Dichloroacetate prevents TGFβ-induced epithelial-mesenchymal transition of retinal pigment epithelial cells

Highlights

• Epithelial-mesenchymal transition (EMT) of retina pigment epithelial (RPE) cells is important in the development of proliferative retinopathies.

• DCA inhibits TGFβ–induced EMT in ARPE-19 cells as evident by reduced wound healing and expression of EMT markers.

• Anti-EMT effect of DCA on ARPE-19 cells is also associated with the modulation of MAPK/Erk and PI3K/Akt pathways.

Abstract

Proliferative retinopathies are associated with formation of fibrous epiretinal membranes. At present, there is no pharmacological intervention for the treatment of retinopathies. Cytokines such as TGFβ are elevated in the vitreous humor of the patients with proliferative vitro-retinopathy, diabetic retinopathy and age-related macular degeneration. TGFβ isoforms lead to epithelial-mesenchymal transition (EMT) or trans-differentiation of the retinal pigment epithelial (RPE) cells. PI3K/Akt and MAPK/Erk pathways play important roles in the EMT of RPE cells. Therefore, inhibition of EMT by pharmacological agents is an important therapeutic strategy in retinopathy. Dichloroacetate (DCA) is shown to prevent proliferation and EMT of cancer cell lines but its effects are not explored on the prevention of EMT of RPE cells. In the present study, we have investigated the role of DCA in preventing TGFβ2 induced EMT of RPE cell line, ARPE-19. A wound-healing assay was utilized to detect the anti-EMT effect of DCA. The expressions of EMT and cell adhesion markers were carried out by immunofluorescence, western blotting, and quantitative real-time PCR. The expression of MAPK/Erk and PI3K/Akt pathway members was carried out using western blotting. We found that TGFβ2 exposure leads to an increase in the wound healing response, expression of EMT markers (Fibronectin, Collagen I, N-cadherin, MMP9, S100A4, α-SMA, Snai1, Slug) and a decrease in the expression of cell adhesion/epithelial markers (ZO-1, Connexin 43, E-cadherin). These changes were accompanied by the activation of PI3K/Akt and MAPK/Erk pathways. Simultaneous exposure of DCA along with TGFβ2 significantly inhibited wound healing response, expression of EMT markers and cell adhesion/epithelial markers. Furthermore, DCA and TGFβ2 effectively attenuated the activation of MAPK/Erk/JNK and PI3K/Akt/GSK3β pathways. Our results demonstrate that DCA has a strong anti-EMT effect on the ARPE-19 cells and hence can be utilized as a therapeutic agent in the prevention of proliferative retinopathies.

Introduction

Dichloroacetate (DCA) is a structural analog of pyruvate, a metabolic intermediate that enters into the mitochondria and metabolized by aerobic reactions particularly the Krebs cycle. DCA stimulates multi-enzyme mitochondrial matrix complex, pyruvate dehydrogenase complex (PDC) which is involved in converting pyruvate, alanine and lactate to acetyl CoA (Crabb et al., 1981; Fouque et al., 2003). The reaction catalyzed by PDC is an important rate-limiting step in the aerobic reaction and hence it is integral to the cellular energetics (Patel and Roche, 1990). DCA stimulates activation of PDC by inhibiting four known pyruvate dehydrogenase kinase (PDK) isoforms involved in inhibition of PDH, thereby maintaining the PDH in its un-phosphorylated active form (Stacpoole, 1989; Michelakis et al., 2008). DCA is not a new drug and it was safely used for the treatment of congenital lactic acidosis (Abdelmalak et al., 2013; Stacpoole et al., 1997). Several studies have found that cancer cells stimulate the Krebs cycle in the mitochondria after the addition of DCA (Li et al., 2018; James et al., 2017). Overstimulation of the Krebs cycle in mitochondria by the addition of DCA leads to the production of reactive oxygen species (ROS), oxidative stress and finally apoptosis in the cancer cells (Madhok et al., 2010; Kinnaird et al., 2016; Ward et al., 2017). By blocking PDK, DCA decreases lactate production, interfere with cell cycle and induce apoptosis in many tumors (Haugrud et al., 2014; Kumar et al., 2012; Anemone et al., 2017). DCA is unable to elicit this response in normal cells because their mitochondria are functioning normally (Michelakis et al., 2008). DCA being small molecule has 100% bioavailability (Curry et al., 1991). DCA is metabolized in the liver and less than 1% is shown to be excreted in the urine (Lukas et al., 1980; Shangraw and Fisher, 1996). DCA is known to inhibit its own metabolism and hence it results in successive higher concentration in the blood after multiple doses and reaches plateau with ongoing use (Barton et al., 1999; Stacpoole et al., 2003). DCA also show sex and gender-specific difference in the absorption and clearance being faster in the women compared to that of men (Schultz and Shangraw, 2006). Like many other drugs, DCA is also not free from side effects. Oral or intravenous administration of DCA is associated with side effects such as peripheral neuropathy, headache, dizziness, etc. (Spruijt et al., 2001).

