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This study aims to elucidate the anti-hypoxia mechanism of sesamoside, an active component of Phlomis younghusbandii Mukerjee, through a network pharmacology approach. Sesamoside has demonstrated potential anti-oxidant and antiglycation activities. The hypoxia-related disease targets were collected from databases like GeneCards and OMIM. Protein-protein interaction (PPI) networks were constructed using the STRING database. GO/KEGG enrichment analysis was performed using the Metascape database to identify biological processes and signaling pathways. Our results indicate that sesamoside interacts with multiple targets related to glucose and lipid metabolism, nucleotide metabolism, and inflammatory, and we find that AKR1B1 (AR) plays a crucial role in sesamoside responses to hypoxia. Molecular docking studies were performed using Autodock software, revealing good binding activity between sesamoside and AR. We then use CCK-8 assay, qPCR, WB, and ELISA analysis to validate the role of sesamoside in regulating AR and participating in anti-hypoxia through cell experiments. The results show that compared with the hypoxia group, sesamoside treatment significantly improves the expression of AR and inflammation cytokines. In summary, this study sheds light on the anti-hypoxia mechanism of sesamoside using a network pharmacology approach, providing a theoretical basis and experimental foundation for its application in the prevention and treatment of hypoxic diseases.

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Gastric cancer is a malignant tumor associated with a high mortality rate. Recently, emerging evidence has shown that ferroptosis, a regulated form of cell death induced by iron (Fe)-dependent lipid peroxidation. Nuclear factor E2 related factor 2 (NRF2) is a key regulator of intracellular oxidation homeostasis that plays a pivotal role in controlling lipid peroxidation, which is closely related to the process of ferroptosis. However, the molecular mechanism of NRF2 on ferroptosis remains to be investigated in gastric cancer. In our study, NRF 2 was found to transcriptionally activate Aldo-keto reductase 1 member B1 (AKR1B1) expression in gastric cancer. AKR1B1 is involved in the regulation of lipid metabolism by removing the aldehyde group of glutathione. We found that AKR1B1 is highly expressed in gastric cancer and is associated with a poor prognosis of the patients. In vitro experiments found that AKR1B1 has the ability to promote the proliferation and invasion of gastric cancer cells. AKR1B1 inhibited RSL3-induced ferroptosis in gastric cancer by reducing reactive oxygen species accumulation and lipid peroxidation, as well as decreasing intracellular ferrous ion and malondialdehyde expression and increasing glutathione expression. Our study demonstrated that AKR1B1 resisted RSL3-induced ferroptosis by regulating GPX4, PTGS2 and ACSL4, which was further demonstrated in a xenograft nude mouse model. Our work reveals a critical role for the AKR1B1 in the resistance to RSL3-induced ferroptosis in gastric cancer.

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A significant variation in chromatin accessibility is an epigenetic feature of leukemia. The cause of this variation in leukemia, however, remains elusive. Here, we identify SMARCA5, a core ATPase of the imitation switch (ISWI) chromatin remodeling complex, as being responsible for aberrant chromatin accessibility in leukemia cells. We find that SMARCA5 is required to maintain aberrant chromatin accessibility for leukemogenesis and then promotes transcriptional activation of AKR1B1, an aldo/keto reductase, by recruiting transcription co-activator DDX5 and transcription factor SP1. Higher levels of AKR1B1 are associated with a poor prognosis in leukemia patients and promote leukemogenesis by reprogramming fructose metabolism. Moreover, pharmacological inhibition of AKR1B1 has been shown to have significant therapeutic effects in leukemia mice and leukemia patient cells. Thus, our findings link the aberrant chromatin state mediated by SMARCA5 to AKR1B1-mediated endogenous fructose metabolism reprogramming and shed light on the essential role of AKR1B1 in leukemogenesis, which may provide therapeutic strategies for leukemia.

