Chloroquine Exerts Anti-metastatic Activities Under Hypoxic Conditions in Cholangiocarcinoma Cells

Cholangiocarcinoma (CCA) is an aggressive tumor with high infiltrating growth and metastasis to lymph node and blood vessels in humans as well as in an animal (Blechacz and Gores, 2008; Songserm et al., 2009; Sripa et al., 2011). CCA patients are predominantly clinically silent and difficult to diagnose until the disease has reached an advanced or metastatic stage, at which the prognosis is poor. Moreover, CCA exerts a poor response to current therapies (Thongprasert, 2005). Hypoxia, a common hallmark of most solid tumors, is known to induce a transcription factor namely, hypoxia inducible factor, HIF-1α (O’Donnell et al., 2006). In normal atmosphere, HIF-1α is rapidly degraded by hydroxylation of prolyl hydroxylases domain proteins (PHDs) in the PHD-pVHL system (Fan et al., 2014). Under hypoxic conditions, the PHDs process is inhibited, the stabilization of HIF-1α accumulates in the nucleus and dimerises with HIF-1b (Zhang et al., 2013), resulting in the activation of various target hypoxia-responsive genes controlling tumor progression, angiogenesis, invasion, metastasis and resistance to chemotherapy (Liu et al., 2008; Sahlgren et al., 2008). One impact of HIF-1α’s target genes is a vascular endothelial growth factor (VEGF), that functions to increase O2 delivery to cells by stimulating 1Department of Biochemistry, 3Liver Fluke and Cholangiocarcinoma Research Center, Faculty of Medicine, 2Department of Biochemistry, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen, Thailand *For correspondence: nisana@kku.ac.th Abstract


Introduction
Cholangiocarcinoma (CCA) is an aggressive tumor with high infiltrating growth and metastasis to lymph node and blood vessels in humans as well as in an animal (Blechacz and Gores, 2008;Songserm et al., 2009;Sripa et al., 2011).CCA patients are predominantly clinically silent and difficult to diagnose until the disease has reached an advanced or metastatic stage, at which the prognosis is poor.Moreover, CCA exerts a poor response to current therapies (Thongprasert, 2005).
Hypoxia, a common hallmark of most solid tumors, is known to induce a transcription factor namely, hypoxia inducible factor, HIF-1α (O'Donnell et al., 2006).In normal atmosphere, HIF-1α is rapidly degraded by hydroxylation of prolyl hydroxylases domain proteins (PHDs) in the PHD-pVHL system (Fan et al., 2014).Under hypoxic conditions, the PHDs process is inhibited, the stabilization of HIF-1α accumulates in the nucleus and dimerises with HIF-1b (Zhang et al., 2013), resulting in the activation of various target hypoxia-responsive genes controlling tumor progression, angiogenesis, invasion, metastasis and resistance to chemotherapy (Liu et al., 2008;Sahlgren et al., 2008).One impact of HIF-1α's target genes is a vascular endothelial growth factor (VEGF), that functions to increase O 2 delivery to cells by stimulating

Conditions in Cholangiocarcinoma Cells
Suyanee Thongchot 1,3 , Watcharin Loilome 1,3 , Puangrat Yongvanit 1,3 , Hasaya Dokduang 1,3 , Raynoo Thanan 1,3 , Anchalee Techasen 2,3 , Nisana Namwat 1,3 * angiogenesis, and intracellular proteins which allow cells to survive in O 2 deprivation by reprogramming their metabolism (Semenza, 2012).Cobalt chloride (CoCl 2 ) is a chemically induced hypoxic-mimicking condition which occurs by inhibiting PHD and stabilizing HIF-1α, and it is frequently used for inducing a hypoxic condition in many in vitro systems (Yuan et al., 2003;Wu and Yotnda, 2011).It has been showed that 100 μM of CoCl 2 treatments to human prostate cancer cell lines resulted in elevated VEGF mRNA and protein secretion, and drove cells to be highly invasive metastatic phenotype (Liu et al., 1999).In addition, a recent study has demonstrated that CCA cells treated with 100 μM of CoCl 2 increased HIF-1α expression that contributed to the progression by metastatic potential (Thongchot et al., 2014).The hypoxic microenvironment is very important in regulating tumor processes such as epithelial-mesenchymal transition (EMT) that is believed to be a major mechanism by which cancer cells become metastatic and invasive (Zhang et al., 2013).Hypoxia stabilizes HIF-1α expression that induces EMT by induction of the mesenchymal markers N-cadherin (Yang et al., 2007) and downregulation of the epithelial marker E-cadherin (Salnikov et al., 2012).
Chloroquine, 7-chloro-4 [4diethylamino1methyl butylamino] quinoline (CQ), is a widely well-known medicine used as a potent treatment of malaria and auto-immune diseases.Recently, several studies have shown that it has extensive biological effects such as inhibiting cell growth and/or inducing cell death in many types of cancer cells (Fan et al., 2006).Although CQ has been shown to inhibit metastasis of breast cancer cells to lung in a xenograft model (Jiang et al., 2010), the mechanism by which CQ suppresses tumor metastasis is still unclear.
In the present study, we investigated the in vitro effect of CQ on CCA cell metastasis under hypoxic-mimicking conditions.Expressions of HIF-1α, PHDs, VEGF, E-cadherin and N-cadherin were determined in CQ treated CCA cells under hypoxia-mediated metastasis.

