feasible using current techniques.     

AKR1B10: A potential target for cancer therapy

Aldo-keto reductase family 1 B10 (AKR1B10, also designated aldose reductase-like-1, ARL-1) is a novel protein identified from human hepatocellular carcinoma (HCC). This protein belongs to aldo-keto reductase superfamily, a group of proteins implicated in intracellular detoxification, cell carcinogenesis, and cancer therapeutics. AKR1B10 is primarily expressed in the colon and small intestine with low levels in the liver, thymus, prostate, and testis but overexpressed in the liver and lung cancer, making it a potential cancer diagnostic and/or prognostic marker. AKR1B10 could reduce retinals to retinols eliminating intracellular retinoic acid, a signaling molecule regulating cell proliferation and differentiation. AKR1B10 may impact the carcinogenesis process through controlling retinoic acid signaling.     

Deregulated expression of homeobox-containing genes, HOXB6, B8, C8, C9, and Cdx-1, in human colon cancer cell lines

Previously we have demonstrated a reciprocal deregulation of various homeobox genes (HOXB6, B8, C8 and C9 vs Cdx-1) in human colorectal cancer (CRC). In the present study, using RT-PCR, we have investigated the expression pattern of these homeobox genes in various human colon cell lines, representing various stages of colon cancer progression and differentiation. Thus, we have tested polyposis coli Pc/AA adenoma cells, Caco-2, HT-29 and LS174T adenocarcinoma cell lines. All cell lines, except LS174T, demonstrated a pattern of deregulated homeobox gene expression which resembled that of CRC. In contrast, the pattern of expression of these genes in the highly oncogenic LS174T cells, as well as in Caco-2 cells transfected with activated Ha-ras or Polyoma middle T oncogene, resembled that of the normal mucosa. The reciprocal deregulation of HOX and Cdx-1 genes in CRC and in CRC-derived cell lines suggests a possible role in human CRC development.     

Epidermal growth factor induces tumour marker AKR1B10 expression through activator protein-1 signalling in hepatocellular carcinoma cells

AKR1B10 (aldo-keto reductase 1B10) is overexpressed in liver and lung cancer, and plays a critical role in tumour development and progression through promoting lipogenesis and eliminating cytotoxic carbonyls. AKR1B10 is a secretory protein and potential tumour marker; however, little is known about the regulatory mechanism of AKR1B10 expression. The present study showed that AKR1B10 is induced by mitogen EGF (epidermal growth factor) and insulin through the AP-1 (activator protein-1) signalling pathway. In human HCC (hepatocellular carcinoma) cells (HepG2 and Hep3B), EGF (50?ng/ml) and insulin (10?nM) stimulated endogenous AKR1B10 expression and promoter activity. In the AKR1B10 promoter, a putative AP-1 element was found at bp -222 to -212. Deletion or mutation of this AP-1 element abrogated the basal promoter activity and response to EGF and AP-1 proteins. This AP-1 element bound to nuclear proteins extracted from HepG2 cells, and this binding was stimulated by EGF and insulin in a dose-dependent manner. Chromatin immunoprecipitation showed that the AP-1 proteins c-Fos and c-Jun were the predominant factors bound to the AP-1 consensus sequence, followed by JunD and then JunB. The same order was followed in the stimulation of endogenous AKR1B10 expression by AP-1 proteins. Furthermore, c-Fos shRNA (short hairpin RNA) and AP-1 inhibitors/antagonists (U0126 and Tanshinone IIA) inhibited endogenous AKR1B10 expression and promoter activity in HepG2 cells cultured in vitro or inoculated subcutaneously in nude mice. U0126 also inhibited AKR1B10 expression induced by EGF. Taken together, these results suggest that AKR1B10 is up-regulated by EGF and insulin through AP-1 mitogenic signalling and may be implicated in hepatocarcinogenesis.     

Loss of AKR1C1 is a good prognostic factor in advanced NPC cases and increases chemosensitivity to cisplatin in NPC cells

Cisplatin resistance is one of the main obstacles in the treatment of advanced nasopharyngeal carcinoma (NPC). AKR1C1 is a member of the Aldo-keto reductase superfamily (AKRs), which converts aldehydes and ketones to their corresponding alcohols and has been reported to be involved in chemotherapeutic resistance of multiple drugs. The expression and function of AKR1C1 in NPC have not been reported until now. The aim of this research was to investigate the expression of AKR1C1 and it is role in cisplatin resistance in NPC. AKR1C1 protein expression was detected by immunohistochemistry in human NPC tissues and by Western blot assays in NPC and immortalized nasopharyngeal epithelial cells. The effects of AKR1C1 knock-down by siRNA on proliferation, migration and invasion in NPC cells were evaluated by CCK8, wound healing and transwell assays. To evaluate the effects of AKR1C1 silencing on cisplatin sensitivity in NPC cells, CCK8 assays were used to detect cell proliferation, flow cytometry was used to detect cell cycle distribution, and flow cytometry and DAPI staining were used to detect cell apoptosis. AKR1C1 down-regulation was associated with advanced clinicopathological characters such as larger tumor size, more lymphatic nodes involvement, with metastasis and later clinical stages, while AKR1C1 down-regulation was a good prognostic factor for overall survival (OS) in NPC patients. In vitro study showed that AKR1C1 was not directly involved in the malignant biological behaviours such as proliferation, cell cycle progression and migration of NPC cells, whereas AKR1C1 knock-down could enhance cisplatin sensitivity of NPC cells. These results suggest that AKR1C1 is a potential marker for predicting cisplatin response and could serve as a molecular target to increase cisplatin sensitivity in NPC.     

Downregulation of <Emphasis Type="Italic">AKR1B10</Emphasis> expression in colorectal cancer

Colorectal cancer is one of the most common cancers in the world. Changes in AKR1B1 and AKR1B10 expression levels, whose diagnostic value was previously shown for several other cancer types, were studied in colorectal tumors. These genes encode aldose reductases, members of the aldo-keto reductase superfamily, which comprises enzymes capable to reduce a range of aromatic and aliphatic aldehydes and ketones. They are also involved in retinoid metabolism and carcinogenesis. AKR1B1 and AKR1B10 mRNA levels were compared in paired specimens of normal and colorectal tumor tissues using RT-PCR and quantitative real-time PCR. For the first time, the downregulation of these genes was demonstrated in colorectal carcinoma. AKR1B10 expression was decreased in most tumor specimens (88%, 65/74) even at the early stages, and in more than 60% of cases mRNA levels were decreased more than 10-fold. AKR1B1 mRNA levels were decreased in 10% of specimens. Therefore, these two structurally similar genes show quite different mRNA expression patterns in colorectal cancer, suggestive of their different functional roles in the intestine. Significant downregulation of AKR1B10 expression can be considered a potential diagnostic marker of colorectal cancer.