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Due to various factors, there is still a lack of effective neuroprotective agents for ischemic stroke in clinical practice. Neuroinflammation and neuronal apoptosis mediated by endoplasmic reticulum stress are some of the important pathological mechanisms in ischemic stroke. Linarin has been reported to have anti-inflammation, antioxidant, and anti-apoptotic effects in myocardial ischemia, osteoarthritis, and kidney disease. Whether it exerts neuroprotective functions in ischemic stroke has not been investigated. The results showed that linarin could reduce the infarct volume in cerebral ischemia animal models, improve the neurological function scores and suppress the expression of inflammatory factors mediating the NF-κB. Meanwhile, it could protect the neurons from OGD/R-induced-apoptosis, which was related to the PERK-eIF2α pathway. Our results suggested linarin could inhibit neuronal inflammation and apoptosis induced by endoplasmic reticulum stress. Furthermore, the neuroprotective effect of linarin may be related to the inhibition of AKR1B1. Our study offers new insight into protecting against ischemia-reperfusion injury by linarin treatment in stroke.

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Background: Sepsis and sepsis-associated acute kidney injury (SA-AKI) pose significant global health challenges, necessitating the development of innovative therapeutic strategies. Dysregulated protein expression has been implicated in the initiation and progression of sepsis and SA-AKI. Identifying potential protein targets and modulating their expression is crucial for exploring alternative therapies. Method: We established an SA-AKI rat model using cecum ligation perforation (CLP) and employed differential proteomic techniques to identify protein expression variations in kidney tissues. Aldose reductase (AKR1B1) emerged as a promising target. The SA-AKI rat model received treatment with the aldose reductase inhibitor (ARI), epalrestat. Blood urea nitrogen (BUN) and creatinine (CRE) levels, as well as IL-1ß, IL-6 and TNF-α levels in the serum and kidney tissues, were monitored. Hematoxylin-eosin (H-E) staining and a pathological damage scoring scale assessed renal tissue damage, while protein blotting determined PKC (protein kinase C)/NF-κB pathway protein expression. Result: Differential proteomics revealed significant downregulation of seven proteins and upregulation of 17 proteins in the SA-AKI rat model renal tissues. AKR1B1 protein expression was notably elevated, confirmed by Western blot. ARI prophylactic administration and ARI treatment groups exhibited reduced renal injury, low BUN and CRE levels and decreased IL-1ß, IL-6 and TNF-α levels compared to the CLP group. These changes were statistically significant (P < 0.05). AKR1B1, PKC-α, and NF-κB protein expression levels were also lowered in the ARI prophylactic administration and ARI treatment groups compared to the CLP group (P < 0.05). Conclusions: Epalrestat appeared to inhibit the PKC/NF-κB inflammatory pathway by inhibiting AKR1B1, resulting in reduced inflammatory cytokine levels in renal tissues and blood. This mitigated renal tissue injuries and improved the systemic inflammatory response in the severe sepsis rat model. Consequently, AKR1B1 holds promise as a target for treating sepsis-associated acute kidney injuries.

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Epidermal growth factor receptor substrate 15 (EPS15) is part of the EGFR pathway and has been implicated in various tumorigenesis. Increasing evidence suggests that long noncoding RNA (lncRNA) plays an essential role in liver hepatocellular carcinoma (LIHC) by regulating the expression of proteins and genes. Through analysis of the cancer genome atlas (TCGA) database, we found that EPS15 is highly expressed in LIHC tissue, and lncRNA EPS15-antisense1 (EPS15-AS1) decreased in LIHC cell lines. However, the function of EPS15-AS1 in LIHC is still unknown. When EPS15-AS1 was overexpressed in HepG2 cell lines, the expression of EPS15 was reduced and cell activity and invasiveness were inhibited. In addition, we observed an increase in Fe2+ ion and lipid peroxidation after overexpression of EPS15-AS1, and further analysis showed that the susceptibility to ferroptosis increased. Aldo-keto reductase family 1 member B 1 (AKR1B1) belongs to the aldo/keto reductase superfamily and is involved in maintaining the cellular redox balance. Survival analysis revealed that patients with a higher level of AKR1B1 have a lower survival rate in the TCGA database. We also found that EPS15 enhanced the AKR1B1 expression in LIHC, and AKR1B1 had the ability to promote cell invasiveness. Moreover, overexpression of AKR1B1 alleviated the promoting effect of EPS15-AS1 on ferroptosis. Therefore, EPS15-AS1 can induce ferroptosis in hepatocellular carcinoma cells by inhibiting the expression of EPS15 and AKR1B1 and disrupting the redox balance. EPS15 and AKR1B1 may serve as biomarkers for diagnosis and lncRNA EPS15-AS1 potential drug for LIHC.

