Inhibition of ALDH1A1 activity decreases expression of drug transporters and reduces chemotherapy resistance in ovarian cancer cell lines

Highlights

• High expression of ALDH1A1 was observed in paclitaxel and topotecan- resistant cancer cell lines.

• Pretreatment with ATRA and DEAB reduced chemotherapy resistance in investigated cell lines.

• ATRA treatment led to downregulation of ALDH1A1, P-gp and BCRP proteins.

• DEAB treatment led to downregulation of P-gp and BCRP proteins.

Abstract

The high mortality of ovarian cancer patients results from the failure of treatment caused by the inherent or acquired chemotherapy drug resistance. It was reported that overexpression of aldehyde dehydrogenase A1 (ALDH1A1) in cancer cells can be responsible for the development of drug resistance. To add the high expression of the drug transporter proteins the ALDHA1 is considered as a molecular target in cancer therapy. Therefore, we analysed drug-resistant ovarian cancer cell lines according to ALDHA1 expression and the association with drug resistance.

The expression of ALDH1A1, P-glycoprotein (P-gp) or breast cancer resistance protein (BCRP) was determined using a microarray and confirmed by Q-PCR, western blot and fluorescence analysis. ALDH1A1 activity was determined using an Aldefluor assay. The impact of all-trans retinoic acid (ATRA) and diethylaminobenzaldehyde (DEAB) on chemotherapy resistance was assessed by the MTT chemosensitivity assay.

The most abundant expression of ALDH1A1 was noted in paclitaxel- and topotecan-resistant cell lines where two populations of ALDH-positive and ALDH-negative cells could be observed. Those cell lines also revealed the overexpression of P-gp and BCRP respectively, and were able to form spheres in non-adherent conditions. Pre-treatment with ATRA and DEAB reduced chemotherapy resistance in both cell lines. ATRA treatment led to downregulation of the ALDH1A1, P-gp and BCRP proteins. DEAB treatment led to downregulation of the P-gp protein and BCRP transcript and protein.

Our results indicate that ALDH1A1-positive cancer cells can be responsible for drug resistance development in ovarian cancer. Developing more specific ALDH1A1 inhibitors can increase chemotherapy effectiveness in ovarian cancer.

Introduction

Ovarian cancer is the most lethal cancer among gynaecological tumours and is the fifth most frequent cause of cancer deaths among women. At the beginning of treatment, ovarian cancer is among the most chemosensitive malignancies. However, during treatment, most patients develop drug resistance leading to the ineffectiveness of further treatment (Hennessy et al., 2009). The first-line treatment includes taxane/platinum-based chemotherapy (Parmar et al., 2003). Unfortunately, most of the ovarian cancer patients with good response to first-line treatment will experience recurrence of the disease. In the second-line treatment, taxane derivatives as well as cisplatin (CIS), topotecan (TOP), doxorubicin (DOX) and gemcitabine are mainly used (Pfisterer et al., 2006, Mutch et al., 2007, Ferrandina et al., 2008, Sehouli et al., 2008, Mahner et al., 2015).

The failure of chemotherapy results from inherent or acquired chemotherapy drug resistance. At least tens of proteins are responsible for the drug resistance of cancer cells. However, the most important and widespread mechanism of drug resistance is the active removal of the cytostatic drugs from the cancer cells by transport proteins. These transport proteins belong to ABC family of transmembrane transporters and use the energy from the hydrolysis of ATP to remove drugs from the cell (Stavrovskaya, 2000). This form of drug resistance is called multiple drug resistance (MDR). The most important ABC protein is P-glycoprotein (P-gp), which is encoded by the ABCB1 (multidrug resistance protein 1-MDR1) gene (Leonard et al., 2003). P-gp overexpression has been reported in more than 50% of cancers with the MDR phenotype (Choi, 2005). Another important MDR protein is the breast cancer resistance protein, BCRP (ABCG2). This drug transporter confers resistance to many cytostatic drugs, including mitoxantrone and TOP (Maliepaard et al., 1999, Robey et al., 2007). Expression of both proteins was also observed by us in drug-resistant ovarian cancer cell lines (Januchowski et al., 2013a).