Cell lines, chemicals, and reagents
Two human CCA cell lines, M139 and M214, which were established at the Liver Fluke and Cholangiocarcinoma Research Center, Khon Kaen University, Thailand, were cultured in Ham's F-12 medium supplemented with 44 mM NaHCO 3 , penicillin (100 units/ml), streptomycin (100 mg/ml) and 10 % fetal bovine serum in a humidified atmosphere containing 5 % CO 2 .Cobalt chloride (CoCl 2 ), and chloroquine (CQ) were purchased from Sigma, St. Louis, MO, United States.All other chemicals used were of analytical grade.

The in vitro cellular transwell migration assay
A total of 4×10 4 CoCl 2 or CQ-treated cells were seeded onto the upper chamber of Transwell ® (8 µm pore size; Corning, NY, USA), and HAM's F-12 medium supplemented with 10% (v/v) FBS was placed in the lower chamber.After incubation at 37°C for 16 or 36 h, cells in the upper surface of the filter were scraped off, and cells which migrated to the underside of the filter were fixed with absolute ethanol for 30 min, stained with hematoxylin solution, and then counted under a microscope.The mean value of all low-power fields (200×) was determined.Assays were done in triplicate and three independent experiments were conducted.

Statistical analysis
Data were presented as the means±standard deviation of three separate experiments.Statistical significance of the differences between the experimental conditions was determined by the SPSS version 17.0 (SPSS Inc., Chicago, IL) and was analyzed using unpaired, two-tailed student's t-test.Data were considered statistically significant when p<0.05 (*) and p<0.001 (**).

CQ suppressed CCA cell migration under the CoCl 2induced hypoxia
Recent studies have demonstrated that the behavior of CCA cell lines is significantly affected by hypoxia (Seubwai et al., 2012;Thongchot et al., 2014).We applied 100 μM CoCl 2 to provide the hypoxic-mimic conditions for CCA cell culture, and trans-migrating cells were evaluated.Upon CoCl 2 treatment for 36 h in M139 cells and 16 h in M214 cells, the results showed that cell migration was 59% increased (p=0.030) in M139 cells (Figure 1A), and 65% increased (p=0.043) in M214 cells (Figure 1B) over the untreated cells.
To elucidate the anti-metastasis effects of CQ on CCA cell lines, the M139 and M214 cell lines were exposed to 50 μM CQ (or not) under 100 μM CoCl 2 and the transmigration assay was determined.The result showed that 50 μM CQ treatment significantly suppressed the number of migrating cells when compared with untreated control cells (6.3 times for M319 cells in Figure 1A and 2.9 times for M214 cells in Figure 1B).The inhibitory effect of CQ on cell migration was markedly enhanced in 100 μM CoCl 2 treatment condition when compared with CoCl 2

CQ altered the expression levels of metastasis-related proteins under CoCl 2 -induced hypoxia in CCA cells
M139 and M214 cells were treated with CoCl 2 to induce hypoxia-mimicking conditions and the hypoxicresponsive proteins were assessed.The results revealed that 100 µM CoCl 2 increased the expression of HIF-1α but completely suppressed the PHD-2 protein level (lane 2 in Figure 2A for M139 and Figure 2B for M214 cells).The VEGF expression was significantly elevated under the CoCl 2 treatment in M139 (lane 2 in Figure 2A) and M214 (lane 2 in Figure 2B) cells over the untreated control cells.The CCA cells treated with 50 µM CQ showed the suppression of protein levels of HIF-1α and VEGF when compared with untreated cells (lane 3 in Figure 2A for M139 and Figure 2B for M214).Cells treated with 50 µM CQ combined with 100 µM CoCl 2 continued to suppress HIF-1α and VEGF but induce PHD-2 when compared with cells treated with CoCl 2 alone (lane 4 in Fig. 2A for M139 and Fig. 2B for M214 cells).Notably, CQ alone did not suppress the level of PHD-2 in both cells (lane 3 in Figure 2A for M139 and Figure 2B for M214).
Our results showed that treatment of 100 µM CoCl 2 reduced the ratio between E-cadherin and N-cadherin in CCA cells (lane 2 in Figure 3A for M139 and Fig. 3B for M214 cells).Interestingly, 50 µM CQ applied in CCA cells increased the ratio between E-cadherin and N-cadherin (lane 3 in Figure 3A for M139 and Figure 3B for M214).Cells treated with 50 µM CQ combined with 100 µM CoCl 2 also increased in the ratio between E-cadherin and N-cadherin when compared with cells treated with CoCl 2 alone (lane 4 in Figure 3A for M139 and Figure 3B for M214 cells).

