Aldo-keto Reductase Family 1B10 Research Articles

Warburg effect enhanced by AKR1B10 promotes acquired resistance to pemetrexed in lung cancer-derived brain metastasis

BackgroundResistance to pemetrexed (PEM), a rare chemotherapeutic agent that can efficiently cross the blood-brain barrier, limits the therapeutic efficacy for patients with lung cancer brain metastasis (BM). Aldo-keto reductase family 1 B10 (AKR1B10) was recently found to be elevated in lung cancer BM. The link between AKR1B10 and BM-acquired PEM is unknown.MethodsPEM drug-sensitivity was assessed in the preclinical BM model of PC9 lung adenocarcinoma cells and the BM cells with or without AKR1B10 interference in vitro and in vivo. Metabolic reprogramming of BM attributed to AKR1B10 was identified by chromatography-mass spectrometry (GC-MS) metabolomics, and the mechanism of how AKR1B10 mediates PEM chemoresistance via a way of modified metabolism was revealed by RNA sequencing as well as further molecular biology experimental approaches.ResultsThe lung cancer brain metastatic subpopulation cells (PC9-BrM3) exhibited significant resistance to PEM and silencing AKR1B10 in PC9-BrM3 increased the PEM sensitivity in vitro and in vivo. Metabolic profiling revealed that AKR1B10 prominently facilitated the Warburg metabolism characterized by the overproduction of lactate. Glycolysis regulated by AKR1B10 is vital for the resistance to PEM. In mechanism, AKR1B10 promoted glycolysis by regulating the expression of lactate dehydrogenase (LDHA) and the increased lactate, acts as a precursor that stimulates histone lactylation (H4K12la), activated the transcription of CCNB1 and accelerated the DNA replication and cell cycle.ConclusionsOur finding demonstrates that AKR1B10/glycolysis/H4K12la/CCNB1 promotes acquired PEM chemoresistance in lung cancer BM, providing novel strategies to sensitize PEM response in the treatment of lung cancer patients suffering from BM.

Amentoflavone-loaded nanoparticles enhanced chemotherapy efficacy by inhibition of AKR1B10

Therapeutic nanoparticles can be combined with different anticancer drugs to achieve a synergistic therapy and avoid the limitations of traditional medicine and thus have clinical prospects for cancer. Herein, an effective nanoplatform was developed for self-assembling AMF@DOX-Fe3+-PEG nanoparticles (ADPF NPs) via the coordination of ferric ions (Fe3+), amentoflavone (AMF), doxorubicin (DOX), and PEG-polyphenol. The ADPF NPs possessed high drug loading efficiency, good stability and dispersion in water, prolonged blood circulation, and pH-dependent release, which leading to targeted drug transport and enhanced drug accumulation in the tumor. The AMF from the ADPF NPs could inhibit the expression of the Aldo-keto reductase family 1B10 (AKR1B10) and nuclear factor-kappa B p65 (NF-κB p65), which reduced the cardiotoxicity induced by DOX and enhanced the chemotherapy efficacy. This study established a new strategy of combining drug therapy with a nanoplatform. This new strategy has a wide application prospect in clinical tumor therapy.

AKR1B10 negatively regulates autophagy through reducing GAPDH upon glucose starvation in colon cancer.

Autophagy is considered to be an important switch for facilitating normal to malignant cell transformation during colorectal cancer development. Consistent with other reports, we found that the membrane receptor Neuropilin1 (NRP1) is greatly upregulated in colon cancer cells that underwent autophagy upon glucose deprivation. However, the mechanism underlying NRP1 regulation of autophagy is unknown. We found that knockdown of NRP1 inhibits autophagy and largely upregulates the expression of aldo-keto reductase family 1 B10 (AKR1B10). Moreover, we demonstrated that AKR1B10 interacts with and inhibits the nuclear importation of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and then subsequently represses autophagy. Interestingly, we also found that an NADPH-dependent reduction reaction could be induced when AKR1B10 interacts with GAPDH, and the reductase activity of AKR1B10 is important for its repression of autophagy. Together, our findings unravel a novel mechanism of NRP1 in regulating autophagy through AKR1B10.

