Hepatocellular Carcinoma Cell Line HCCLM3 Research Articles

LASS2 enhances p53 protein stability and nuclear import to suppress liver cancer progression through interaction with MDM2/MDMX

LASS2 functions as a tumor suppressor in hepatocellular carcinoma (HCC), the most common type of primary liver cancer, but the underlying mechanism of its action remains largely unknown. Moreover, details on its role and the downstream mechanisms in Cholangiocarcinoma (CCA) and hepatoblastoma (HB), are rarely reported. Herein, LASS2 overexpression was found to significantly inhibit proliferation, migration, invasion and induce apoptosis in hepatoma cells with wild-type (HB cell line HepG2) and mutated p53 (HCC cell line HCCLM3 and CCA cell line HuCCT1). Gene set enrichment analysis determined the enrichment of the differentially expressed genes caused by LASS2 in the p53 signaling pathway. Moreover, the low expression of LASS2 in HCC and CCA tumor tissues was correlated with the advanced tumor-node-metastasis (TNM) stage, and the protein expression of LASS2 positively correlated with acetylated p53 (Lys373) protein levels. At least to some extent, LASS2 exerts its tumor-suppressive effects in a p53-dependent manner, in which LASS2 interacts with MDM2/MDMX and causes dual inhibition to disrupt p53 degradation by MDM2/MDMX. In addition, LASS2 induces p53 phosphorylation at ser15 and acetylation at lys373 to promote translocation from cytoplasm to nucleus. These findings provide new insights into the LASS2-induced tumor suppression mechanism in liver cancer and suggest LASS2 could serve as a potential therapeutic target for liver cancer.

Effect of zebularine on apoptotic pathways in hepatocellular carcinoma cell lines

The alteration of DNA cytosine methylation is one of the most common epigenetic changes that can play a significant role in human cancers. The enzymes involved in DNA methylation of promoter regions of the genes are DNA methyltransferases (DNMTs). The therapeutic activities and apoptotic effects of DNA methyltransferase inhibitors (DNMTIs) have been reported in various cancers. This study was assigned to assess the effect of zebularine on intrinsic and extrinsic pathways, DNAT 1, 3a, and 3b, p21, and p53, viability, and apoptosis in hepatocellular carcinoma (HCC) cell lines.Hepatocellular carcinoma cell lines (HCCLM3, MHCC97H, and MHCC97L) were purchased from the National Cell Bank of Iran, Pasteur Institute, treated with zebularine, and the MTT assay was performed. Then, flow cytometry assay and real-time RT-PCR analysis were performed with zebularine. Statistical comparisons between groups were made using GraphPad Prism software version 8.0. A significant difference was considered as P < 0.05.Zebularine up-regulated DR4, DR5, FAS, FAS-L, TRAIL, Bax, Bak, Bim, p21WAF/CIP1 (p21), and p53 and down-regulated DNMTs (DNAT 1, 3a, and 3b), Bcl-2, Bcl-xL, and Mcl-1, significantly resulting in apoptosis induction in HCC cell lines. Maximal and minimal apoptosis was seen in HCCLM3 and MHCC97L cell lines, respectively.Our findings indicated that DNMTI zebularine can induce apoptosis and inhibit cell growth through both pathways (extrinsic and intrinsic) in HCC cell lines HCCLM3, MHCC97H, and MHCC97L.

Dexmedetomidine promotes apoptosis and suppresses proliferation of hepatocellular carcinoma cells via microRNA-130a/EGR1 axis

