Identifying Clonal Origin of Multifocal Hepatocellular Carcinoma and Its Clinical Implications.

Hepatocellular carcinoma (HCC) is characterized by high prevalence of multifocality. Multifocal HCC can arise synchronously or metachronously either from intrahepatic metastasis (IM) or multicentric occurrence (MO). To date, there have been no established criteria to accurately distinguish whether multifocal HCC originates from IM or MO. Histopathological features remain the most convenient strategy but with subjectivity and limited accuracy. Various molecular biological techniques involving assessment of TP53 mutation status, hepatitis B virus integration sites, and chromosomal alterations have been applied to determine the clonal origin. The introduction of next-generation sequencing facilitates a more comprehensive annotation of intertumor heterogeneity, resulting in more sensitive and accurate clonal discrimination. Generally, MO-HCC has better overall survival than IM-HCC after curative resection. Adjuvant antiviral treatment has been proved to decrease post-treatment recurrence probably by reducing MO-HCC recurrence, whereas adjuvant sorafenib treatment targeting prior micrometastasis failed to reduce IM-HCC recurrence. Recent studies recommended transcatheter arterial chemoembolization (TACE) and traditional Chinese medicine Huaier granule as effective adjuvant treatments probably by preventing IM and both types of recurrences respectively. Immunotherapy that inhibits immune checkpoint interaction may be an optimal choice for both MO- and IM-HCC. In the future, effective personalized therapy against multifocal HCC may be achieved.

Extracellular matrix collagen I promotes the tumor progression of residual hepatocellular carcinoma after heat treatment.

BACKGROUND: Accelerated malignant behaviors induced by insufficient thermal ablation have been increasingly reported, however, the exact mechanisms are still unclear. Here, we investigated the importance of the extracellular matrix (ECM) in modulating the progression of residual hepatocellular carcinoma (HCC) after heat treatment. METHODS: Heat-exposed residual HCC cells were cultured in different ECM gels. We used basement membrane gel (Matrigel) to simulate the normal microenvironment and collagen I to model the pathological stromal ECM. The alterations of morphology and parameters of proliferation, epithelial-mesenchymal transition (EMT) and stemness were analyzed in vitro and in vivo. RESULTS: Increased collagen I deposition was observed at the periablational zone after incomplete RFA of HCC in a xenograft model. The markers of cell proliferation, EMT, motility and progenitor-like traits of heat-exposed residual HCC cells were significantly induced by collagen I as compared to Matrigel (p values all < 0.05). Importantly, collagen I induced the activation of ERK phosphorylation in heat-exposed residual HCC cells. ERK1/2 inhibitor reversed the collagen I-promoted ERK phosphorylation, cell proliferative, protrusive and spindle-like appearance of heat-treated residual HCC cells in vitro. Moreover, collagen I promoted the in vivo tumor progression of heat-exposed residual HCC cells, and sorafenib markedly reversed the collagen I-mediated protumor effects. CONCLUSIONS: Our findings demonstrate that collagen I could enhance the aggressive progression of residual HCC cells after suboptimal heat treatment and sorafenib may be a treatment approach to thwart this process.

Increased matrix stiffness promotes tumor progression of residual hepatocellular carcinoma after insufficient heat treatment.

Aggravated behaviors of hepatocellular carcinoma (HCC) will occur after inadequate thermal ablation. However, its underlying mechanisms are not fully understood. Here, we assessed whether the increased matrix stiffness after thermal ablation could promote the progression of residual HCC. Heat-treated residual HCC cells were cultured on tailorable 3D gel with different matrix stiffness, simulating the changed physical environment after thermal ablation, and then the mechanical alterations of matrix stiffness on cell phenotypes were explored. Increased stiffness was found to significantly promote the proliferation of the heat-treated residual HCC cells when the cells were cultured on stiffer versus soft supports, which was associated with stiffness-dependent regulation of ERK phosphorylation. Heat-exposed HCC cells cultured on stiffer supports showed enhanced motility. More importantly, vitamin K1 reduced stiffness-dependent residual HCC cell proliferation by inhibiting ERK phosphorylation and suppressed the in vivo tumor growth, which was further enhanced by combining with sorafenib. Increased matrix stiffness promotes the progression of heat-treated residual HCC cells, proposing a new mechanism of an altered biomechanical environment after thermal ablation accelerates HCC development. Vitamin K1 plus sorafenib can reverse this protumor effect.

Astragaloside IV inhibits metastasis in hepatoma cells through the suppression of epithelial-mesenchymal transition via the Akt/GSK-3ß/ß-catenin pathway.