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Sperm survival and activity depend on the provision of energy and nutrients from seminal plasma (SP). This study aimed to investigate the variations of metabolites within SP before and after freezing and subsequently explore the potential regulatory mechanisms affecting yak sperm cryodamage due to changes in metabolites in the SP. Untargeted metabolomics analysis was performed to screen for differential metabolites, followed by KEGG analysis to identify enriched signaling pathways. The combinatorial analysis of metabolomics and sperm proteomics revealed the influence of key SP metabolites on sperm proteins. Subsequently, the relevant differentially expressed proteins were verified by Western blot analysis. Finally, the mechanism underlying the positive effect of galactose on sperm motility was determined by assessing the change in ATP content in sperm before and after freezing and thawing. The data showed that a total of 425 and 269 metabolites were identified in the positive and negative ion modes, respectively. Freezing and thawing resulted in the up-regulation of 70 metabolites and the down-regulation of 29 metabolites in SP. The primary impact of freezing and thawing was observed in carbohydrate metabolism, including pyruvate metabolism, pentose phosphate pathway, galactose metabolism, the TCA cycle, and butanoate metabolism. In the combined analysis and Western blot results, a significant positive correlation was observed between galactose and Aldo-keto reductase family 1 member B1 (AKR1B1) (P < 0.05), which has the ability to convert galactose into galactol. Furthermore, the addition of galactose to thawed semen improved sperm motility by increasing AKR1B1 protein in sperm and was associated with the content of ATP. These data identify differential metabolites between fresh and frozen-thawed SP and suggest that galactose is a valuable additive for cryopreserved sperm, providing a theoretical basis for further exploration of the refrigerant formula for yak sperm cryopreservation.

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Gastric cancer (GC) is a common malignant tumor in the digestive tract and a major cause of global cancer death. Due to the limited access to early screening, many patients are diagnosed with advanced GC. Therefore, postoperative radiotherapy and chemotherapy possess limited efficacy in treating GC. AKR1B1 has been associated with tumorigenesis and metastasis across various tumors, becoming a potential therapeutic target for GC. However, its role and mechanism in GC remain unclear. In this study, AKR1B1 was elevated in GC tissue, depicting a poor prognosis. AKR1B1 is closely related to age, vascular and neural invasion, lymph node metastasis, and the TNM stage of GC. The developed survival prediction model suggested that AKR1B1 expression level is crucial in the prognosis of GC patients. Moreover, the expression level of AKR1B1 in GC tissues is closely associated with the AKT-mTOR pathway. In vitro and in vivo assays functional assays helped determine the oncogenic role of AKR1B1. Additionally, the knockdown of AKR1B1 expression level in GC cell lines could effectively suppress the AKT-mTOR pathway and inhibit the proliferation and migration of tumor cells. In conclusion, this study provides a theoretical basis to establish the potential association and regulatory mechanism of AKR1B1 while offering a new strategy for GC-targeted therapy.

