Aldo-keto reductase 1B10 promotes development of cisplatin resistance in gastrointestinal cancer cells through down-regulating peroxisome proliferator-activated receptor-γ-dependent mechanism

Highlights

• AKR1B10 is up-regulated with CDDP resistance in gastrointestinal cancer cells.

• PPARγ is down-regulated in the CDDP-resistant cells.

• AKR1B10 detoxifies reactive aldehydes and suppresses PPARγ expression.

• Treatment with AKR1B10 inhibitor and PPARγ ligand overcomes the CDDP resistance.

Abstract

Cisplatin (cis-diamminedichloroplatinum, CDDP) is one of the most effective chemotherapeutic drugs that are used for treatment of patients with gastrointestinal cancer cells, but its continuous administration often evokes the development of chemoresistance. In this study, we investigated alterations in antioxidant molecules and functions using a newly established CDDP-resistant variant of gastric cancer MKN45 cells, and found that aldo-keto reductase 1B10 (AKR1B10) is significantly up-regulated with acquisition of the CDDP resistance. In the nonresistant MKN45 cells, the sensitivity to cytotoxic effect of CDDP was decreased and increased by overexpression and silencing of AKR1B10, respectively. In addition, the AKR1B10 overexpression markedly suppressed accumulation and cytotoxicity of 4-hydroxy-2-nonenal that is produced during lipid peroxidation by CDDP treatment, suggesting that the enzyme acts as a crucial factor for facilitation of the CDDP resistance through inhibiting induction of oxidative stress by the drug. Transient exposure to CDDP and induction of the CDDP resistance decreased expression of peroxisome proliferator-activated receptor-γ (PPARγ) in MKN45 and colon cancer LoVo cells. Additionally, overexpression of PPARγ in the cells elevated the sensitivity to the CDDP toxicity, which was further augmented by concomitant treatment with a PPARγ ligand rosiglitazone. Intriguingly, overexpression of AKR1B10 in the cells resulted in a decrease in PPARγ expression, which was recovered by addition of an AKR1B10 inhibitor oleanolic acid, inferring that PPARγ is a downstream target of AKR1B10-dependent mechanism underlying the CDDP resistance. Combined treatment with the AKR1B10 inhibitor and PPARγ ligand elevated the CDDP sensitivity, which was almost the same level as that in the parental cells. These results suggest that combined treatment with the AKR1B10 inhibitor and PPARγ ligand is an effective adjuvant therapy for overcoming CDDP resistance of gastrointestinal cancer cells.

Introduction

Cisplatin (cis-diamminedichloroplatinum, CDDP), a first member of platinum-based chemotherapy drugs, is widely used to treat various types of cancer, such as malignant lymphomas and lung, gastrointestinal, esophageal, ovarian, bladder, prostate and cervical cancers. The platinum drug interferes with growth of the cancer cells through intra- and inter-strand crosslinking of the DNA helix and the resulting inhibition of replication and synthesis of nucleic acids, hereby leading to the cancer cell death [1], [2]. Besides inhibiting the cancer cell proliferation, CDDP is considered to damage the cancer cells by the enhanced formation of reactive oxygen species (ROS), which trigger apoptosis towards cancer cells through DNA injury, mitochondrial dysfunction, activation of p53- and caspase-signaling cascades, and cell cycle arrest [2], [3], [4]. Despite the potent anticancer action of CDDP, the continuous exposure easily gives rise to hyposensitivity to the drug, so-called chemoresistance, as well as onset and development of severe side-effects including nephrotoxicity [4]. Induction of chemoresistance in cancer cells limits efficacy of the anticancer drug and consequently imposes complexed multidrug therapy on the cancer patients. In many cases, it also forms the incurable malignant cancer cells featuring overgrowth and elevation of invasive and metastatic capacities. Therefore, it is hoped that novel medicine to inhibit function and/or expression of molecules requisite for the gain of CDDP resistance is developed. Mechanisms of chemoresistance including cellular uptake and efflux of CDDP and potentiation of the DNA-repair and cytoprotective capacity have been suggested so far [2], [5], [6]. In addition, caudal type homeobox 2 [7], autophagy-related gene-5 [8] and syndecan-1 [9] have been more recently proposed as the target molecules for developing the drug resistance.

