Xiao-Wu Huang

Effect of Rapamycin Alone and in Combination with Sorafenib in an Orthotopic Model of Human Hepatocellular Carcinoma

Purpose: Novel therapeutic strategies are needed to prevent the tumor recurrence or metastasis after liver transplantation for hepatocellular carcinoma (HCC). This study was to investigate the effect of rapamycin, alone and in combination with sorafenib, on HCC in vivo. Experimental Design: Xenograft of a highly metastatic human HCC tumor (LCI-D20) was used to evaluate primary tumor growth and lung metastasis after treatment with rapamycin alone or in combination with sorafenib. Tumor cell proliferation was determined by Ki-67 immunostaining. To detect tumor cell apoptosis, the terminal deoxynucleotidyl-transferase–mediated dUTP nick-end labeling assay was used. Tumor angiogenesis was investigated by using a monoclonal anti-CD31 antibody. A vascular endothelial growth factor ELISA kit was used to measure vascular endothelial growth factor protein levels in the mice serum. Results: Rapamycin, alone and in combination with sorafenib, strongly inhibited primary tumor growth and lung metastases in LCI-D20 model. Furthermore, the combination therapy significantly enhanced the effect of antitumor on primary tumor growth compared with single treatment with either rapamycin (P < 0.001) or sorafenib (P < 0.001). Rapamycin alone inhibited HCC cell proliferation, induced apoptosis, and decreased tumor angiogenesis. Nevertheless, the combination therapy showed a significant inhibition of tumor cell proliferation (P < 0.05). Additionally, the combination therapy also further enhanced suppression of tumor cell angiogenesis compared with rapamycin treatment (P < 0.01). However, the induction of apoptosis in combination therapy group was not significantly higher than in the rapamycin-treated group (P > 0.05). Conclusions: The combination therapy of rapamycin and sorafenib could be a new and promising therapeutic approach to the treatment of HCC and prevention of HCC recurrence after liver transplantation.

Overexpression of CXCL5 mediates neutrophil infiltration and indicates poor prognosis for hepatocellular carcinoma

CXCL5 (epithelial neutrophil‐activating peptide‐78) is a member of a proangiogenic subgroup of the CXC‐type chemokine family of small, secreted proteins. Recently, evidence that CXCL5 is involved in carcinogenesis and cancer progression has emerged. To investigate the role of CXCL5 in tumor growth, invasion, and prognosis of hepatocellular carcinoma (HCC), we examined CXCL5 messenger RNA (mRNA) and protein levels in HCC cell lines with various metastatic potentials and in three independent cohorts of 919 HCC patients. We found that CXCL5 expression was increased in the highly metastatic HCC cell lines and in tumor tissues from patients with recurrent HCC compared to controls. CXCL5 activated the PI3K‐Akt and ERK1/2 signaling pathways in HCC cells and promoted proliferation, migration, and invasion. Furthermore, we found that CXCL5 had a direct chemoattractant effect on neutrophils in vitro. In animal studies, the up‐regulation of CXCL5 in HCC cells promoted tumor growth, lung metastasis, and intratumoral neutrophil infiltration. Conversely, down‐regulation of CXCL5 in HCC cells reduced tumor growth, metastasis, and intratumoral neutrophil infiltration. Immunohistochemical analysis in HCC samples showed that overexpression of CXCL5 was well correlated with intratumoral neutrophil infiltration, shorter overall survival, and tumor recurrence. Multivariate analysis revealed that CXCL5 overexpression alone, or combined with the presence of intratumoral neutrophils, was an independent prognostic indicator for overall survival and cumulative recurrence. Conclusion: CXCL5 promotes HCC cell proliferation, invasion, and intratumoral neutrophil infiltration. CXCL5 overexpression, alone or combined with intratumoral neutrophil presence, is a novel prognostic predictor, and CXCL5 is a potential therapeutic target for HCC. (HEPATOLOGY 2012;56:2242–2254)

Sirolimus inhibits the growth and metastatic progression of hepatocellular carcinoma

PurposeImmunosuppressive therapy after liver transplantation for hepatocellular carcinoma (HCC) is one of the major contributory factors for HCC recurrence and metastasis. Sirolimus, a potent immunosuppressant, has been reported to be an effective inhibitor in a variety of tumors. The present study is designed to explore whether sirolimus could block the growth and metastatic progression of HCC.MethodsMHCC97H cells were used as targets to explore the effect of sirolimus on cell cycle progression, apoptosis, proliferation, and its antiangiogenic mechanism. LCI-D20, a highly metastatic model of human HCC in nude mice, was also used as the model tumor to explore the effect of sirolimus on tumor growth and metastatic progression.ResultsIn vitro, sirolimus induced cell cycle arrest at the G1 checkpoint and blocked proliferation of MHCC97H cells but did not induce apoptosis. In vivo, sirolimus prevented tumor growth and metastatic progression in LCI-D20. Intratumoral microvessel density and circulating levels of VEGF in tumor-bearing mice were also significantly reduced in sirolimus treatment group. Quantitative RT-PCR showed that sirolimus down-regulated the mRNA expression of VEGF and HIF-1a, but not of bFGF, and TGF-b in MHCC97H cells. Furthermore, western blot analysis confirmed that sirolimus also decreased expression of HIF-1a at protein level, in parallel with the down-regulation of the levels of VEGF protein excretion in a time-dependent manner as compared to untreated control cells following anoxia.ConclusionsThe immunosuppressive macrolide sirolimus prevents the growth and metastatic progression of HCC, and suppresses VEGF synthesis and secretion by downregulating HIF-1a expression. Sirolimus may be useful for clinical application in patients who received a liver transplant for HCC.

