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

Chadamas Sakonsinsiri; Waleeporn Kaewlert; Napat Armartmuntree; Raynoo Thanan; Poungrat Pakdeechote

doi:10.14715/cmb/2018.64.10.5

Issue

Vol. 64 No. 10: Issue 10

Issue Published : August 3, 2018

The undersigned hereby assign all rights, included but not limited to copyright, for this manuscript to CMB Association upon its submission for consideration to publication on Cellular and Molecular Biology. The rights assigned include, but are not limited to, the sole and exclusive rights to license, sell, subsequently assign, derive, distribute, display and reproduce this manuscript, in whole or in part, in any format, electronic or otherwise, including those in existence at the time this agreement was signed. The authors hereby warrant that they have not granted or assigned, and shall not grant or assign, the aforementioned rights to any other person, firm, organization, or other entity. All rights are automatically restored to authors if this manuscript is not accepted for publication.

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

https://doi.org/10.14715/cmb/2018.64.10.5

Chadamas Sakonsinsiri

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

Waleeporn Kaewlert

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

Napat Armartmuntree

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

Raynoo Thanan

Department of Biochemistry, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

Poungrat Pakdeechote

Department of Physiology, Faculty of Medicine, Khon Kaen University, Khon Kaen 40002, Thailand.

Corresponding Author(s) : Chadamas Sakonsinsiri

schadamas@kku.ac.th

Cellular and Molecular Biology, Vol. 64 No. 10: Issue 10
Article Published : July 30, 2018

Abstract

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.

Keywords

Cholangiocarcinoma Asiatic acid Plant-derived compound Cytotoxicity Apoptosis.

Sakonsinsiri, C., Kaewlert, W., Armartmuntree, N., Thanan, R., & Pakdeechote, P. (2018). Anti-cancer activity of asiatic acid against human cholangiocarcinoma cells through inhibition of proliferation and induction of apoptosis. Cellular and Molecular Biology, 64(10), 28–33. https://doi.org/10.14715/cmb/2018.64.10.5

