Liver cancer cells are sensitive to Lanatoside C induced cell death independent of their PTEN status
Graphical abstract
Introduction
Hepatocellular carcinoma is the sixth most common and one of the most fatal cancer type worldwide (Ferlay et al. 2012). Furthermore, incidence of HCC has been recently increased among individuals with obesity related liver disease in developed countries. Although the etiological factors that are thought to be fundamentally related to HCC are well-known (such as viral infections, toxic injury and recently obesity), there are still limited treatment options (Baffy et al. 2012). This is due to the fact that liver cancer cells are extremely resistant to conventional chemotherapy and radiotherapy. With the exception of Sorafenib, which generated notably modest clinical response, no well-known target-specific drug provided satisfactory clinical response to HCC in clinical assays (El-Serag et al. 2008). The five-year survival rate of HCC is only about seven percent and the recurrence rate is very high. Therefore, it is essential to recommend novel preventive or therapeutic approaches towards HCC (Yau et al. 2009).
Cancer cells acquire survival capacities by altering more than a single protein/pathway in parallel. Hence in these cancerous cells, there are proteins which exhibit altered functions in favor of cell survival and anti-proliferative cellular activities. Therefore, molecules targeting these signaling pathways are good candidates for targeted therapies (Ersahin et al. 2015). A good example to the alteration of mechanism in cancer cells would be the hyper-activation of the PI3K/Akt signaling pathway and hyperactivity of this pathway is frequently associated with numerous cancer types. PI3K/Akt pathway is involved in cell survival, proliferation, growth and metabolism and hence any changes in this pathway can alter key mechanisms required for cell survival and lead to various cancers (Ke et al. 2011). In HCC, constitutively active PI3K/Akt signaling due to loss of tumor suppressor protein PTEN is significantly associated with aggressive tumor behavior (Buontempo et al. 2011). Activation of PI3K/Akt/GSK-3β pathway in several cases of liver cancer was reported to induce EMT which promoted tumor invasion and metastasis (Shearn et al. 2011). Ras/Raf/MEK/ERK pathway, which also controls of cell survival mechanisms in liver cancer, has cross-talks with PI3K/Akt/GSK-3β pathway. Activation of ERK protein has dual effects on cell survival and death. Stress induced ERK activation can also promote cell death; therefore in some conditions where there are cross-talks between these pathways, Akt and ERK proteins have anti- and pro-apoptotic actions respectively (Cagnol and Chambard 2010).
Cardiac glycosides are steroid-like compounds and their main source is the Digitalis genus and many cell-signaling mechanisms have been described for their anticancer activities (Prassas and Diamandis 2008). One of the main mechanisms is based on the inhibition of Na+/K+-ATPase by cardiac glycosides and involves Ras pathway activation, which leads to the release of ROS, resulting in apoptosis. Bufalin, a member of cardiac glycoside family, was previously shown to induce ROS and SAPK/JNK pathway activation in human colon cancer cells (Xie et al. 2011). Another cardiac glycoside ouabain was also described to induce ROS in lung cancer cells, activate JNK and PI3K/Akt signaling in myocyte proliferation (Liu et al. 2007). Even though there are various studies, which show the effects of these natural drugs in various cancer cells, their effects on HCC are still not known. In this study, cytotoxicities of pure glycosides, which were originated from D. ferruginea, in liver cancer cells were investigated. The study also analyzed the underlying molecular cellular pathways for this activity in drug resistant PTEN deficient mesenchymal like Mahlavu liver cancer cells and drug sensitive PTEN adequate epithelial like Huh7 cells (Buontempo et al. 2011). It was demonstrated that ROS induced actions of glycosides alternatively modified Akt and ERK pathways in these two cells with different genetic backgrounds.
This study investigates the antitumor activity of cardiac glycosides, particularly Lanatoside C, in liver cancer cells both in vitro and in vivo. For this purpose, initially the cytotoxic activities of Lanatoside C along with other glycosides were tested on liver cancer cells. Lanatoside C was further analyzed at molecular level in order to identify the cellular pathways involved in this activity. Finally, the antitumor activity was exploited in nude mice xenograft models with PTEN deficient Mahlavu cell lines.
Section snippets
Extraction and fractionation of glycosides
Glycosides were obtained from Digitalis ferruginea (also known as “rusty foxglove”, Aladağ – Bolu, Turkey). Initial and majority of in vitro experiments were done with pure Lanatoside C (D. ferruginea) and further in vivo and in vitro studies were performed with Lanatoside C from Sigma Aldrich (≥95% purity by TLC, CID 656630) for versatility. Extraction and fractionation of cardioactive glycosides were done as described in the previous study (Calis et al. 1999a, 1999b). The air-dried and
Cytotoxicities of glycosides purified from Digitalis ferruginea
Cytotoxic activities of purified glycosides obtained from the D. ferruginea (Fig. 1) were initially investigated on both epithelial-like (Huh7, HepG2) and mesenchymal-like (FOCUS, Mahlavu) liver cancer cell lines. The cardiac glycosides Lanatoside A, Lanatoside C and Glucogitoroside showed significant levels of cytotoxicity whereas the phenylpropanoid glycosides had no notable effects on cell growth (Fig. 2a).