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Accumulating evidence attributes the role of aldose reductase (AR) in modulating ROS and inflammation which are the main factor responsible for cancer progression and drug resistance. Epalrestat is the only AR inhibitor being used in Asian countries. It did not make it to the markets of the USA and Europe due to marginal efficacy as an antioxidant and anti-inflammatory agent owing to difficulty reaching intracellular targets. In our previous studies, we attempted to synthesize the epalrestat analogs and reported that the compound 4-((Z)-5-((Z)-2-Cyano-3-phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) benzoic acid named as NARI-29 has potent AR inhibition compared to epalrestat. In the current study, we aimed to find the effect of NARI-29 on ROS-induced cancer progression and TRAIL resistance in colon cancer in vitro models. In the first part of the study, we demonstrated that the NARI-29 has specific AKR1B1 inhibition and superior drug-like properties than epalrestat using bioinformatics tools. In the second part of the study, it was proven that NARI-29 has induced the hydrogen peroxide-triggered TRAIL-induced apoptosis in the colon cancer cells via modulating the AKR1B1/4HNE/FOXO3a/DR axis. The selective cytotoxicity of NARI-29 (10-fold) compared to epalrestat (4-fold) toward cancer cells is due to its differential ROS regulation and anti-inflammatory activities. Altogether, these data show that NARI-29 may be a potential candidate for AR inhibitors, which will be used to prevent colon cancer progression and as adjuvant therapy for preventing TRAIL resistance.

AKR1B1 is over-expressed in advanced-stage human colon cancer tissuesAKR1B1 mediates resistance to H₂O₂ and TRAIL in human CRC cell linesA co-activation loop exists between NF-κB and AKR1B1 in CRC cell lines to counteract ROSEstablishing epalrestat analog, NARI-29 (4-((Z)-5-((Z)-2-Cyano-3-phenylallylidene)-4-oxo-2-thioxothiazolidin-3-yl) benzoic acid) as potent anti-colon cancer agentsNARI-29 induced selective apoptosis in colon cancer cells by differentially modulating the ROS and sensitizing TRAIL.

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Infiltrated immune cells are an important constitute of tumor microenvironment, which exert complex effects on gastric cancer (GC) pathogenesis and progression. By using weighted gene co-expression network analysis, integrating the data from The Cancer Genome Atlas-stomach adenocarcinoma and GSE62254, we identify Aldo-Keto Reductase Family 1 Member B (AKR1B1) as a hub gene for immune regulation in GC. Notably, AKR1B1 is associated with higher immune infiltration and worse histologic grade of GC. In addition, AKR1B1 is an independent factor for predicting the survival rate of GC patients. In vitro experiments further demonstrated that AKR1B1-overexpressed THP-1-derived macrophages promoted the proliferation and migration of GC cells. Taken together, AKR1B1 plays an important role in GC progression by regulating immune microenvironment, which could be a biomarker for predicting GC prognosis as well as a potential therapeutic target for GC treatment.

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Glioma is a fatal primary brain tumor. Improved glioma treatment effectiveness depends on a better understanding of its underlying mechanisms. Glioblastoma (GBM), was classified as high-grade glioma with the most lethality and therapeutic resistance. Herein, we reported LINC00978 overexpressed in high-grade gliomas. Down-regulation of LINC00978 in glioblastoma cells inhibited cell proliferation, invasion, migration, and induced apoptosis. In vivo experiments confirmed that the CamK-A siRNA of LINC00978 could effectively inhibit the proliferation of glioblastoma cells. The main pathway and genes regulated by LINC00978 were detected using RNA sequencing to elucidate the molecular mechanism. The results suggest that LINC00978 regulates the expression of genes related to metabolic pathways, including aldo-keto reductase family 1 member B (AKR1B1), which mediates the cytotoxicity of 2-deoxyglucose. LINC00978 positively regulated AKR1B1 expression, and 2-deoxyglucose induced AKR1B1 expression via a LINC00978-dependent mechanism. This research has revealed that LINC00978 promotes the sensitivity of glioblastoma cells to 2DG. LINC00978 is highly expressed in most high-grade glioma patients. Thus, understanding the anticancer mechanism identified in this study may contribute to treating the majority of glioma patients. This study clarified the function and molecular mechanism of LINC00978 in glioblastoma and provided a study basis for LINC00978 to guide the clinical treatment of glioblastoma.