Download Citation

References

Srivatanakul P, Ohshima H, Khlat M, Parkin M, Sukaryodhin S, Brouet I, et al. Opisthorchis viverrini infestation and endogenous nitrosamines as risk factors for cholangiocarcinoma in Thailand. Int J Cancer. 1991;48:821-5.
Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, et al. Liver Fluke Induces Cholangiocarcinoma. PLoS Med. 2007;4:e201.
Kaewpitoon N, Kaewpitoon SJ, Pengsaa P, Sripa B. Opisthorchis viverrini: the carcinogenic human liver fluke. World J Gastroenterol. 2008;14:666-74.
Parkin DM. The global health burden of infection-associated cancers in the year 2002. Int J Cancer. 2006;118:3030-44.
Sithithaworn P, Yongvanit P, Duenngai K, Kiatsopit N, Pairojkul C. Roles of liver fluke infection as risk factor for cholangiocarcinoma. J Hepatobiliary Pancreat Sci. 2014;21:301-8.
Thanan R, Oikawa S, Hiraku Y, Ohnishi S, Ma N, Pinlaor S, et al. Oxidative stress and its significant roles in neurodegenerative diseases and cancer. Int J Mol Sci. 2014;16:193-217.
Malhi H, Gores GJ. Cholangiocarcinoma: Modern advances in understanding a deadly old disease. J Hepatol. 2006;45:856-67.
Hyder O, Hatzaras I, Sotiropoulos GC, Paul A, Alexandrescu S, Marques H, et al. Recurrence after operative management of intrahepatic cholangiocarcinoma. Surgery. 2013;153:811-8.
Eckmann KR, Patel DK, Landgraf A, Slade JH, Lin E, Kaur H, et al. Chemotherapy outcomes for the treatment of unresectable intrahepatic and hilar cholangiocarcinoma: A retrospective analysis. Gastrointest Cancer Res. 2011;4:155-60.
Okusaka T, Nakachi K, Fukutomi A, Mizuno N, Ohkawa S, Funakoshi A, et al. Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan. Br J Cancer. 2010;103:469-74.
Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010;362:1273-81.
Marica Bakovic NH. Biologically Active Triterpenoids and Their Cardioprotective and Anti- Inflammatory Effects. J Bioanal Biomed. 2015;01.
Parmar SK, Sharma TP, Airao VB, Bhatt R, Aghara R, Chavda S, et al. Neuropharmacological effects of triterpenoids. Phytopharmacology. 2013;4:354-72.
Yadav VR, Prasad S, Sung B, Kannappan R, Aggarwal BB. Targeting inflammatory pathways by triterpenoids for prevention and treatment of cancer. Toxins. 2010;2:2428-66.
Somboonwong J, Kankaisre M, Tantisira B, Tantisira MH. Wound healing activities of different extracts of Centella asiatica in incision and burn wound models: an experimental animal study. BMC Complement Altern Med. 2012;12:1115-.
Huang GJ, Huang SS, Chiu CS, Chen HJ, Hou WC, Sheu MJ, et al. Antinociceptive activities and the mechanisms of anti-inflammation of asiatic acid in mice. Evid Based Complement Alternat Med. 2011;2011.
Pakdeechote P, Bunbupha S, Kukongviriyapan U, Prachaney P, Khrisanapant W, Kukongviriyapan V. Asiatic acid alleviates hemodynamic and metabolic alterations via restoring eNOS/iNOS expression, oxidative stress, and inflammation in diet-induced metabolic syndrome rats. Nutrients. 2014;6:355-70.
Maneesai P, Bunbupha S, Kukongviriyapan U, Prachaney P, Tangsucharit P, Kukongviriyapan V, et al. Asiatic acid attenuates renin-angiotensin system activation and improves vascular function in high-carbohydrate, high-fat diet fed rats. BMC Complement Altern Med. 2016;16:123.
Zhang W, Men X, Lei P. Review on anti-tumor effect of triterpene acid compounds. J Cancer Res Ther. 2014;10 Suppl 1:14-9.
Wang X, Lu Q, Yu DS, Chen YP, Shang J, Zhang LY, et al. Asiatic acid mitigates hyperglycemia and reduces islet fibrosis in Goto-Kakizaki rat, a spontaneous type 2 diabetic animal model. Chin J Nat Med. 2015;13:529-34.
Lv H, Qi Z, Wang S, Feng H, Deng X, Ci X. Asiatic Acid Exhibits Anti-inflammatory and Antioxidant Activities against Lipopolysaccharide and d-Galactosamine-Induced Fulminant Hepatic Failure. Front Immunol. 2017;8:785.
Hsu YL, Kuo PL, Lin LT, Lin CC. Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells. J Pharmacol Exp Ther. 2005;313:333-44.
Park BC, Bosire KO, Lee ES, Lee YS, Kim JA. Asiatic acid induces apoptosis in SK-MEL-2 human melanoma cells. Cancer Lett. 2005;218:81-90.
Lee YS, Jin DQ, Kwon EJ, Park SH, Lee ES, Jeong TC, et al. Asiatic acid, a triterpene, induces apoptosis through intracellular Ca2+ release and enhanced expression of p53 in HepG2 human hepatoma cells. Cancer Lett. 2002;186:83-91.
Cho CW, Choi DS, Cardone MH, Kim CW, Sinskey AJ, Rha C. Glioblastoma cell death induced by asiatic acid. Cell Biol Toxicol. 2006;22:393-408.
Tang X-L, Yang X-Y, Jung H-J, Kim S-Y, Jung S-Y, Choi D-Y, et al. Asiatic acid induces colon cancer cell growth inhibition and apoptosis through mitochondrial death cascade. Biol Pharm Bull 2009;32:1399-405.
Gurfinkel DM, Chow S, Hurren R, Gronda M, Henderson C, Berube C, et al. Disruption of the endoplasmic reticulum and increases in cytoplasmic calcium are early events in cell death induced by the natural triterpenoid Asiatic acid. Apoptosis. 2006;11:1463-71.
Ren L, Cao Q-X, Zhai F-R, Yang S-Q, Zhang H-X. Asiatic acid exerts anticancer potential in human ovarian cancer cells via suppression of PI3K/Akt/mTOR signalling. Pharm Biol. 2016;54:2377-82.
Wu T, Geng J, Guo W, Gao J, Zhu X. Asiatic acid inhibits lung cancer cell growth in vitro and in vivo by destroying mitochondria. Acta Pharm Sin B. 2017;7:65-72.
Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82:1107-12.
Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. 2007;35:495-516.
Wu Q, Lv T, Chen Y, Wen L, Zhang J, Jiang X, et al. Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway. Mol Med Rep. 2015;12:1429-34.
Yongvanit P, Pinlaor S, Bartsch H. Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis. Parasitol Int. 2012;61:130-5.
Pinlaor S, Ma N, Hiraku Y, Yongvanit P, Semba R, Oikawa S, et al. Repeated infection with Opisthorchis viverrini induces accumulation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanine in the bile duct of hamsters via inducible nitric oxide synthase. Carcinogenesis. 2004;25:1535-42.
Pinlaor S, Hiraku Y, Ma N, Yongvanit P, Semba R, Oikawa S, et al. Mechanism of NO-mediated oxidative and nitrative DNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis. Nitric Oxide. 2004;11:175-83.
Thanan R, Pairojkul C, Pinlaor S, Khuntikeo N, Wongkham C, Sripa B, et al. Inflammation-related DNA damage and expression of CD133 and Oct3/4 in cholangiocarcinoma patients with poor prognosis. Free Radic Biol Med. 2013;65:1464-72.
Armartmuntree N, Murata M, Techasen A, Yongvanit P, Loilome W, Namwat N, et al. Prolonged oxidative stress down-regulates Early B cell factor 1 with inhibition of its tumor suppressive function against cholangiocarcinoma genesis. Redox Biol. 2018;14:637-44.