Members of the aldo-keto reductase (AKR) superfamily are NAD(P)(H)-dependent oxidoreductases that metabolize carbohydrates, steroids, and prostaglandins, as well as exogenous carbonyl compounds such as drugs and toxicants [10]. Among human 15 members of this superfamily, AKR1C1, AKR1C2 and AKR1C3 have been shown to be highly expressed and associated with resistance against anticancer drugs such as anthracyclines, CDDP and methotrexate [11], [12], [13]. The three AKR1C isoforms are hydroxysteroid dehydrogenases with broad substrate specificity for carbonyl compounds including 4-hydroxy-2-nonenal (HNE), a major product of lipid peroxidation through oxidative stress, but it remains unclear as to how the enzymes confer the drug resistance. In addition, AKR1B10, i.e. human aldose reductase-like protein, is up-regulated in many types of solid tumors [14], [15], [16], [17], [18], and its gene silencing inhibits replication of colorectal cancer cells [19] and tumor growth in vivo [18]. Although detailed mechanisms of the AKR1B10-initiated tumorigenic action are still unclear, it is inferred to be due to metabolizing retinoids [20] and isoprenoids [21], regulating fatty acid synthesis [22], and detoxifying cytotoxic aldehydes such as acrolein [19] and HNE [23]. Thus, AKR1B10 has been recognized as a potential molecular target for preventing cancer proliferation as well as a diagnostic tumor marker. Besides, AKR1B10 was found to be overexpressed in medulloblastoma cell lines resistant to cyclophosphamide [24]. We also showed that mitomycin C (MMC) [25] or doxorubicin (DOX) resistance in gastrointestinal cancer [26] as well as CDDP resistance in lung cancer cells [27] are mediated by up-regulation of AKR1B10, suggesting a close association of the enzyme up-regulation with cancer chemoresistance.

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear receptor superfamily [28]. PPARs regulate transcription of target genes by binding to their peroxisome proliferator-response elements as heterodimers with retinoid X receptor. The PPAR receptor family is comprised of the three subtypes, PPARα, PPARβ/δ, and PPARγ, each of which mediates important physiological actions in metabolism of lipids and sugars. In addition to the energy homeostasis, ligands for PPARs are considered to act as key regulators for proliferation of gastrointestinal cancer cells. For example, treatment with ligands of PPARα and PPARγ inhibits proliferation of the cancer cells [29], [30]. Especially, PPARγ is closely linked to growth suppression and death of cancer cells, and incubation with the ligands appears to potently induce apoptosis in many kinds of cancer cells such as breast, thyroid, bladder, esophageal, gastric, colon, lung, and prostate cancers and lymphomas [31]. In addition, PPARγ is also associated with CDDP resistance of ovarian cancer [32], [33], bladder cancer [34] and malignant glioma [35], and that treatment with its ligands sensitizes the cancer cells to the drug. Furthermore, AKR1C3 is reported to facilitate proliferation of cancer cells such as myeloid leukemia cell and skin squamous cell carcinoma via metabolizing prostaglandin D₂ into prostaglandin F_2α, not 15-deoxy-Δ^12,14-prostaglandin J₂, an endogenous PPARγ agonist [36], [37], [38], [39], [40]. However, it is uncertain whether the PPARγ-dependent mechanism is involved in acquisition of CDDP resistance and expression of other AKRs, except for AKR1C3, in gastrointestinal cancer cells.

In this study, we have generated CDDP-resistant variants of gastrointestinal cancer (MKN45 and LoVo) cells, monitored alterations in factors involved in antioxidant response of the cells with development of CDDP resistance, and found a significant up-regulation of AKR1B10 as a pivotal process for promoting the chemoresistance. We have also investigated an association of PPARγ with the CDDP resistance, and finally evaluated efficacy of combined treatment with AKR1B10 inhibitor and PPARγ ligand for overcoming resistance in the resistant cells.