Infiltrating regulatory T cells promote invasiveness of liver cancer cells via inducing epithelial-mesenchymal transition.

BACKGROUND: Regulatory T (Treg) cells are a major component of the microenvironment of hepatocellular carcinoma (HCC) contributing to immunosuppression. The present study aimed to evaluate the effects of Treg cells on the invasion potential of HCC. METHODS: Infiltrating Treg cells were isolated from fresh HCC tissues by immunomagnetic bead separation and detected by flow cytometry. Circulating tumor cells (CTCs) were detected using the CellSearch platform. The cell migration and invasion potentials were evaluated by Transwell assays. The cell viability was tested by the cell counting kit-8 (CCK8) approach, and the apoptosis rates were determined by flow cytometry. The concentrations of active transforming growth factor-ß1 (TGFß1) were measured by enzyme-linked immunosorbent assay. RESULTS: Infiltrating Treg cells significantly correlated with the number of CTCs and vascular invasion (both P<0.05). Moreover, these cells could greatly promote HCC migration, invasion, and proliferation, and inhibit HCC apoptosis. Polymerase chain reaction and Western blot assays revealed that Treg cells significantly decreased the expression levels of epithelium-related molecules and increased the expression levels of mesenchyme-related molecules. Treg cells could activate Smad2/3 via secreting TGFß1, and these effects could be impaired by knocking down the expression of TGFß1 in Treg cells. CONCLUSIONS: The involvement of infiltrating Treg cells in triggering the TGFß1 signaling pathway and promoting the epithelial-mesenchymal transition (EMT) of cancer cells during tumor hematogenous dissemination is presumably responsible for increasing the invasiveness potential of HCC cells. Targeting Treg cells in microenvironments can be a promising therapeutic strategy to improve the prognosis for patients with HCC undergoing resection.

A mouse radiation-induced liver disease model for stereotactic body radiation therapy validated in patients with hepatocellular carcinoma.

PURPOSE: Lower radiation tolerance of the whole liver hinders dose escalations of stereotactic body radiation therapy (SBRT) in hepatocellular carcinoma (HCC) treatment. This study was conducted to define the exact doses that result in radiation-induced liver disease (RILD) as well as to determine dose constraints for the critical organs at risk (OARs) in mice; these parameters are still undefined in HCC SBRT. METHODS: This study consisted of two phases. In the primary phase, mice treated with helical tomotherapy-based SBRT were stratified according to escalating radiation doses to the livers. The pathological differences, signs [such as mouse performance status (MPS)], and serum aspartate aminotransferase (AST)/alanine aminotransferase (ALT)/albumin levels were observed. Radiation-induced disease severities of the OARs were scored using systematic evaluation standards. In the validation phase in humans, 13 patients with HCC who had undergone radiotherapy before hepatectomy were enrolled to validate RILD pathological changes in a mouse study. RESULTS: The evaluation criteria of the mouse liver radiotherapy-related signs were as follows: MPS ≥ 2.0 ± 0.52, AST/ALT ≥ 589.2 ± 118.5/137.4 ± 15.3 U/L, serum albumin ≤ 16.8 ± 2.29 g/L. The preliminary dose constraints of the OARs were also obtained, such as those for the liver (average dose ≤ 26.36 ± 1.71 Gy) and gastrointestinal tract (maximum dose ≤ 22.63 Gy). Mouse RILD models were able to be developed when the livers were irradiated with average doses of ≥31.76 ± 1.94 Gy (single fraction). RILD pathological changes in mice have also been validated in HCC patients. CONCLUSIONS: Mouse RILD models could be developed with SBRT based on the dose constraints for the OARs and evaluation criteria of mouse liver radiotherapy-related signs, and the authors' results favor the study of further approaches to treat HCC with SBRT.

A polymeric nanoparticle formulation of curcumin in combination with sorafenib synergistically inhibits tumor growth and metastasis in an orthotopic model of human hepatocellular carcinoma.