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This study aimed to investigate the regulatory role of Aldo-keto reductase family 1 member B1 (AKR1B1) in glioma cell proliferation through p38 MAPK activation to control Bcl-2/BAX/caspase-3 apoptosis signaling. AKR1B1 expression was quantified in normal human astrocytes, glioblastoma multiforme (GBM) cell lines, and normal tissues by using quantitative real-time polymerase chain reaction. The effects of AKR1B1 overexpression or knockdown and those of AKR1B1-induced p38 MAPK phosphorylation and a p38 MAPK inhibitor (SB203580) on glioma cell proliferation were determined using an MTT assay and Western blot, respectively. Furthermore, the AKR1B1 effect on BAX and Bcl-2 expression was examined in real-time by Western blot. A luminescence detection reagent was also utilized to identify the effect of AKR1B1 on caspase-3/7 activity. The early and late stages of AKR1B1-induced apoptosis were assessed by performing Annexin V-FITC/PI double-staining assays. AKR1B1 expression was significantly downregulated in glioma tissues and GBM cell lines (T98G and 8401). Glioma cell proliferation was inhibited by AKR1B1 overexpression but was slightly increased by AKR1B1 knockdown. Additionally, AKR1B1-induced p38 MAPK phosphorylation and SB203580 reversed AKR1B1's inhibitory effect on glioma cell proliferation. AKR1B1 overexpression also inhibited Bcl-2 expression but increased BAX expression, whereas treatment with SB203580 reversed this phenomenon. Furthermore, AKR1B1 induced caspase-3/7 activity. The induction of early and late apoptosis by AKR1B1 was confirmed using an Annexin V-FITC/PI double-staining assay. In conclusion, AKR1B1 regulated glioma cell proliferation through the involvement of p38 MAPK-induced BAX/Bcl-2/caspase-3 apoptosis signaling. Therefore, AKR1B1 may serve as a new therapeutic target for glioma therapy development.

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Objectives: To investigate the association of polymorphism in rs752010122 in aldose reductase gene with the pathogenesis of diabetic retinopathy, and to determine the association and allelic frequency between the variant and the disease. METHODS: The cross-sectional study was conducted from June 2021 to March 2022 at Centre for Research in Experimental and Applied Medicine (CREAM) Laboratory, Department of Biochemistry and Molecular Biology, Army Medical College, in collaboration with the Armed Forces Institute of Ophthalmology, Rawalpindi, Pakistan, and comprised blood samples from subjects of either gender aged 40-70 years. The samples were divided into group I having diabetic retinopathy patients, group II having diabetics without retinopathy, and group III having healthy controls matched for age and gender. The samples were subjected to molecular analysis. Gene sequence was downloaded from the Human Genome Database and Ensemble. Data was analysed using SPSS 22. RESULTS: Of the 150 subjects, there were 50(33.3%) in each of the 3 groups. Variants of aldose reductase rs752010122 polymorphism were significantly associated with a lower risk of diabetic retinopathy (p<0.05). An odds ratio of 1 was noted for both heterozygous and homozygous genotypes (95% confidence interval: 1). CONCLUSIONS: Aldose reductase was associated with lower risk of the disease.