Surgical treatment for early hepatocellular carcinoma: comparison of resection and liver transplantation

PurposeThe optimum strategy, hepatic resection (HR) or liver transplantation (LT), for treatment of early hepatocellular carcinoma (HCC) associated with liver diseases of Child–Pugh A is far from established. The aim of this study was to compare and determine which strategy is optimal for HCC fulfilling the Milan criteria.MethodsConsecutive data were collected in 1,018 HCC patients treated with HR and 89 HCC patients listed for LT (1 drop out for HCC progression) between January of 2003 and December of 2007.ResultsThe independent prognostic factors identified by multivariate analysis were tumor size-plus-number, microscopic venous invasion, and operation type (LT or HR). When tumor size-plus-number was ≤4 or microscopic venous invasion was absent, there was no significant difference in overall survival (OS) between the LT and HR group. When tumor size-plus-number was >4 or microscopic venous invasion was present, OS was higher in the LT group.ConclusionsSince the pathological microscopic venous invasion was not easily available before operation which is limitation for widespread clinical use, thus in practice, we concluded that, for early HCC associated with Child–Pugh A cirrhosis, when tumor size-plus-number is >4, LT provides the best cure; when it is ≤4, HR remains the initial treatment of choice.

Oxaliplatin induces apoptosis in hepatocellular carcinoma cells and inhibits tumor growth.

Objective: The platinum-based chemotherapeutic agent oxaliplatin displays a wide range of antitumor activities. To date, no detailed data are available about the effects of oxaliplatin on hepatocellular carcinoma (HCC) cells. Herein, the anti-proliferation effects of oxaliplatin on HCCLM3 and Hep3B cells in vitro and in vivo are studied. Research methods: Cell viability was assessed by an MTT assay and apoptosis by flow cytometry and transmission electron microscopy. Apoptosis-related proteins in HCCLM3 cells were evaluated by microarray analysis, quantitative reverse transcriptase-PCR assay and western blotting. The effect of oxaliplatin was also studied in vivo using a xenograft model. Results: Oxaliplatin inhibited the growth of HCCLM3 and Hep3B cells. Using flow cytometry, transmission electron microscopy and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay, we found that apoptosis was the main mechanism by which oxaliplatin inhibited tumor progression. Microarray analysis, quantitative reverse transcriptase-PCR and western blot analysis further demonstrated downregulation of the anti-apoptotic proteins Bcl-2 and Bcl-xL and upregulation of the pro-apoptotic protein Bax during oxaliplatin-induced apoptosis. Conclusions: The anti-proliferation effect of oxaliplatin in HCC cells is due to induction of apoptosis. Therefore, oxaliplatin may be an effective treatment for HCC and its use merits further in-depth investigation.

Prognostic significance and clinical relevance of Sprouty 2 protein expression in human hepatocellular carcinoma.

BACKGROUND In vitro experiments and mice models have confirmed the importance of Sprouty 2 (Spry2) in inhibiting tumorigenesis and the progression of human cancer. However, the prognostic value of Spry2 in cancer patients remains unknown. This study is aimed to investigate the clinical relevance and prognostic significance of Spry2 expression in patients with hepatocellular carcinoma (HCC). METHODS With samples from 240 randomly-selected HCC patients who underwent surgery, immunohistochemistry was used to investigate Spry2 expression on tissue microarrays. The correlation of Spry2 expression with survival was estimated by the Kaplan-Meier method and univariate/multivariate Cox proportional hazard regression analysis. Spry2, ERK and phospho-ERK expression in HCC cell lines was detected by Western blotting. RESULTS Among the patients, 86.3% (207 of 240) exhibited down-regulation of Spry2 expression. Patients negative for Spry2 showed poorer survival (P=0.002) and increased recurrence (P=0.003). Multivariate analysis further established Spry2 as an independent predictor of postoperative recurrence in HCC patients (HR=1.47; 95% CI, 1.02-2.08; P=0.037). Down-regulation of Spry2 was associated with highly malignant phenotypes like vascular invasion and advanced tumor stages, and was positively correlated with the metastatic potential of HCC cell lines. CONCLUSION In the era of molecular targeted therapy, the expression of Spry2 in HCC may have relevant clinical significance and turn out to be a key factor in prognostic assessment and in treatment planning.