Diagnostic Role of Aldoketoreductase family 1B10 (AKR1B10) in Hepatocellular Carcinoma and Benign Hepatic Lesions

Abstract Background Hepatocellular carcinoma is the most common primary liver cancer. It is the second most common cause of cancer deaths. The majority of HCCs arise in chronically diseased livers with cirrhotic background. Several benign lesions might be found in a cirrhotic liver along with hepatocellular carcinoma (HCC), and may exhibit typical or atypical imaging features. Distinction between benign hepatocellular nodular lesions, such as hepatic adenoma, and focal nodular hyperplasia, from HCC is of crucial clinical importance in determining appropriate therapy and assessing the prognosis.AKR1B10 is a promising marker that is recently used in the HCC diagnosis. Aim of the Work is to study the immuno-histochemical expression of Aldoketoreductase family 1B10 (AKR1B10) in hepatocellular carcinoma and benign hepatic lesions, to assess its role in the diagnosis of hepatocellular carcinoma and in differentiating it from different benign hepatic lesions. Materials and Methods Hepatocellular carcinoma (HCC(T)):(109), corresponding non tumor tissue with and without cirrhosis (NT):(80), benign hepatic lesions (Focal Nodular Hyperplasia(FNH):(6) and Hepatic Adenoma(HCA)) (6) and Hepatic Cirrhosis (Cs) without HCC (16). Results AKR1B10 was over expressed in 82.6% (90/109) of studied HCC cases, negative expression in studied corresponding non-tumor hepatic tissue (NT) in 92.5% (74/80), complete negativity of AKR1B10 was observed in studied benign hepatic lesions 100% (0/12) and also negative expression in 81.3% (13/16) of included hepatic cirrhosis without HCC. Conclusion AKR1B10 is a promising marker in the diagnosis of HCC and distinguishing it from other benign hepatic lesions.

AKR1B10 (Aldo-keto reductase family 1 B10) promotes brain metastasis of lung cancer cells in a multi-organ microfluidic chip model

Brain metastasis (BM) is a leading cause of mortality in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanisms underlying BM of NSCLC remain largely unknown because of the lack of models to accurately investigate such a dynamic and complex process. Here we developed a multi-organ microfluidic chip as a new methodological platform to study BM. The chip consisted of two bionic organ units – an upstream “lung” and a downstream “brain” characterized by a functional “blood–brain barrier (BBB)” structure, allowing real-time visual monitoring of the entire BM process, from the growth of primary tumor to its breaking through the BBB, and finally reaching the brain parenchyma. The chip was verified by lung cancer cell lines with differing metastatic abilities and then applied for the BM research where we first demonstrated that the protein expression of Aldo-keto reductase family 1 B10 (AKR1B10) was significantly elevated in lung cancer BM. Silencing AKR1B10 in brain metastatic tumor cells suppressed their extravasation through the BBB in the in vitro Transwell model, in our ex vivo microfluidic chip, as well as the in vivo model of brain metastasis in nude mice. Moreover, AKR1B10 downregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-9 via MEK/ERK signaling in metastatic lung cancers. These data suggest that our multi-organ microfluidic chip is a practical alternative to study BM pathogenesis, and AKR1B10 is a diagnostic biomarker and a prospective therapeutic target for NSCLC BM. Statement of SignificanceBrain metastasis (BM) of non-small cell lung cancer (NSCLC) is a complex cascade, and in particular, the process of lung cancer cells penetrating the blood–brain barrier (BBB) is very unique. However, due to the lack of reliable models that can faithfully mimic the dynamic process of BBB breaking, its molecular mechanisms have not well elucidated so far. In addition, although Aldo-keto reductase family 1 B10 (AKR1B10) has been implicated to the tumor development of liver cancer and many other cancers, little is known on its roles in the BM. Here, we established a multi-organ microfluidic bionic chip platform to recapitulate the entire BM process, and applied it to the BM pathology research, especially BBB extravasation. By using the chip and traditional models synergistically, we first demonstrated that AKR1B10 was significantly elevated in lung cancer BM, and defined the value of AKR1B10 as a diagnostic serum biomarker for lung cancer patients suffering from BM. Further, we investigated the role and mechanisms of AKR1B10 in BM that it promotes the extravasation of cancer cells through the BBB.

AKR1B10 Promotes Brain Metastasis of Lung Cancer Cells in a Multi-Organ Microfluidic Chip Model

