Anti-cancer activity of asiatic acid against human cholangiocarcinoma cells through inhibition of proliferation and induction of apoptosis

Plant-derived anti-cancer agents have been of considerable interest due to their promising effectiveness with low side effects. Asiatic acid, the main constituent of the medicinal plant Centella asiatica (L.) Urban, has a wide range of biological properties such as antioxidant, anti-inflammatory and anti-cancer activities. Cholangiocarcinoma (CCA), which is a malignant tumor of bile duct epithelium, is one of the leading cancers in Southeast Asia, notably the northeast of Thailand where the liver fluke, Opisthorchis viverrini predominates. Many in vitro and in vivo studies have provided evidence supporting that oxidative stress induced by chronic inflammation is involved in CCA genesis with aggressive clinical outcomes. This study was performed to evaluate the cytotoxic effects of asiatic acid on two human CCA cell lines (KKU-156 and KKU-213). Cell viability was determined by a sulforhodamine B (SRB) assay. Morphological changes of the cells were observed by microscopy. Cell apoptosis was detected by flow cytometry using annexin V and propidium iodide (PI) staining. Messenger RNA (mRNA) expression levels of BAX, BCL2 and Survivin/BIRC5 were analyzed by real-time polymerase chain reaction (PCR). It was found that asiatic acid efficiently suppressed CCA cellular viability via induction of apoptosis. In addition, the occurrence of asiatic acid-induced apoptosis was confirmed by microscopic observation of apoptotic vesicles, down-regulation of anti-apoptotic genes (BCL2 and Survivin/BIRC5) and increased early and late apoptotic cells. Our results showed the chemotherapeutic activities of asiatic acid, suggesting the anti-cancer properties of this compound should be clinically assessed and its supplementation may lead to an improvement of survival of CCA patients.


Introduction
The northeast of Thailand, which includes Khon Kaen Province, has the highest prevalence of the liver fluke, Opisthorchis viverrini, infection and the highest incidence of cholangiocarcinoma (CCA − cancer of the bile ducts) worldwide (1). Infection of O. viverrini is associated with the custom(s) of consuming raw, fermented or partially cooked fresh-water fish, notably fish belonging to the Cyprinid family, which contains the infective life cycle stage called metacercariae. After ingestion, the metacercariae excyst in the duodenum whereupon they migrate to the hepatobiliary tracts where they become egg-producing adult worms (2). The International Agency for Research on Cancer (IARC) has classified O. viverrini as a Group 1 carcinogen (i.e., carcinogenic to humans), which represents a direct risk factor for CCA development (3). Chronic inflammation and oxidative stress caused by O. viverrini has been shown to be implicated in the pathogenesis of CCA (2,(4)(5)(6). CCA is one of the most difficult diseases to diagnose and treat because signs and symptoms of early stage are subtle and non-specific. Most patients present with advanced stages of the disease, leading to a poor prognosis and a very high mortality rate. Cure is possible with surgical resection of patients with initial stages of CCA, who do not have underlying liver or biliary tract disease. However, the 5-year survival rate is still poor, with 60 to greater than 90% post-operative recurrence rates (7,8). At present, CCA treatments including chemotherapy and radiation therapy are not sufficiently effective. The use of fluorouracil (5-FU), gemcitabine or capecitabine, either alone or in combination with a platinum analogue (i.e., oxaliplatin or cisplatin) has been suggested for CCA treatment. Nevertheless, those regimens have yielded low response rates (9)(10)(11). Additionally, elderly patients with CCA are likely to refuse chemotherapy, surgery and radiation therapy, but instead seek out other treatment options, e.g., herbal remedies. Therefore, there is an urgent need to find alternative ways to prevent and treat this silent killer disease and, hence, improve the quality of life of the people, notably poverty-stricken people in the northeast of Thailand and elsewhere in Southeast Asia where O. viverrini is distributed.
Accumulated evidence has shown the anti-cancer activities of asiatic acid against various types of cancer cell lines, including breast cancer, colon cancer, prostate cancer, lung cancer, melanoma, hepatoma, glioblastoma and ovarian cancer cells. Different mechanisms have been reported to account for the anti-cancer action of asiatic acid. For instance, asiatic acid has been reported to induce S-G2/M phase cell cycle arrest and apoptosis of human breast cancer cell lines via activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase (MAPK) pathways (22). Asiatic acid-induced apoptosis in human melanoma cells is mediated by reactive oxygen species (ROS) generation and BAX/BCL2 regulation (23), and can promote the apoptosis of human hepatoma and malignant glioma cells through increasing intracellular Ca 2+ levels (24,25). In colon cancer cells, asiatic acid has been reported to inhibit cell growth and induce apoptosis via mitochondria-dependent pathway by increasing mitochondrial membrane permeability and cytochrome c release (26). Asiatic acid could also perturb the endoplasmic reticulum and alterations in calcium homeostasis to induce cell death of prostate cancer cells (27) and it has been demonstrated to promote the pro-apoptotic effects in human ovarian cancer cells via the suppression of PI3K/ Akt/mTOR signaling cascades (28). In lung cancer cells, asiatic acid inhibited cell proliferation and caused cell death mainly through a mitochondrion-mediated pathway. This triterpene also significantly decreased weight and tumor volume in a mouse xenograft model of lung cancer (29). Asiatic acid-induced apoptosis has been proposed to be associated with the modulation of cell cycle progression and induction of apoptosis by targeting multiple molecules, e.g., NF-κB, p38 MAP and ERK kinases, caspases, PARP and BCL2/BAX in the aforementioned cancer types (22)(23)(24)(25)(26)(27)(28).
One of the significant hallmarks of cancer cells is to evade apoptosis − a process of programmed cell death. Deregulation of apoptotic pathways is thought to be a significant factor in development and progression of cancer cells. Alterations in the expression of proteins which can either promote or prevent apoptotic cell death such as BCL2, BAX and Survivin/BIRC5 can be used for the detection of apoptosis. In the present study, cytotoxicity effects of asiatic acid on two human CCA cell lines (KKU-156 and KKU-213) were evaluated by sulforhodamine B (SRB) assay, morphological obser-vations and flow cytometry. Messenger RNA (mRNA) expression levels of three genes, including BAX, BCL2, and Survivin/BIRC5 were determined by means of realtime polymerase chain reaction (PCR).