The dynamic changes of T-bet+/GATA-3+ and RORγt+/FOXP3+ cells in recipient spleens and grafts after rat orthotopic liver transplantation

BACKGROUND Rejection of transplanted liver occurs when the host generates alloantigen-reactive T cells, and CD4(+) T-cell subsets, including Th1, Th2, T17 and iTreg, could be involved in changing the dynamics of graft rejection onset. In the current immunosuppressive strategies, rejection is treated as an undifferentiated process that is uniform, which results in the failure of tolerance induction. Here, we established a rejection model to observe the reciprocal interaction of CD4(+) T-cell subsets in the complex networks of the immune system. METHODS Orthotopic liver transplantation (OLT) from male inbred Lewis rats (n=15) to male inbred Brown Norway (BN) rats was performed by Kamadas two-cuff technique without reconstruction of the hepatic artery. OLT from BN to BN rats (n=5) was performed as a control. Recipients were sacrificed on postoperative days 3, 5, 7, 9, 11, 13 and 15. Recipient spleens and grafts were harvested, fixed in 10% neutral formalin, and embedded in paraffin. Meanwhile, hematoxylin and eosin and immunohistochemical staining was done, acute rejection was graded by the Banff scheme, and the number of T-bet(+), GATA-3(+), RORgammat(+) and FOXP3(+) cells in the spleen and grafts were counted. RESULTS In recipient spleens, the T-bet(+) and RORgammat(+) cells were increased more significantly in the mild acute rejection (AR) group than in the control group (P=0.016, P=0.009, respectively), while both cell types were decreased in the moderate AR group. Compared with the control group, the RORgammat(+) cells did not differ significantly in the severe AR group, while the T-bet(+) cells were significantly decreased (P=0.465, P=0.009, respectively). The GATA-3(+) cells were significantly decreased in the mild AR group compared with the control group (P=0.009). With regard to the FOXP3(+) cells, there was no significant difference between the control and mild AR groups (P=0.754), while they were significantly decreased in the moderate and severe AR groups (P=0.028, P=0.009, respectively). The ratio of T-bet(+)/GATA-3(+) cells was more associated with AR than was the RORgammat(+)/FOXP3(+) cell ratio in the early stage. In the graft, the T-bet(+) and RORgammat(+) cells were significantly increased in mild, moderate and severe AR groups compared with the control group (P<0.009). The expression of GATA-3(+) cell was not significantly increased in the mild AR group compared with the control group, while it was significantly increased in the moderate and severe AR groups (P=0.028, P=0.009, respectively). Concerning the FOXP3(+) cells, no significant difference was found between the control and mild AR groups (P=0.347), while they were significantly increased in the moderate and severe AR groups (P<0.009). CONCLUSIONS Our study revealed the dynamic changes of T-bet(+), GATA-3(+), RORgammat(+) and FOXP3(+) cells in the spleen and grafts of recipient rats. It seems that the ratio of T-bet(+)/GATA-3(+) cells was more associated with AR than was RORgammat(+)/FOXP3(+) cells in the early stage of rejection, while the ratio of RORgammat(+)/FOXP3(+) cells was associated more with the later but more persistent stage of rejection. This study could make a contribution to the optimal selection of immunosuppressive regimens according to the dynamic changes in CD4(+) T-cell subsets at different stages of rejection.

An investigation of the effect of sorafenib on tumour growth and recurrence after liver cancer resection in nude mice independent of phosphorylated extracellular signal-regulated kinase levels

Objective: The goal of this study is to investigate the effects of sorafenib on tumor growth, recurrence and metastasis after curative resection of liver cancer. Research methods: SMMC-7721 and HCCLM3 liver tumors, each with different metastatic potential and basal phosphorylated extracellular signal-regulated kinase (pERK) levels, were orthotopically implanted into 56 nude mice. Mice were divided into a treatment sub study and a prevention sub study. Results: In the treatment sub study, tumor volumes in the high pERK-expressing HCCLM3 model were 2.58 ± 0.83 and 0.38 ± 0.09 cm3 without and with sorafenib, respectively (p < 0.001). The corresponding volumes in the low pERK-expressing SMMC-7721 model were 1.36 ± 0.24 and 0.24 ± 0.14 cm3 (p < 0.001), respectively. Sorafenib inhibited HCCLM3 cell proliferation and decreased tumor angiogenesis, but did not inhibit proliferation in the SMMC-7721 model. In the prevention sub study, intrahepatic recurrent tumor volumes were 1.96 ± 0.45 and 0.18 ± 0.24 cm3 (p < 0.001); lung metastasis frequencies were 100 and 0% (p = 0.005); and lifespans were 36 ± 3 and 46 ± 5 days (p = 0.002) in the control and sorafenib subgroups, respectively. Conclusions: Sorafenib inhibits tumor growth and prevents metastatic recurrence after resection of hepatocellular carcinoma in nude mice. The effect of sorafenib does not exclusively depend on high levels of pERK in tumors.