Brain metastasis (BM) is a leading cause of mortality in patients with non-small cell lung cancer (NSCLC). However, the molecular mechanisms underlying BM of NSCLC remain largely unknown because we lack models to accurately investigate such a dynamic and complex process. Here we developed a multi-organ microfluidic chip and provided a new methodological platform to study BM. The chip consisted of two bionic organ units – an upstream “lung” and a downstream “brain” characterized by a functional “blood-brain barrier (BBB)” structure. The chip allowed real-time visual monitoring of the entire BM process, from the growth of primary tumor to its breaking through the BBB, and finally reaching the brain parenchyma. We verified this model using lung cancer cell lines with differing metastatic abilities. We first demonstrated that the protein expression of Aldo-keto reductase family 1 B10 (AKR1B10) was significantly elevated in lung cancer cell lines with higher metastatic ability and clinical brain metastases of lung cancers. Silencing AKR1B10 in brain metastatic tumor cells suppressed their extravasation through the BBB in the in vitro Transwell model and in our ex vivo microfluidic chip, as well as the in vivo model of metastasis to the brain in nude mice. Moreover, AKR1B10 downregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-9 via MEK/ERK signaling in metastatic lung cancers. These data suggest that our multi-organ microfluidic chip is a practical alternative to study BM pathogenesis, and AKR1B10 is a potential diagnostic biomarker and a prospective therapeutic target for NSCLC BM.

Prognostic Significance of 14-3-3ε, Aldo-keto Reductase Family 1 B10 and Metallothionein-1 in Hepatocellular Carcinoma.

Expression of 14-3-3ε is associated with prognostic outcomes of hepatocellular carcinoma (HCC) patients. Metallothionein-1 (MT-1) proteins and aldo-keto-reductase family 1 B10 (AKR1B10) are considered potential tumor regulators of HCC. The aim of this study, was to examine the prognostic value of 14-3-3ε, MT-1 and AKR1B10 expression in HCC. The expression levels of 14-3-3ε, MT-1 and AKR1B10 in HCC cell lines and paraffin-embedded tissues were examined by western blotting and immunohistochemical analysis. 14-3-3ε positivity was significantly associated with decreased MT-1 expression in HCC. Patients with decreased MT-1 expression had worse survival rates and a higher risk of metastasis than 14-3-3ε-positive HCC patients with unchanged MT-1 expression. Distinct expression patterns of 14-3-3ε/MT-1/AKR1B10 were significantly associated with the metastatic incidence and survival rates of HCC patients. Patients with negative 14-3-3ε staining in primary tumors had better prognostic outcomes. In contrast, patients with positive 14-3-3ε staining, decreased MT-1 expression and no increase in AKR1B10 expression in primary tumors had the worst overall and disease-free survival rates and the highest metastatic risk. 14-3-3ε, AKR1B10, and MT-1 act as potential prognostic biomarkers of HCC.

Abstract 2237: Diclofenac as a dual inhibitor of cyclooxygenase and aldo-keto-reductase 1B10 inhibits pancreatitis-enhanced carcinogenesis in LSL-Kras G12D -Pdx-1-Cremice

Abstract Diclofenac is one of non-steroid anti-inflammatory drugs and is recently identified as a competitive inhibitor of aldo-keto reductase family 1B10 (AKR1B10). AKR1B10 is an NADPH-dependent enzyme that exhibits lipid substrate specificity, especially for farnesyl and geranylgeranyl involved in protein prenylation (such as for Kras) and plays an oncogenic role in pancreatic carcinogenesis. In the present study, we determined the chemopreventive effects and mechanism of diclofenac on inhibiting pancreatitis-carcinogenesis in LSL-KrasG12D/Pdx1-Cre mice (called Pankras mice), particularly on Kras prenylation. Pancreatitis was induced by single i.p. injection of Cearulein (250ug/Kg body weight) for 5-week old mice, and one week after caerulein treatment, Pankras mice were fed an AIN-93M diet or a diet with 5 or 20ppm diclofenac (n=12 mice/group) for 7 months. The results showed that Diclofenac significantly reduced the severity of chronic pancreatitis, as measured by the extent of acini loss, inflammatory cell infiltration and stromal fibrosis. The progression of low-grade mPanIN I to high-grade mPanIN II/III and to invasive carcinoma was significantly suppressed and the tumor incidence significantly reduced to 50% and 17% in the mice treated with either 5ppm or 20 diclofenac compared to 86% in Pankras mice. Inhibition of membrane-bound mutant Kras (prenylated) and its transmitted phosphorylation of mitogen-activated protein kinase's kinase/extracellular signal-regulated kinases were demonstrated in pancreatic tissues by western blot assay. Analysis of the eicosanoid profile revealed a significant reduction of prostaglandin E2. These results indicate that diclofenac is a highly potential agent for preventing chronic pancreatitis and carcinogenesis, mainly via targeting mutant Kras and inflammation (Supported by NIH R01 CA164041). Citation Format: Jie Liao, Leyu Sun, Haonan Li, Rong Xia, Xiaoming You, Dandan Xu, Guang-Yu Yang. Diclofenac as a dual inhibitor of cyclooxygenase and aldo-keto-reductase 1B10 inhibits pancreatitis-enhanced carcinogenesis in LSL-KrasG12D-Pdx-1-Cremice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2237.

