|Ahead of print publication
Blocking of methionine aminopeptidase-2 by TNP-470 induces apoptosis and increases chemosensitivity of cholangiocarcinoma
Sonexai Kidoikhammouan1, Wunchana Seubwai2, Atit Silsirivanit1, Sopit Wongkham1, Kanlayanee Sawanyawisuth1, Chaisiri Wongkham1
1 Department of Biochemistry; Cholangiocarcinoma Research Institute, Khon Kaen University, Khon Kaen, Thailand
2 Cholangiocarcinoma Research Institute, Khon Kaen University; Department of Forensic Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen, Thailand
Department of Forensic Medicine, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002
Source of Support: None, Conflict of Interest: None
Context: Resistance of cancer cells to chemotherapeutic drugs is a major pitfall of the failure of chemotherapy treatment for cholangiocarcinoma (CCA). A new therapeutic strategy that can improve treatment efficacy is mandatory for CCA patients. Our previous findings demonstrated the overexpression of methionine aminopeptidase-2 (MetAP2) in CCA patients. In addition, supplementation of TNP-470, a MetAP2 inhibitor, significantly inhibited the growth and metastatic activities of CCA cell lines. However, the molecular mechanism of antitumor activity of TNP-470 and the synergistic antitumor activity of TNP-470 combined with chemotherapeutic drugs are still unknown.
Aims: The aim of this study is to evaluate the molecular mechanism of anticancer activity and the potential use of TNP-470 as a chemosensitizing agent in CCA cell lines.
Materials and Methods: Cell cycle and apoptosis of CCA cell lines were evaluated using flow cytometry with propidium iodide staining. Expression of apoptosis regulatory proteins was measured by Western blotting. The chemosensitizing effect of TNP-470 was determined using combination index.
Results: TNP-470 inhibited the growth of CCA cells via induction of apoptosis through activation of the p38-phosphorylation and up- and down-regulation of Bax and Bcl-xL, respectively. Furthermore, TNP-470 significantly enhanced the antitumor activity of 5-fluorouracil, cisplatin, doxorubicin, and gemcitabine.
Conclusions: The present results show that TNP-470 could be a potential therapeutic or adjuvant agent for CCA.
Keywords: Chemosensitization, cholangiocarcinoma, methionine aminopeptidase 2, TNP-470
|How to cite this URL:|
Kidoikhammouan S, Seubwai W, Silsirivanit A, Wongkham S, Sawanyawisuth K, Wongkham C. Blocking of methionine aminopeptidase-2 by TNP-470 induces apoptosis and increases chemosensitivity of cholangiocarcinoma. J Can Res Ther [Epub ahead of print] [cited 2019 Feb 19]. Available from: http://www.cancerjournal.net/preprintarticle.asp?id=251399
| > Introduction|| |
Cholangiocarcinoma (CCA) is a rare cancer developed from biliary epithelium. Recently, the incidence of CCA is increasing globally, and the highest incidence of CCA has been reported in the Northeast Thailand. Liver fluke infection was identified as a strong risk factor of CCA in this region. In the Northeast Thailand and in Lao People's Democratic Republic, 6 and 2 million people, respectively, are estimated to be infected with the carcinogenic liver fluke, Opisthorchis viverrini. Under such circumstances, the high incidence of liver fluke-associated CCA is endemic in Southeast Asia. As it is difficult to diagnose CCA in the early stage, most of the CCA patients are diagnosed at the advanced stage. Thus, surgical cure cannot be offered to them and chemotherapy is generally a treatment of choice. Many chemotherapeutic drugs such as gemcitabine, cisplatin, and 5-fluorouracil (5-FU) have been used clinically either as single agent or combination for CCA patients. Nevertheless, the outcome of all chemotherapies is unsatisfactory with a low response rate and short median survival time., In addition to the side effects of chemotherapy, the frequent acquisition of drug-resistant phenotypes and the occurrence of secondary malignancies associated with chemotherapy remain serious problems in the treatment of CCA patients. Thus, there is a need for developing a new or an alternative approach to treat CCA patients.