Cell cultures
Two human CCA cell lines, KKU-213 and KKU-156, were obtained from Cholangiocarcinoma Research Institute (CARI, Khon Kaen University, Thailand). Both cell types were cultured in Ham's F-12 medium (Invitrogen, CA, U.S.A.) supplemented with 10% fetal calf serum, 100 U/ml penicillin and 100 g/ml streptomycin (complete media) at 37 °C in a humidified atmosphere of 5% CO 2 . A subculture was performed when the cells reached the confluent stage and the media were changed every two days.

Asiatic acid solution preparation
Asiatic acid (Sigma-Aldrich, MO, U.S.A) was dissolved in 100% dimethyl sulfoxide (DMSO) to obtain 2 × 10 4 µM stock solution, aliquoted and stored at -20 °C until used. An aliquot of the stock solution (2 × 10 4 µM) was diluted 100-fold with complete media to make 200 µM asiatic acid containing 1% DMSO, which was subsequently diluted 2-fold to with complete media to make 100 µM asiatic acid containing 0.5% DMSO. Each concentration of asiatic acid was prepared by diluting with complete media containing 0.5% DMSO. Final concentration of DMSO was adjusted to 0.5% in all conditions.

Sulforhodamine B (SRB) cell cytotoxicity assay
CCA cell lines (3 × 10 3 cells in 100 µl/well) were seeded into 96-well plates for 24 h. Both KKU-213 and KKU-156 cells were treated with various concentrations (20-55 µM) of asiatic acid in complete media containing 0.5% DMSO in triplicate for 24 and 48 h. Cell images were taken by an inverted microscope (Zeiss Axiovert 40 CFL) every 24 h. Cytotoxicity was determined using SRB assay (30,31). Cells were washed with ice-cold phosphate-buffered saline (PBS), fixed by adding 100 µL of chilled 10% trichloroacetic acid (TCA) in PBS to each well and incubated at 4 °C for 24 h. After removal of the TCA, cells were washed with deionized water three times and dried at 60 °C for 30 min. The cells were then incubated with SRB (50 µl per well, 0.4% in 1% acetic acid) in the dark for 45 min at room temperature. The SRB was removed, and the wells washed three times with 1% acetic acid (200 µl) to remove excess stains. The plate was then allowed to dry at 60 °C for 30 min. A volume of 200 µl of Tris-base (10 mM, pH 10.5) was added to each well, followed by incubation for 1 h at room temperature with gentle shaking. The absorbance was determined spectrophotometrically at 540 nm using an ELISA microplate reader (Sunrise, Tecan Austria GmbH). Asiatic acid concentrations required to inhibit 50% cell growth (IC 50 ) were calculated from concentration-effect curves after linear regression analysis.