Discussion
From accumulating evidence hypoxia has emerged as a pivotal factor of tumor development since it can activate the related genes of the tumor cells in order to adapt to the microenvironment to promote tumor metastatic progression and resistance to therapy (Vaupel and Mayer, 2007).Metastasis is the most lethal characteristic of CCA and it is a major problem in CCA treatment.Elucidation of molecular mechanisms that are involved in metastasis is still a challenge for developing new effective therapeutic drugs and improving the clinical outcome of patients with CCA.This experimental study points to a fundamental effect of CQ on CCA cell metastasis under hypoxiamimicking condition.We created an in vitro hypoxia-like state by treatment of two CCA cell lines with 100 μM of CoCl 2 (Liu et al., 1999;Law et al., 2012;Thongchot et al., 2014).We demonstrated that CoCl 2 -induced hypoxia significantly increased metastases in both CCA cell lines, using a transwell migration assay.We also confirmed that CoCl 2 truly induces hypoxic conditions as demonstrated by the suppressing the protein level of PHD-2 and the increased level of hypoxia responsive elements HIF-1α and VEGF (Wu and Yotnda, 2011).In addition, we  revealed that CoCl 2 -induced hypoxia facilitates CCA cells to metastasize by altering the level of proteins involved in EMT-mediated metastasis, i.e., reduction of E-cadherin to N-cadherin ratio, a hallmark of EMT (Gravdal et al., 2007;Chen et al., 2014) were observed for M139 and M214 cells.Our study suggests that the HIF-1α/VEGF/ EMT is likely to play a critical role in the metastatic potential in CCA.
Chloroquine (CQ) is a well-tolerated, safe drug which is already in clinical use for treatment of malaria and other types of parasitic infections (Winstanley, 2003).Our results showed that CQ (50 µM) significantly delayed CCA cell migration and completely inhibited the effect of CoCl 2 enhanced metastasis by suppressing the protein levels of HIF-1α.Interestingly, we also demonstrated that CQ stabilized the protein level of the HIF-1α inhibitor PHD-2 in CCA cells under hypoxic condition.Consequently, the reduction of HIF-1α potentially leads to a decrease in VEGF expression, resulting in metastasis suppression.Although CQ has been reported to inhibit tumor cell growth, it also inhibits tumor metastasis which has been previously demonstrated in breast cancer cells by suppressing matrix metalloproteinase (MMP-2 and MMP 9) mRNA expression and protein activities (Tuomela et al., 2013).Interestingly, our results showed that CQ disrupted EMT-mediated hypoxia in CCA cells by reducing N-cadherin but induced E-cadherin upon CoCl 2 treatment.To gain further knowledge of the mechanisms by which CQ inhibit EMT-mediated metastasis in CCA needs to be further investigated.
In conclusion, our study supports the role of hypoxia that induces tumor metastasis, possibly mediated by EMT and CQ which can inhibit CCA metastasis.

Figure 1 .
Figure 1.Effect of Hypoxia and CQ on in vitro M139 and M214 CCA Cells Migration; A transwell migration assay without matrigel was used to detect the migration activity of M139 and M214 CCA cells in the exposed to 50 μM CQ (or not) under 100 μM of CoCl 2 .Means ± SEM. * P <0.05 and ** p<0.001.Results are expressed as the means ± SD error of the mean of three separated experiments

Figure 3 .
Figure 3.Western Blot Analysis of the Expression of HIF-1α, PHD-2, VEGF and EN Switch in Each Group Treated in in vitro M139 and M214 CCA Cell Line; Representative EN switch expression.Western blotting images are shown of CCA with untreated (Lane 1), 100 μM of CoCl 2 treatment (lane 2), 50 μM of CQ treatment (lane 3), and 100 μM of CoCl 2 with CQ 50 μM treatment (lane 4).Data are expressed as ratio of all proteins to β-actin, and are means±SEM.*p<0.05 and **p<0.001