Epithelial-mesenchymal transition (EMT) is a process in which epithelial cells loses its epithelial properties and acquires mesenchymal properties including migration and contraction. Progression of EMT involves loss of apical–basal polarity, acquisition of front-rear polarity, cytoskeletal reorganization and activation of signaling mechanisms that control the shape and migratory/invasive properties of cells (Dongre and Weinberg, 2019). Significant gene expression changes take place during EMT at both the transcription and translational levels. These differentially expressed genes involve epithelial marker proteins (E-cadherin, ZO-1, Connexin 43); mesenchyme marker proteins (α-SMA, Vimentin, Fibronectin); extracellular matrix components (Collagen 1, MMP2, MMP9), and transcription factors (Snai1, Slug, Zeb1, Zeb2, Twist, etc.) (Lamouille et al., 2014). Alteration in the expression of above listed genes is due to signaling induced by cytokines/growth factors such as TGFβ, FGF, HGF, and EGF (Sistigu et al., 2017). EMT is essential process during the development, tissue repair and wound healing (Stone et al., 2016; Barriere et al., 2015). However, the abnormal EMT is shown to be associated with fibrosis of several organs including eyes and metastasis of cancer cells (Nisticò et al., 2012; Takahashi, 2016; Roche, 2018).

EMT plays an important role in the development of many eye diseases including retinopathies (Yang et al., 2015), cataract (Johar et al., 2007), posterior capsular opacification (Raj et al., 2007), etc. The retinal pigment epithelium (RPE) is a highly specialized innermost monolayer of cells in the retina. RPE plays a very important and indispensable role in absorbing scattered light to improve spatial resolution in vision, recycling visual pigments to ensure light sensitivity of photoreceptors, and transporting nutrients and metabolites between the choriocapillaris and the neural retina as a blood-retinal barrier (Strauss, 2005). RPE cells are usually quiescent however, due to physical or physiological trauma; RPE cells rapidly proliferate and undergo epithelial-mesenchymal transition (EMT). EMT of RPE cells is involved in the development of proliferative vitreo-retinopathy (PVR) (Yang et al., 2015), diabetic retinopathy (DR) (Chen et al., 2014b) and age-related macular degeneration (AMD) (Ghosh et al., 2018). EMT is associated with the down-regulation of mitochondria, mitochondrial membrane potential and mitochondrial DNA copy number in various cells and cell lines including RPE cells and its cell line, ARPE-19 (Zhang et al., 2018; Hu et al., 2019; Miceli and Jazwinski, 2005). DCA is shown to inhibit the TGFβ mediated EMT of rat renal cells and kidney cells of obstructed mouse kidney model (Wei et al., 2019; Diamond et al., 2003). Hence DCA can be utilized to prevent the EMT of ARPE-19 cells.

In the present study we have stimulated EMT in the ARPE-19 cells by exposing them to TGFβ, a growth factor found to be abundant in the vitreous humor of the patients suffering from PVR (Esser et al., 1997), DR (Hirase et al., 1998) and AMD (Tosi et al., 2017). The inhibitory effect of DCA on the EMT was studied by analyzing the expression of various EMT and cell adhesion markers and MAPK/Erk and PI3K/Akt pathway which are shown to be associated with EMT of RPE cells (Chen et al., 2014a; Yokoyama et al., 2012; Saika et al., 2009).