Methionine aminopeptidase 2 (MetAP2) is a member of dimetallohydrolase family ubiquitously distributed in both eukaryotes and prokaryotes. It plays a biological role in removing methionine residue from newly synthesized polypeptide chains. Apart from physiological roles, MetAP2 plays an important role in the growth and angiogenesis of different types of cancers., The upregulation of MetAP2 has been reported in several cancers including colon cancer and B-cell lymphoma. Inhibition of MetAP2 using specific inhibitors–fumagillin, ovalicin, PPI-2458, and TNP-470–is lethal for cancer cells. Inhibition of cell growth and angiogenesis are the postulated mechanisms of tumor suppression. At present, the anticancer activity of TNP-470 is examined in vitro, in vivo, and in clinical trials.,,,,
We have previously demonstrated the overexpression of MetAP2 in human CCA tissues with close association to metastasis. Moreover, a subtoxic dose of TNP-470 significantly reduced the migration and invasion of CCA cells via inhibition of the suppression of c-MYC, MMP2, and MMP9 expression. However, the effects of TNP-470 on cell growth, apoptosis, and chemosensitization on CCA cells remained unexplored. In this study, we aimed to investigate the antitumor effect of TNP-470 using CCA cell lines with special emphasis on the molecular mechanisms underlying the antitumor effect of TNP-470, and the possibility of using TNP-470 as a chemosensitizing agent was explored.
| > Materials and Methods|| |
CCA cell lines, KKU-213 and KKU-214, were developed from primary tumors obtained from Thai CCA patients and registered in the Japanese Collection of Research Bioresources Cell Bank, Osaka, Japan. All cell lines were cultured in Dulbecco's Modified Eagle's Medium supplemented with 10% heat-inactivated fetal calf serum and 1% antimycotic-antibiotic (Gibco, Grand Island, NY, USA) at 37°C, 5% CO2.
Cell cycle and apoptosis analyses
The effect of TNP-470 on cell cycle progression was assessed using flow cytometry. CCA cells at 1 × 105 cell/well were grown in a 6-well plate with different concentrations of TNP-470. Cells were harvested, washed with cold phosphate-buffered saline (PBS), trypsinized, and centrifuged. Cells were suspended in 50 μl cold PBS, 450 μl of cold ethanol was added, and incubated at 4°C for 1 h. After centrifugation at 110 ×g for 5 min, the pellet was washed with cold PBS, resuspended in 500 μl PBS, and then incubated with 5 μl RNase (20 μg/ml final concentration) at 37°C for 30 min. The cells were chilled on ice for 10 min and incubated with propidium iodide at a final concentration of 50 μg/ml for 1 h in the dark. Cell cycle distribution was determined by flow cytometry, and DNA histograms for cell cycle analysis were determined using the FlowJo software (Tree Star, San Jose, CA, USA).
Western blot analysis
CCA cells were lysed with NP-40 lysis buffer (50 mM Tris, pH 7.4; 150 mM NaCl; 1% NP-40), and proteins from the whole cell lysate were separated in a 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The electrophoresed proteins were blotted onto a polyvinylidene fluoride membrane (GE Healthcare, Tokyo, Japan) and incubated with the primary antibodies such as rabbit anti-Caspase 3 (1:1000), rabbit anti-Caspase 9 (1:1000), rabbit anti-Bax (1:1000), mouse anti-Bcl-xL (1:1000), mouse anti-p38 and pp38α/β (1:1000) at 4°C overnight, and mouse anti-β-actin (1:40,000) (Santa Cruz Biotechnology, Santa Cruz, CA, USA) at room temperature for 1 h. Quantification of the Western blot bands was performed using GelPro 32 (Media Cybernetics®, Bethesda, MD, USA). The relative density of each band was evaluated and normalized with β-actin.
CCA cell lines (3 × 103 cells/well) were incubated with different concentrations (IC25, IC50, and IC75) of TNP-470 and chemotherapeutic drugs such as 5-FU, cisplatin, doxorubicin, and gemcitabine, simultaneously in a 96-well plate for 72 h. The cell numbers in the treated and nontreated groups was determined using MTT assay. The combination index (CI) theorem of Chou–Talalay offers quantitative definition for additive effect (CI = 1), synergism (CI <1), and antagonism (CI >1) in drug combinations. All data were analyzed using the COMPUSYN software (ComboSyn Inc., NY, USA).