Time-dependent cytotoxic activities of cardiac glycosides were examined with the use
Discussion
Cardiac glycosides are among one of the oldest medicines and have been in use for centuries to treat heart failure (Prassas and Diamandis 2008). They are steroid-like compounds, which inhibit Na+/K+-ATPase and indirectly regulate cell signaling. This study investigated the molecular mechanisms of the cytotoxic effects of cardiac glycosides obtained from D. ferruginea on liver cancer cells in which the treatment required novel chemotherapeutic agents. Due to various etiological factors, liver
Conflict of interest
The authors declare no conflicts of interest.
Acknowledgments
The authors thank Dr. Ali Osmay Gure for discussions about the final version of the manuscript, Bilge Ozturk and Gamze Aykut, DVM for laboratory assistance and Ms. R. Nelson, M. Atalay (U. Chicago) and A.S. Kurt (U. King's College London) for edition of the manuscript in English.
References (26)
- et al.
Hepatocellular carcinoma in non-alcoholic fatty liver disease: an emerging menace
J. Hepatol.
(2012) Diagnosis and treatment of hepatocellular carcinoma
Gastroenterology
(2008)Bufalin induces autophagy-mediated cell death in human colon cancer cells through reactive oxygen species generation and JNK activation
Free Radic. Biol. Med.
(2011)- et al.
Superoxide production in rat hippocampal neurons: selective imaging with hydroethidine
J. Neurosci.
(1996) Inhibition of Akt signaling in hepatoma cells induces apoptotic cell death independent of Akt activation status
Investig. New Drugs
(2011)- et al.
ERK and cell death: mechanisms of ERK-induced cell death – apoptosis, autophagy and senescence
FEBS J.
(2010) Phenylethanoid and cardioactive glycosides from Digitalis ferruginea
Pharmazie
(1999)Phenylethanoid glycosides from Digitalis ferruginea subsp. ferruginea
Chem. Pharm. Bull.
(1999)- et al.
GSK-3: tricks of the trade for a multi-tasking kinase
J. Cell Sci.
(2003) - et al.
The PI3K/AKT/mTOR interactive pathway
Mol. Biosyst.
(2015)
Extrinsic versus intrinsic apoptosis pathways in anticancer chemotherapy
Oncogene
GSK3beta is involved in JNK2-mediated beta-catenin inhibition
PLoS One
Cited by (33)
Novel indole-pyrazole hybrids as potential tubulin-targeting agents; Synthesis, antiproliferative evaluation, and molecular modeling studies
2023, Journal of Molecular StructureNovel therapeutic strategies targeting UCP2 in uterine leiomyosarcoma
2023, Pharmacological ResearchTargeting the Otub1/c-Maf axis for the treatment of multiple myeloma
2021, BloodCitation Excerpt :LanC is a generic cardiac glycoside that could be used for congestive heart failure and cardiac arrythmia.25 Recent studies show that LanC induces apoptosis of several types of cancer cells, including gastric,26 liver,27 and colorectal cancers,28 and glioblastomas.29 The present study is the first report identifying LanC as an inhibitor of the Otub1/c-Maf axis.
Synthesis of new derivatives of boehmeriasin A and their biological evaluation in liver cancer
2019, European Journal of Medicinal ChemistryCitation Excerpt :In addition, these Boehmeriasin derivatives induced oxidative stress, that was assessed by evaluating the phosphorylation status of the proteins involved in the ROS pathway via western blot [25]. It was found that Mahlavu and Huh7 cells treated with 1 and 19 led to a decrease of the phosphorylated Ser-966-ASK1 levels which is associated with cellular oxidative stress, though the effect on Mahlavu is being more prominent [25] (Fig. 6B). Mahlavu cells are characterized by a hyperactive PI3K/AKT pathway due to PTEN protein deletion and therefore we observe differential downstream P-SAPK/JNK protein levels.
Cardenolides: Insights from chemical structure and pharmacological utility
2019, Pharmacological ResearchCitation Excerpt :In addition to the well-characterized pharmacological use of CGs in treating heart failure, cardenolides have been reported to have strong cytotoxic effects towards tumours. CGs were previously reported to possess anti-tumour activities against breast cancer, colon cancer, bladder cancer, liver cancer, gastric cancer, pancreatic cancer and lung cancer, and notably, safer CGs with fewer signs of toxicity have been prepared [101,102]. Oleandrin, ouabain, and digoxin induce cell death in human prostate cancer cell lines.
Cytotoxic activity of medicinal plants of the Kakamega County (Kenya) against drug-sensitive and multidrug-resistant cancer cells
2018, Journal of EthnopharmacologyCitation Excerpt :Another possibility to kill MDR cells is to by bypassing drug efflux by using cytotoxic natural compounds, which are not substrates of P-glycoprotein or BCRP and which are therefore not transported out of the cells by ABC transporters (Kuete et al., 2015, 2016b). Phytochemicals are appealing to kill drug-resistant cells, because they may reveal novel chemical structures that enlarge the chemical space (Durmaz et al., 2016; Shrestha et al., 2016; Juengel et al., 2017) and phytotherapy and natural products are frequently considered as being well tolerated without severe side effects. Instead of screening plants in a non-directed manner, it may be more promising to focus on plants from traditional medicine.