Adenovirus Mediated Co-expression Of ING4 And PTEN Cooperatively Enhances Their Antitumor Activity In Human Hepatic Carcinoma Cells

Objective:The objective of this study is to explore the combined effect of ING4 and PTEN tumor suppressors,on cell growth,apoptosis,migration,invasion and cell cycle in HepG2 and SMMC-7721 cell line of the human hepatic carcinoma cells.This study is based on previous antitumor feature studies of combined suppressors ING4 and PTEN in human nasopharyngeal carcinoma cells,which demonstrate that co-expression of ING4 and PTEN enhance growth inhibition,apoptosis stimulation and suppression of tumor angiogenesis.Methods:1.Recombinant adenoviruses co-expressing ING4 and PTEN(Ad-ING4-PTEN)were constructed using the Internal Ribosome Entry Site(IRES)strategy.2.The SMMC-7721 and HepG2 cells were infected with Ad-ING4-PTEN,Ad-ING4,Ad-PTEN,and Ad.3.The adenoviral infection efficiency was examined according to GFP expression using fluorescence microscopy and the ING4 and PTEN trans-gene expression was determined using western blot.4.Cell proliferation of SMMC-7721 and HepG2 cell line of Human HCC was measured by MTT assay.5.Cell apoptosis rate and cell cycle analysis in each cell line was measured by Flow Cytometry.6.The effect of combined tumor suppressors on the cell migration and invasion on both the cell lines was measured by Wound Healing assay and Trans-well assay respectively.7.Western Blot was applied to analyze the role of the combined tumor suppressors on apoptotic and cell-cycled related proteins.Results:1.The MTT assay showed that the infection of recombinant adenovirus co-expressing ING4 and PTEN prolongs the cell proliferation of human HCC cells,longer than individual tumor suppressors.2.Flow cytometry showed that combination of ING4 and PTEN induced synergistic apoptosis stimulation and co-operatively regulated the cell cycle with more cells in the G2/M phase arrest.3.Wound healing and Trans-well assay demonstrated Ad-ING4-PTEN promoted migration and invasion suppression in both the HCC cell lines.4.Western blot showed that Ad-ING4-PTEN induced an enhanced effect on the altered expression of cell cycle-related(P53,P21 and Cyclin-D1)and apoptosis-related proteins(Bad,Bcl-2,BCL-XL and BAX)Conclusion:The above results prove that infection of adenovirus mediated co-expressed tumor suppressors ING4 and PTEN can induce enhanced synergistic cancer cell growth inhibition,invasion suppression and may lead to enhancement of cell cycle alteration and cooperative activation of apoptotic pathways,and be responsible for the synergistic antitumor effect.These results indicate that cancer gene therapy,combining two or more tumor suppressors such as ING4 and PTEN,may constitute a novel and effective therapeutic strategy for hepatocellular carcinoma.Introduction:Hepatocellular carcinoma(HCC)is the fifth most common cancer in the world,and is the third common cause of cancer-related mortality(1).It is responsible for 600,000 deaths annually(53).In most countries,HCC accounts for 70%-85%of primary liver cancer cases(54);this disease burden is expected to increase over the next few years(55).In the recently published GLOBOCAN(2012),which is a useful cited source of information in worldwide incidence on common cancer types,it was estimated that 55%of HCC cases were recorded in China alone(94)and 82%of total HCC cases were recorded in developing countries.Liver cancer incidences are moderately high(11-20 cases per 100,000 males)or very high(>20 per 100,000)in China,Southeastern Asia,and Sub-Saharan,Western&Eastern Africa(94,95).In most developed areas of the world,including North America and much of Europe,liver cancer incidences are at low(<5 per 100,000)or intermediate(5-10 per 100,000)levels.However,southern Europe and Japan are notable exceptions,with higher incidence rates(11.6 per 100,000 and 23.1 per 100,000,respectively).A myriad of diseases contributes to its etiology such as viral hepatitis(56,57),alcoholic hepatitis(58),non-alcoholic fatty liver disease,NAFLD(59,60)and metabolic syndrome including diabetes mellitus(61,62).High vascularity,which is one of the characteristic pathological features of HCC,is necessary to increase cell proliferation and vascularity of the tumor.Growth factors facilitate cancer cell proliferation and high vascularity,e.g.VEGF,PDGF,EGF,FGF and IGF.These are expressed in cancer cells as well as in surrounding cells.Tumor invasion and portal thrombosis also exhibits high growth factor expression(63,64,65),e.g.EGF expression is higher in differentiation and invasion by cancer cells(66)whereas PDGF is related to metastasis of HCC cells(64).The proliferation of cancer cells by growth factors occurs by autocrine and paracrine fashion through the surrounding cells(67).In addition,the serum and tissues of HCC patients have decreased anti-vascular