Accumulating evidence has revealed the role of microRNAs (miRs) in hepatocellular carcinoma (HCC). Dexmedetomidine, a highly selective α2-adrenergic agonist, is widely used in perioperative settings for analgesia and sedation. Herein, we aimed to determine whether dexmedetomidine might directly regulate miR-130a/early growth response 1 (EGR1) axis in HCC and explore the related mechanisms. miR-130a and EGR1 expression were determined in HCC tissues and their correlation was evaluated. Human HCC cell line HCCLM3 was selected. Upon the determination of the optimal concentration of dexmedetomidine, HCCLM3 cells were treated with dexmedetomidine, miR-130a- or EGR1-related oligonucleotides or plasmids were transfected into cells to explore their functions in cell biological behaviors. miR-130a and EGR1 levels in cells were tested. The targeting relationship between miR-130a and EGR1 was verified. miR-130a was inhibited while EGR1 was elevated in HCC tissues and they were negatively correlated. EGR1 was targeted by miR-130a. With the increase of dexmedetomidine concentration, HCCLM3 cell viability was correspondingly inhibited, miR-130a expression was elevated and EGR1 expression was decreased. Dexmedetomidine, upregulating miR-130a or downregulating EGR1 inhibited proliferation, invasion and migration, and promoted apoptosis of HCCLM3 cells. MiR-130a upregulation/downregulation enhanced/impaired the effect of dexmedetomidine on cell biological behaviors. Our study provides evidence that raising miR-130a enhances the inhibitory effects of dexmedetomidine on HCC cellular growth via inhibiting EGR1. Thus, miR-130a may be a potential candidate for the treatment of HCC.

Development and validation of LRP1B mutation-associated prognostic model for hepatocellular carcinoma.

Purpose: To develop a lipoprotein receptor-related protein 1B (LRP1B) gene mutation-based prognostic model for hepatocellular carcinoma (HCC) patients risk prediction. Methods: The LRP1B gene mutation rate was calculated from HCC patient samples. Meanwhile, differentially expressed genes according to LRP1B mutant were screened out for prognostic model establishment. Based on this innovative model, HCC patients were categorized into high- and low-risk groups. The immune status including immune cell infiltration ratio and checkpoints have been explored in two groups. The functions of LRP1B and risk factors in the model were verified using both in vivo and in vitro experiments. Results: It could be demonstrated that LRP1B was a potential negative predictor for HCC patients prognosis with high mutation frequency. The functions of LRP1B were verified with ELISA and Quantitative Real-time PCR method based on clinic-recruited HCC participants. Eleven genes displayed significant differences according to LRP1B status, which could better predict HCC patient prognosis. The functions of these genes were examined using HCC cell line HCCLM3, suggesting they played a pivotal role in determining HCC cell proliferation and apoptosis. From the immune cell infiltration ratio analysis, there was a significant difference in the infiltration degree of seven types of immune cells and two immune checkpoints between high- and low-risk HCC patients. Conclusion: The present study hypothesized a potential prognostic biomarker and developed a novel LRP1B mutation-associated prognostic model for HCC, which provided a systematic reference for future understanding of clinical research.

GPER-Induced ERK Signaling Decreases Cell Viability of Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) is an aggressive malignancy with a poor prognosis. Effective biomarkers and specific therapeutic targets for HCC are therefore urgently needed. G protein-coupled estrogen receptor (GPER) plays a crucial role in numerous cancer types; however, its functions in HCC require further exploration. In the present study, we found a remarkable difference in GPER staining between tumor tissue (100/141, 70.9%) and matched non-tumor tissue (27/30, 90.0%). Compared with the GPER-negative patients, the GPER-positive patients with HCC were closely associated with female sex, negative hepatitis B surface antigen, small tumor size, low serum alpha fetoprotein level, and longer overall survival. Treatment with GPER-specific agonist G1 led to the sustained and transient activation of the EGFR/ERK and EGFR/AKT signaling pathways, respectively, in the HCC cell lines HCCLM3 and SMMC-7721, which express high levels of GPER. Interestingly, G1-induced EGFR/ERK signaling, rather than EGFR/AKT signaling mediated by GPER, was involved in decreasing cell viability by blocking cell cycle progression, thereby promoting apoptosis and inhibiting cell growth. Clinical analysis indicated that simultaneous high expression of GPER and phosphorylated-ERK (p-ERK) predicted improved prognosis for HCC. Finally, the activation of GPER/ERK signaling remarkably suppressed tumor growth in an HCC xenograft model, and this result was consistent with the in vitro data. Our findings suggest that specific activation of the GPER/ERK axis may serve as a novel tumor-suppressive mechanism and that this axis could be a therapeutic target for HCC.