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AIMS: The study aims to find a new functional additive for diabetic liver injury. BACKGROUND: It is well-established that type 2 diabetes mellitus (T2DM) is a metabolic disease with multiple complications and places a significant health and economic burden on modern society. Linarin is a natural flavonoid isolated from Asteraceae and Lamiaceae, which has beneficial effects in preventing and treating metabolic diseases such as nonalcoholic steatohepatitis and diabetes. OBJECTIVE: We aimed to investigate the pharmacological effect and underlying mechanism of linarin on T2DM-associated liver injury in vivo and in vitro. METHODS: Using a high-glucose and high-palmitic acid-induced hepatocyte injury model and a type 2 diabetic rat model. Following linarin treatment, serum biochemical parameters, liver histology, and lipid profiles of rats were examined. Oxidative stress index and inflammatory response were detected in vivo and in vitro. The expression level of AKR1B1 in rat liver tissues and in vitro cells was detected by western blot and by real-time fluorescent quantitative PCR. RESULTS: The present study found that linarin could prevent oxidative stress and inflammation. In high-fat-fed diabetic rats, linarin administration (15, 30, and 60 mg/kg/day) reduced hepatic lipid accumulation, oxidative stress, and inflammation. Linarin (20 µM) significantly alleviated oxidative stress, inflammation, and apoptosis induced by high glucose combined with palmitic acid in LX-2 cells. Western blotting and overexpression experiments showed that these effects were related to AKR1B1 inhibition in vivo and in vitro. CONCLUSION: This study indicated that linarin could protect against liver injury in T2DM by alleviating oxidative stress and inflammation mediated by AKR1B1 and may be a promising additive for diabetic liver injury therapy.

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Identifying the target linking inflammation and oxidative stress to aggravate the disease progression will help to prevent colitis associated carcinogenesis. Since AKR1B1 overexpression is observed in inflammatory diseases and various cancers, we have investigated the role of AKR1B1 in colitis-associated colon carcinogenesis with the aid of epalrestat and its potent analogue NARI-29 (investigational molecule) as pharmacological probes. A TNF-α inducible NF-κB reporter cell line (GloResponse™ NF-κB-RE-luc2P HEK293) and dextran sodium sulfate (DSS) and 1,2 dimethyl hydrazine (DMH))-induced mouse model was used to investigate our hypothesis in vitro and in vivo. Clinically, an increased expression of AKR1B1 was observed in patients with ulcerative colitis. Our in vitro and in vivo findings suggest that the AKR1B1 modulated inflammation and ROS generation for the progression of colitis to colon cancer. AKR1B1 overexpression was observed in DSS + DMH-treated mice colons. Moreover, we could observe histopathological changes like immune cell infiltration, aberrant crypt foci, and tumour formation in DC groups. Mechanistically, we have witnessed modulation of the IKK/IκB/NF-κB and Akt/FOXO-3a/DR axis, increased inflammatory cytokines, increased expression of proliferative markers, Ki-67 and PCNA, and accumulation of ß-catenin in the colon epithelium. However, pharmacological inhibition of AKR1B1 using NARI-29 or EPS has reversed the clinical, histopathological, and molecular alterations induced by DSS + DMH, confirming the obvious role of AKR1B1 in the promotion of colitis-associated carcinogenesis. In conclusion, our findings suggest that AKR1B1 targeted therapy could be a promising strategy for preventing CA-CRC and NARI-29 could be developed as a potent AKR1B1 inhibitor.

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Inhibition of aldose reductase (AKR1B1) is a promising option for the treatment of diabetic complications. However, most of the developed small molecule inhibitors lack selectivity or suffer from low bioactivity. To address this limitation, a novel series of quinazolin-4(1H)-one derivatives as potent and selective inhibitors of AKR1B1 were designed and synthesized. Aldose reductase inhibitory activities of the novel compounds were characterized by IC50 values ranging from 0.015 to 31.497 µM. Markedly enhanced selectivity of these derivatives was also recorded, which was further supported by docking studies. Of these inhibitors, compound 5g exhibited the highest inhibition activity with selectivity indices reaching 1190.8. The structure-activity relationship highlighted the importance of N1-acetic acid and N3-benzyl groups with electron-withdrawing substituents on the quinazolin-4(1H)-one scaffold for the construction of efficient and selective AKR1B1 inhibitors.

