Targeting Hepatocellular Carcinoma With Natural Sesquiterpene Lactone Compounds

Liver cancer is the 5th most frequently diagnosed cancer worldwide and is 2nd and 6th leading cause of cancer-related deaths in men and women respectively.Liver cancer incidence has increased from 748,300 in 2008 to 782500 in 2012 and liver cancer-related deaths have risen from 695,900 in 2008 to 745,500 in 2012 worldwide.Only in China,383,000 deaths occurred due to liver cancer which accounts for 50 % of all deaths occurred worldwide.There are various treatment options and strategies to cure hepatocellular carcinoma and selection of treatment options,strategies and clinical presentation of HCC depends on the conditions,stages of diagnosis,liver function and extent of portal hypertension,Child-Pugh and overall health status of the patients.At present,the available treatment options for liver cancer include surgery,radiotherapy and chemotherapy.Despite advancements in the existing therapies,the overall prognosis of advanced hepatocellular carcinoma still remains very poor with 5 year survival rate about 10 %.Heterogeneity of liver cancer makes the selection of treatment option more difficult.Currently,sorafenib is the only FDA-approved drug for advanced stage HCC patients.However,drug resistance and severe toxicity has become the major limitation of this drug.In addition to sorafenib,several other chemotherapeutic drugs such as doxorubicin,5-fluorouracil and cisplatin have been tested against HCC,however,these drugs failed to improve the survival rate of patients with HCC.As cancer development and progression result from the aberrations in multiple signaling cascades,it has been therefore,an ongoing challenge for pharmacologist to develop a multi-target medicine to cure cancers due to complexity of molecular pathogenesis.However,cancer is a preventable ailment and concept of chemoprevention by natural products especially of plant origin,is gaining more importance as it is believed to be safe,cost effective and alternative approach for cancer treatment.Natural products are the richest source of outstanding chemical and structural diversity and represent the most fruitful authenticated approach of small molecule drug discovery.Throughout the human history,natural products particularly plant-based systems have been extensively practiced in the treatment of wide spectrum ailments.Plants are considered as the basic milestone to lay the foundation of traditional medicine systems.The medicinal use of 1000 plant-derived formulations which are still in use for the treatment of parasitic infections and inflammations has been documented,dating back around 2600 BC,in Mesopotamia.The importance of natural products can be valued by the fact that 87% of all categorized human disease including cancer,bacterial/parasitic infections and inflammations are being treated with natural product formulations.According to the WHO report,80% of global population still relies on plant-derived medicines for their primary health care.Statistical reports show that more than 60% FDA approved commercially available anticancer drugs are derived from natural sources including plants,marine organisms and micro-organisms.More than 3000 plant species have been reported to treat cancers.Moreover,about 20% cancer incidence and 200,000 cancer-related deaths can be prohibited with more consumption of fruits and vegetables.Phyto-compounds such as nutraceuticals including curcumin,epigallocatechingallate,quercetin,genisteinandcapsaicin,and sesquiterpene lactones including parthenolide,dihydroartemisinin,artemisinin,and artesunate,costunolide and dehydrocostuslactonehave have been shown to exhibit anti-inflammatory and antitumor activities against various cancers including esophageal cancer,leukemia,lung cancer,pancreatic cancer,breast cancer,colon and liver cancer.Sesquiterpene lactones represent a large number of naturally occurring and biologically active phytocompounds,“terpenoids”,with a skeleton of 15 carbons.More than 5000 SLs have been identified in various plant families including Apiaceae,Anacardiaceae,Acanthaceae,Araceae,Cactaceae,Euphorbiaceae,Hepatideae,Lauraceae,Magnoliaceae,Menispermaceae,Rutaceae,Solanaceaeand Winteraceae.The presence of ?-methylene-?-lactone entity attributes to broad spectrum of several biological activities of sesquiterpene lactone compounds such as antifungal,antibacterial,antiviral,cytotoxic,antimalarial and anticancer activity.Several studies have shown that sesquiterpene lactone compounds induce apoptosis in cancer cells through multiple pathways.Some of them have reached cancer clinical trial.Thus,it is necessary to explore new sesquiterpene lactones and their underlying mechanism for cancer treatments.The study was therefore,aimed to identify novel sesquiterpene lactones with promising anti-hepatoma activity.We have identified several sesquiterpene lactones with anticancer activity,however,due to time limitation,two compounds: deoxyelephantopin and santamarine were selected for further study to find out their cellular targets and underlying molecular mechanisms in HepG2 liver cancer cells.In the first project,we have investigated the anti-hepatoma mechanism of Deoxyelephantopin(DET).DET a naturally occurring sesquiterpene lactone present in Chinese medicinal herbs,Elephantopus scaber and Elephantopus carolinianus,has been shown to exert anti-inflammatory as well as anticancer effects in various cancer cells of human origin in vitro.However,the exact molecular mechanism underlying DET-induced apoptosis remains largely unexplored,particularly in human hepatocellular carcinoma G2(HepG2)cells.We found that DET inhibits proliferation and induces apoptosis in HepG2 cells in a dose-dependent manner.This DET-mediated apoptosis was found to be associated with reactive oxygen species(ROS)generation,glutathione(GSH)depletion and decreased activity of thioredoxin reductase(TrxR),mitochondrial membrane potential(MMP)disruption,Bcl-2 family proteins modulation,cytochrome c release,caspases-3 activation,PARP cleavage and inhibition of NF-?B activation.DET inhibited the constitutive as well as induced-translocation of NF-?B into nucleus and augmented the apoptotic effect of Gemcitabine.IKK-16(IKK inhibitor)further enhanced the cytotoxicity of DET and gemcitabine indicating that DET induces apoptosis in HepG2 cells at least partially through inhibition of NF-?B activation.Further mechanistic study demonstrated that DET inhibits the translocation of constitutive as well as induced-NF-?B into nucleus by decreasing phosphorylation of IkB?.Moreover,pretreatment of cells with 3 mM NAC reversed DET-mediated cell death and NF-?B inhibition,indicating that DET exerts its anticancer effects mainly through oxidative stress.Therefore,DET may be developed into a lead chemotherapeutic drug as a single agent or in combination with clinical drugs for the effective treatment of liver cancer.In the second research report,we have identified and demonstrated the potential anticancer mechanism of novel sesquiterpene lactone “santamarine” in HepG2 cells.Santamarine is a component of Saussurealappa,Costusspeciosus and Ambrosia confertiflora and it has been shown to exhibits anti-bacterial and anti-inflammatory activity.Here,we found that it inhibited proliferation and induced apoptosis dosedependently with IC50 about 70 ?M.Induction of apoptosis was found to be linked with increased reactive oxygen species(ROS)generation,decreased activity of thioredoxin reductase(TrxR),glutathione(GSH)depletion,mitochondrial membrane potential(??m)dissipation,Bcl-2 family proteins modulation,cytochrome c release,caspases-8,-9 and-3 activation and PARP cleavage.Further mechanistic study demonstrated that STM inhibited the constitutive and TNF-?-induced translocation of NF-?B into nucleus by decreasing phosphorylation of IkB-?.Moreover,STM inhibited STAT3 activation by decreasing phosphorylation at tyrosine-705.NAC pretreatment reversed the effect of STM-mediated cell death,NF-?B inhibition and blockage of STAT3 activity,indicating the involvement of oxidative stress in STM-mediated anticancer activity.IKK-16 inhibited the activation of STAT3 indicating that NF-?B might act as an upstream regulator of STAT3 activation.Further studies are needed to explore the exact molecular mechanism of STM-induced apoptosis to develop it into a lead for treatment of liver cancer in future.