Our previous studies demonstrated that traditional Chinese herbal medicine 'Songyou Yin' inhibited the growth and invasion of hepatocellular carcinoma (HCC) cells, and altered epithelial­mesenchymal transition (EMT) markers in oxaliplatin­treated HCC tissues and cell lines. In the present study, we aimed to explore whether astragaloside IV (AS-IV), a component of 'Songyou Yin', can affect the growth and invasion of HCC cells and the underlying mechanism involved. Human HCC cell lines Huh7 and MHCC97-H, with low and high metastatic potential, respectively, were treated with increasing doses of AS-IV. The Cell Counting Kit-8 (CCK-8), plate clone formation, Transwell, wound healing and immunofluorescence assays were used to investigate the effects of AS-IV on HCC cell proliferation, migration and invasion. The protein expression levels were analyzed by western blotting and immunofluorescence assay. The CCK-8 and plate clone formation assays showed that AS-IV had little effect on the proliferation of HCC cells in vitro. However, the Transwell and wound healing assays demonstrated that AS-IV inhibited the migration and invasion of HCC cells in a dose-dependent manner and the morphology of HCC cells was altered from spindle into oval shaped in the AS-IV pretreated groups. The upregulation of E-cadherin and downregulation of N-cadherin, vimentin, α-SMA and Slug were also observed in the AS-IV pretreated groups. Additionally, AS-IV treatment resulted in a profound decrease in the phosphorylated forms of Akt and GSK-3ß, which in turn inhibited the expression of ß-catenin. Thus, we conclude that AS-IV attenuates the invasive and migratory abilities of HCC cells through the inhibition of EMT by targeting the Akt/GSK-3ß/ß-catenin pathway.

Herbal Compound Songyou Yin and Moderate Swimming Suppress Growth and Metastasis of Liver Cancer by Enhancing Immune Function.

Objective Both the Chinese herbal compound Songyou Yin (SYY) and swimming exercise have been shown to have protective effects against liver cancer in animal models. In this study, we investigated whether SYY and moderate swimming (MS) have enhanced effect on suppressing progression of liver cancer by immunomodulation. Methods C57BL/6 mice were transplanted with Hepa1-6 murine liver cancer cell lines and received treatment with SYY alone or SYY combined with MS. The green fluorescent protein (GFP)-positive metastatic foci in lungs were imaged with a stereoscopic fluorescence microscope. Flow cytometry was used to test the proportion of CD4 +, CD8 + T cells in peripheral blood and the proportions of CD4 + CD25 + Foxp3 + Treg cells in peripheral blood, spleen, and tumor tissues. Cytokine transforming growth factor (TGF)-ß1 level in serum was detected by ELISA. Results SYY plus MS significantly suppressed the growth and lung metastasis of liver cancer and prolonged survival in tumor-burdened mice. SYY plus MS markedly raised the CD4 to CD8 ratio in peripheral blood and lowered the serum TGF-ß1 level and the proportions of Treg cells in peripheral blood, spleen, and tumor tissue. The effects of the combined intervention were significantly superior to SYY or MS alone. Conclusion The combined application of SYY and MS exerted an enhanced effect on suppressing growth and metastasis of liver cancer by strengthening immunity.

The herbal compound Songyou Yin (SYY) inhibits hepatocellular carcinoma growth and improves survival in models of chronic fibrosis via paracrine inhibition of activated hepatic stellate cells.

Chronic fibrosis is a major risk factor for the development of hepatocellular carcinoma (HCC). The pathological progression of hepatic fibrosis has been linked to cellular processes that promote tumor growth and metastasis. Several recent studies have highlighted the cross-talk between tumor cells and activated hepatic stellate cells (aHSCs) in HCC. The herbal compound Songyou Yin (SYY) is known to attenuate hepatoma cell invasion and metastasis via down-regulation of cytokine secretion by aHSCs. However the underlying mechanism of SYY treatment in reversal of hepatic fibrosis and metastasis of liver cancers is not known. In the current study, a nude mouse model with liver fibrosis bearing orthotopic xenograft was established and we found that SYY could reduce associated fibrosis, inhibit tumor growth and improve survival. In the subcutaneous tumor model with fibrosis, we found that SYY could inhibit liver cancer. In vitro, hepatoma cells incubated with conditioned media (CM) from SYY treated aHSCs showed reduced proliferation, decrease in colony formation and invasive potential. SYY treated group showed altered gene expression, with 1205 genes up-regulated and 1323 genes down-regulated. Gene cluster analysis indicated that phosphatidylinositol-3-kinase (PI3K) was one of the key genes altered in the expression profiles. PI3K related markers were all significantly down-regulated. ELISA also indicated decreased secretion of cytokines which were regulated by PI3K/AKT signaling after SYY treatment in the hepatic stellate cell line, LX2. These data clearly demonstrate that SYY therapy inhibits HCC invasive and metastatic potential and improves survival in nude mice models with chronic fibrosis background via inhibition of cytokine secretion by activated hepatic stellate cells.