Curcumin, a yellow polyphenol extracted from the rhizome of turmeric root (Curcuma longa) has potent anti-cancer properties in many types of tumors with ability to reverse multidrug resistance of cancer cells. However, widespread clinical application of this agent in cancer and other diseases has been limited due to its poor aqueous solubility. The recent findings of polymeric nanoparticle formulation of curcumin (NFC) have shown the potential for circumventing the problem of poor solubility, however evidences for NFC's anti-cancer and reverse multidrug resistance properties are lacking. Here we provide models of human hepatocellular carcinoma (HCC), the most common form of primary liver cancer, in vitro and in vivo to evaluate the efficacy of NFC alone and in combination with sorafenib, a kinase inhibitor approved for treatment of HCC. Results showed that NFC not only inhibited the proliferation and invasion of HCC cell lines in vitro, but also drastically suppressed primary tumor growth and lung metastases in vivo. Moreover, in combination with sorafenib, NFC induced HCC cell apoptosis and cell cycle arrest. Mechanistically, NFC and sorafenib synergistically down-regulated the expression of MMP9 via NF-κB/p65 signaling pathway. Furthermore, the combination therapy significantly decreased the population of CD133-positive HCC cells, which have been reported as cancer initiating cells in HCC. Taken together, NanoCurcumin provides an opportunity to expand the clinical repertoire of this agent. Additional studies utilizing a combination of NanoCurcumin and sorafenib in HCC are needed for further clinical development.

TLR4-dependant immune response, but not hepatitis B virus reactivation, is important in radiation-induced liver disease of liver cancer radiotherapy.

Toll-like receptor 4 (TLR4) is an important trigger of the immune response against hepatitis B virus (HBV) infection and liver injuries. The roles of HBV reactivation versus TLR4-dependant immune response may be critical factors in preventing radiation-induced liver diseases (RILDs) after liver cancer radiotherapy. This study consists of three phases. In the primary phase, livers of mutant TLR4 (TLR4(-)) mice were irradiated with 30 Gy in either the absence or presence of HBV infection. The latter was done by introduction of plasmid pAAV/HBV 1.2. In the advanced phase, RILDs were compared in normal TLR4 (TLR4(+)) versus TLR4(-) mice. In the validation phase, 28 liver cancer patients who had undergone radiotherapy before hepatectomy were enrolled. Liver biopsies near tumors, irradiated with 35-48 Gy, were used to construct tissue microarrays. HBV reactivation, TLR4 expression, and severity of RILDs were studied in both mouse and human. More HBV reactivation, without significant RILD, was observed in irradiated versus unirradiated TLR4(-) mice. RILD scores of TLR4(+) mice were higher than TLR4(-) mice. In humans, serious RILDs tended to develop in patients with high TLR4 expression, but not in patients with low TLR4 or high HBV surface antigen expression. High TLR4 expression was seen in only 2 of 12 HBV-reactive patients, but in HBV-nonreactive patients, it was seen in 6 of 9 (P < 0.03). In summary, RILDs correlated with high TLR4 expression, but not with HBV reactivation, which is inhibited in liver with high TLR4 expression after liver cancer radiotherapy.

Enhanced production of CTGF and IL-11 from highly metastatic hepatoma cells under hypoxic conditions: an implication of hepatocellular carcinoma metastasis to bone.

PURPOSE: The biology underlying bone-specific metastasis (BM) of hepatocellular carcinoma (HCC) is poorly understood. The goal of the present study is to further elucidate the molecular and cellular mechanisms underlying HCC with BM. METHODS: The expression of connective tissue growth factor (CTGF) and interleukin-11 (IL-11) in RNA extracted from 127 formalin-fixed, paraffin-embedded HCC specimens was examined by quantitative real-time polymerase chain reaction. A cellular hypoxic model was established in vitro to investigate CTGF and osteoprotegerin (OPG) expression and roles in hypoxia-induced tumor aggressiveness. RESULTS: The mean CTGF expression in BM versus non-metastatic samples was 3.63-times higher, and IL-11 expression was detected in 62.5 % (10/16) of BM samples versus only in 18.9 % (21/111) of the non-metastatic ones. Highly metastatic HCC cell lines tended to show strong expression of CTGF and IL-11, but low expression of OPG. Hypoxic stimulation of HCC 97L cells increased the level of CTGF mRNA by 2.80-fold within 1.5 h, and hypoxia-inducible factor-1α mRNA levels in these cells could be increased by stimulation with recombinant CTGF protein. Furthermore, OPG and matrix metalloproteinase-2 and -9 levels were also induced under hypoxic conditions. CONCLUSIONS: Expression levels of intratumoral CTGF or IL-11 were independent prognostic factors for the development of BM in HCC patients. Tumor hypoxia enhanced the expression of CTGF, which initiates the invasive angiogenesis cascade and enhances expression of many hypoxia-associated genes. Cellular release of OPG may play a role in tumor cell survival. The hypoxia-induced cascade in HCC cells may contribute to invasion and metastasis in vivo.

Stimulation of hepatoma cell invasiveness and metastatic potential by proteins secreted from irradiated nonparenchymal cells.