References

Srivatanakul P, Ohshima H, Khlat M, Parkin M, Sukaryodhin S, Brouet I, et al. Opisthorchis viverrini infestation and endogenous nitrosamines as risk factors for cholangiocarcinoma in Thailand. Int J Cancer. 1991;48:821-5.

Sripa B, Kaewkes S, Sithithaworn P, Mairiang E, Laha T, Smout M, et al. Liver Fluke Induces Cholangiocarcinoma. PLoS Med. 2007;4:e201.

Kaewpitoon N, Kaewpitoon SJ, Pengsaa P, Sripa B. Opisthorchis viverrini: the carcinogenic human liver fluke. World J Gastroenterol. 2008;14:666-74.

Parkin DM. The global health burden of infection-associated cancers in the year 2002. Int J Cancer. 2006;118:3030-44.

Sithithaworn P, Yongvanit P, Duenngai K, Kiatsopit N, Pairojkul C. Roles of liver fluke infection as risk factor for cholangiocarcinoma. J Hepatobiliary Pancreat Sci. 2014;21:301-8.

Thanan R, Oikawa S, Hiraku Y, Ohnishi S, Ma N, Pinlaor S, et al. Oxidative stress and its significant roles in neurodegenerative diseases and cancer. Int J Mol Sci. 2014;16:193-217.

Malhi H, Gores GJ. Cholangiocarcinoma: Modern advances in understanding a deadly old disease. J Hepatol. 2006;45:856-67.

Hyder O, Hatzaras I, Sotiropoulos GC, Paul A, Alexandrescu S, Marques H, et al. Recurrence after operative management of intrahepatic cholangiocarcinoma. Surgery. 2013;153:811-8.

Eckmann KR, Patel DK, Landgraf A, Slade JH, Lin E, Kaur H, et al. Chemotherapy outcomes for the treatment of unresectable intrahepatic and hilar cholangiocarcinoma: A retrospective analysis. Gastrointest Cancer Res. 2011;4:155-60.

Okusaka T, Nakachi K, Fukutomi A, Mizuno N, Ohkawa S, Funakoshi A, et al. Gemcitabine alone or in combination with cisplatin in patients with biliary tract cancer: a comparative multicentre study in Japan. Br J Cancer. 2010;103:469-74.

Valle J, Wasan H, Palmer DH, Cunningham D, Anthoney A, Maraveyas A, et al. Cisplatin plus gemcitabine versus gemcitabine for biliary tract cancer. N Engl J Med. 2010;362:1273-81.

Marica Bakovic NH. Biologically Active Triterpenoids and Their Cardioprotective and Anti- Inflammatory Effects. J Bioanal Biomed. 2015;01.

Parmar SK, Sharma TP, Airao VB, Bhatt R, Aghara R, Chavda S, et al. Neuropharmacological effects of triterpenoids. Phytopharmacology. 2013;4:354-72.

Yadav VR, Prasad S, Sung B, Kannappan R, Aggarwal BB. Targeting inflammatory pathways by triterpenoids for prevention and treatment of cancer. Toxins. 2010;2:2428-66.

Somboonwong J, Kankaisre M, Tantisira B, Tantisira MH. Wound healing activities of different extracts of Centella asiatica in incision and burn wound models: an experimental animal study. BMC Complement Altern Med. 2012;12:1115-.

Huang GJ, Huang SS, Chiu CS, Chen HJ, Hou WC, Sheu MJ, et al. Antinociceptive activities and the mechanisms of anti-inflammation of asiatic acid in mice. Evid Based Complement Alternat Med. 2011;2011.

Pakdeechote P, Bunbupha S, Kukongviriyapan U, Prachaney P, Khrisanapant W, Kukongviriyapan V. Asiatic acid alleviates hemodynamic and metabolic alterations via restoring eNOS/iNOS expression, oxidative stress, and inflammation in diet-induced metabolic syndrome rats. Nutrients. 2014;6:355-70.

Maneesai P, Bunbupha S, Kukongviriyapan U, Prachaney P, Tangsucharit P, Kukongviriyapan V, et al. Asiatic acid attenuates renin-angiotensin system activation and improves vascular function in high-carbohydrate, high-fat diet fed rats. BMC Complement Altern Med. 2016;16:123.

Zhang W, Men X, Lei P. Review on anti-tumor effect of triterpene acid compounds. J Cancer Res Ther. 2014;10 Suppl 1:14-9.