Chinese herbal formula QHF inhibits hepatocellular carcinoma metastasis via HGF/c-Met signaling pathway

Hepatocellular Carcinoma (HCC) is one of the most common malignant tumors, and high recurrence and metastasis are the major obstacles to successful treatment of HCC. Traditional Chinese medicine has little known and unique advantages in the treatment of HCC. Previous studies have confirmed that Chinese herbal formula Qingrejiedu (clears away heat and toxins), Huoxuehuayu (promotes blood flow to remove stasis) and Fuzhengguben (strengthens healthy qi and root) (QHF) has a significant effect on patients with advanced HCC, improves the quality of life and prolongs the survival time of patients significantly. In this study, we investigated the effect of QHF on proliferation, migration and invasion of human high metastatic hepatocellular carcinoma cell line HCCLM3 and its underlying mechanism. The results from our in vitro experiments showed that QHF has the ability to inhibit the proliferation by inducing G2/M phase cell cycle arrest and induce apoptosis. Moreover, QHF can also inhibit migration and invasion of HCCLM3 cells and the expression of the p-c-Met protein in HCCLM3 cells was down-regulated. c-Met is closely related to the metastasis of HCC, then we constructed a stable transfected cell line HepG2-met with high expression of c-Met by transfection. Further study in vivo revealed that c-Met gene will promote the growth of tumors and lung metastases in nude mice, and QHF intervention can reduce tumor lung metastases by inhibiting the HGF/c-Met signaling pathway. In conclusion, our study reveals that QHF can inhibit the proliferation, migration and invasion of HCCLM3, and this effect may be related to inhibiting HGF/c-Met signaling pathway.

Increased Oxidative Phosphorylation Is Required for Stemness Maintenance in Liver Cancer Stem Cells from Hepatocellular Carcinoma Cell Line HCCLM3 Cells.

Cancer stem cells (CSCs) are considered to be the main cause of tumor recurrence, metastasis, and an unfavorable prognosis. Energy metabolism is closely associated with cell stemness. However, how the stemness of liver cancer stem cells (LCSCs) is regulated by metabolic/oxidative stress remains poorly understood. In this study, we compare the metabolic differences between LCSCs and the hepatocellular carcinoma cell line HCCLM3, and explore the relationship between metabolism and LCSC stemness. We found that LCSCs from the hepatocellular carcinoma cell HCCLM3 exhibited more robust glucose metabolism than HCCLM3, including glycolysis, oxidative phosphorylation (OXPHOS), and pyruvate produced by glycolysis entering mitochondria for OXPHOS. Moreover, 2-deoxy-D-glucose (2-DG) enhanced the LCSC stemness by upregulating OXPHOS. In contrast, Mdivi-1 reduced the levels of OXPHOS and weakened the stemness by inhibiting mitochondrial fission. Together, our findings clarify the relationship between energy metabolism and LCSC stemness and may provide theoretical guidance and potential therapeutic approaches for liver cancer.

MiR-449a Is Related to Short-Term Recurrence of Hepatocellular Carcinoma and Inhibits Migration and Invasion by Targeting Notch1.

PurposeTo explore the effect of miR-449a inhibits migration and invasion by targeting Notch1 and regulating epithelial–mesenchymal transition (EMT) in hepatocellular carcinoma (HCC), and further study on the molecular mechanism.Patients and methodsThe expression of miR-449a and Notch1 in HCC cells and tissues was detected by qRT-PCR. The HCC cell line HCCLM3 and SMMC-7721 were transfected with miR-449a. The invasion and migration of HCC cell lines were detected by transwell assay and wound healing assay. The Notch pathway and EMT related protein were detected with Western Blotting. The specific binding site of mir-449a on notch1 gene was detected by luciferase assay.ResultsWe found the expression of miR-449a was related to short-term recurrence of hepatocellular carcinoma after hepatectomy and acted as independent risk factors of DFS and OS. The expression of miR-449a decreased in tumor tissues and HCC cell lines, but the expression of Notch1 increased. The overexpressed miR-449a promoted the invasiveness in vitro by regulating EMT via Notch pathway. Mechanically, miR-449a inhibited the translation of Notch1 protein by binding to 3ʹ UTR of its mRNA directly.ConclusionmiR-449a is short-term recurrence-related miRNA and inhibits the invasion and metastasis ability of HCC cells by regulating EMT via Notch pathway. miR-449a may be a new effective therapeutic target for HCC.