PURPOSE: To determine whether factors secreted by irradiated liver nonparenchymal cells (NPCs) may influence invasiveness and/or metastatic potential of hepatocellular carcinoma (HCC) cells and to elucidate a possible mechanism for such effect. METHODS AND MATERIALS: Primary rat NPCs were cultured and divided into irradiated (10-Gy X-ray) and nonirradiated groups. Forty-eight hours after irradiation, conditioned medium from irradiated (SR) or nonirradiated (SnonR) cultures were collected and added to sublethally irradiated cultures of the hepatoma McA-RH7777 cell line. Then, hepatoma cells were continuously passaged for eight generations (RH10Gy-SR and RH10Gy-SnonR). The invasiveness and metastatic potential of McA-RH7777, RH10Gy-SnonR, and RH10Gy-SR cells were evaluated using an in vitro gelatinous protein (Matrigel) invasion and an in vivo metastasis assay. In addition, SR and SnonR were tested using rat cytokine antibody arrays and enzyme-linked immunosorbent assay (ELISA). RESULTS: In vitro gelatinous protein invasion assay indicated that the numbers of invading cells was significantly higher in RH10Gy-SR (40 ± 4.74) than in RH10Gy-SnonR (30.6 ± 3.85) cells, and lowest in McA-RH7777 (11.4 ± 3.56) cells. The same pattern was observed in vivo in a lung metastasis assay, as evaluated by number of metastatic lung nodules seen with RH10Gy-SR (28.83 ± 5.38), RH10Gy-SnonR (22.17 ± 4.26), and McA-RH7777 (8.3 ± 3.8) cells. Rat cytokine antibody arrays and ELISA demonstrated that metastasis-promoting cytokines (tumor necrosis factor-α and interleukin-6), circulating growth factors (vascular endothelial growth factor and epidermal growth factor), and metalloproteinases (MMP-2 and MMP-9) were upregulated in SR compared with SnonR. CONCLUSIONS: Radiation can increase invasiveness and metastatic potential of sublethally irradiated hepatoma cells, and soluble mediators released from irradiated NPCs promote this potential. Increased secretion of metastasis-related cytokines and factors from NPCs after irradiation may be a possible mechanism for the radiation-induced invasiveness and metastatic potential of HCC.

Expression of deleted in malignant brain tumours 1 (DMBT1) relates to the proliferation and malignant transformation of hepatic progenitor cells in hepatitis B virus-related liver diseases.

AIMS: The molecular mechanisms underlying proliferation and malignant transformation of hepatic progenitor cells (HPCs) remain largely unknown. The purpose of this study was to evaluate the correlation between the expression of deleted in malignant brain tumours 1 (DMBT1) and the biological behaviour of HPCs in different hepatitis B virus (HBV)-related human liver diseases. METHODS AND RESULTS: Expression of DMBT1 in HPCs was investigated by double immunofluorescence labelling in control-group and HBV-related liver diseases, including hepatitis, hepatocellular carcinoma (HCC), non-tumoral liver tissue away from HCC, non-tumoral cirrhotic tissue adjacent to HCC, and non-HCC cirrhosis. DMBT1-positive HPCs were isolated by laser capture microdissection and subjected to duplex polymerase chain reaction in order to detect homozygous deletion of DMBT1. The number of DMBT1-positive HPCs increased in direct proportion to inflammation severity. Loss of heterozygosity for DMBT1 was more frequent in HCC tumour area and non-tumoral cirrhotic tissue adjacent to HCC, compared with other HBV-related liver diseases (P < 0.05). CONCLUSIONS: DMBT1 may play an important role in the proliferation of HPCs in HBV-related liver diseases. Moreover, down-expression of DMBT1 might enhance the risk of malignant transformation of HPCs.

Hepatic progenitor cell represents a transitioning cell population between liver epithelium and stroma.

