Background
Cholangiocarcinoma is a cancer characterized by early vascular invasion and metastasis. Patients with cholangiocarcinoma are often diagnosed at advanced stage. Threeyear survival rates of 35% to 50% can be achieved only in a subset of patients, who have negative histological margins at the time of surgery [
1]. Palliative therapeutic approaches consisting of percutaneous and endoscopic biliary drainage have usually been used for these patients, since there is no effective chemotherapeutic treatment for this type of cancer [
2]. A novel agent, oxaliplatin, has been extensively used as chemotherapeutic agent in treating solid tumors [
3,
4]. Oxaliplatin is a diaminocyclohexane platinum compound that acts like cisplatin to induce DNA adducts formation. Although early studies suggested that oxaliplatin might be used as an active agent against cholangiocarcinoma [
5,
6], more recent data indicated that cholangiocarcinoma cells were resistant to oxaliplatin [
7]. Therefore, elucidating the mechanism of resistance to oxaliplatin in cholangiocarcinoma cells is crucial to improve the treatment of patients with advanced cholangiocarcinoma.
Activation of the phosphoinositide-3-kinase (PI3K)/Akt signaling pathway is frequently found in cholangiocarcinoma cells [
8]. It has been suggested to be a key step leading to the resistance of cancer cells to chemotherapy, especially when using DNA-damaging agents such as cisplatin and oxaliplatin [
9,
10]. Furthermore, previous studies have demonstrated that PI3K/Akt activation regulates sensitivity of cells to G1 arrest induced by mTOR inhibitors [
11]. Taken together, these data indicate that chemotherapeutic agents might function better in killing cancer cells if the PI3K pathway is blocked. In this study, we hypothesize that inhibition of PI3K or its downstream target, mTOR, may be increase oxaliplatin efficacy in treating cholangiocarcinoma. The effect of PI3K and mTOR inhibition on oxaliplatin sensitivity of cholangiocarcinoma cells is examined.
Methods
Cell culture and Materials
Ham's F12 medium and fetal bovine serum (FBS) were purchased from Gibco (Gibco, Grand Island, NY, USA). Polyclonal antibodies to Akt (phosphorylated at Ser473 and total), mTOR, PP70S6K and P38 MAPK (phosphorylated at Thr180/Tyr182 and total) were purchased from Cell Signaling (Cell Signaling Technology, Beverly, MA, USA). Oxaliplatin was purchased from Sanofi Aventis (Sanofi Aventis, Bridgewater, NJ, USA). Cell culture plastic plates were obtained from Nunc (Thermo Fisher, Rochester, NY, USA). LY294002 (PI3K inhibitor) was purchased from Calbiochem (EMD Chemicals, Gibbstown, NJ, USA). RAD001 (everolimus), an oral derivative of rapamycin, was generously provided by Novartis Pharma AG (Novartis International AG, Basel, Switzerland). Stock solutions (10 mmol/L) were dissolved in DMSO (Sigma-Aldrich, St. Louis, MO, USA), stored at -80°C, and diluted in fresh medium immediately before use.
The human intrahepatic cholangiocarcinoma cell lines RMCCA1 [
12] and KKU100 (kindly provided by Dr. Banchob Sripa, Department of Pathology, Faculty of Medicine, Khon Kaen University) were grown in Ham's F12 medium supplemented with 10% FBS at 37°C in a 5% CO
2 humidified atmosphere. For experiments, cells were grown in Ham's F12 medium supplemented with 1% FBS.
