Influence of adriamycin on changes in Nanog, Oct-4, Sox2, ARID1 and Wnt5b expression in liver cancer stem cells

AIM: To determine the influence of Adriamycin (ADM) on the changes in Nanog, Oct4, Sox2, as well as, in ARID1 and Wnt5b expression in liver cancer stem cells.

Liver cancer stem cell markers: Progression and therapeutic implications

Cancer stem cells(CSCs) are a small subpopulation in cancer, have been proposed to be cancer-initiating cells, and have been shown to be responsible for chemotherapy resistance and cancer recurrence. The identification of CSC subpopulations inside a tumor presents a new understanding of cancer development because it implies that tumors can only be eradicated by targeting CSCs. Although advances in liver cancer detection and treatment have increased the possibility of curing the disease at early stages, unfortunately, most patients will relapse and succumb to their disease. Strategies aimed at efficiently targeting liver CSCs are becoming important for monitoring the progress of liver cancer therapy and for evaluating new therapeutic approaches. Herein, we provide a critical discussion of biological markers described in the literature regarding liver cancer stem cells and the potential of these markers to serve as therapeutic targets.

Research progress and prospects of markers for liver cancer stem cells

Liver cancer is a common malignancy and surgery is the main treatment strategy. However, the prognosis is still poor because of high frequencies of postoperative recurrence and metastasis. In recent years, cancer stem cell(CSC) theory has evolved with the concept of stem cells, and has been applied to oncological research. According to cancer stem cell theory, liver cancer can be radically cured only by eradication of liver cancer stem cells(LCSCs). This notion has lead to the isolation and identification of LCSCs, which has become a highly researched area. Analysis of LCSC markers is considered to be the primary method for identification of LCSCs. Here, we provide an overview of the current research progress and prospects of surface markers for LCSCs.

Lentivirus vectors construction of SiRNA targeting interference GPC3 gene and its biological effects on liver cancer cell lines Huh-7

Objective:To build GPC3 gene short hairpin interference RNA(shRNA)slow virus veclor.observe expression of Huh-7 GPC3 gene in human liver cell line proliferation apoptosis and the effect of GPC3 gene influencing on liver cancer cell growth,and provide theoretical basis for genc therapy of liver cancer.Methods:Hepatocellular carcinoma cell line Huh-7 wsa transfected by a RNA interference technique.GPC3 gene expression in a variety of liver cancer cell lines was detected by fluorescence quantitative PCR.Targeted GPC3 gene seqnences of small interfering RNA(siRNA)PGC-shRNA-GPC3 were restructured.Stable expression cell linse of siRNA were screened and established with the heplp of liposomes(lipofectamine^(TM2000))as carrier transfcetion of human liver cell lines.In order to validate siRNA interference efficiency.GPC3 siRNA mRNA expression was detected after transfection by using RT-PCR and Western blot.The absorbance value of the cells of blank group,untransfection group and transfection group,the cell cycle and cell apoptosis were calculated,and effects of GPC3 gene nn Huh-7 cell proliferation and apoptosis were observed.Results:In the liver cancer cell lines Huh-7 GPC3 gene showed high expression.PGC-shRNA-GPC3 recombinant plasmid was constructde successfully via sequencing validation.Stable recombinant plasmid transfected into liver cancer cell linse Huh-7can obviously inhibit GPC3 mRNA expression level.Conclusions:The targeted GPC3 siRNA can effectively inhibit the expression of GPC3.

Clinical implications of microRNAs in liver cancer stem cells

The prognosis of patients diagnosed with hepatocellular carcinoma (HCC) is often dismal, mainly due to late presentation, high recurrence rate, and frequent resistance to chemotherapy and radiotherapy. Accumulating evidence on the differential microRNA (miRNA) expression patterns between non-tumor and HCC tissues or between liver cancer stem cells (CSCs) and non-CSC subsets and the significant clinical implications of these differences suggest that miRNAs are a promising, non-invasive marker for the prognosis and diagnosis of the disease. This perspective article summarizes the current knowledge of miRNAs in liver CSCs and highlights the need for further investigations of the role of miRNAs in regulating liver CSC subsets for possible future clinical applications.