Insufficient radiofrequency ablation promotes the metastasis of residual hepatocellular carcinoma cells via upregulating flotillin proteins

PurposeRadiofrequency ablation (RFA) therapy has proven to be effective and feasible for early-stage hepatocellular carcinoma (HCC); however, rapid progression of residual tumor cells after RFA has been confirmed, but the molecular mechanisms of this phenomenon are poorly understood. This study evaluated the effect of the lipid raft proteins known as flotillins on the invasive and metastatic potential of residual HCC.MethodsThe human HCC cell line HCCLM3 was used to establish insufficient RFA models in vivo and in vitro. Changes in cellular morphology, soft agar colony formation, motility, metastasis, and epithelial–mesenchymal transition (EMT) markers after insufficient RFA intervention in vitro and in vivo were detected by real-time PCR, western blotting, immunohistochemistry and transwell assays.ResultsThe results showed that flotillin-1 and flotillin-2 expression were upregulated in HCCLM3 cells following 45 °C heat treatment and in residual HCCLM3 xenografts cells after insufficient RFA. Knocking down flotillin-1 or flotillin-2 in HCCLM3 cells by shRNA significantly lowered insufficient RFA-induced tumor growth, EMT changes, and metastasis in vitro and in vivo. Furthermore, mechanism studies indicated that flotillins altered the EMT status and metastatic potential of heat-treated HCCLM3 cells by activating the Akt/Wnt/β-catenin signaling pathway.ConclusionsOur findings present new evidence that flotillins play a key role in the aggressive behaviors of residual cancer cells after insufficient RFA and provide new insights into the regulatory mechanism of Wnt/β-catenin signaling.

Peroxiredoxin2 downregulation enhances hepatocellular carcinoma proliferation and migration, and is associated with unfavorable prognosis in patients.

It has been revealed by our previous proteomic study that the expression profile is different between well-differentiated and poorly differentiated hepatocellular carcinoma (HCC). Among those differently expressed proteins, peroxiredoxin2 (PRDX2) was our protein of interest. The present study aimed to further investigate the value of PRDX2 as a prognostic factor in HCC. Tissue microarrays were used to investigate the expression difference between HCC tissues and their adjacent normal liver tissues. The expression of PRDX2 at both mRNA and protein levels was examined by q-RT-PCR, western blotting and immunohistochemical assessment in HCC tissues and cell line HCCLM3. Silencing of PRDX2 in HCCLM3 was achieved usingpGMLV-SC1 lentiviral vectors. Cell Counting Kit-8 (CCK-8) and Transwell migration assays were used to assess cell proliferation and migration, respectively. Categorical variables were assessed using the Chi-square test, and ordinal variables were examined using the Mann-Whitney U test. The difference of continuous variables between groups were compared with t-tests. The Kaplan-Meier method was used to calculate the overall survival (OS) and disease-free survival (DFS) of patients, and the log-rank test was used to analyze the differences between groups. The results revealed that the expression of PRDX2 was decreased at both the mRNA and protein levels in an HCC cell line compared to that of a normal human liver cell line. PRDX2 protein expression levels were significantly downregulated in HCC tissues and were positively linked to overall survival (OS) and disease-free survival (DFS) of HCC patients. Patients with high PRDX2 expression levels had longer OS and DFS times than those with lower PRDX2 expression. Silencing of PRDX2 in the HCC cell line HCCLM3 promoted cancer cell proliferation and migration. Our findings indicated that PRDX2 may play an important role in HCC development; PRDX2 may serve as a useful prognostic factor and a therapeutic target.

Hepatitis B virus X protein promotes epithelial-mesenchymal transition and metastasis in hepatocellular carcinoma cell line HCCLM3 by targeting HMGA2.