AKR1B10-inhibitory Selaginella tamariscina extract and amentoflavone decrease the growth of A549 human lung cancer cells in vitro and in vivo

Ethnopharmacological relevanceSelaginella tamariscina (P.Beauv.) Spring is a traditional medicinal plant used to treat various human diseases, including cancer, in Asia. The detailed molecular mechanism underlying the anti-cancer effects of this plant and the anti-cancer action of the combinatorial treatment of S. tamariscina and doxorubicin have not yet been investigated. Aim of the studyWe evaluated the inhibitory activity of S. tamariscina extract (STE) and its major compound, amentoflavone, on human aldo-keto reductase family 1B10 (AKR1B10), which is a detoxification enzyme involved in drug resistance, to evaluate their anti-cancer effects and their potential as adjuvant agents for doxorubicin cancer chemotherapy. Materials and methodsWe tested the AKR1B10 inhibitory activity of STE and amentoflavone via an in vitro biochemical assay using recombinant human AKR1B10. We tested the anti-proliferative activity in A549, NCI-H460, SKOV-3, and MCF-7 human cancer cells, which contain different expression levels of AKR1B10, and determined the combination index to evaluate whether the addition of STE and amentoflavone is synergistic or antagonistic to the anti-cancer action of doxorubicin. We finally evaluated the in vivo anti-tumor effects of STE in a nude mouse xenograft model of A549 cells. ResultsSTE and amentoflavone potently inhibited human AKR1B10 and synergistically increased the doxorubicin anti-proliferative effect in A549 and NCI-H460 human lung cancer cells that express a high level of AKR1B10 mRNA and protein. STE also significantly inhibited A549 tumor growth in animal experiments. ConclusionOur results suggest that STE and amentoflavone could be potential anti-cancer agents that target AKR1B10 and might be candidate adjuvant agents to boost the anti-cancer effect of doxorubicin.

Novel chemical scaffolds of the tumor marker AKR1B10 inhibitors discovered by 3D QSAR pharmacophore modeling.

Recent evidence suggests that aldo-keto reductase family 1 B10 (AKR1B10) may be a potential diagnostic or prognostic marker of human tumors, and that AKR1B10 inhibitors offer a promising choice for treatment of many types of human cancers. The aim of this study was to identify novel chemical scaffolds of AKR1B10 inhibitors using in silico approaches. The 3D QSAR pharmacophore models were generated using HypoGen. A validated pharmacophore model was selected for virtual screening of 4 chemical databases. The best mapped compounds were assessed for their drug-like properties. The binding orientations of the resulting compounds were predicted by molecular docking. Density functional theory calculations were carried out using B3LYP. The stability of the protein-ligand complexes and the final binding modes of the hit compounds were analyzed using 10 ns molecular dynamics (MD) simulations. The best pharmacophore model (Hypo 1) showed the highest correlation coefficient (0.979), lowest total cost (102.89) and least RMSD value (0.59). Hypo 1 consisted of one hydrogen-bond acceptor, one hydrogen-bond donor, one ring aromatic and one hydrophobic feature. This model was validated by Fischer's randomization and 40 test set compounds. Virtual screening of chemical databases and the docking studies resulted in 30 representative compounds. Frontier orbital analysis confirmed that only 3 compounds had sufficiently low energy band gaps. MD simulations revealed the binding modes of the 3 hit compounds: all of them showed a large number of hydrogen bonds and hydrophobic interactions with the active site and specificity pocket residues of AKR1B10. Three compounds with new structural scaffolds have been identified, which have stronger binding affinities for AKR1B10 than known inhibitors.

Aldoketoreductase family 1B10 (AKR1B10) as a biomarker to distinguish hepatocellular carcinoma from benign liver lesions

