The Mechanism Of Herbal Compounds Exhibiting Antitumor Activity Via Induction Of Premature Senescence In Hepatocellular Carcinoma

Liver cancer is one of the most common cancers and the leading causes of cancer death worldwide.Hepatocellular carcinoma(HCC),accounting for 70-90%of primary liver cancers,is highly malignant with poor prognosis.Currently,surgical resection and transplantation remain the main approaches to treat HCC.However,most HCCs are inoperable due to advanced stages when surgery is no longer applicable.Advanced stage HCC always shows weak response to traditional radiotherapy and chemotherapy.Targeted therapy of sorafenib is the first choice for advanced HCC.However,the targeted therapy has the problem of unclear indications.In addition,acquired drug resistance and drug toxicity in patients limited long-term usage of sorafenib and other chemotherapeutic drugs.Therefore,there is an urgent need to study the mechanisms of the pathogenesis and drug resistance of HCC and explore alternative approaches using effective and less toxic anti-HCC agents.Cancer cells are driven to face their death and choose their subsequent fate following chemotherapy.These cell fate determinations include classical types of cell death,apoptosis and necroptosis,and therapy-induced cellular senescence.Senescence is defined as a state in which an irreversible permanent growth arrest occurs accompanied by changes in cell morphology and physiology.Senescent cells remain viable and metabolically active but losing proliferation potential and the ability for DNA synthesis and further cell division,thereby distinguishing senescence from cell death.Senescent cells display characteristic changes,typically including cell cycle arrest and increased senescence-associated-p-galactosidase(SA-?-Gal)activity,along with distinctive changes in morphology to flat and enlarged cell shape,the senescence-associated secretory phenotype(SASP),and expression alterations in the molecular biomarkers.Senescence can be prematurely triggered by various inappropriate stresses which include telomere dysfunction,oncogene activation,DNA damage,ROS and chromatin perturbation.Recently,accumulating data have demonstrated that senescence is a potent tumor suppressive mechanism to bar the initiation and development of cancer,and senescence-based chemotherapy,as a possible therapeutic option,has been receiving increasing attention.As a novel strategy,cellular senescence plays a positive role in many aspects.It can be induced when drug concentrations are significantly lower than those of traditional treatments,providing the possibility to reduce drug toxicity.At the same time,since the mechanism involved in cellular senescence is obviously different from apoptosis,it can provide new therapeutic targets for tumor cells that have been resistant to apoptosis.Therefore,it has become a research hotspot of anti-tumor research via inducing senescence of tumor cells by chemotherapy drugs.Novel natural anti-tumor drugs with high efficiency,low toxicity and multi-mechanism anti-cancer activity from natural resources provide a new idea for cancer treatment.Natural materials,as an important source of drugs,are one of the main strategies for the prevention and treatment of human diseases.Natural compounds have abundant sources,diverse structures and activities,among which many active ingredients in traditional Chinese medicine provide new ideas for the discovery of anti-tumor,anti-drug resistance and chemotherapeutic combination strategies.In this study,through evaluating pharmacological activities of natural compounds extracted from natural resources,we found the marked anti-tumor and senescence-inducing activities of acetyl-11-keto-?-boswellic acid(AKBA),a pentacyclic triterpenic acid isolated from the fragrant gum resin of the Boswellia serrate tree,and icaritin(ICT),a prenylflavonoid derivative from Epimedium brevicornu Maxim.Both Epimedium brevicornu Maxim and the fragrant gum resin are traditional Chinese Herbal medicines.We further explored the mechanisms of the two compounds to induce senescence and in vivo activities.In addition,due to sorafenib-resistance in HCC and lack of effective chemotherapy drugs,the drug-resistance inhibition effect of the two natural compounds was studied,thus laying the foundation for the discovery and clinical application of novel therapeutic strategies for HCC.Part ?,Antitumor and