Cell proliferation assay
For proliferation assay, cells were seeded in 96-well culture plastic plates at a density of 10,000 cells per well. Vehicle (PBS) or oxaliplatin in various concentrations (0–200 μM) were added to each well. For the Akt or mTOR inhibition studies, cells were treated with Vehicle (DMSO), LY294002 (PI3K inhibitor) or RAD001 (mTOR inhibitor), respectively, for 1 hour before the addition of oxaliplatin. Cells were then incubated for 48 hours before applying the WST-1 cell proliferation assay reagent (Roche Diagnostics, Laval, Quebec, Canada), according to the recommendation of the manufacturer. The amount of cell proliferation was assessed by determining the A450 nm of the cell culture media after addition of WST-1 for 2 hours. Results were reported as percentage of the inhibition of cell proliferation, where the optical density measured from vehicle-treated cells was considered to be 100% of proliferation. Percentage of inhibition of cell proliferation was calculated as follows: (1-Aexp group/Acontrol) × 100.
Cell apoptosis assay
The number of apoptotic cells was determined with the Apo-BrdU TUNEL assay kit (Invitrogen, Carlsbad, CA, USA), following manufacturer's instructions. Briefly, cells were washed with cold PBS and then fixed with 1% paraformaldehyde and ice-cold 70% ethanol for 30 minutes. Fixed cells were labeled with BrdUTP using terminal deoxynucleotide transferase (TdT) at 37°C for 60 minutes and stained with Alexa Fluor 488-labeled anti-BrdU antibody for 30 minutes at room temperature. To score for apoptosis, cells were counterstained with DAPI, and at least 200 cells were counted under fluorescent microscope at 400× magnification. The percentage of apoptotic cells per experimental condition was then determined.
Western blotting analyses
Approximately 500,000 cells were seeded in a six-well culture plate, followed by treatment with vehicle (PBS), or oxaliplatin for 12 hours. Cells were collected, washed with PBS and lysed in lysis buffer. Western blot analyses were performed as previously described [
8]. The blots were first probed with antibodies against phospho-Akt, phospho-mTOR, phospho-P70S6K or cleaved caspase-3 and then reprobed with antibodies against total Akt, mTOR, P70S6K or caspase-3. Bound antibodies were detected using chemiluminescence.
Statistical analysis
The experiments were all performed in triplicate, and each result is reported as the mean with SD. Data between three or more groups were compared using the one-way analysis of variance, followed by Dunnett's post hoc test. A p-value of less than 0.05 was considered statistically significant.
Discussion
Cholangiocarcinoma is a rapidly lethal disease and generally considered to be incurable. One of the main reasons for its low survival rate is that cholangiocarcinoma exhibits extensive local invasion and frequent regional lymph node metastasis. Most patients are not candidates for curative surgical resection [
13]. Until recently, there has been no effective chemotherapeutic drug for this disease.
Oxaliplatin has been used for the treatment of a number of solid tumors including lung, gastric, and colorectal cancer [[
3,
6], and [
14]]. Recently, a prospective multicenter phase II study focused on capecitabine and oxaliplatin (CAPOX) combination therapy in advanced cholangiocarcinoma [
3,
7]. Unfortunately, the results suggested that this regimen produced poor results for intrahepatic cholangiocarcinoma [
7]. An alternative strategy is then needed to evaluate the efficacy of oxaliplatin as chemotherapeutic agent. We used two cholangiocarcinoma cell lines, RMCCA1 and KKU100, derived from cholangiocarcinoma patients to study the effect of oxaliplatin
in vitro. These cell lines exhibited resistance to oxaliplatin, even at high concentrations (100–200 μM). In addition, we demonstrated that oxaliplatin-treated cholangiocarcinoma cells exhibit high levels of Akt and mTOR phosphorylation as a result of PI3K activation. Thus, we hypothesized that activation of the PI3K pathway in cholangiocarcinoma cells may, in turn, protect the cells from oxaliplatininduced cytotoxicity. Our results indeed showed that inhibition of Akt by LY294002 significantly increased oxaliplatin efficacy in inhibiting cell proliferation. This finding suggests that Akt phosphorylation might be attributed to oxaliplatin resistance in cholangiocarcinoma cells. This result is also consistent with recent evidence showing that the mechanism of drug resistance in cancer cells was primarily through the induction of PI3K/Akt pathways [
15].