Hepatocellular carcinoma (HCC) is one of the most common highly aggressive malignant tumors worldwide. Aldoketoreductase 1B10 (AKR1B10) was first isolated from HCC and further identified to be over-expressed in many cancers from various organs. AKR1B10 contributes to detoxification of xenobiotics by lipid peroxidation and metabolizes physiological substrates such as farnesal, retinal, and carbonyls. Metabolizing these lipid substrates plays a crucial role in promoting carcinogenesis. In the present study, immunohistochemical analysis was performed to determine the prevalence/pattern of AKR1B10 expression in HCC and its usefulness to differentiate benign liver lesions from HCC. Oncogenic function of AKR1B10 was examined in hepatocellular carcinoma cells in vitro using Western blotting and shRNA knockdown approaches, with emphasis on cell apoptosis and response to chemotherapy. Immunohistochemistry analysis revealed AKR1B10 was overexpressed in 97% (86/89) of hepatocellular carcinomas, with minimal to no expression in adjacent hepatic tissue, while hepatic adenomas and focal nodular hyperplasia did not exhibit expression of AKR1B10. shRNA-mediated silencing of AKR1B10 expression in hepatocellular carcinoma cells resulted in (1) increased cell apoptosis, (2) decreased colony formation and size, and (3) enhanced cytoreductive response following exposure to doxorubicin chemotherapy. Our findings provide first time evidence that AKR1B10 is a unique biomarker involved in hepatocellular carcinogenesis via modulation of proliferation, cell apoptosis and chemoresistance and is a potential promising biomarker to differentiate HCCs from benign hepatic lesions.

Abstract 190: Sulindac inhibits pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice by suppressing aldo-keto reductase family 1B10 (AKR1B10).

Abstract Sulindac inhibits carcinogenesis via the mechanism that appears beyond cyclooxygenase inhibition. Recently sulindac has been identified as competitive inhibitors of aldo-keto reductase family 1B10 (AKR1B10). AKR1B10 is involved in the process of carbonyls, farnesyl and geranylgeranyl as well as retinal metabolisms, and these metabolic activities are important to carcinogenesis. However, there is no evidence on if inhibition of pancreatic carcinogenesis by sulindac is via inhibition of AKR1B10 and protein prenylation (i.e., Kras protein). To determine the effect of sulindac on pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice, five-week-old mice were fed with either AIN-93M diet or AIN-93M diet contained 200ppm sulindac (n=20 mice/group). Kaplan-Meier survival analysis with Log-Rank test was performed to analyze the effect of sulindac on animal survival from the development of pancreatic carcinoma. The results showed that average animal survival in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice was 143.7 ± 8.8 days; and with the sulindac 200ppm treatment, animal survival significantly increased to 168.0 ± 8.8 days (p<0.005). Extensively histopathological analyses revealed that 90% of mice developed malignant pancreatic carcinomas, of which 10% invasive pancreatic adenocarcinoma had metastasis to liver and lymph nodes. With 200ppm sulindac treatment, incidence of pancreatic cancer and its metastasis were significantly reduced to 56% and 0%, respectively (P<0.01). Immunchemical analysis showed that sulindac treatment significantly decreased Ki-67-labeled cell proliferation, increased caspase-3-labeled cell apoptosis, and suppressed phosphorylated ERK1/2. In vitro mouse pancreatic carcinoma cells derived from the LSL-KrasG12D/+-LSL-Trp53R172/+-Pdx-1-Cre mice were further treated with sulindac and revealed that sulindac inhibited AKR1B10 enzyme activity, suppressed Kras and HDJ2 protein prenylation, and further inhibited phosphylation of C-raf, ERK1/2 and MEK1/2. Similar to sulindac treatment, siRNA-silencing AKR1B10 expression resulted in a significant reduction of Kras protein prenylation and its downstream signals. Our results demonstrate that sulindac inhibits pancreatic carcinogenesis, mainly via inhibition of kras protein prenylation and its activated signals by targeting AKR1B10. (supported NCI R01 CA164041) Citation Format: Jie Liao, Yeon Tae Chung, Haonan Li, Wanying Zhang, Guang-Yu Yang. Sulindac inhibits pancreatic carcinogenesis in LSL-KrasG12D-LSL-Trp53R172H-Pdx-1-Cre mice by suppressing aldo-keto reductase family 1B10 (AKR1B10). [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 190. doi:10.1158/1538-7445.AM2013-190

Abstract 3702: Aldo-keto reductase family 1B10 (AKR1B10) knockdown or inhibition suppresses pancreatic tumorigenesis in vitro and in vivo.