8-bromo-7-methoxychrysin inhibits properties of liver cancer stem cells via downregulation of β-catenin

AIM:To evaluate whether 8-bromo-7-methoxychrysin(BrMC),a synthetic analogue of chrysin,inhibits the properties of cancer stem cells derived from the human liver cancer MHCC97 cell line and to determine the potential mechanisms.METHODS:CD133+cells were sorted from the MHCC97 cell line by magnetic activated cell sorting,and amplified in stem cell-conditioned medium to obtain the enriched CD133+sphere forming cells(SFCs).The stem cell properties of CD133+SFCs were validated by the tumorsphere formation assay in vitro and the xenograft nude mouse model in vivo,and termed liver cancer stem cells(LCSCs).The effects of BrMC on LCSCs in vitro were evaluated by MTT assay,tumorsphere formation assay and transwell chamber assay.The effects of BrMC on LCSCs in vivo were determined using a primary and secondary xenograft model in Balb/c-nu mice.Expressions of the stem cell markers,epithelialmesenchymal transition(EMT)markers andβ-catenin protein were analyzed by western blotting or immunohistochemical analysis.RESULTS:CD133+SFCs exhibited stem-like cell properties of tumorsphere formation and tumorigenesis capacity in contrast to the parental MHCC97 cells.We found that BrMC preferentially inhibited proliferation and self-renewal of LCSCs(P<0.05).Furthermore,BrMC significantly suppressed EMT and invasion of LCSCs.Moreover,BrMC could efficaciously eliminate LCSCs in vivo.Interestingly,we showed that BrMC decreased the expression ofβ-catenin in LCSCs.Silencing ofβ-catenin by small interfering RNA could synergize the inhibition of self-renewal of LCSCs induced by BrMC,while Wnt3a treatment antagonized the inhibitory effects of BrMC.CONCLUSION:BrMC can inhibit the functions and characteristics of LCSCs derived from the liver cancer MHCC97 cell line through downregulation ofβ-catenin expression.

Regulators of liver cancer stem cells

Hepatocellular carcinoma(HCC)is a leading cause of cancer deaths.It is often detected at a stage when there are few therapeutic options.Liver cancer stem cells(LCSCs)are highly tumorigenic and resistant to chemotherapy and radiation therapy.Their presence in HCC is a major reason why HCC is difficult to treat.The development of LCSCs is regulated by a variety of factors.This review summarizes recent advances on the factors that regulate the development of LCSCs.Due to the importance of LCSCs in the development of HCC,a better understanding of how LCSCs are regulated will help to improve the treatments for HCC patients.

Cytotoxic Effect of Chitosan Based Nanocomposite Synthesized by Radiation： In Vitro Liver and Breast Cancer Cell Line

A silver nanoparticle （AgNP） is likely to provide an attractive object for combining a variety of biochemical properties with great therapeutic potential by using radiation. The present study explores the ICs0 value of chitosan-poly （vinyl alcohol） hydrogel （Cs/PVA） and Ag-doped chitosan-poly （vinyl alcohol） （Cs/PVA/Ag） nanocomposite in view of their anticancer application. The aim was to develop （Cs/PVA） based hydrogel synthesized by gamma radiation which could behave both as a nanoreactor for Ag nanoparticle with promising anticancer applications. The （Cs/PVA/Ag） nanocomposite was confirmed by FTIR （Fourier transform infrared） spectroscopy, XRD （X-ray diffraction） and EDX （energy dispersive X-ray） analysis. The anti-cancer activity of the prepared nanocomposites was demonstrated in human liver cancer cell line （HEPG2） and breast cancer cell lines （MCF7）. It has significant effects against human liver cancer cell line HEPG2 compared to breast cancer cell line MCF7. Further quantitative analysis on the molecular and protein levels is still required to confirm the impact of chitosan on genotoxic effect before reaching a final conclusion and starting its biomedical application.