Oxaliplatin and 5-fluorouracil hepatic infusion with lipiodolized chemoembolization in large hepatocellular carcinoma.

AIM: To investigate transarterial chemoembolization (TACE) with hepatic infusion of oxaliplatin and 5-fluorouracil and Lipiodol chemoembolization in large hepatocellular carcinoma (HCC). METHODS: In this retrospective study, 132 patients with unresectable HCCs larger than 10 cm were treated with hepatic infusion of oxaliplatin and 5-fluorouracil followed by Lipiodol chemoembolization. The primary endpoint was overall survival (OS). Sixteen-week disease-control rate, time to progression (TTP), and major complications were also studied. Univariate and multivariate analyses were performed to identify prognostic factors affecting OS and TTP. RESULTS: A total of 319 procedures were performed in the 132 patients. Eleven (8.3%) patients received radical resection following TACE treatment (median time to initial TACE 4.3 ± 2.3 mo). The median OS and TTP were 10.3 and 3.0 mo respectively, with a 50.0% 16-wk disease-control rate. Major complications were encountered in 6.0% (8/132) of patients following TACE and included serious jaundice in 1.5% (2/132) patients, aleukia in 1.5% (2/132), and hepatic failure in 3.0% (4/132). One patient died within one month due to serious hepatic failure and severe sepsis after receiving the second TACE. The risk factor associated with TTP was baseline alpha-fetoprotein level, and vascular invasion was an independent factor related to OS. CONCLUSION: Hepatic infusion of oxaliplatin and 5-fluorouracil followed by lipiodolized-chemoembolization is a safe and promising treatment for patients with HCCs larger than 10 cm in diameter.

Herbal compound "Songyou Yin" attenuates hepatoma cell invasiveness and metastasis through downregulation of cytokines secreted by activated hepatic stellate cells.

BACKGROUND: Activated hepatic stellate cells (aHSCs) play an important role in the progression of hepatocellular carcinoma (HCC). Here, we determined if cytokines secreted in response to the herbal compound "Songyou Yin" (SYY) treatment of aHSCs could influence invasiveness and metastatic capabilities of hepatoma cells. METHODS: Primary rat hepatic stellate cells (HSCs) were isolated, activated, divided into SYY treated and untreated (nSYY) groups, and conditioned media (CM-SYY and CM-nSYY, respectively) were collected. The hepatoma cell line, McA-RH7777 was cultured for 4 weeks with SYY, CM-SYY, and CM-nSYY, designated McA-SYY, McA-SYYCM and McA-nSYYCM. The invasiveness and metastatic capabilities were evaluated using Matrigel invasion assay in vitro and pulmonary metastasis in vivo. Matrix metalloproteinase-2 (MMP-2), MMP-9, E-cadherin, N-cadherin, and vimentin protein levels in McA-SYYCM and McA-nSYYCM were evaluated by Western blot. Cytokine levels in conditioned media were tested using enzyme-linked immunosorbent assay (ELISA). RESULTS: Matrigel invasion assay indicated that the number of McA-SYYCM cells passing through the basement membrane was less than in McA-nSYYCM cells (P < 0.01). Similar results were also observed in vivo for lung metastasis. McA-SYYCM cells showed less pulmonary metastasis capabilities than McA-nSYYCM cells (P < 0.001). The reduced expression of MMP-2 and reversed epithelial to mesenchymal transition with E-cadherin upregulation, and N-cadherin and vimentin downregulation were also found in McA-SYYCM compared to McA-nSYYCM. Metastasis-promoting cytokines hepatocyte growth factor, interleukin-6, transforming growth factor-ß1, and vascular endothelial growth factor were markedly decreased in CM-SYY compared to CM-nSYY. CONCLUSIONS: SYY attenuates hepatoma cell invasiveness and metastasis capabilities through downregulating cytokines secreted by activated hepatic stellate cells.

[Effects of the Chinese herbal extract Songyou Yin on the residual hepatocellular carcinoma after chemotherapy in nude mice].