Abstract AKR1B10 is a member of aldo-keto reductase family and has relatively specific lipid substrates including carbonyls, retinal and farnesal/geranylgeranial. Metabolizing these substracts is crucial in carcinogenesis, particularly for the reduction of farnesal/geranylgeranial to farnesol/ geranylgeraniol that is an important step for recycling the intermediates of cholesterol synthesis and for protein prenylation. Mutant KRAS is a most common oncogene involved in the development of pancreatic cancer, and KRAS requires protein prenylation for its activity. The aims of this study were to determine oncogenic role of AKR1B10 in pancreatic tumorigenesis. Using shRNA knockdown approach and AKR1B10 inhibitor oleanoic acid (OA) in CD18 human pancreatic carcinoma cells, silencing of AKR1B10 resulted in a significant inhibition of anchor-dependent growth (170±3.7colonies/HPF vs. 67±9.5colonies/HPF, P<0.01), and AKR1B10 inhibtor OA showed a dose-dependent inhibition of cell growth with IC50 of 30μM. Cell invasion study showed a significant decreased invasion index in shRNA AKR1B10 knockdown cells compared to vector-control cells (0.27 vs. 1.00). A significant inhibition of cell migration in shRNA AKR1B10 knockdown cells compared to vector control cells was observed and showed cell migration index14.0±1.8% vs. 9.2±1.2% at 16 hours, 30.5±3.5% vs. 21.0±1.1% at 24 hours, and 88.9±3.0% vs. 51.9±5.7% at 48 hours (P<0.01). AKR1B10 enzyme activity was also inhibited by OA as dose- dependent pattern. Western blot analysis revealed a significant down-regulation of active form KRAS and its downstream signals including phosphorylated C-Raf, ErK, and MEK1/2 as well as an up-regulation of E-cadherin. A significant reduction of in vivo tumor growth was observed in shRNA AKR1B10 knockdown CD18 pancreatic carcinoma cells implanted in nude mice as compared to vector-control CD18 human pancreatic carcinoma cells (tumor volume: 275.9±68.9mm3 vs. 582.7±10.5.6mm3, P=0.03; and tumor weight: 0.25±0.06g vs. 0.52±0.07g, P=0.01). Implanted CD18 pancreatic carcinomas in nude mice were further treated AKR1B10 inhibitor OA and showed a significant smaller tumor size (315.6±50.8mm3 vs. 582.7±10.5.6mm3, P=0.03) and tumor weight (0.35±0.05g vs. 0.52±0.07g, P=0.05) as compared to non OA treated tumor. With immunohistochemical approach, Ki67-labeled cell proliferation and phosphorylated ERK1/2 exhibited a significant lower in CD18 pancreatic carcinoma with either shRNA AKR1B10 knockdown or AKR1B10 inhibition. Our findings provide evidence that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis and in modulation of protein prenylation (KRAS). Thus, AKR1B10 could be a potential target for prevention and treatment of highly lethal pancreatic carcinoma. (supported NCI R01 CA164041) Citation Format: Wanying Zhang, Haonan Li, Jie Liao, Guang-Yu Yang. Aldo-keto reductase family 1B10 (AKR1B10) knockdown or inhibition suppresses pancreatic tumorigenesis in vitro and in vivo. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3702. doi:10.1158/1538-7445.AM2013-3702

Overexpression and oncogenic function of aldo-keto reductase family 1B10 (AKR1B10) in pancreatic carcinoma

AbstractAldo-keto reductase family 1B10 (AKR1B10) exhibits more restricted lipid substrate specificity (including farnesal, geranylgeranial, retinal and carbonyls), and metabolizing these lipid substrates has a crucial role in promoting carcinogenesis. Overexpression of AKR1B10 has been identified in smoking-related carcinomas such as lung cancer. As development of pancreatic cancer is firmly linked to smoking, the aim of the present study was to examine the expression and oncogenic role of AKR1B10 in pancreatic adenocarcinoma. AKR1B10 expression was analyzed in 50 paraffin-embedded clinical pancreatic cancer samples using immunohistochemistry. Oncogenic function of AKR1B10 was examined in pancreatic carcinoma cells in vitro using western blotting and siRNA approaches, mainly on cell apoptosis and protein prenylation including KRAS protein and its downstream signals. Immunohistochemistry analysis revealed that AKR1B10 overexpressed in 70% (35/50) of pancreatic adenocarcinomas and majority of pancreatic intraepithelial neoplasia, but not in adjacent morphologically normal pancreatic tissue. Compared with a normal pancreatic ductal epithelial cell (HPDE6E7), all of the six cultured pancreatic adenocarcinoma cell lines had an overexpression of AKR1B10 using immunoblotting, which correlated with increase of enzyme activity. siRNA-mediated silencing of AKR1B10 expression in pancreatic cancer cells resulted in (1) increased cell apoptosis, (2) increased non-farnesyled HDJ2 protein and (3) decreased membrane-bound prenylated KRAS protein and its downstream signaling molecules including phosphorylated ERK and MEK and membrane-bound E-cadherin. Our findings provide first time evidence that AKR1B10 is a unique enzyme involved in pancreatic carcinogenesis possibly via modulation of cell apoptosis and protein prenylation.