Effects of adenoviral vector-mediated transduction of human p53,B7-1 and GM-CSF genes on liver cancer cells

The potential efficacy and clinical feasibility of gene therapy for liver cancer were tested through therecombinant adenovirus-mediated (Ad-multigenes ) co-transfer of human wild-type p53, B7-l co-stimulation(CD8o) and granulocyte-macrophage colony-stimulating factor (GM-CSF) genes into human hepatocellular carcinoma cell lines. The treated cells underwent apoptosis with specific DNA fragmentation and became more sensitiveto cisplatin, a chemotherapeutic drug. Their growth was partly inhibited. Efficient proliferation and generation ofCTLs and cytokine production were induced in mixed lymphocytes through tumor cell reaction (MLTR) using peripheral blood T lymphocytes from donors as effector cells and Ad-multigenes or Ad-p53-transfected human hepatocellular carcinoma cells (HepG2 or BEL7402) as stimulator cells. Ad-multigenes-transfected rat carcinosarcomaWalker 256 cells were inoculated subcutaneously into normal rats. Fourteen days later, the activity of spleen cellsin rats inoculated with Ad-multigenes-transduced Walker 256 cells was higher than that in Ad-p53-transducedones. These findings suggest that adenovirus-mediated multigenes p53, B7-1 and GM-CSF can induce apoptosis ofliver cancer cells and initiate a potent antitumor immune response against them.

Establishment and identification of induced pluripotent stem cells in liver cancer patients

Objective:To induce pluripotent stem(IPS)cells from fibrocytes that are separated from liver cancer patients.Methods:The fibrocytes were reprogrammed to IPS cells by lentiviral vector,stained and identified by immunohistochemistry.Results:The IPS cells were successfully established from fibrocytes after infection,and IPS cell clones formed in round shape under a microscopy.The induction rate was 0.013%±0.007%.No tumor formed at the back of nude mice within 8 weeks after the inoculation of cell clone.However,tetatoma appeared in nude mice within 1 week after IPS inoculation.A few tumors formed in nude mice within 4 weeks after the inoculation of cell clones.However,subcutaneous tumors formed within 1 week after IPS inoculation.The induced IPS cells showed three germ layers in tetatoma.Nanog and OCT4 in the induced IPS cells showed hypomethylation.SSEA-A,TRA-1-6-,TRA-1-81 and Nanog were highly expressed in the induced IPS cells,indicating the IPS cells possessed the similar ability as the stem cells.Conclusion:The IPS cells of liver cancer patients can be established effectively from fibrocytes and can be cultured stably in vitro,which provides an approach for the treatment of intermediate or advanced stage liver cancer.

Chimeric antigen receptor-engineered T-cell therapy for liver cancer

Background: Chimeric antigen receptor-engineered T-cell(CAR-T) therapy is a newly developed immunotherapy used in the treatment of cancers. Because CAR-T therapy has shown great success in treating CD19-positive hematological malignancies, its application has been explored in the treatment of solid tumors, such as liver cancer. In this review, we discuss the immune characteristics of liver cancer, the obstacles encountered during the application of CAR-T therapy, and preclinical and clinical progress in the use of CAR-T therapy in patients with liver cancer.Data sources: The data on CAR-T therapy related to liver cancers were collected by searching Pub Med and the Web of Science databases prior to December 2017 with the keywords "chimeric antigen receptor","CAR-T", "liver cancer", "hepatocellular carcinoma", and "solid tumor". Additional articles were identified by manual search of references found in the primary articles. The data for clinical trials were collected by searching Clinical Trials.gov.Results: The liver has a tolerogenic nature in the intrahepatic milieu and its tumor microenvironment significantly affects tumor progression. The obstacles that reduce the efficacy of CAR-T therapy in solid tumors include a lack of specific tumor antigens, limited trafficking and penetration of CAR-T cells to tumor sites, and an immunosuppressive tumor microenvironment. To overcome these obstacles, several strategies have emerged. In addition, several strategies have been developed to manage the side effects of CAR-T, including enhancing the selectivity of CARs and controlling CAR-T activity. To date, no clinical trials of CAR-T therapy against HCC have been completed. However, preclinical studies in vitro and in vivo have shown potent antitumor efficacy. Glypican-3, mucin-1, epithelial cell adhesion molecule, carcinoembryonic antigen, and other targets are currently being studied.Conclusions: The application of CAR-T therapy for liver cancer is just beginning to be explored and more research is needed. However, we are optimistic that CAR-T therapy will offer a new approach for the treatment of liver cancers in the future.