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Although the incidence and death rates of gastric cancer (GC) are decreasing, approximately one million new cases and 800,000 GC-related deaths were reported worldwide in 2018. Currently, the oncogenesis of GC remains unclear, and the demand for novel treatment options are unmet. Here, we explored the role of aldo-keto reductase family 1 member B (AKR1B1) in the progression of GC. The proliferation, migration, and invasion of GC cells were evaluated by CCK-8 assay, wound healing assay, and transwell assay, respectively. The interaction between EBF transcription factor 1 (EBF1) and the promoter region of AKR1B1 was determined by luciferase reporter assay and chromatin immunoprecipitation (ChIP). Gene expression levels were measured by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blotting assay. The expression of AKR1B1 was elevated in GC cells, resulting in increased cell proliferation, migration, and invasion. Meanwhile, EBF1 was a negative regulator of AKR1B1; its overexpression suppressed AKR1B1 expression and GC progression. Furthermore, knockdown of ZNF521 induced EBF1 expression, thus suppressing AKR1B1 expression and resulting in attenuated GC growth and invasiveness. Notably, knockdown of ZNF521 attenuated GC progression and was rescued by overexpression of AKR1B1. Our current study revealed a novel ZNF521/EBF1/AKR1B1 axis in GC and elaborated its important role in promoting GC progression, providing potential therapeutic targets for anti-GC treatments.

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Synthetic trans-(±)-kusunokinin ((±)KU), a potential anticancer substance, was revealed to have an inhibitory effect on breast cancer. According to the computational modeling prediction, AKR1B1, an oxidative stress and cancer migration protein, could be a target protein of trans-(-)-kusunokinin. In this study, we determined the binding of (±)KU and AKR1B1 on triple-negative breast and non-serous ovarian cancers. We found that (±)KU exhibited a cytotoxic effect that was significantly stronger than zopolrestat (ZP) and epalrestat (EP) (known AKR1B1 inhibitors) on breast and ovarian cancer cells. (±)KU inhibited aldose reductase activity that was stronger than trans-(-)-arctiin ((-)AR) but weaker than ZP and EP. Interestingly, (±)KU stabilized AKR1B1 on SKOV3 and Hs578T cells after being heated at 60 and 75 °C, respectively. (±)KU decreased malondialdehyde (MDA), an oxidative stress marker, on Hs578T cells in a dose-dependent manner and the suppression was stronger than EP. Furthermore, (±)KU downregulated AKR1B1 and its downstream proteins, including PKC-δ, NF-κB, AKT, Nrf2, COX2, Twist2 and N-cadherin and up-regulated E-cadherin. (±)KU showed an inhibitory effect on AKR1B1 and its downstream proteins, similar to siRNA-AKR1B1. Interestingly, the combination of siRNA-AKR1B1 with EP or (±)KU showed a greater effect on the suppression of AKR1B1, N-cadherin, E-cadherin and NF-κB than single treatments. Taken together, we concluded that (±)KU-bound AKR1B1 leads to the attenuation of cellular oxidative stress, as well as the aggressiveness of breast cancer cell migration.

