ORIGINAL ARTICLE Cytotoxic, apoptotic, and sensitization properties of ent-kaurane-type diterpenoids from Croton tonkinensis Gagnep on human liver cancer HepG2 and Hep3b cell lines Minh Quan Pham a,b,1 , Anne Laure Iscache c , Quoc Long Pham b , Jean Edouard Gairin a * a Facult  e des Sciences Pharmaceutiques, UPS, UMR 152 Pharma-DEV, Universit  e de Toulouse,Universit  e Toulouse 3, 35 Chemin des Mara ^ ıchers, Toulouse Cedex 9 F-31062, France b Institute of Natural Products Chemistry, Vietnam Academy of Science and Technology, Building 1H, 18 Hoang Quoc Viet, Hanoi, Vietnam c Plateau technique de cytom etrie et tri cellulaire, UMR INSERM 1043-CNRS 5282- Universit  e Toulouse 3, CHU Purpan, BP3028, 31024 Toulouse Cedex 3, France Keywords apoptosis, cytotoxicity, ent-kaurane type diterpenoid, Hepatocellular carcinoma, HepG2 and Hep3b cell lines, sensitization Received 12 October 2015; revised 16 December 2015; accepted 23 December 2015 *Correspondence and reprints: jean-edouard.gairin@univ-tlse3. fr 1 Recipient of a Vietnam government – Campus France USTH fellowship ABSTRACT Human hepatocellular carcinoma (HCC) is the most common type of liver cancer, the second most common cause of death from cancer worldwide. A very poor prognosis and a lack of effective treatments make liver cancer a major public health problem, notably in less developed regions, particularly in eastern Asia. This fully justifies the search of new molecules and therapeutic strategies against HCC. Ent-kaurane diterpenoids are natural compounds displaying a broad spec- trum of potential therapeutic effects including anticancer activity. In this study, we analyzed the pharmacological properties of a family of ent-kaurane diter- penoids from Croton tonkinensis Gagnep in human HepG2 and Hep3b cell lines, used as cellular reference models for in vitro evaluation of new molecules active on HCC. A structure-related cytotoxicity was observed against both HCC cell lines, enlighting the role of the 16-en-15-one skeleton of ent-kaurane diter- penoids. Cytotoxicity was closely correlated to apoptosis, evidenced by concentra- tion-dependent subG1 cell accumulation, and increased annexin V expression. In addition, subtoxic concentration of ent-kaurane diterpenoid dramatically enhanced the sensitivity of HCC cells to doxorubicin. All together, our data bring strong support to the potential interest of ent-kaurane diterpenoids, alone or in combina- tion with a cytotoxic agent, in cancer and more precisely against HCC. INTRODUCTION Hepatocellular carcinoma (HCC) is the most common type (>80%) of liver cancer which has become the sec- ond most common cause of death from cancer world- wide, with estimated 746 000 deaths and 782 000 new cases in 2012, according to the most recent GLOBOCAN data from the International Agency for Research on Cancer (http://www.globocan.iarc.fr/) [1]. More than 80% of these deaths and new cases occur in the less developed regions, particularly in eastern Asia, making liver cancer a major public health problem in these countries. The very poor prognosis for liver cancer (over- all ratio mortality/incidence: 0.95) together with the lack of effective treatments makes the search of new molecules and/or new therapeutic strategies a challeng- ing goal. As a part of our ongoing program aimed at identify- ing Vietnamese natural compounds endowed with antiproliferative properties against HCC, we focused our ª 2015 Soci et e Franc ßaise de Pharmacologie et de Th erapeutique Fundamental & Clinical Pharmacology 30 (2016) 137–146 137 doi: 10.1111/fcp.12176