Estabishment of A Human Liver Cancer Cell Line Transfected with IL-2 cDNA and Its Biologic Activity

Objective To obtain IL 2 gene transfected human liver cancer cells and study IL 2 expression and its biologic activity in vivo. Methods\ Human liver cancer cells SMMC 7721 were cocultured with recombinant retroviral vector LNC IL 2,and screening was performed in G418 medium.The exogenous IL 2 cDNA at the DNA,RNA,and protein levels were determined by using dot hybridization,PR PCR and MTT methods respectively.The tumorigenesis and antitumorigenesis of the screened liver cancer cell with subcutaneous injection in nude mice were observed. Results and Conclusion\ The IL 2 cDNA was successfully integrated into SMMC 7721 cell genomic DNA and continuously expressed for more than 88 days.Subcutaneous vaccination of the nude mice with transfected cells revealed an obvious suppression of its tumorigenicity,and could induce antitumor activity in vivo. \ \

Investigating the cytotoxic effect of ibuprofen concentration in liver cancer cells(HepG2)and normal fibroblast(AGO)

Objective:Although many studies have reported that nonsteroidal anti-inflammatory drugs can have anticancer effects,the results are still challenging.The aim of this research is to Mechanism and effect of anti-inflammatory drugs in cancer treatment.Methods:In this laboratory study,cell lines were randomly divided into control group(no exposure to ibuprofen and groups exposed to ibuprofen concentrations of 10,1,0.1,and 0.001 mg/mL.The cytotoxic effect of ibuprofen was measured at 24 and 72 hours using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT).Data were compared between groups using a one-way variance test.Results:The results showed that the viability rate of HepG2 cancer cells at concentrations of 1 and 10 mg/mL decreased significantly compared to the control group in 24 hours(P<0.0001).Also,the viability rate at concentrations of 1,10,0.1,and 0.001 mg/mL decreased significantly compared to the control group in 72 h(P<0.0001).Only the concentration of 10 mg/mL ibuprofen decreased the viability of normal cells compared to the control(P<0.05).Conclusion:Overall,the results of this research showed that different concentrations of ibuprofen had a cytotoxic effect on liver cancer cells,and except for the concentration of 10 mg/mL,the other concentrations did not have a cytotoxic effect on normal cells.

A STUDY OF THE FORMATION OF LIVER METASTASIS AND ITS MECHANISM USING THE INTRASPLENIC INOCULATION OF CANCER CELLS

In order to establish an animal model with hepatic metastasis intrasplenic inoculation of carcinoma cells from murine uterine cervical carcinoma (U14) was employed. Results showed a high incidence of hepatic metastasis could be obtained through the intrasplenic inoculation of 1 × 106 carcinoma cells. Removal of the primary carcinoma through splenec-tomy at different intervals after intrasplenic inoculation proved that the hepatic metastatic mechanism was not due to mechanical pressure but occurred spontaneously. This experimental model provides a useful means for studying the mechanism and prevention of hepatic metastasis.

A novel animal model for in vivo study of liver cancer metastasis

AIM:To establish an animal model with human hepatocyte-repopulated liver for the study of liver cancer metastasis.METHODS:Cell transplantation into mouse livers was conducted using alpha-fetoprotein(AFP)-producing hu-man gastric cancer cells(h-GCCs) and h-hepatocytes as donor cells in a transgenic mouse line expressing urokinase-type plasminogen activator(uPA) driven by the albumin enhancer/promoter crossed with a severe combined immunodeficient(SCID) mouse line(uPA/SCID mice).Host mice were divided into two groups(A and B).Group A mice were transplanted with h-GCCs alone,and group B mice were transplanted with h-GCCs and h-hepatocytes together.The replacement index(RI),which is the ratio of transplanted h-GCCs and h-hepatocytes that occupy the examined area of a histological section,was estimated by measuring h-AFP and h-albumin concentrations in sera,respectively,as well as by immunohistochemical analyses of h-AFP and human cytokeratin 18 in histological sections.RESULTS:The h-GCCs successfully engrafted,repopulated,and colonized the livers of mice in group A(RI = 22.0% ± 2.6%).These mice had moderately differentiated adenocarcinomatous lesions with disrupted glandular structures,which is a characteristics feature of gastric cancers.The serum h-AFP level reached 211.0 ± 142.2 g/mL(range,7.1-324.2 g/mL).In group B mice,the h-GCCs and h-hepatocytes independently engrafted,repopulated the host liver,and developed colonies(RI = 12.0% ± 6.8% and 66.0% ± 12.3%,respectively).h-GCC colonies also showed typical adenocarcinomatous glandular structures around the h-hepatocyte-colonies.These mice survived for the full 56 day-study and did not exhibit any metastasis of h-GCCs in the extrahepatic regions during the observational period.The mice with an h-hepatocyte-repopulated liver possessed metastasized h-GCCs and therefore could be a useful humanized liver animal model for studying liver cancer metastasis in vivo.CONCLUSION:A novel animal model of human liver cancer metastasis was established using the uPA/SCID mouse line.This model could be useful for in vivo testing of anti-cancer drugs and for studying the mechanisms of human liver cancer metastasis.