OBJECTIVE: To investigate the effects of a Chinese herbal extract Songyou Yin on residual hepatocellular carcinoma after chemotherapy in nude mice and the relevant mechanisms. METHODS: Orthotopic nude mouse models bearing residual hepatocellular carcinoma after chemotherapy was established using human liver carcinoma MHCC97L cells. Three different doses of Songyon Yin (2.1 g/kg, 4.2 g/kg and 8.4 g/kg) were administered to the mice in the trial groups by intragastric gavage, respectively. The mice in the control group were administered physiological saline. The tumor growth, metastasis and survival in the mice of each group were recorded. The corresponding mechanisms were studied. RESULTS: The pulmonary metastasis rates of the control group and 2.1g/kg, 4.2g/kg, 8.4g/kg Songyou Yin treatment group were 86.7%, 73.3%, 40.0%, and 20.0%, respectively, and the survivals of these groups were 53.83 ± 4.71, 56.50 ± 6.09, 66.67 ± 5.61, 81.17 ± 7.36 days, respectively. Compared with the mice in the control group, mice in the 4.2 g/kg, 8.4 g/kg Songyou Yin treatment groups had a lower pulmonary metastasis rate (P = 0.021 and P = 0.001, respectively) and longer survival (P = 0.002 and P = 0.001, respectively). A restoration of E-cadherin expression and a concomitant reduction of N-cadherin expression were detected in the tumors of the 4.2 g/kg and 8.4 g/kg Songyou Yin treatment groups. CONCLUSIONS: Songyou Yin effectively inhibits the invasion and metastasis of the residual hepatocellular carcinoma after chemotherapy in nude mice through attenuating the epithelia-mesenchymal transition and prolongs the survival. Songyon Yin may have potential to promote the efficacy of chemotherapy in hepatocellular carcinoma.

[Evaluation of the efficacy and prognostic factors for colorectal liver metastases treated with transcatheter arterial chemoembolization].

OBJECTIVE: The aim of this study was to evaluate the therapeutic efficacy and to determine the prognostic factors of TACE in patients with colorectal liver metastases (CRLM). METHODS: The clinical data of 183 patients with unresectable CRLM treated with TACE from Jan. 2002 to Dec. 2008 were retrospectively reviewed. Log-rank method was used for univariate analysis and Cox proportional hazard model was used for multivariate analysis of the prognostic factors. RESULTS: The median survival time was 22 months, and the 0.5-, 1-, 2-, 3-, 5-year survival rates were 93.9%, 81.1%, 39.8%, 18.2%, and 3.9%, respectively. Multivariate analysis showed that tumor involved more than one lobe of the liver, and elevated CEA and CA19-9 levels were independent risk factors for the overall survival (P < 0.01). Females, more times of TACE, combination with regional therapy and received phase II resection were related with a good survival (P < 0.01) in CRLM patients after TACE treatment. CONCLUSIONS: Transcatheter arterial chemoembolization is an effective therapy for unresectable colorectal liver metastases. Patients with tumor spread more than one lobe of the liver, high CEA and CA19-9 levels are independent poor prognostic factors. Females, patients received more times of TACE, combined with regional therapy and received phase II resection may have a good survival.

Herbal Compound "Songyou Yin" Renders Hepatocellular Carcinoma Sensitive to Oxaliplatin through Inhibition of Stemness.

We investigated the effect of Chinese herbal compound Song-you Yin on HCC stemness. MHCC97H and Hep3B cell lines were pretreated with SYY for 4 weeks, and their chemosensitivity to oxaliplatin was evaluated. The expression of CSC-related markers, cell invasion and migration, and colony formation were also examined. SYY-treated orthotopic nude mouse models of human HCC were developed to explore the effect of oxaliplatin on tumor growth, metastasis, and survival. The CSC-related molecular changes in vivo were also evaluated. The result showed that MHCC97H and Hep3B cells pretreated with SYY showed significantly increased chemosensitivity to oxaliplatin and the downregulation of CSC-related markers CD90, CD24, and EPCAM. SYY also attenuated cell motility, invasion, and colony formation in MHCC97H and Hep3B cell lines. The reduced tumorigenicity and pulmonary metastasis were observed in SYY-pretreated cell lines. Combination treatment with oxaliplatin and SYY significantly reduced tumor volume and pulmonary metastasis and prolonged survival compared with oxaliplatin treatment alone. Immunohistochemical analysis showed reduced expression of CD90, ABCG2, ALDH, CD44, EPCAM, vimentin, and MMP-9 and increased the expression of E-cadherin, in HCC cells following combination treatment. These data clearly demonstrate that SYY renders hepatocellular carcinoma sensitive to oxaliplatin through the inhibition of stemness.

[Therapeutic efficacy and prognostic factors of sorafenib treatment in patients with unresectable primary hepatocellular carcinoma].