Wang X, Lu Q, Yu DS, Chen YP, Shang J, Zhang LY, et al. Asiatic acid mitigates hyperglycemia and reduces islet fibrosis in Goto-Kakizaki rat, a spontaneous type 2 diabetic animal model. Chin J Nat Med. 2015;13:529-34.

Lv H, Qi Z, Wang S, Feng H, Deng X, Ci X. Asiatic Acid Exhibits Anti-inflammatory and Antioxidant Activities against Lipopolysaccharide and d-Galactosamine-Induced Fulminant Hepatic Failure. Front Immunol. 2017;8:785.

Hsu YL, Kuo PL, Lin LT, Lin CC. Asiatic acid, a triterpene, induces apoptosis and cell cycle arrest through activation of extracellular signal-regulated kinase and p38 mitogen-activated protein kinase pathways in human breast cancer cells. J Pharmacol Exp Ther. 2005;313:333-44.

Park BC, Bosire KO, Lee ES, Lee YS, Kim JA. Asiatic acid induces apoptosis in SK-MEL-2 human melanoma cells. Cancer Lett. 2005;218:81-90.

Lee YS, Jin DQ, Kwon EJ, Park SH, Lee ES, Jeong TC, et al. Asiatic acid, a triterpene, induces apoptosis through intracellular Ca2+ release and enhanced expression of p53 in HepG2 human hepatoma cells. Cancer Lett. 2002;186:83-91.

Cho CW, Choi DS, Cardone MH, Kim CW, Sinskey AJ, Rha C. Glioblastoma cell death induced by asiatic acid. Cell Biol Toxicol. 2006;22:393-408.

Tang X-L, Yang X-Y, Jung H-J, Kim S-Y, Jung S-Y, Choi D-Y, et al. Asiatic acid induces colon cancer cell growth inhibition and apoptosis through mitochondrial death cascade. Biol Pharm Bull 2009;32:1399-405.

Gurfinkel DM, Chow S, Hurren R, Gronda M, Henderson C, Berube C, et al. Disruption of the endoplasmic reticulum and increases in cytoplasmic calcium are early events in cell death induced by the natural triterpenoid Asiatic acid. Apoptosis. 2006;11:1463-71.

Ren L, Cao Q-X, Zhai F-R, Yang S-Q, Zhang H-X. Asiatic acid exerts anticancer potential in human ovarian cancer cells via suppression of PI3K/Akt/mTOR signalling. Pharm Biol. 2016;54:2377-82.

Wu T, Geng J, Guo W, Gao J, Zhu X. Asiatic acid inhibits lung cancer cell growth in vitro and in vivo by destroying mitochondria. Acta Pharm Sin B. 2017;7:65-72.

Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, et al. New colorimetric cytotoxicity assay for anticancer-drug screening. J Natl Cancer Inst. 1990;82:1107-12.

Elmore S. Apoptosis: A Review of Programmed Cell Death. Toxicol Pathol. 2007;35:495-516.

Wu Q, Lv T, Chen Y, Wen L, Zhang J, Jiang X, et al. Apoptosis of HL-60 human leukemia cells induced by Asiatic acid through modulation of B-cell lymphoma 2 family proteins and the mitogen-activated protein kinase signaling pathway. Mol Med Rep. 2015;12:1429-34.

Yongvanit P, Pinlaor S, Bartsch H. Oxidative and nitrative DNA damage: key events in opisthorchiasis-induced carcinogenesis. Parasitol Int. 2012;61:130-5.

Pinlaor S, Ma N, Hiraku Y, Yongvanit P, Semba R, Oikawa S, et al. Repeated infection with Opisthorchis viverrini induces accumulation of 8-nitroguanine and 8-oxo-7,8-dihydro-2'-deoxyguanine in the bile duct of hamsters via inducible nitric oxide synthase. Carcinogenesis. 2004;25:1535-42.

Pinlaor S, Hiraku Y, Ma N, Yongvanit P, Semba R, Oikawa S, et al. Mechanism of NO-mediated oxidative and nitrative DNA damage in hamsters infected with Opisthorchis viverrini: a model of inflammation-mediated carcinogenesis. Nitric Oxide. 2004;11:175-83.

Thanan R, Pairojkul C, Pinlaor S, Khuntikeo N, Wongkham C, Sripa B, et al. Inflammation-related DNA damage and expression of CD133 and Oct3/4 in cholangiocarcinoma patients with poor prognosis. Free Radic Biol Med. 2013;65:1464-72.

Armartmuntree N, Murata M, Techasen A, Yongvanit P, Loilome W, Namwat N, et al. Prolonged oxidative stress down-regulates Early B cell factor 1 with inhibition of its tumor suppressive function against cholangiocarcinoma genesis. Redox Biol. 2018;14:637-44.

Author biographies is not available.