Terpenoids as potential chemopreventive and therapeutic agents in liver cancer

Despite significant advances in medicine, liver cancer, predominantly hepatocellular carcinoma remains a major cause of death in the United States as well as the rest of the world. As limited treatment options are currently available to patients with liver cancer, novel preventive control and effective therapeutic approaches are considered to be reasonable and decisive measures to combat this disease. Several naturally occurring dietary and non-dietary phytochemicals have shown enormous potential in the prevention and treatment of several cancers, especially those of the gastrointestinal tract. Terpenoids, the largest group of phytochemicals, traditionally used for medicinal purposes in India and China, are currently being explored as anticancer agents in clinical trials. Terpenoids (also called "isoprenoids") are secondary metabolites occurring in most organisms, particularly plants. More than 40 000 individual terpenoids are known to exist in nature with new compounds being discovered every year. A large number of terpenoids exhibit cytotoxicity against a variety of tumor cells and cancer preventive as well as anticancer efficacy in preclinical animal models. This review critically examines the potential role of naturally occurring terpenoids, from diverse origins, in the chemoprevention and treatment of liver tumors. Both in vitro and in vivo effects of these agents and related cellular and molecular mechanisms are highlighted. Potential challenges and future directions involved in the advancement of these promising natural compounds in the chemoprevention and therapy of human liver cancer are also discussed.

Hepatic cancer stem cells and drug resistance: Relevance in targeted therapies for hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is one of most common malignancies in the world. Systemic treatments for HCC, particularly for advanced stages, are limited by the drug resistance phenomenon which ultimately leads to therapy failure. Recent studies have indicated an association between drug resistance and the existence of the cancer stem cells (CSCs) as tumor initiating cells. The CSCs are resistant to conventional chemotherapies and might be related to the mechanisms of the ATP Binding Cassette (ABC) transporters and alterations in the CSCs signaling pathways. Therefore, to contribute to the development of new HCC treatments, further information on the characterization of CSCs, the modulation of the ABC transporters expression and function and the signaling pathway involved in the self renewal, initiation and maintenance of the cancer are required. The combination of transporters modulators/inhibitors with molecular targeted therapies may be a potent strategy to block the tumoral progression. This review summarizes the association of CSCs, drug resistance, ABC transporters activities and changes in signaling pathways as a guide for future molecular therapy for HCC.

Tripartite motif.containing 3(TRIM3)inhibits tumor growth and metastasis of liver cancer