factors(68).Activation of the RAF/MEK/ERK pathway by tyrosine kinase type receptors,such as VEGFR,PDGFR,EGFR,FGFR and IGFR,in-turn activate intracellular RAS,which is also related to the disease progression of HCC(69)and HBV-related HCC development(70).Furthermore,RAF,the core protein of HCV,plays an important role in the development of HCC(71).Phosphatedylinositol-3 kinase(PI3K)pathway plays an important role in the proliferation and survival of cancer cells in various solid tumors,including HCC(72)as the(PI3K)pathway activates AKT,which in-turn phosphorylates intracellular proteins like mTOR(73),induces cell proliferation and inactivates BAD(74).Inactivation of BAD is important for cancer cells to survive by regulating apoptosis(74).Phosphatase and Tensin homolog,which is deleted on chromosome 10(PTEN)negatively regulates the PI3K pathway and it is suppressed in half of HCC cells clinically(75).This suppression is due to HBx protein in HBV hepatitis patients(76),and the down regulation of PTEN is associated with tumor grade progression,tumor stage and poor overall prognosis(75).Biomarkers like CD133(77),CD90(78),CD44(78)and EPCAM(79,80)have been revealed to be markers for cancer stem cells in HCC patients,which are very useful in helping estimate the prognosis of HCC.CD133 antigen is perceived to be related to prognosis and metastasis in HCC patients(81)as these cells increase in PTEN deleted mice(82),which also indicates PTEN's important role in regulating CD 13 3-positive cancer stem cells.Expression of CD90 in oval cells and progenitor cells is related to tumor development(83).The receptor of hyaluronate,CD44,is expressed on the cell surface and is often co-expressed with CD90(80,84).EPCAM,the cell marker for progenitor cells is a direct target of the Wnt/beta catenin pathway(79,85).CD 13 positive cells can be found in the peripheral areas of HCC after TACE treatment and are considered to be related to tumor recurrence(86).Surgical resection is the mainstay of curative treatment for early HCC;however,prognosis remains unsatisfactory due to frequent recurrence(2).Liver transplantation is another potentially curative treatment for HCC,but its application is limited,owing to a shortage of grafts(3).For unresectable HCC,various loco-regional therapies may be used to palliate symptoms and prolong survival(4).Furthermore,conventional chemotherapy hasn't yet proved to be effective in HCC,and a proven effective systemic therapy for HCC patients with metastatic disease does not exist(5).Hence,the search for novel therapeutic modalities for HCC is of paramount importance.Gene therapy is a promising therapeutic modality for cancers.Gene therapy for HCC is dependent on the transfer of genetic material to tumor cells,to induce a therapeutic effect.It could complement or substitute current treatment options for HCC.In gene therapy,it is imperative to efficiently transfer the gene to tumor cells and minimize its transfer to normal cells(87).Owing to the impaired liver function typically observed in the HCC patient population,it is particularly important to avoid the expression of the transferred gene in the normal hepatocytes;as direct toxic effects of gene therapy might pose a serious threat to the already impaired liver function(88,89).For efficient gene transfer,several fundamental factors should be integrated including the appropriate choice of a therapeutic gene(i.e.tumor suppressor genes,suicide genes,immune-modulatory genes etc.)and the selection of a safe and effective cell entry strategy(i.e.viral vector or non-viral vector);while the optimal delivery technique remains fundamentally important(90).Based on reports that suggest the role that growth factors play in the HCC cell proliferation,invasion and metastasis,it can be assumed that specific growth factors can be targets for HCC treatment.Cancer biomarkers could be useful for specific targeted therapy for cancer cells as well.E.g.NSC74859,a specific inhibitor of signal transducer and activator of transcription-3(STAT3)activation,decreases CD133-positive cells with suppression of cancer development(91),which suggests that CD 133 can be a molecular target for HCC treatment.The knockdown of the Wnt/beta-catenin by small interfering RNA(siRNA)of Wnt/beta-catenin decreases the number of EPCAM-positive cells along with suppression of tumor development,while inducing apoptosis(92).Furthermore,inhibitors of CD 13 such as 24F can suppress the invasion and angiogenesis of HCC(93).One of the gene therapy-based anticancer strategies is to replace or modify tumor suppressor gene.However,carcinogenic processes are multistep-in terms of their etiology and multifactor contribution.Tumor cells often undergo multiple genetic abnormalities,which limit efficacy of a single gene-mediated cancer therapy.Therefore,multi-gene based combination therapy may be an