Chronic hepatitis B virus (HBV) infection is a major risk factor for hepatocellular carcinoma (HCC), and HBV X protein (HBx) serves an essential role in the development of HCC. However, its mechanism remains to be elucidated. The aim of the present study was to investigate the role and mechanism of the HBx protein in the epithelial-mesenchymal transition (EMT) and metastasis of HCC. The HCCLM3 cell line was transfected with a HBx-expressing vector. The effects of HBx overexpression on proliferation, migration, invasion and EMT capacities of the HCCLM3 cell line were evaluated using MTT, migration and invasion assays, and western blotting, respectively. Furthermore, the impact of High mobility group AT-hook 2 (HMGA2) knockdown on HBx-mediated metastasis was investigated in the HCC cell line HCCLM3. The results demonstrated that HBx significantly upregulated HMGA2 expression, and enhanced the proliferation, EMT, invasion and migration in HCC cells. Furthermore, HMGA2 knockdown almost abolished HBx-induced EMT and metastasis in HCC. The results of the present study suggest that HBx promotes the proliferation, EMT, invasion and migration of HCC cells by targeting HMGA2. HMGB2 may serve as a potential therapeutic target for HBV-associated HCC.

CD8+ T cells mediate the antitumor activity of frankincense and myrrh in hepatocellular carcinoma

BackgroundTumor-promoting inflammation is an emerging hallmark of cancer, which participates in both cancer progression and immune escape. Hepatocellular carcinoma (HCC) is a typical inflammation-related cancer with an extremely poor prognosis. Frankincense and myrrh are anti-inflammation agents commonly used in clinic. The purpose of this study is to investigate whether extract of frankincense and myrrh (FM) downregulates inflammatory microenvironment of HCC and thereby restores antitumor immune responses.MethodsThe water-decocting FM was obtained and quantified. HCC cell lines HCCLM3 and Hepa1-6 were used to evaluate the efficacy of FM targeting NF-κB and STAT3 signaling with western blot and qRT-PCR analysis. CD8+NKG2D+ cells were derived from human peripheral blood and were used for evaluation of immune cells-mediated inflammation and oncolysis on HCCLM3 cells. The antitumor efficacy of FM was investigated both in immune compromised and immune competent mice bearing subcutaneous HCC. Mice received daily oral gavage of FM at 60 mg/kg. Immune activity within tumor microenvironment (TME) was assessed by ELISpot assay and flow cytometry, respectively. Depletion of CD8+ T cells or NK cells was achieved by intraperitoneal injection of respective neutralizing antibody.ResultsFM significantly inhibited the activation of NF-κB and STAT3 signaling in HCC cells induced by cytokines (TNF-α or IL-6) and in co-culture system with CD8+NKG2D+ cells. Furthermore, FM sensitized HCC cells to CD8+NKG2D+ cells-mediated oncolysis. In HCC-bearing mice, FM at a non-toxic dose failed to reduce tumor growth in immune compromised mice, whereas it significantly inhibited tumor growth and prolonged life span in immune competent mice. While the number of IFN-γ-producing cells within TME was increased in mice treated with FM, the infiltration of CD8+ T cells and NK cells was not increased. Finally, we identified that depletion of CD8+ T cells rather than NK cells abrogated the antitumor activity of FM.ConclusionsOur results show for the first time that CD8+ T cells mediate the antitumor activity of FM at a non-toxic dose. This may provide new insights to this ancient mysterious prescription in cancer therapy, which offers a novel and practical therapeutic strategy and the possibilities of combined immunotherapy for HCC as well as other inflammation-related cancers in clinic.