Following an acute injury, the liver may maintain its structure and function through mitotic division of mature hepatocytes (i.e. hepatic regeneration). However, the regeneration ability of hepatocytes can be impaired in chronic liver diseases including chronic viral infection and alcohol abuse. Hepatic progenitor cells/oval cells (HPCs/OCs), capable of differentiation into both hepatocytes and cholangiocytes, occur and proliferate during chronic injury. Unfortunately, a use of HPCs for clinical therapy is blocked by the difficulty of exact identity of HPCs in liver. Focusing on the links between phenotype of HPCs and real stem cells originating from fetal liver or bone marrow (BM), the recent studies of HPCs neglect functional analysis and the close relationship between activation of HPCs and extracellular matrix (ECM) remodeling. It is currently widely accepted that mesenchymal-epithelial transition (EMT) and epithelial-mesenchymal transition (MET) play important roles not only in liver development but also in healing of chronic injured adult liver. Co-expression of epithelial/mesenchymal and HPCs markers has been demonstrated in cells undergoing EMT/MET. These cells led to hepatic regeneration after transplanted into rats with chronic liver injury. Notably, there is an increased expression of mesenchymal markers in HPCs after exposure to transforming growth factor-beta1 (TGF-ß1). Based on these evidences, we hypothesize that HPCs represent a transitioning cell population undergoing EMT/MET, both parenchymal and mesenchymal cells of liver may be the direct sources of HPCs.

Aberrant expression and demethylation of gamma-synuclein in colorectal cancer, correlated with progression of the disease.

Recent evidence suggests that gamma-synuclein is abnormally expressed in a high percentage of tumor tissues of diversified cancer types, but rarely expressed in tumor-matched non-neoplastic adjacent tissues (NNAT). The molecular mechanism of CpG island demethylation may underlie aberrant gamma-synuclein expression. To fully understand the roles of aberrant gamma-synuclein expression and demethylation in the development of colorectal cancer (CRC), we examined the expression and methylation status of gamma-synuclein in 67 CRC samples, 30 NNAT samples, and five CRC cell lines as well. By using reverse transcription-polymerase chain reaction (RT-PCR), western blot, and immunohistochemistry analyses, gamma-synuclein expression was detected in both HT-29 and HCT116 cells, and was much higher in CRC samples than in NNAT samples (P < 0.05). The demethylating agent, 5-aza-2 cent-deoxycytidine, can induce re-expression of gamma-synuclein in COLO205, LoVo, and SW480 cells. Unmethylated gamma-synuclein alleles were detected in HT-29, HCT116, and LoVo cells by nested methylation-specific PCR, and the demethylated status of gamma-synuclein was much higher in CRC samples than in NNAT samples by real-time quantitative methylation-specific PCR (P < 0.05). The results of genomic bisulfite DNA sequencing further confirmed that the aberrant gamma-synuclein expression in CRC was primarily attributed to the demethylation of CpG island. The protein expression and demethylation status of gamma-synuclein in 67 CRC samples correlated with clinical stage, lymph node involvement, and distant metastasis. These findings suggest an involvement of aberrant gamma-synuclein expression and demethylation in progression of CRC, especially in advanced stages.

Preliminary study on the clinical and pathological relevance of gastric hepatoid adenocarcinoma.

OBJECTIVE: To appraise the histological characteristics and clinical features of gastric hepatoid adenocarcinoma (GHAC) and their relevance with prognosis. METHODS: From January 2001 to December 2003, six patients were diagnosed and confirmed pathologically in Ruijin Hospital as having a gastric hepatoid carcinoma. All these six patients, together with 30 randomly selected patients with gastric poorly differentiated adenocarcinoma (GPDA) and 30 with a primary hepatocellular carcinoma (HCC) who served as controls, were studied and analyzed clinically, histologically and immunohistologically. RESULTS: The average age of the six patients with GHAC was 66.8 years, and their serum alpha fetoprotein (AFP) level was 84-2230 ng/mL. Of these six patients, two had a recurrence of cancer and two had liver metastasis. Their average survival period was 17 months. Morphologically, the histological appearance was similar to that of HCC, with glycogen granules and hyaline globules arranged in a solid or trabecular pattern and an abundance of blood vessels and sinusoids, while blood vessel or lymphatic invasion were more often seen. Immunohistological staining showed that the tumor cells were positive for AFP and negative for Hepatocyte paraffin 1 (HepPar1), and the tumor cells were separated by CD34-positive blood vessels into a small trabecular pattern. The GPDA had a solid nest or diffuse distribution pattern and was negative for both AFP and HepPar1. In the HCC, the histological feature was manifested as solid, small and large trabecular patterns with abundance of blood vessels and sinusoids; immunohistochemical staining showed the tumor cells were positive for HepPar1. CONCLUSION: Serum AFP-positive GHAC occurred more frequently in older patients with higher rates of recurrence and liver metastasis and a poor prognosis. Histologically, GHAC was similar to HCC. The tumor cells that were positive for AFP but negative for HerPar1 could be used to differentiate with GPDA (both AFP and HerPar1 negative) and primary HCC (most AFP and HerPar1 positive).