The difference of continuous data between the two groups as treatment and nontreatment groups was analyzed using an unpaired t-test (parametric test). All analyses were performed using the SPSS software version 16.0 (SPSS IBM Inc., Chicago, IL, USA) and the GraphPad Prism software (GraphPad Software Inc., La Jolla, CA, USA). P < 0.05 was considered statistically significant.
| > Results|| |
TNP470 inhibited the growth of cholangiocarcinoma cell lines via induction of apoptosis
Our previous report demonstrated the antitumor activity of TNP-470 in two CCA cell lines, KKU-213 and KKU-214, which were established from CCA patients, in that TNP-470 significantly reduced the growth of these cell lines. In the present study, we further investigated the effect of TNP-470 on cell cycle distribution and apoptosis of those CCA cell lines. KKU-213 and KKU-214 cell lines were incubated for 48 h (this optimized incubation time was obtained from our previous study) with various concentrations of TNP-470 (0, 2.5, 5, 10, and 20 μg/ml) or with vehicle control (0.05% dimethyl sulfoxide). Cells were stained with propidium iodide and then analyzed by flow cytometry. The results showed that TNP-470 did not affect the cell cycle progression of CCA cells [Figure 1]a. However, TNP-470 induced apoptosis of both KKU-213 and KKU-214 cells in a dose-dependent manner [Figure 1]b.
|Figure 1: The effect of TNP-470 on the cell cycle and apoptosis of cholangiocarcinoma cell lines. KKU-213 and KKU-214 were treated with TNP-470 for 48 h and stained with propidium iodide, then analyzed by flow cytometry. (a) Relative cell numbers are illustrated at each phase of the cell cycle. (b) Percent of apoptotic cells.*P < 0.05|
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Apoptosis induction by TNP470 is mediated by p38 pathway
To investigate the mechanism by which TNP-470 induced apoptosis of CCA cell lines, KKU-213 and KKU-214 were treated with various concentrations of TNP-470 for 48 h. The expression of apoptotic regulatory proteins including caspase 9 and 3, Bcl-xL, Bax, p38, and phosphorylated p38 was determined by Western blot technique. The results are depicted in [Figure 2]. TNP-470 treatment did not affect the expression of p38. By contrast, TNP-470 treatment induced the increase of phosphorylated p38 in a dose-dependent manner. The degree of TNP-470-induced p38 phosphorylation paralleled with the induction of apoptosis, indicating that p38 pathway might be involved in the regulation of apoptosis. TNP-470 also induced the pro-apoptotic protein, Bax. By contrast, TNP-470 suppressed the expression of the anti-apoptotic protein, Bcl-xL, in a dose-dependent manner, resulting into the activation of caspase 9 and 3.
|Figure 2: Effect of TNP-470 on the expression of apoptotic-related proteins. Cholangiocarcinoma cell lines treated by TNP-470 with various concentrations (2.5, 5, 10, and 20 μg/ml) or control (0.05% dimethyl sulfoxide) for 48 h. Then, total proteins were collected and subjected to Western blot analysis for p38, pp38, Bcl-xL, Bax, caspase 3 and 9. β-actin was used as internal control|
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TNP-470 enhances the antitumor activity of chemotherapeutic drugs on cholangiocarcinoma cell lines
To examine the chemosensitizing effect of TNP-470 on CCA cell lines, KKU-213 and KKU-214 were treated with varying concentrations (IC25, IC50, and IC75) of four chemotherapeutic drugs (5-FU, cisplatin, doxorubicin, and gemcitabine) in combination with IC25, IC50, and IC75 of TNP-470 for 72 h. Cell viability was determined using MTT assay, and the CI was calculated using the COMPUSYN software. The results showed that the combination of chemotherapeutic drugs with TNP-470 gave higher degree of growth inhibition of the two CCA cell lines compared with a single-agent treatment of either TNP-470 or chemotherapeutic drug alone. All combinations of TNP-470 and chemotherapeutic drugs gave synergistic effects on both KKU-213 and KKU-214 as indicated by the CI <1, except for the combination of IC75 of TNP-470 and IC25 of doxorubicin [Table 1] and [Table 2].