The downregulation of NCRUPAR is associated with the clinical characteristics of hepatocellular carcinoma

Background: Long non-coding RNAs (lncRNAs) appear to be a new class of regulators of cellular processes, such as cell growth, apoptosis, and carcinogenesis. However, the clinical significance of most lncRNAs in screening for hepatocellular carcinoma (HCC) is largely unknown. Recently, a novel lncRNA upstream from the coagulation factor II thrombin receptor ( F2R/PAR1 ) gene, called NCRUPAR, was found to be involved in the tumorigenesis of colorectal cancer and gastric cancer. However, the expression of NCRUPAR and its clinical significance in HCC have not yet been reported. Methods: We collected 137 samples of HCC tissues compared with paired adjacent nontumor tissues and measured the NCRUPAR levels in tissues and cell lines using real-time reverse transcription-polymerase chain reaction, and then the associations between NCRUPAR expression and the clinicopathological features of HCC. Results: The expression of NCRUPAR in the HCC cell lines HCCLM3, HUH7, MHCC97H, SK-Hep1 and Hep3B was significantly downregulated compared with the normal liver cell line QSG-7701. It was downregulated in 73.7% (101/137) of the HCC tissues compared with paired adjacent normal tissues (P Conclusions: Our data suggest that NCRUPAR may plays crucial roles during cancer occurrence and progression and is a potential new biomarker of hepatocellular carcinoma.

Insufficient Radiofrequency Ablation Treated Hepatocellular Carcinoma Cells Promote Metastasis by Up-Regulation ITGB3.

Radiofrequency ablation (RFA) is one of the standards of care for early stage hepatocellular carcinoma (HCC). However, rapid progression of residual tumor after RFA has been confirmed. The aim of this study was to investigate the underlying mechanism of this phenomenon. Human HCC cell lines HCCLM3 and HepG2 were employed to establish insufficient RFA models in vivo and in vitro, respectively. The effects of insufficient RFA on metastatic potential of residual tumors were evaluated. The molecular changes after insufficient RFA were evaluated by PCR array, western blot, immunofluorescence, and immunohistochemistry. Results showed that insufficient RFA significantly promoted lung and intrahepatic residual tumor cells in vivo, and heat intervention promoted migration and invasion of hepatoma cells in vitro. PCR array revealed that the expression of integrin β3 (ITGB3) and MMP2 were up-regulated in the residual tumors of HCCLM3 xenograft model. The up-regulation of ITGB3 was confirmed by qRT-PCR, Western blot and immunohistochemistry. Knockdown ITGB3 expression in HCCLM3 cells by shRNA significantly lowered the pro-metastatic effects of insufficient RFA. Mechanism studies indicated that ITGB3 mediated the expression of MMP2 by activing FAK/PI3K/AKT signaling pathway. The up-regulation of ITGB3 contributed to enhanced metastatic potential of residual cancer in HCCLM3 model after insufficient RFA. Targeting ITGB3 expression may further improve the clinical effects of RFA.

Protrusion-localized STAT3 mRNA promotes metastasis of highly metastatic hepatocellular carcinoma cells in vitro.

Recent evidence shows that localization of mRNAs and their protein products at cellular protrusions plays a decisive function in the metastasis of cancer cells. The aim of this study was to identify the variety of proteins encoded by protrusion-localized mRNAs and their roles in the metastasis and invasion of liver cancer cells. Highly metastatic hepatocellular carcinoma cell line HCCLM3 and non-metastatic hepatocellular carcinoma cell line SMMC-7721 were examined. Cell protrusions (Ps) were separated from cell bodies (CB) using a Boyden chamber assay; total mRNA population in CB and Ps fractions was analyzed using high-throughput direct RNA sequencing. The localization of STAT3 mRNA and protein at Ps was confirmed using RT-qPCR, RNA FISH, and immunofluorescence assays. Cell migration capacity and invasiveness of HCCLM3 cells were evaluated using MTT, wound healing migration and in vitro invasion assays. The interaction between Stat3 and growth factor receptors was explored with co-immunoprecipitation assays. In HCCLM3 cells, 793 mRNAs were identified as being localized in the Ps fraction according to a cut-off value (Ps/CB ratio) >1.6. The Ps-localized mRNAs could be divided into 4 functional groups, and were all closely related to the invasive and metastatic properties. STAT3 mRNA accumulated in the Ps of HCCLM3 cells compared with non-metastatic SMMC-7721 cells. Treatment of HCCLM3 cells with siRNAs against STAT3 mRNA drastically decreased the cell migration and invasion. Moreover, Ps-localized Stat3 was found to interact with pseudopod-enriched platelet-derived growth factor receptor tyrosine kinase (PDGFRTK) in a growth factor-dependent manner. This study reveals STAT3 mRNA localization at the Ps of metastatic hepatocellular carcinoma HCCLM3 cells by combining application of genome-wide and gene specific description and functional analysis.