Previous studies demonstrated that exposure of cancer cells to oxaliplatin induced protein misfolding. These misfolded proteins are prone to oxidative stress as a result of better accessibility of reactive oxygen species (ROS) to the protein structure [
16]. As a consequence, recruitment of Bax to the mitochondria, release of cytochrome c to the cytosol, activation of caspase-3 and apoptotic cell death take place in cancer cells treated with oxaliplatin. Recently, Kim et al. reported that the activation of Akt could inhibit oxaliplatininduced apoptosis through maintaining XIAP protein levels [
10]. In this study, we demonstrate that inhibition of Akt by LY294002 increases the percentage of apoptotic cells after oxaliplatin treatment. In addition, activation of caspase-3 was clearly observed in cholangiocarcinoma cells treated with both LY294002 and oxaliplatin. These data indicate that activation of Akt in cholangiocarcinoma cells may be the key mechanism in inhibiting oxaliplatin-induced apoptosis.
PI3K and Akt regulate the processes of cellular glucose metabolism. Inactivation of PI3K and Akt may have deleterious effects on normal cell metabolism [
17]. Therefore, only inhibitors of those downstream molecules of PI3K and Akt that are not involved in glucose metabolism should be considered for clinical treatment. The mammalian target of rapamycin is mTOR, a 289 kDa serine/threonine kinase. mTOR is a downstream effector of the PI3K/Akt signaling pathway involved in the regulation of many transduction processes of cell growth as well as cell cycle progression, membrane trafficking, protein degradation, and protein kinase C signaling and transcription [
18].
Recently, a derivative of rapamycin, RAD001 (everolimus), has been developed. RAD001 has been shown to inhibit mTOR activity, thereby halting the proliferation of cancer cells, both
in vitro and
in vivo. Phase II clinical trials with RAD001 are currently being performed for many types of cancers [
18,
19]. Based on the results of our study, the 0.5 μM RAD001 alone did not inhibit the proliferation of cholangiocarcinoma cells. This is consistent with a previous study, which demonstrated that RAD001 has only cytostatic effects in cancer cells. To induce cytotoxicity of RAD0001 in cancer cells, other chemotherapeutic drugs should be combined with RAD0001 [
18,
20]. For example, pretreating ovarian cancer cells with RAD001 can increase their sensitivity to cisplatin [
21]. In this study, we found that RMCCA1 and KKU100 displayed high levels of Akt and mTOR phosphorylation after treatment with oxaliplatin. Pretreatment of cholangiocarcinoma cells with 0.5 μM RAD001 significantly increased the sensitivity of oxaliplatin when used at 200 μM. However, pretreatment with 0.5 μM RAD001 did not significantly increase the efficacy of oxaliplatin when used at 100 μM. In addition, the number of apoptotic cells and the activation of caspase-3 did not significantly increase when the cells were exposed to both RAD001 and oxaliplatin. This might be explained by the fact that inhibition of P70S6K by RAD001 induces IGF-IR/IRS-1/PI3K signaling, eventually increasing the level of Akt phosphorylation [
22]. This feedback mechanism might be responsible for the decrease in sensitivity to oxaliplatin, leading to a reduction in the inhibition of cell proliferation. These results are consistent with the recent report that inhibition of mTOR resulted in Akt activation in several human cancer cell lines [
22].
In summary, this study presents the possible mechanism in oxaliplatin resistance in cholangiocarcinoma cells. As proof-of-concept, we are able to show that activation of the Akt signaling pathway has a potent effect on oxaliplatin resistance. The model presented here may serve as a practical tool for identifying the molecular mechanism of chemotherapeutic drug-resistance in cholangiocarcinoma cells.
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
KL conceived of the study, designed, coordinated the study, statistical analysis and drafted the manuscript, SN carried out the proliferation and western blotting assays and helped with the statistical analysis, WU carried out the TUNEL and western blotting assays, SL revised the manuscript critically for important intellectual content, and helped draft the manuscript, ST also gave final approval for the paper to be submitted for publication.