Quantification of BCL2, BAX and Survivin/BIRC5 mRNA levels by real-time polymerase chain reaction (PCR)
CCA cell lines (1 × 10 5 cells in 2 ml/well) were plated into 6-well plates for 24 h. Cells were treated with 20 µM asiatic acid for 24 h (KKU-213) and 48 h (KKU-156). Cells were washed three times with ice-cold PBS and harvested by trypsinization. Cell pellets were stored at -70 °C until used. Total RNA was isolated from cell pellets with TRIzol ® reagent (Invitrogen, CA, U.S.A.) according to the manufacturer's protocol. RNA quality was assessed using a NanoDrop ND-2000 spectrophotometer (NanoDrop Technologies, DE, U.S.A.). Total RNA (2 µg) was converted to cDNA using High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, CA, U.S.A). Real-time PCR was conducted by using ABI-7500 Real-Time PCR system (Applied Biosystems, CA, U.S.A.). Primer and probe sequences for real-time detection of BAX (catalog no. Hs00180269-m1), BCL2 (catalog no. Hs00608023-m1), Survivin/ BIRC5 (catalog no. Hs04194392-s1) and endogenous control gene ACTB (catalog no. Hs99999903-m1) mRNA were purchased from Applied Biosystems. Realtime PCR was performed by using human Taqman predeveloped assay reagents (Applied Biosystems, CA, U.S.A.). Relative mRNA expression levels were analyzed with a cycle threshold (Ct) in the linear range of amplification to β-actin as an internal control.

Statistical analysis
Statistical analysis was performed using the SPSS software version 17.0 (IBM Corporation, U.S.A.). Levels of mRNA expressions were compared by Student's t-test. A p<0.05 was considered as statistical significance.

Cytotoxicity of asiatic acid on cholangiocarcinoma cell lines
The cytotoxic effect of asiatic acid on KKU-156 and KKU-213 CCA cells was determined using the SRB method. The principle of SRB assay is based on the abi-

Analysis of apoptosis by flow cytometry
To further evaluate the ability of asiatic acid to induce apoptosis in KKU-156 and KKU-213 CCA cells, flow cytometric analysis was performed using annexin V/PI staining. Annexin V is a protein that binds to phosphatidylserine residues, which are present predominantly in at the inner leaflet of the plasma membrane. In apoptotic cells, the translocation of phosphatidylserine from the inner to the outer surfaces can be detected by staining with fluorochrome-conjugated annexin V. Using annexin V in combination with propidium idodide (PI), a cellular DNA dye, can distinguish live cells, early apoptotic cells, late apoptotic cells and necrotic cells (33). As shown in Figure 3, following asiatic acid treatment, flow cytometric results revealed that apoptosis was induced in a dose-dependent manner in both CCA cell lines, particularly at asiatic acid concentration of 45 µM. In the case of KKU-156 cell line (Figure 3a (Figure 3b), the total population of early and late apoptotic cells was found to be increased from 8.9% in untreated cells to 9.9%, 35.8% and 86.4% for cells subjected to 35, 40 and 45 µM of asiatic acid treatment, respectively. These results showed the ability of asiatic acid to induce apoptosis in human CCA cells.

Effects of asiatic acid on apoptosis-related gene expression of CCA cell lines
Changes in mRNA expression levels of three apoptosis-related genes (BAX, BCL2, and Survivin/BIRC5) in human CCA cell lines during asiatic acid treatment were investigated by real-time PCR. The mRNA expression levels of BAX tended to be increased after 48 h treatment with 20 μM asiatic acid in KKU-156 cells as well as after 24 h treatment with 20 μM asiatic acid in KKU-213 cells, in compared with that in untreated cells (Figure 4). On the other hand, in both CCA cells, the mRNA expression levels of BCL2 and Survivin/BIRC5 significantly decreased after 24 h or 48 h treatment with 20 μM asiatic acid. These findings showed that the treatment of asiatic acid against CCA cell lines affected the balance of pro-and anti-apoptotic signaling pathways towards apoptosis via down-regulation of anti-apoptotic genes. Moreover, after treatment with 20 μM asiatic acid, increases in BAX/BCL2 ratios were observed in both cell line types. The BAX/BCL2 ratios were determined to be 3.3 and 1.6 in the case of KKU-156 and KKU-213 cell lines, respectively.