senescence-inducing activity screening of herbal compoundsObjective:To screen natural compounds with antitumor and senescence-inducing activity in HCC,and to conduct a detailed study on their pharmacological activity.Methods:1.The proliferation inhibition effects of various natural compounds on different HCC cells were detected by MTT assay;clone formation assay was used to further clarify the effects of the selected compounds on cell proliferation.2.Cell senescence was analyzed by SA-P-Gal staining and cell morphology changes to determine the cell senescence-inducing activity and optimal concentrations of the compounds.3.Apoptosis was detected by AnnextinV-FITC/PI staining,to determine the effects of different concentrations of compounds on apoptosis.4.The effects of selected compounds on cell cycle were determined by flow cytometry.5.The effects of selected compounds on cell DNA synthesis were determined through 5-Bromo-2-deoxyuridin(BrdU)incorporation assay.6.Expression changes of cell cycle regulators and inflammatory factors were detected by Western blotting and qRT-PCR.Results:1.It was determined for the first time that AKBA and ICT,the two natural compounds from herbals,had significant effects on proliferation inhibition and senescence induction in HCC cells.2.It was demonstrated that both AKBA and 1CT,induced the occurrence of cell senescence at low concentrations,but not induced significant apoptosis;however,both of them triggered apoptotic cell death at high concentrations.3.The concentrations of AKBA and ICT to induce senescence were identified:3-5?M and 1-2 ?M for AKBA and ICT respectively.At the present range of concentrations,both of the compounds were able to arrest cell cycle at G0/G1 phase,inhibit cell clone formation,and block DNA synthesis,thus further confirming their senescence-inducing activity.Conclusion:The discovery and clarification of the senescence-inducing activity of natural compounds AKBA and ICT suggested a novel anti-tumor mechanism,thus providing a theoretical basis for further research of AKBA and ICT as a new anti-HCC drug.Part ?.The mechanisms of acetyl-11-keto-?-boswellic acid and icaritin inducing cellular senescence in HCC and in vivo activity analysisObjective:Based on the results of the compound screening results of part I,to further explore the regulatory mechanism of AKBA and ICT in inducing cellular senescence and their pharmacological activities in vivo to induce cancer cell senescence.Methods:1.The effects of low concentrations of AKBA and ICT on DNA damage were analyzed by immunofluorescence and western blotting of yH2AX.2.Western blotting and qRT-PCR were used to detect the expression changes of DNA damage,DNA repair and cell cycle regulation related genes and proteins.3.The effects of AKBA and ICT on intracellular ROS production were determined by flow cytometry.4.p21CIP1 gene knockout was performed with siRNA to analyze the effect of p53/p21CIP1 pathway on AKBA-induced senescence.5.N-acetylcysteine(NAC)was used to pretreat cells to inhibit ROS production,then the role of ROS on ICT-induced DNA damage and cell senescence was analyzed.6.HCC xenografts were established by subcutaneously injecting HepG2 cells into 6-week-old nude mice.The antitumor effects of the compounds were determined by detecting the tumor weight.In addition,SA-?-Gal staining and changes in cell morphology were used to analyze induction of cell senescence in HCC cells in vivo,and the expression changes of key proteins were analyzed by immunofluorescence.Results:1.The results of immunofluorescence and western blotting showed that low concentrations of AKBA could significantly cause the formation and accumulation of yH2AX fluorescence punctuate,and the expression of yH2AX was significantly upregulated,indicating that low concentrations of AKBA could induce DNA damage in HCC cells.At the same time,AKBA can significantly inhibit the expression of multiple DNA repair genes involved in multiple DNA damage repair pathways,including homologous recombination and non-homologous end junctions(e.g.RAD51,RAD51AP1,RPA1.XRCC2,XRCC5,XRCC6,FENI),mismatch repair(e.g.MSH2,MSH6,EXO1),and nucleotide excision repair(e.g.DDB1,ERCC1,GTF2H3,RPA1).These results indicate that AKBA can induce DNA damage and simultaneously inhibit DNA repair,thus functioning together to potentiate its activity of promoting cellular senescence.Further,the signaling mechanism of AKBA inducing cell