effective practice in cancer gene therapy,which can achieve greater therapeutic benefit by targeting multiple pathways.To find pivotal genes closely associated with tumor,occurrence and progression are key steps for this promising therapy.Inhibitor of growth(ING)is a type II tumor suppressor family implicated in carcinogenesis,apoptosis,cell cycle control,DNA repair and senescence.ING family includes five known members(ING1-ING5)(6-8).The five ING genes are located on five different chromosomes,and they are all close to the telomere/sub-telomere region except for ING3.ING4,as a novel member of ING family,is located at chromosome 12p13.31 and encodes a 249 amino acid(96,97).It is frequently found to be deleted or down-regulated in many cancers,viz.breast carcinoma(9),head and neck carcinoma(10),glioblastoma(11),hepatocellular carcinoma(HCC)(12),melanoma(13)and gastric carcinoma(14).The close correlation of ING4 with tumor progression and prognosis has attracted much attention.ING4 has been reported to significantly induce growth inhibition and apoptosis in a variety of tumor cells(11,15-19)and enhance chemosensitivity of cancer cells to chemotherapy drugs such as CDDP and doxorubicin in HepG2 hepatocarcinoma cells(16,20).It also has a specific role in restraining additional responses relevant to tumor progression such as anchorage-independent growth(98),angiogenesis(99,100),or response to hypoxia(99,101,102).ING4 functions mainly as a chromatin regulator,like other ING genes,linking histone marks to complexes with histone-modifying activities.It specifically binds H3K4me3(histone H3 trimethylated at lysine 4,a distinctive mark of euchromatin)through its plant homeodomain(PHD)domain(103-104),and it is also a component of complexes with histone acetyltransferase activity,in connection with HBO1(100,105,106).ING4 can also cooperate with p53 and nuclear factor-kappa B in transcriptional control,contributing to p53 posttranslational modifications or as a cofactor for both(107-108).Reduced levels of ING4 messenger RNA or protein are the most prevalent alterations of ING4 in tumors.In addition,there are also examples of short deletions and insertions or point mutations that lead to truncations or amino acid changes in the ING4 protein sequence(98,109,110).Furthermore,ING4 can inhibit tumor cell spreading,migration,and invasion by co-localizing and interacting with liprin a1 at lamellipodia16 and down-regulating the expression and activity of matrixmetalloproteinase-2(MMP-2)and MMP-9(13,17).These findings revealed that ING4 as a potent tumor suppressor negatively modulates tumor growth via multiple pathways.Phosphatase and Tensin homologue deleted on chromosome ten(PTEN)is well-defined as a tumor suppressor(21),which is frequently lost or genetically altered in a variety of human tumors(22,23),including endometrial and prostate carcinomas,glioblastomas and melanomas(111)at a frequency on par with the DNA damage sensor P53.Currently,over 2700 mutations in PTEN from 28 different tumor types have been listed in the COSMIC cancer database(24,25).PTEN is a dual specificity protein and lipid phosphatase,its primary cellular substrate is the second messenger phosphate-dyl-inositol(3,4,5)-tri-phosphate(PIP3),which it hydrolyzes to phosphate-dyl-inositol(4,5)-bi-phosphate(PIP2)(26-29).PTEN blocks phosphate-dyl-inositol 3 kinase(PI3K)signaling by inhibiting PIP3 dependent processes such as the membrane recruitment and activation of AKT,therefore inhibiting cell survival,growth,and proliferation.PTEN also has significant PIP3-independent functions.Specifically,PTEN protein phosphatase activity is critical for PTEN mediated inhibition of cellular migration(30).PTEN has also been reported to exhibit protein phosphatase activity.In-vitro studies have shown that PTEN de-phosphorylates tyrosine,serine,and threonine residues on phosphor-peptides(112),and it interacts with and de-phosphorylates focal adhesion kinase(FAK)and Shc(114,115).The protein phosphatase activity of PTEN also reduces Cyclin-D1 levels,preventing cell cycle progression(113).While cytoplasmic PTEN is primarily involved in regulating PI3K/PIP3 signaling,nuclear PTEN exhibits phosphatase-independent tumor suppressive functions,including regulation of chromosome stability,DNA repair,and apoptosis(15,16).Based on the antitumor features of ING4 and PTEN,the combined double tumor suppressors have been studied in human nasopharyngeal carcinoma cell.It was demonstrated that the co-expression of ING4 and PTEN enhanced growth inhibition,apoptosis stimulation and the suppression of tumor angiogenesis(31).To develop better therapeutic strategies for HCC,in this study,recombinant adenoviruses co-expressing ING4 and PTEN(Ad-ING4-PTEN)were constructed and its potential combined antitumor effect on SMMC-7721 and HepG2 human hepatocarcinoma cells was evaluated.