Background:Reduced expression of tripartite motif-containing 3(TRIM3) has been reported to be involved in the pathogenesis of human glioblastoma.In our previous research,we found that TRIM3 expression was markedly reduced in human primary hepatocellular carcinoma(HCC) tissues and that low TRIM3 expression was associated with short survival of HCC patients.However,the role of TRIM3 in liver cancer remains unknown.This study aimed to investigate the function of TRIM3 in liver cancer cells.Methods:The protein levels of TRIM3 in five liver cancer cell lines(SK-Hep1,Hep3 B,Huh7,HepG2,Bel-7402) and one normal liver cell line(L02) were detected with Western blotting.HepG2 and Bel-7402 cells with low TRIM3 expression were infected with recombinant lentiviruses overexpressing TRIM3(LV-TRIM3),whereas Huh7 and Hep3 B cells with high TRIM3 expression were transfected with TRIM3-targeted small interfering RNA(siTRIM3).The functions of TRIM3 in the proliferation,colony formation,cell cycle,migration,invasion,and apoptosis of the above cell lines were examined.The effect ofTRIM3 on tumor growth and metastases in nude mice was also investigated.Results:TRIM3 was overexpressed in HepG2 and Bel-7402 cells with LV-TRIM3 infection,which further reduced proliferation,colony formation,migration,and invasion of both cell lines.Cell cycle analysis showed that TRIM3 overexpression induced G_0/G_1 phase arrest in HepG2 and Bel-7402 cells.Moreover,apoptosis was not increased in HepG2 or Bel-7402 cells overexpressing TRIM3.Contrarily,silencing TRIM3 expression in Huh7 and Hep3 B cells by siTRIM3 led to significantly decreased percentages of both cells in the G_0/G_1 phase and promoted cell proliferation,colony formation,migration,and invasion.In vivo experiment results confirmed that TRIM3 overexpression suppressed tumor growth and metastasis.Conclusions:TRIM3 plays a tumor-suppressing role in the regulation of liver cancer development by reducing cell proliferation through cell cycle arrest at the G_0/G_1 phase.

Does autologous blood transfusion during liver transplantation for hepatocellular carcinoma increase risk of recurrence?

AIM: To analyze outcomes in patients who underwent liver transplantation(LT) for hepatocellular carcinoma(HCC) and received autologous intraoperative blood salvage(IBS). METHODS: Consecutive HCC patients who underwent LT were studied retrospectively and analyzed according to the use of IBS or not. Demographic and surgical data were collected from a departmental prospective maintained database. Statistical analyses were performed using the Fisher's exact test and the Wilcoxon rank sum test to examine covariate differences between patients who underwent IBS and those who did not. Univariate and multivariate Cox regression models were developed to evaluate recurrence and death,and survival probabilities were estimated using the Kaplan-Meier method and compared by the log-rank test.RESULTS: Between 2002 and 2012,158 consecutive patients who underwent LT in the same medical center and by the same surgical team were identified. Among these patients,122(77.2%) were in the IBS group and 36(22.8%) in the non-IBS group. The overall survival(OS) and recurrence free survival(RFS) at 5 years were 59.7% and 83.3%,respectively. No differences in OS(P=0.51) or RFS(P=0.953) were detected between the IBS and non-IBS groups. On multivariate analysis for OS,degree of tumor differentiation remained as the only independent predictor. Regarding patients who received IBS,no differences were detected in OS or RFS(P=0.055 and P=0.512,respectively) according to the volume infused,even when outcomes at 90 d or longer were analyzed separately(P=0.518 for both outcomes).CONCLUSION: No differences in RFS or OS were detected according to IBS use. Trials addressing this question are justified and should be designed to detect small differences in long-term outcomes.

Incomplete radiofrequency ablation provokes colorectal cancer liver metastases through heat shock response by PKCα/Fra-1 pathway

Objective: Incomplete radiofrequency ablation(ICR) has been proposed as a major cause of recurrence in the treatment of hepatic metastatic tumors.We tried to determine the mechanisms of this progression in colorectal cancer(CRC) liver metastasis(CRLMs)Methods: We have established a mouse model of radiofrequency ablation(RFA) therapy to demonstrate increased risk of recurrence of CRLMs with ICR.Here we focused on heat shock-induced CRC malignancy.Sub-lethal heat shock(HS) in CRC cell lines provoked cell growth, invasion, and tumor initiation in vitro and in vivo.Results: We found that Fra-1, which lies downstream of PKCα-ERK1/2 signaling, was significantly increased by HS compared with the untreated CRC cells.Silencing Fra-1 reversed the tumor promoting effects of HS.Furthermore, proliferation and tumor initiation inducer c-Myc, together with tumor invasion inducer matrix-metalloprotase 1(MMP-1) expression were up-regulated by AP-1/Fra-1 induced genes transcription.Conclusions: Our study demonstrated that ICR generated HS induces CRC malignancy by targeting Fra-1, which could be a potential prognostic marker and a promising therapeutic strategy to prevent disease recurrence after radiofrequency ablation treatment.