Aldehyde dehydrogenases (ALDHs) are a family of NAD(P)⁺-dependent enzymes involved in detoxifying a wide variety of endogenous and exogenous aldehydes to their corresponding weak carboxylic acids (Sladek, 2003). The ALDH1A1 isoform is involved in metabolism of vitamin A (retinol) and oxidizes retinaldehyde to retinoid acid (RA) (Duester et al., 2003). This is related to its role in cell differentiation. The results of many studies indicate that ALDH1A1 is involved in the biology of normal stem cells (NSCs) as well as cancer stem cells (CSCs) and that ALDH1A1 plays a role in the drug resistance of these cells (Ma and Allan, 2011, Januchowski et al., 2013b). Sun et al. conducted a proteomics analysis study and showed that one of the proteins overexpressed in the A549-Taxol lung cancer cell line, which is resistant to paclitaxel, was ALDH1 (Sun et al., 2011). In 2009, Tanei et al., using immunohistochemical staining of breast tumour tissue obtained before and after neoadjuvant chemotherapy (DOX + paclitaxel (PAC)) demonstrated that tissue from patients with poor response to chemotherapy had an increased level of ALDH1 expression. Furthermore, this expression increased after chemotherapy (Tanei et al., 2009). It was observed that patients with pancreatic cancer had ALDH-negative primary tumours but ALDH-positive metastases (Rasheed et al., 2010). Expression of ALDH1 has also been reported in ovarian cancers and ovarian cancer cell lines (Landen et al., 2010, Silva et al., 2011, Wang et al., 2012). Furthermore, taxane and platinum resistance correlated with ALDH1A1 expression in ovarian cancer cell lines (Landen et al., 2010). The role of ALDH1 in resistance to cytostatic drugs was also confirmed in breast cancer. In 2011, Croker et al., using a cell sorter, isolated cells with overexpression of ALDH from MDA-MB-231 and MDA-MB-468 breast cancer cell lines (Croker and Allan, 2012). These cells demonstrated significant resistance to DOX and PAC in comparison to cells with low ALDH expression. All of these results suggest that cancer cells with overexpression of ALDH1 (ALDH1A1) can be responsible for the development of drug resistance both in vivo and in vitro.

Currently, two major, contrasting models exist to explain the development of cancer drug resistance at the cellular level. The classical theory of therapy resistance involves cells developing acquired resistance following a particular therapy (Gerlinger and Swanton, 2010, Greaves and Maley, 2012). The CSCs model postulates that a small population of cancer cells is intrinsically resistant to chemotherapy and that these cells are responsible for drug resistance in primary tumours as well as for metastasis (Clevers, 2011). One of the characteristic features of CSCs in the context of drug resistance is the high expression levels of ABC transporters (Moitra et al., 2011) and other self-protection mechanisms (Alison et al., 2012). The other characteristic features of these cells are high expression levels of ALDH enzymes (especially the ALDH1A1 and/or ALDH3A1 isoforms) (Alison et al., 2010, Januchowski et al., 2013b).

Increased expression of ALDH1A1 in tissues from chemotherapy resistant patients⿿ tumours as well as in many drug-resistant cell lines inspired researchers to determine whether ALDH can be used as a molecular target in cancer therapy (Alison et al., 2012, Januchowski et al., 2013b). Downregulation of ALDH1 by antisense RNA in the A549 lung cancer cell line resulted in decreased ALDH1 expression and increased sensitivity of cancer cells to 4-hydroperoxycyclophosphamide (4-HC) (Moreb et al., 2000). Landen et al. showed that silencing of ALDH1A1 using siRNA in ovarian cancer cell lines resistant to taxane and platinum sensitized these cells to chemotherapy (Landen et al., 2010). It has been shown that treatment of the A549 lung cancer cell line with the differentiation agent All Trans Retinoid Acid (ATRA) led to the downregulation of ALDH1A1 and ALDH3A1 and increased sensitivity to 4-hydroperoxycyclophosphamide (4-HC) (Moreb et al., 2005). In 2011, Croker et al. showed that treatment of ALDH-positive breast cancer cells with ATRA or Diethylaminobenzaldehyde (DEAB), a specific ALDH inhibitor, sensitizes these cells to PAC and epirubicin (Croker and Allan, 2012). The effect of ATRA can result from its differentiation activity. ATRA is generally used in stem cell research because of its differentiation properties (Su et al., 2010, Tonge and Andrews, 2010). In the clinic, ATRA is used in the treatment of acute promyelocytic leukaemia (APL) (Petrie et al., 2009). ATRA treatment increases the level of intracellular retinoid acid (RA). Consequently, the levels of ALDH1A1 and ALDH3A1 are decreased and the malignant promyelocytes differentiate into mature neutrophils with enhanced sensitivity to chemotherapy. The role of DEAB in increasing the sensitivity of ALDH-positive cancer cells to chemotherapy is unclear. Because DEAB inhibits ALDH activity it has been suggested that ALDH, especially ALDH1A1, can play a direct role in drug resistance. However, the mechanism of its action in resistance to drugs other than cyclophosphamide is unknown.

Because ALDH-positive cells play a role in drug resistance in many cancers and are present in ovarian cancer tumours (Silva et al., 2011) as well as cell lines (Landen et al., 2010), we analysed our drug-resistant ovarian cancer cell lines according to different ALDH isoform expression and their association with drug resistance. Using microarray analysis of gene expression in six drug-resistant ovarian cancer cell lines, we found that cells resistant to PAC and TOP have very high expression levels of ALDH1A1. Furthermore, we were able to confirm these results using RQ-PCR, western blot, fluorescence microscopy and Aldefluor assay. Using an MTT assay, we showed that blocking ALDH1A1 by ATRA or DEAB treatment sensitizes these cells to cytostatic drugs. The mechanism responsible for cell sensitization resulted from decreased P-gp and BCRP protein levels after ATRA or DEAB treatment.

Our results confirm that ALDH1A1 plays a role in drug resistance and can be a molecular target in ovarian cancer chemotherapy. We have shown for the first time that ATRA or DEAB treatment decreases P-gp and BCRP protein levels. This leads to increased sensitivity of multidrug resistant cancer cells to cytostatic drugs.