senescence was analyzed through activation of downstream signaling of DNA damage.It was found that p53/p21CIP1 played a key role,and the activity of AKBA induced cell senescence was significantly reduced by using p21CIP1-targeted siRNA.Thus,these results suggested a possible mechanism of AKBA suppressing cell growth,namely by promoting p53/p21CIP1 and in turn cell cycle arrest,thereby contributing to AKBA-driven cellular senescence upon DNA damage.The results of animal experiments showed that AKBA significantly inhibited the tumor growth.The AKBA group also showed increased positive rate of SA-P-Gal staining,the upregulated expression of yH2AX protein,and the activation of p53/p21CIP1 signaling pathway.These data suggested that AKBA can also induce cellular senescence through DNA damage and p53/p21CIP1 activation in vivo.2.After low concentrations of ICT treatment of HCC cells,ROS production increased significantly.with 30%increase for I ?M ICT,and 80%increase for 2 ?M ICT,respectively.yH2AX fluorescence punctuate formation and protein expression analysis showed that ROS induced by ICT led to DNA damage response.After NAC pretreatment to eliminate ROS,ICT-mediated ?H2AX expression and the positive rate of SA-P-Gal staining was significantly reduced,confirming that ICT-induced ROS overproduction and subsequent DNA damage caused senescence of HCC cells.Conclusion:Both AKBA and ICT have significant effects on inducing cell senescence,and their regulatory mechanisms are closely related to DNA damage,but there are differences between the two:AKBA-induced DNA damage and DNA repair inhibition activated p53/p21CIP1 pathway,which caused cell cycle arrest and loss of cell proliferation ability,ultimately leading to cell senescence;ICT induces and cell senescence by stimulating ROS overproduction and subsequent DNA damage.These results indicated the possibility and potential of their application as senescence-inducing drugs in the clinical treatment of HCC.Part ?.The mechanism of acetyl-11-keto-p-boswellic acid and icaritin inhibiting drug-resistant HCCObjective:Through the construction of sorafenib-resistant cell model,to discuss the drug-resistance inhibition activity of AKBA and ICT and the mechanism of action.Methods:1.The cell proliferation was detected by MTT assay.2.The invasion ability of cells was detected by Transwell assay.3.Cell senescence was analyzed by SA-?-Gal staining and cell morphology changes.4.The stem cell phenotype of drug-resistant cells was analyzed by cell mammosphere formation assay and stem cell-related gene expression.5.Cellular redox status was determined by ROS level,superoxide anion level and glutathione reduced/oxidized ratio(GSH/GSSG).6.Key signaling proteins affected by AKBA and ICT were identified by transfection of expression plasmids.Results:1.Through molecular event tracking and relevant regulatory signal analysis of HCC cells in the process of drug resistance,it was found that HCC cells escaped cell senescence during the continuous screening process of sorafenib,and the escaped cells developed to stably proliferative clones,which eventually developed into sorafenib-resistant cells Huh7/Sor.The resistant cells showed significant stem cell-like phenotypes in addition to drug resistance and aggressive characteristics;JAK/STAT3 and PI3K/AKT signaling pathways were activated.Meanwhile,the resistant cells showed low ROS levels,and GSH/GSSG ratio was significantly up-regulated.2.AKBA and ICT exerted significant inhibitory activity on drug-resistant cells Huh7/Sor.Specific mechanism studies revealed that AKBA and ICT can inhibit the stem cell phenotypes and induce cell senescence in resistant cells.Their sternness inhibitory activities were confirmed by mammosphere formation assay,sternness gene expression analysis and stem cell antibody array analysis.Further analysis of the signal regulation mechanism identified that JAK/STAT3 and PI3K/AKT were the main signaling pathways of AKBA and ICT affecting sternness and drug resistance.AKBA and ICT could also induce senescence in drug-resistant cells.Conclusion:AKBA and ICT have significant inhibitory activity on drug-resistant cells by inhibiting the stem cell phenotypes and inducing cell senescence.These findings further expanded the potential use of AKBA and ICT,providing a theoretical and material basis for their application in drug-resistant HCC or in combination with other chemotherapy drugs.