OBJECTIVE: To evaluate the efficacy and analyze the prognostic factors of sorafenib treatment in patient with unresectable primary hepatocellular carcinoma (HCC). METHODS: During the period from December 2005 to March 2009, 50 patients with unresectable primary HCC of Child-Pugh status A were treated with sorafenib (400 mg, Bid). The tumor response was evaluated with CT or MRI imaging every 6 - 8 weeks according to the RECIST criteria. The overall survival (OS) and time to progression (TTP) were defined as the time from administration of sorafenib to the death or the last follow up and were evaluated by Kaplan-Meier method. RESULTS: There was no PR or CR, but 28 patients (56.0%) achieved stable disease. The median follow up time was 15 months with a median OS of 14 months and median TTP of 4 months. The common adverse events were dermal reaction (68.0%, 34/50), diarrhea (52.0%, 26/50), hypertension (4.0%, 2/50), hair loss (14.0%, 7/50), myelosuppression (16.0%, 8/50), and liver dysfunction (20.0%, 10/50). However, most of the drug-related adverse events were grade I-II and reversible. The patients with lower tumor burden and without distant metastasis had better prognosis. CONCLUSION: Soafenib is effective for unresectable primary HCC with tolerable toxicity. Tumor stage is a predominant prognostic factor.

Residual hepatocellular carcinoma after oxaliplatin treatment has increased metastatic potential in a nude mouse model and is attenuated by Songyou Yin.

BACKGROUND: The opposite effects of chemotherapy, which enhance the malignancy of treated cancers such as hepatocellular carcinoma (HCC), are not well understood. We investigated this phenomenon and corresponding mechanisms to develop a novel approach for improving chemotherapy efficacy in HCC. METHODS: Human hepatocellular carcinoma cell lines HepG2 (with low metastatic potential) and MHCC97L (with moderate metastatic potential) were used for the in vitro study. An orthotopic nude mouse model of human HCC was developed using MHCC97L cells. We then assessed the metastatic potential of surviving tumor cells after in vitro and in vivo oxaliplatin treatment. The molecular changes in surviving tumor cells were evaluated by western blot, immunofluorescence, and immunohistochemistry. The Chinese herbal extract Songyou Yin (composed of five herbs) was investigated in vivo to explore its effect on the metastatic potential of oxaliplatin-treated cancer cells. RESULTS: MHCC97L and HepG2 cells surviving oxaliplatin treatment showed enhanced migration and invasion in vitro. Residual HCC after in vivo oxaliplatin treatment demonstrated significantly increased metastasis to the lung (10/12 vs. 3/12) when re-inoculated into the livers of new recipient nude mice. Molecular changes consistent with epithelial-mesenchymal transition (EMT) were observed in oxaliplatin-treated tumor tissues and verified by in vitro experiments. The Chinese herbal extract Songyou Yin (4.2 and 8.4 g/kg) attenuated EMT and inhibited the enhanced metastatic potential of residual HCC in nude mice (6/15 vs. 13/15 and 3/15 vs. 13/15, respectively). CONCLUSIONS: The surviving HCC after oxaliplatin treatment underwent EMT and demonstrated increased metastatic potential. Attenuation of EMT by Songyou Yin may improve the efficacy of chemotherapy in HCC.

[Transcatheter hepatic arterial chemoembolization on recurrent hepatocellular carcinoma after resection].

OBJECTIVE: To investigate the effect of transcatheter hepatic arterial chemoembolization (TACE) therapy on the survival and prognosis of recurrent hepatocellular carcinoma (HCC) after surgical resection. METHODS: The data of 130 surgically resected but recurrent HCC patients treated by TACE were reviewed retrospectively. The survival and influencing factors on the prognosis were analyzed. RESULTS: The overall 1-, 3-, 5-year survival rates of these 130 patients were 83.0%, 45.5% and 17.6% respectively (median survival time 2.4 years). Ninty-four of the series were treated with TACE alone, which gave the 1-, 3- year survival rates of 76.4% and 37.1%, respectively (median survival time 2.1 years). Thirty-six out of 130 patients treated with TACE plus percutaneous ethanol injection (PEI), the 1-, 3-year survival rates were 100.0% and 66.5% respectively with a median survival time (MST) of 3.5 years. The survival of TACE plus PEI group was significantly better, and the mortality risk was significantly lower than that of TACE alone group (P < 0.05). The mortality risk of those with > 5 cm diameter recurrent tumor or with distant metastasis was significantly higher than those with < or = 5 cm diameter tumor or without metastasis (P < 0.05). CONCLUSION: TACE combined with PEI may improve the survival of recurrent HCC patients.