Animal and cellular models of hepatocellular carcinoma bone metastasis: establishment and characterisation.

An increasingly high occurrence of bone metastases in hepatocellular carcinoma (HCC) patients highlights the importance of fundamental research on HCC bone metastasis, which has been limited in its success due to the lack of a model system. Establishment of animal and cellular models of HCC bone metastasis and discovery of HCC bone metastasis-related genes. Luciferase-transfected HCC cell lines HCCLM3, MHCC97H, and SMMC-7721 were used to inoculate nude mice intracardially. Formation of bone metastases was examined by bioluminescence imaging, SPECT, and pathology study. Metastatic cells in bone were isolated and subcultured. Differences between bone metastatic cells and their parental cells were studied by in vitro/in vivo assays. Mouse model of HCC bone metastasis was successfully established. Injected tumour cells formed metastases in the skull, the spine, the hind limbs, and the sternum, causing osteolytic lesions via act of MMP-1 and recruitment of osteoclasts. Four bone metastatic cell lines were extracted from HCCLM3-inoculated mice and were demonstrated to exhibit a much stronger ability to form bone metastases as well as other phenotypes, including enhanced in vitro migration/invasion and colony formation. Moreover, the expression of PTHrP, MMP-1, and CTGF was significantly elevated in bone metastatic cells compared to parental HCC cells. The nude mouse model and bone metastatic cell lines together provide an effective simulation of HCC bone metastasis. This model system will become powerful tool with which to explore the mechanisms and therapies of HCC bone metastasis. Additionally, PTHrP, MMP-1, and CTGF are candidate genes related to HCC bone metastasis.

Incomplete radiofrequency ablation enhances invasiveness and metastasis of residual cancer of hepatocellular carcinoma cell HCCLM3 via activating β-catenin signaling.

BackgroundRadiofrequency ablation (RFA) is one of the curative therapies for hepatocellular carcinoma (HCC), however, accelerated progression of residual HCC after incomplete RFA has been reported more frequently. The underlying molecular mechanism of this phenomenon remains to be elucidated. In this study, we used an incomplete RFA orthotopic HCC nude mouse model to study the invasive and metastatic potential of residual cancer as well as the correlated mechanism.MethodsThe incomplete RFA orthotopic nude mouse models were established using high metastatic potential HCC cell line HCCLM3 and low metastatic potential HCC cell line HepG2, respectively. The changes in cellular morphology, motility, metastasis and epithelial–mesenchymal transition (EMT), and HCC cell molecular markers after in vitro and in vivo incomplete RFA intervention were observed.ResultsPulmonary and intraperitoneal metastasis were observed in an in vivo study. The underlying pro-invasive mechanism of incomplete RFA appeared to be associated with promoting EMT, including down-regulation of E-cadherin and up-regulation of N-cadherin and vimentin. These results were in accordance with the in vitro response of HCC cells to heat intervention. Further studies demonstrated that β-catenin was a pivotal factor during this course and blocking β-catenin reduced metastasis and EMT phenotype changes in heat-treated HCCLM3 cells in vitro.ConclusionIncomplete RFA enhanced the invasive and metastatic potential of residual cancer, accompanying with EMT-like phenotype changes by activating β-catenin signaling in HCCLM3 cells.