Abstract 717: Aldo-keto reductase family 1B10 (AKR1B10): Novel biomarker and molecular target of hepatocellular carcinoma

Abstract Aldo -keto reductase family 1B10 (AKR1B10) is a member of aldo-keto reducase (AKR) superfamily with biologic functions involved in osmolytic regulation, carbonyl detoxification, and carcinogenesis. Overexpression of AKR1B10 is identified in tumors of liver, lung, cervix and pancreas. However, the functional role of AKR1B10 in cancer remains unclear. In the present studies, we determined the expression and function of AKR1B10 in human hepatocellular carcinoma (HCC) using ex vivo HCC tissues and HCC cell lines. Thirty nine of HCC surgical resected specimens have been analyzed for the expression of AKR1B10 using immunohistochemistry method and revealed that 92.3% (36/39) of HCC expressed AKR1B10, including 100% (10/10) well differentiated, 95.2% (20/21) moderate differentiated, and 75% (6/8) poor differentiated HCC. No expression was observed in the adjacent morphologically normal liver. Expression of AKR1B10 in HCC confined to the cytoplasm. In order to investigate the oncogenic role of AKR1B10 in HCC, silence or inhibition of AKR1B10 in HCC cells (Huh 7) was approached using lentivirus mediated shRNA method or AKR1B10 inhibitor epalrestat. The results showed that the expression of AKR1B10 protein was significantly decreased in the AKR1B10 silenced cell lines with three different shRNA sequences using western blot approach. Knockdown AKR1B10 resulted in a significant inhibition of Huh 7 cell proliferation/tumorigenesis by colony formation assay and showed the average clone number was 174±25 of pGIPZ vector control Huh7 cell, and 86±32 of the AKR1B10 shRNA silencing cells (p<0.05). The average clone size was also significantly decreased in AKR1B10 shRNA silencing cells. A slightly increased cell apoptosis was found in AKR1B10 silenced cells as detected with both caspase-3 immunohistochemistry (IHC) and Annexin V flow cytometry. Furthermore, AKR1B10 silenced cells or pGIPZ vector control Huh7 cells pretreated with AKR1B10 inhibitor epalrestat significantly increased sensitivity to doxorubicin and 4-hydroxy Hexenal-induced cell death as measured with Annexin V flow cytometry. Reduction of GTP-KRAS activity and its downstream MAPK signals were observed in AKR1B10 silencing cells as measured by western bolt approach. Our results indicated that AKR1B10 is involved in the process of cell proliferation, apoptosis, and Kras activation, and further imply that AKR1B10 is a novel biomarker and crucial molecular target of HCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 717. doi:1538-7445.AM2012-717

AKR1B10 induces cell resistance to daunorubicin and idarubicin by reducing C13 ketonic group

Daunorubicin, idarubicin, doxorubicin and epirubicin are anthracyclines widely used for the treatment of lymphoma, leukemia, and breast, lung, and liver cancers, but tumor resistance limits their clinical success. Aldo-keto reductase family 1 B10 (AKR1B10) is an NADPH-dependent enzyme overexpressed in liver and lung carcinomas. This study was aimed to determine the role of AKR1B10 in tumor resistance to anthracyclines. AKR1B10 activity toward anthracyclines was measured using recombinant protein. Cell resistance to anthracycline was determined by ectopic expression of AKR1B10 or inhibition by epalrestat. Results showed that AKR1B10 reduces C13-ketonic group on side chain of daunorubicin and idarubicin to hydroxyl forms. In vitro, AKR1B10 converted daunorubicin to daunorubicinol at V max of 837.42 ± 81.39 nmol/mg/min, K m of 9.317 ± 2.25 mM and k cat / K m of 3.24. AKR1B10 showed better catalytic efficiency toward idarubicin with V max at 460.23 ± 28.12 nmol/mg/min, K m at 0.461 ± 0.09 mM and k cat / K m at 35.94. AKR1B10 was less active toward doxorubicin and epirubicin with a C14-hydroxyl group. In living cells, AKR1B10 efficiently catalyzed reduction of daunorubicin (50 nM) and idarubicin (30 nM) to corresponding alcohols. Within 24 h, approximately 20 ± 2.7% of daunorubicin (1 μM) or 23 ± 2.3% of idarubicin (1 μM) was converted to daunorubicinol or idarubicinol in AKR1B10 expression cells compared to 7 ± 0.9% and 5 ± 1.5% in vector control. AKR1B10 expression led to cell resistance to daunorubicin and idarubicin, but inhibitor epalrestat showed a synergistic role with these agents. Together our data suggest that AKR1B10 participates in cellular metabolism of daunorubicin and idarubicin, resulting in drug resistance. These data are informative for the clinical use of idarubicin and daunorubicin.