|Table 1: Combination index (CI) of chemotherapeutic drugs and TNP-470 on KKU-213|
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|Table 2: Combination index (CI) of chemotherapeutic drugs and TNP-470 on KKU-214|
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| > Discussion|| |
We have previously reported the growth inhibitory effect of TNP-470, a MetAP2 inhibitor, on CCA cell lines. Furthermore, a sublethal dose of TNP-470 significantly reduced the migration and invasion of CCA cells via suppression of cMYC, MMP-2, and MMP-9 expression. In this study, we further demonstrated the molecular mechanism underlying the growth inhibitory effect of TNP-470 on CCA cell lines, KKU-213 and KKU-214. The synergistic effect of TNP-470 on chemotherapeutic drugs for CCA treatment was also demonstrated for its further application.
MetAPs are bifunctional proteins that play a critical role in the regulation of protein synthesis and posttranslational processing. These enzymes are responsible for the removal of methionine from the amino-terminus of newly synthesized proteins. Many reports documented that inhibition of MetAP2 action by its inhibitors, for example, fumagillin and sulfonamides, was lethal for cancer cells.,,,, Among these MetAP2 inhibitors, TNP-470 is more attractive because it is being tested in clinical trials. In addition, TNP-470 not only inhibits cancer cell growth, but also suppresses endothelial cell growth and angiogenesis. In the present study, the molecular mechanism by which TNP-470 exhibited growth inhibitory effects on CCA cell lines was shown to be the induction of apoptosis. The flow cytometry analysis revealed that TNP-470 did not affect cell cycle progression, but could induce apoptosis of CCA cells in a dose-dependent manner. TNP-470-mediated apoptosis has been reported in several cancers in vitro and in vivo.,
In this study, p38 MAPK activation is shown to be a key molecular mechanism of TNP-470-induced cell apoptosis of CCA cells. TNP-470 is a potent stimulator of p38 MAPK, a member of mitogen-activated protein kinases that plays an important role in the regulation of growth inhibition and differentiation, apoptosis, and autophagy., p38 MAPK activation is critically necessary for the induction of apoptosis by several anticancer agents., p38-mediated enhancement of pro-apoptotic response can be conducted via several pathways. p38 can stimulate the phosphorylation of Bcl-2 family proteins such as Bax and induce translocation of them from the cytosol to mitochondria, leading to the release of cytochrome C from the mitochondria., In this study, TNP-470 treatment activated p38 phosphorylation and consequently increased Bax and decreased Bcl-xL expressions. The mechanisms by which p38 induces Bax and inhibits Bcl-xL are presently unknown. Alterations of Bax and Bcl-xL in the mitochondria are known to reduce mitochondrial membrane potential, enhance cytochrome C release from the mitochondria, and activate the caspase cascade. The increased activity of caspase 3 and caspase 9 as shown by the increase of pro-caspases 3 and 9 after TNP-470 treatment supports this assumption. Taken all these together, the present results suggest that the antitumor action of TNP-470 is mediated via activation of p38, which modulates the cellular levels of Bax/Bcl-xL and subsequently activates programmed cell death.
In the present study, TNP-470 could substantially enhance the antitumor efficiency of four chemotherapeutic drugs (5-FU, doxorubicin, cisplatin, and gemcitabine) against two CCA cell lines, KKU-213 and KKU-214. The CI showed synergistic effect (CI <1) in combination between TNP-470 and chemotherapeutic drugs. Similar synergistic effect of TNP-470 with chemotherapeutic drugs has been reported on colon and gastric cancers., These findings suggest the possible use of TNP-470 as a potential chemotherapy sensitizer for clinical application in cancer, including CCA.
This work was supported by the TRF Senior Research Scholar Grant to S. Wongkham, Thailand Research Fund and Khon Kaen University (RTA5780012) and for the Higher Education Research Promotion and National Research University Project of Thailand, Office of the Higher Education Commission, through the Health cluster (SHeP-GMS, NRU582021) to W. Seubwai. We also acknowledge and thank Professor Yukifumi Nawa for editing this manuscript via the English Editing Publication Clinic, Faculty of Medicine, Khon Kaen.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2]
[Table 1], [Table 2]