The study of AGO1 regulates p53 expression and the prognosis of patients with hepatocellular carcinoma after curative resection

Objective To investigate AGO1 expression,functions and prognostic value in Hepatocellular carcinoma patients underwent radical resection.Methods AGO1 expression was measured in HCCLM3,MHCC97L,and HepG2 cell lines.Using small interfering RNA,AGO1 functions and its effects on p53 were investigated in HCCLM3 and HepG2 cell lines.Immunohistochemistry in tissue microarrays consisting of 200 tumor tissue from hepatocellular carcinoma (HCC) patients underwent radical resection was used to analyze the AGO1 expression with prognosis.Results AGO1 mRNA expression in HCC cell lines HCCLM3,MHCC97L and HepG2 is 0.400 ± 0.025,0.188 ± 0.022 and 0.154 ± 0.019,increased in parallel with the metastatic potential of the HCC cell lines (P ＜ 0.05).Cell proliferation,invasion,and p53 expression significantly decreased after AGO1 depletion in the HCC cell lines.Intratumoral AGO1 was correlated with younger age (P ＜ 0.05),higher α-fetoprotein (P ＜ 0.01),HBeAg-positive status (P ＜0.05),liver cirrhosis (P ＜ 0.05),intrahepatic and extrahepatic recurrence (P ＜ 0.01),and was an independent risk factor for overall survival (P ＜ 0.01) and recurrence-free survival (P ＜ 0.01).Conclusion These findings indicate that AGO1 may promote HCC proliferation and metastasis through the P53 pathways,and AGO1 may be a reliable prognostic factor in HCC patients after resection. Key words: AGO1 ; Hepatocellular carcinoma; Prognosis; p53

Reduced High-Mobility Group Box 1 Expression Induced by RNA Interference Inhibits the Bioactivity of Hepatocellular Carcinoma Cell Line HCCLM3

Increased expression of high-mobility group box 1 (HMGB1) has been observed in many tumor types, but the role of HMGB1 in hepatocellular carcinoma (HCC) is unknown. To examine the effects of RNA interference HMGB1 on the bioactivity of HCC cell line HCCLM3. We synthesized three specific small interfering RNAs of HMGB1 (HMGB1-siRNAs) and transfected these into HCCLM3 cells by use of Lipofectamine 2000. RT-PCR and Western blot were performed to determine the effects of HMGB1-siRNAs on HMGB1 expression and to detect NF-κB/p65 and VEGF-C expression after transfection of HMGB1-siRNAs into HCCLM3. In vitro proliferation was assessed by MTT assay. Migration and invasive ability were determined by use of the Transwell assay. Apoptosis was demonstrated by flow cytometry. RT-PCR and Western blotting showed that all three specific HMGB1-siRNAs significantly inhibited HMGB1 expression, with inhibition by HMGB1-siRNA-1 being highest (70-80%). MTT assay demonstrated that the growth of cells transfected with HMGB1-siRNA-1 was significantly lower than that of control cells (P < 0.01). The Transwell assay showed that cell migration and invasion were significantly inhibited in HMGB1 knockdown cells compared with control cells (P < 0.01). FCM revealed that apoptosis was significantly increased in HMGB1-siRNA-1-transfected cells compared with control cells (P < 0.01). Expression of NF-κB/p65 and VEGF-C was inhibited in HCCLM3 cells transfected with HMGB1-siRNA-1 compared with control cells (P < 0.01). Downregulation of HMGB1 could obviously inhibit the growth of HCCLM3 cells, and their migration and invasion ability. HMGB1 may serve as a potential target for treatment of HCC.

Fiber Confocal Back-Scattering Micro-Spectral Analysis for Single Cell

A fiber confocal back scattering micro-spectrometer (FCBS) was established, which combined fiber confocal microscopy with light scattering spectroscopy (LSS) for early diagnosis of the cancer cell at cellular level. An adherent monolayer human normal gastric epithelium line GES-1 and a carcinoma cell line NCI-N87 as well as a normal liver cell line L02 and a high-metastatic-potential hepatocellular carcinoma cell line HCC-LM3 were measured respectively. The spectral results showed that micro-back-scattering intensity from GES-1 cell and L02 cell possessed interesting oscillations in contrast to NCI-N87 and HCC-LM3 cells. There was significant difference between the spectra of the normal and the cancer cells (p<0.001). This demonstrates that the FCBS system here is able to distinguish dysplastic cells from normal cells at cellular level.