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BACKGROUND: Epithelial ovarian cancer is the most lethal gynaecological cancer worldwide. Chemotherapy resistance represents a significant clinical challenge and is the main reason for poor ovarian cancer prognosis. We identified novel expression of markers related to epithelial mesenchymal transitions (EMT) in a carboplatin resistant ovarian cancer cell line by proteomics. This was validated in the platinum resistant versus sensitive parental cell lines, as well as platinum resistant versus sensitive human ovarian cancer patient samples. The prognostic significance of the different proteomics-identified marker proteins in prognosis prediction on survival as well as their correlative association and influence on immune cell infiltration was determined by public domain data bases. METHODS: We explored the proteomic differences between carboplatin-sensitive OVCAR5 cells (parental) and their carboplatin-resistant counterpart, OVCAR5 CBPR cells. qPCR and western blots were performed to validate differentially expressed proteins at the mRNA and protein levels, respectively. Association of the identified proteins with epithelial-mesenchymal transition (EMT) prompted the investigation of cell motility. Cellular bioenergetics and proliferation were studied to delineate any biological adaptations that facilitate cancer progression. Expression of differentially expressed proteins was assessed in ovarian tumors obtained from platinum-sensitive (n = 15) versus platinum-resistant patients (n = 10), as well as matching tumors from patients at initial diagnosis and following relapse (n = 4). Kaplan-Meier plotter and Tumor Immune Estimation Resource (TIMER) databases were used to determine the prognostic significance and influence of the different proteomics-identified proteins on immune cell infiltration in the tumor microenvironment (TME). RESULTS: Our proteomics study identified 2422 proteins in both cell lines. Of these, 18 proteins were upregulated and 14 were downregulated by ≥ twofold (p < 0.05) in OVCAR5 CBPR cells. Gene ontology enrichment analysis amongst upregulated proteins revealed an overrepresentation of biological processes consistent with EMT in the resistant cell line. Enhanced mRNA and/or protein expression of the identified EMT modulators including ITGA2, TGFBI, AKR1B1, ITGAV, ITGA1, GFPT2, FLNA and G6PD were confirmed in OVCAR5 CBPR cells compared to parental OVCAR5 cell line. Consistent with the altered EMT profile, the OVCAR5 CBPR cells demonstrated enhanced migration and reduced proliferation, glycolysis, and oxidative phosphorylation. The upregulation of G6PD, AKR1B1, ITGAV, and TGFß1 in OVCAR5 CBPR cells was also identified in the tumors of platinum-resistant compared to platinum-sensitive high grade serous ovarian cancer (HGSOC) patients. Matching tumors of relapsed versus newly diagnosed HGSOC patients also showed enhanced expression of AKR1B1, ITGAV, TGFß1 and G6PD protein in relapsed tumors. Among the identified proteins, significant enhanced expression of GFPT2, FLNA, TGFBI (CDGG1), ITGA2 predicted unfavorable prognosis in ovarian cancer patients. Further analysis suggested that the expression of TGFBI to correlate positively with the expression of identified and validated proteins such as GFPT2, FLNA, G6PD, ITGAV, ITGA1 and ITGA2; and with the infiltration of CD8+ T cells, macrophages, neutrophils, and dendritic cells in the TME. CONCLUSIONS: Our research demonstrates proteomic-based discovery of novel EMT-related markers with an altered metabolic profile in platinum-resistant versus sensitive ovarian cancer cell lines. The study also confirms the expression of selected identified markers in the tumors of platinum-resistant versus sensitive, and in matching relapsed versus newly diagnosed HGSOC patients. The study provides insights into the metabolic adaptation of EMT-induced carboplatin resistant cells that confers on them reduced proliferation to provide effective migratory advantage; and the role of some of these identified proteins in ovarian cancer prognosis. These observations warrant further investigation of these novel target proteins in platinum-resistant patients.

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Epalrestat (EPA) is a potent inhibitor of aldose reductases AKR1B1 and AKR1B10, used for decades in Japan for the treatment of diabetic peripheral neuropathy. This orally-active, brain-permeable small molecule, with a relatively rare and essential 2-thioxo-4-thiazolidinone motif, functions as a regulator intracellular carbonyl species. The repurposing of EPA for the treatment of pediatric rare diseases, brain disorders and cancer has been proposed. A detailed analysis of the mechanism of action, and the benefit of EPA to combat advanced malignancies is offered here. EPA has revealed marked anticancer activities, alone and in combination with cytotoxic chemotherapy and targeted therapeutics, in experimental models of liver, colon, and breast cancers. Through inhibition of AKR1B1 and/or AKR1B10 and blockade of the epithelial-mesenchymal transition, EPA largely enhances the sensitivity of cancer cells to drugs like doxorubicin and sorafenib. EPA has revealed a major anticancer effect in an experimental model of basal-like breast cancer and clinical trials have been developed in patients with triple-negative breast cancer. The repurposing of the drug to treat chemo-resistant solid tumors seems promising, but more studies are needed to define the best trajectory for the positioning of EPA in oncology.

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