Lanatoside C, a cardiac glycoside, acts through protein kinase Cδ to cause apoptosis of human hepatocellular carcinoma cells

www.nature.com/scientificreports OPEN received: 18 August 2016 accepted: 13 March 2017 Published: 07 April 2017 Lanatoside C, a cardiac glycoside, acts through protein kinase Cδ to cause apoptosis of human hepatocellular carcinoma cells Min-Wu Chao1,*, Tzu-Hsuan Chen2,*, Han-Li Huang1,*, Yu-Wei Chang3, Wei-Chun HuangFu1, Yu-Ching Lee4,5, Che-Ming Teng3,6 & Shiow-Lin Pan1,7 Recent studies have revealed that cardiac glycosides, such as digitalis and digoxin, have anticancer activity and may serve as lead compounds for the development of cancer treatments. The poor prognosis of hepatocellular carcinoma (HCC) patients reflects the development of resistance to current chemotherapeutic agents, highlighting the need for discovering new small-molecule therapeutics. Here, we found that lanatoside C, an anti-arrhythmic agent extracted from Digitalis lanata, inhibited the growth of HCC cells and dramatically decreased tumor volume as well as delayed tumor growth without obvious body weight loss. Moreover, lanatoside C triggered mitochondrial membrane potential (MMP) loss, activation of caspases and translocation of apoptosis-inducing factor (AIF) into the nucleus, which suggests that lanatoside C induced apoptosis through both caspase-dependent and -independent pathways. Furthermore, we discovered that lanatoside C activated protein kinase delta (PKCδ) via Thr505 phosphorylation and subsequent membrane translocation. Inhibition of PKCδ reversed lanatoside C-induced MMP loss and apoptosis, confirming that lanatoside C caused apoptosis through PKCδ activation. We also found that the AKT/mTOR pathway was negatively regulated by lanatoside C through PKCδ activation. In conclusion, we provide the first demonstration that the anticancer effects of lanatoside C are mainly attributable to PKCδ activation. Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is the fifth-leading cause of cancer-related death worldwide1. The incidence of HCC is not uniformly distributed geographically, with the highest frequency found in developing countries2. HCC often results from chronic liver diseases that accompany hepatitis B or C infection. Clinical management options for HCC, including surgical resection, liver transplantation, percutaneous ethanol injection, radiofrequency ablation, transarterial embolization as well as systemic chemotherapy, depend on tumor staging3. Unfortunately, HCC exhibits a high recurrence rate and relatively poor overall survival owing to late-stage disease at presentation, liver dysfunction, cellular resistance to conventional cytotoxic agents, and ineffectiveness of drugs4. Thus, developing an effective therapeutic strategy for HCC patients with an advanced form of the disease has always been a crucial issue. Cardiac glycosides comprise a large family of naturally derived compounds that share a common structural motif. Their core structure consists of a steroidal framework, which is considered the pharmacophoric moiety responsible for the activity of these compounds5. Lanatoside C and digoxin are structurally closely related in that digoxin can be obtained from lanatoside C by hydrolytic removal of the acetyl and glucose moieties (Fig. 1A)6. The major pharmacological effect of cardiac glycosides, still widely used clinically, is inhibition of the plasma membrane Na+/K+ ATPase, which indirectly enhances cardiac muscle contractile force. However, recent studies 1 The Ph.D. Program for Cancer Biology and Drug Discovery, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan. 2Division of Industrial Promotion, Development Center for Biotechnology, Taipei, Taiwan. 3Pharmacological Institute, College of Medicine, National Taiwan University, Taipei, Taiwan. 4The Center of Translational Medicine, Taipei Medical University, Taipei, Taiwan. 5Ph.D. Program for Biotechnology in Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan. 6School of Pharmacy, College of Pharmacy, Taipei Medical University, Taipei, Taiwan. 7Department of Pharmacology, College of Medicine, Taipei Medical University, Taipei, Taiwan. *These authors contributed equally to this work. Correspondence and requests for materials should be addressed to S.-L.P. (email: ) Scientific Reports | 7:46134 | DOI: 10.1038/srep46134 1 www.nature.com/scientificreports/ Figure 1. Effect of lanatoside C on cell proliferation, cell cycle of HCC cell lines, and Hep3B xenograft model. (A) Structure of lanatoside C. (B) Hep3B and HA22T cells were exposed to lanatoside C for 48 h, and then detected % of control cell growth by SRB assay. Lanatoside C induced Hep3B cell apoptosis in a concentrationdependent (C) and time-dependent (D) manner by FACScan flow cytometry analysis with propidium iodide (PI) staining. (E) Hep3B cells were incubated with lanatoside C in indicated concentration for 18 hr. Cells were stained with TUNEL assay (green fluorescence) and DAPI (blue fluorescence) in the same area. Magnification of TUNEL staining was 200X. (F) SCID mice were ectopically implanted with Hep3B cells. The upper curves show the effect of lanatoside C (2.5 mg/kg, ip, q3d or 2.5 mg/kg, ip, twice a week) on tumor volume and percentage of tumor growth delay (TGD), which was calculated for treatment groups relative to control group; the lower curves show the body weight of mice after indicated treatment. Data are expressed as means ± SEM of three independent determinations. *P < 0.05 and **P < 0.01, untreated cell versus lanatoside C-treated groups. Scientific Reports | 7:46134 | DOI: 10.1038/srep46134 2 www.nature.com/scientificreports/ suggest that several cardiac glycosides has been reported to exert anticancer activity through different mechanisms, such as the SRC/EGFR/RAS/ERK signaling pathway, p21, NF-κB, AP-1, topoisomerase and HIF-1, some of which do not involve targeting the Na+/K+ ion pump7–12. These observations suggest that cardiac glycosides could be repurposed as promising anticancer candidates. The mammalian protein kinase C (PKC) family of serine/threonine kinases is classified into three subfamilies based on calcium dependence and activators: conventional PKC (cPKC), novel PKC (nPKC), and atypical PKC (aPKC). PKC acts through a number of pathways to regulate the expression of genes that control cell survival, apoptosis, tumorigenesis, and/or metastasis. Diverse PKC isoenzymes act via complex mechanisms to exert variable responses in different cell types13. PKCδ, a member of the nPKC subfamily, was first isolated from the particulate fraction of mouse epidermis14. It has been reported that PKCδactivation, triggered by translocation to the cell membrane, is involved in many essential cellular processes, including regulation of cell growth and apoptosis in cells exposed to stimuli such as Fas, ultraviolet (UV) radiation, or etoposide-class chemotherapeutic drugs. In addition, PKCδis proteolytically activated by caspase-3. Sub (...truncated)