Inhibitory Effects of Dicaffeoylquinic Acids from Artemisia dubia on Aldo-keto Reductase Family 1B10

The ethyl acetate fraction of Artemisia dubia showed a potent inhibitory activity on recombinant human AKR1B10 (rh AKR1B10). Due to its potency, subsequent purification of ethyl acetate fraction led to seven compounds including five dicaffeoylquinic acid (DCQA) derivatives. Compounds were identified as 3,4-dihydroxybenzoic acid (1), quercetin 3-O-β-glucose (2), 3,4-DCQA (3), 3,5-DCQA (4), 4,5-DCQA (5), 3,5-DCQA methyl ester (6), and 4,5-DCQA methyl ester (7). A series of DCQA derivatives (3-7) showed inhibitory activity on rhAKR1B10 in the range of IC50 1.24-2.29 μM, whereas 3,4-dihydroxybenzoic acid and quercetin 3-O-β-glucose showed no inhibitory activity on rhAKR1B10.

Inhibitory effects of polyphenols isolated from Rhus verniciflua on Aldo-keto reductase family 1 B10

Aldo-keto reductase family 1 B10 (AKR1B10) is a member of the NADPH-dependent aldo-keto reductase (AKR) superfamily, and has been considered to be a potential cancer therapeutic target. Total extract from the bark of Rhus verniciflua (Toxicodendron vernicifluum (Stokes)) showed AKR1B10 inhibitory activity. To identify the active compounds from R. verniciflua responsible for AKR1B10 inhibition, nine compounds were isolated via bioactivity-guided isolation and tested for their effects against recombinant human AKR1B10 (rhAKR1B10). Results showed that butein, isolated from the ethyl acetate fraction, was most able to inhibit rhAKR1B10. The inhibitory rate of butein against rhAKR1B10 was 42.86% at 1 microM with an IC(50) value of 1.47 microM, and enzyme kinetic analysis revealed its inhibition mode to be uncompetitive.

한국 자생식물 추출물의 항산화 및 Aldo-keto Reductase Family 1 B10 효소 억제 효과

Aldo-keto reductase family 1 B10 (AKR1B10) has been considered as a potential cancer therapeutic target. Ethanol extracts prepared from 82 Korean local plants were examined for their antioxidant activity and inhibitory effects on recombinant human AKR1B10 (rhAKR1B10) in vitro. 21 extracts showed more than 80% of ABTS radical scavenging activity at <TEX>$100\;{\mu}g/mL$</TEX> and 11 extracts inhibited more than 50% of rhAKR1B10 activity at <TEX>$10\;{\mu}g/mL$</TEX>. Especially, 9 extracts showed potent inhibition on rhAKR1B10 activity compared with positive control tetramethylene glutaric acid.

Aldo-keto Reductase Family 1 B10 Affects Fatty Acid Synthesis by Regulating the Stability of Acetyl-CoA Carboxylase-α in Breast Cancer Cells

Recent studies have demonstrated that aldo-keto reductase family 1 B10 (AKR1B10), a novel protein overexpressed in human hepatocellular carcinoma and non-small cell lung carcinoma, may facilitate cancer cell growth by detoxifying intracellular reactive carbonyls. This study presents a novel function of AKR1B10 in tumorigenic mammary epithelial cells (RAO-3), regulating fatty acid synthesis. In RAO-3 cells, Sephacryl-S 300 gel filtration and DEAE-Sepharose ion exchange chromatography demonstrated that AKR1B10 exists in two distinct forms, monomers (approximately 40 kDa) bound to DEAE-Sepharose column and protein complexes (approximately 300 kDa) remaining in flow-through. Co-immunoprecipitation with AKR1B10 antibody and protein mass spectrometry analysis identified that AKR1B10 associates with acetyl-CoA carboxylase-alpha (ACCA), a rate-limiting enzyme of de novo fatty acid synthesis. This association between AKR1B10 and ACCA proteins was further confirmed by co-immunoprecipitation with ACCA antibody and pulldown assays with recombinant AKR1B10 protein. Intracellular fluorescent studies showed that AKR1B10 and ACCA proteins co-localize in the cytoplasm of RAO-3 cells. More interestingly, small interfering RNA-mediated AKR1B10 knock down increased ACCA degradation through ubiquitination-proteasome pathway and resulted in >50% decrease of fatty acid synthesis in RAO-3 cells. These data suggest that AKR1B10 is a novel regulator of the biosynthesis of fatty acid, an essential component of the cell membrane, in breast cancer cells.