Discussion
The main finding of our study was that asiatic acid inhibited the growth of human CCA cells. In both CCA cell lines, IC 50 values obtained in our study are in the same range with those previously reported in other cancer cell types, e.g. skin, breast and lung, all with IC 50 values ranging from 8-80 µM (26)(27)(28)(29). KKU-156 cells showed higher sensitivity and lower IC 50 values than KKU-213 cells when they were treated with asiatic acid. The different IC 50 in the two different cell lines may be due to differences in cell characteristics and distinct target sites of action as reported in the case human glioma cells (34). It is well established that asiatic acid treatment can affect cancer cell growth by inducing apoptosis in many cell lines (23,26). In this study, we found that asiatic acid induced apoptosis as clearly shown by increased apoptotic cells detected by flow cytometry using annexin V/PI staining. The different responses detected using flow cytometry based on annexin-V/PI staining were related to microscopic observation of apoptotic vesicles. Our results confirmed that asiatic acid possesses potent growth inhibitory and pro-apoptotic effects against both types of CCA cell lines. In both normal and cancer cells, cell survival and death are delicately controlled by an intricate balance between anti-apoptotic and pro-apoptotic regulators. Molecular analyses were performed to confirm the differential expression of apoptosis-regulatory proteins, including BAX, BCL2 and survivin/BIRC5. Following asiatic acid treatment, real-time PCR showed that there was a slight increase in the expression of the pro-apoptotic molecule BAX while there was a significant decrease in the expression of the anti-apoptotic molecules BCL2 and Survivin/BIRC5. Moreover, the ratio of BAX/ BCL2 of each cell line was significantly elevated after exposure to asiatic acid. Changes in expression levels of genes involved in regulation of apoptosis indicate that  asiatic acid induced apoptotic cell death. The asiatic acid-induced apoptosis in CCA can be mediated through alteration of the BAX/BCL2 ratio and down-regulation of BCL2 and Survivin/BIRC5, and growth suppression by asiatic acid can be due to apoptosis. Our results for CCA cells are in concordance with the previous findings that showed variations of response of apoptosis regulators BCL2, BAX and Survivin/BIRC5 to asiatic acid, for instance in human leukemia, human melanoma and human breast cancer cells (22,23,35).
Chronic inflammation and oxidative stress was found to play a key role in CCA carcinogenesis via the induction of DNA damage and cell proliferation, leading to CCA initiation and promotion in animal models by infection with liver fluke (36)(37)(38). Moreover, oxidative stress is also involved in CCA progression with poor prognosis and metastasis status of the patients (39,40). On the other hand, asiatic acid has been shown to have an anti-oxidative effect due to the presence of several hydroxyl groups, an olefin group and a carboxylic acid group (41). Due to its abilities to decrease inflammatory, reduce oxidative stress and induce apoptosis in cancers, asiatic acid which have shown in this study to possess anti-CCA activity may be a good candidate for treatment of this fatal cancer caused by chronic inflammation with liver flukes and oxidative stress. A number of studies have indicated that plant-derived products manifest potent anti-cancer potential via their anti-proliferative and pro-apoptotic effects on CCA, such as curcumin, luteolin, quercetin and EGCG, and β-eudesmol by regulating different signaling modulators, e.g. NF-κB and STAT3, involved in tumor development and progression (42)(43)(44)(45). Interestingly, in this study, for the first time, we evaluated the anti-cancer activity of asiatic acid on this type of cancer.
In conclusion, this study showed that asiatic acid could inhibit proliferation and induce apoptotic cell death in human CCA cell lines, KKU-213 and KKU-156, as evidenced by: (i) observation of the increased apoptotic vesicles (ii) in vitro cytotoxic study (iii) flow cytometry and (iv) decreasing the expression levels of anti-apoptotic signaling molecules BCL2 and Survivin/ BIRC5. Our findings provide evidence that the natural product asiatic acid is a promising anti-tumor agent against CCA, suggesting the anti-cancer properties of this compound should be clinically assessed and its supplementation may lead to an improvement of survival in CCA patients.