IL­6 plays a crucial role in epithelial­mesenchymal transition and pro­metastasis induced by sorafenib in liver cancer.

Interleukin­6 (IL­6) is involved in various biological responses, including tumor progression, metastasis and chemoresistance. However, the role and molecular mechanism of IL­6 in the treatment of sorafenib in liver cancer remain unclear. In the present study, through western blot analysis, Transwell assay, flow cytometric assay, ELISA analysis and immunohistochemistry it was revealed that sorafenib promoted metastasis and induced epithelial­mesenchymal transition (EMT) in liver cancer cells in vitro and in vivo, and significantly increased IL­6 expression. Endogenous or exogenous IL­6 affected metastasis and EMT progression in liver cancer cells through Janus kinase 2/signal transducer and activator of transcription 3 (STAT3) signaling. Knocked out IL­6 markedly attenuated the pro­metastasis effect of sorafenib and increased the susceptibility of liver cancer cells to it. In conclusion, the present results indicated that IL­6/STAT3 signaling may be a novel therapeutic strategy for liver cancer.

Effect of TALEN-mediated IL-6 knockout on cell proliferation, apoptosis, invasion and anti-cancer therapy in hepatocellular carcinoma (HCC-LM3) cells.

PURPOSE: To determine the exact effect of Interleukin-6 (IL-6) on tumor cell proliferation, apoptosis, invasion, and anti-cancer therapy in hepatocellular carcinoma (HCC). EXPERIMENTAL DESIGN: IL-6 was disrupted by transcription activator-like effector nucleases (TALEN) in HCCLM3 cells, and was used to evaluate the role of IL-6 on tumor cell proliferation, apoptosis, invasion and key signaling pathways involved in sorafenib and/or IFNα therapy. RESULTS: IL-6 has no direct effect on cell proliferation and invasion but promotes cell apoptosis and up-regulate IL-33 and VEGF-A expression. IL-6 could attenuate the anti-proliferation effect by sorafenib and combination therapy but facilitate the pro-apoptosis of the combination therapy and augment the pro-invasive effect induced by single treatment. IL-6 could down-regulate p-STAT3, however up-regulate the p-MEK/p-ERK and NF-kB/iNOS expression, and it also facilitated the promotion on p-JAK2 and p-MEK/p-ERK by either sorafenib or IFN-α. in vivo study, IL-6 significantly promotes tumor growth. The combination treatment showed the highest inhibition on tumor growth which is derived from HCCLM3-IL6(-) cells. CONCLUSIONS: IL-6 has no direct effect on cell proliferation and invasion but promotes tumor cell apoptosis in vitro study. Sorafenib and combination therapies are suitable for HCC cells with low or no IL-6 expression confirmed in vivo study.

Higher proliferation of peritumoral endothelial cells to IL-6/sIL-6R than tumoral endothelial cells in hepatocellular carcinoma.

BACKGROUND: This study aimed to explore the responses to the interleukin-6 (IL-6)/soluble interleukin-6 receptor (sIL-6R) complex in peritumoral endothelial cells (PECs) and tumor endothelial cells (TECs), as well as determine the signaling pathways in the angiogenesis of hepatocellular carcinoma (HCC). METHODS: The expression of IL-6, IL-6R, gp130, CD68, HIF-1α, and microvessel density (MVD) were assessed with an orthotopic xenograft model in nude mice. ECs were incubated under hypoxic conditions to detect IL-6 and gp130. The proliferation of PECs and TECs in the presence of IL-6 and sIL-6R, as well as the expression of gp130, JAK2/STAT3, PI3K/AKT in endothelial cells were measured. RESULTS: Peritumoral IL-6, IL-6R, gp130, CD68, and HIF-1α expression, as well as MVD, gradually increased during tumor growth. Hypoxia could directly induce IL-6 expression, but not gp130 in PECs. The co-culture of IL-6/sIL-6R induced much higher PEC proliferation and gp130 expression, as well as the elevated phosphorylation of JAK2 and STAT3, however not the phosphorylation of PI3K and AKT. CONCLUSIONS: PECs exhibited higher proliferation in response to IL-6/sIL-6R co-treatment compared with TECs in HCC via the up-regulation of gp130 /JAK2/STAT3. PEC and its associated peritumoral angiogenesis microenvironment may be a potential novel target for anti-angiogenic treatment.

Peritumoral Neuropilin-1 and VEGF receptor-2 expression increases time to recurrence in hepatocellular carcinoma patients undergoing curative hepatectomy.

PURPOSE: To determined Neuropilin-1 (NRP-1) and vascular endothelial growth factor receptor 2 (VEGFR-2) expression in the tumoral and peritumoral tissues of 214 treatment-naïve HCC patients and its correlation with overall survival (OS) and time to recurrence (TTR). EXPERIMENTAL DESIGN: NRP-1 and VEGFR-2 expression were examined by tissue microarray and peritumoral hypoxia by pimonidazole staining and angiogenesis by microvessel density (MVD). OS and TTR were evaluated by Kaplan-Meier analysis and log-rank test. RESULTS: Peritumoral NRP-1 and VEGFR-2 expression were significantly higher than that of the tumoral tissue (p < 0.001 for both), and high peritumoral expression of both factors was negatively associated with tumor size (p < 0.001 for both). Patients with high peritumoral expression of both proteins had the longest median OS (>94.0 months) and TTR (>84.0 months). The multivariate Cox proportional hazards analysis revealed that patients with high peritumoral expression of both NRP-1 and VEGFR-2 were more than 4 times less likely to have recurrence (p = 0.004) and more than 10 times likely to survive (p < 0.001). CONCLUSIONS: Peritumoral NRP-1 and VEGFR-2 expression is associated with prolonged TTR and extended OS of HCC patients and both may be useful as predictors of surgical outcome of HCC patients and explored as potential therapeutic targets.

ZBTB20 is involved in liver regeneration after partial hepatectomy in mouse.

BACKGROUND: A better understanding of the molecular mechanisms in liver regeneration holds promise for exploring the new potential therapy for liver failure. The present study was to investigate the role of zinc finger and BTB domain-containing protein 20 (ZBTB20), a potential factor associated with liver regeneration, in a model of 70% hepatectomy in mice. METHODS: Parameters for liver proliferation such as liver/body ratio and BrdU positivity were obtained via direct measurement and immunohistochemistry. The levels of zinc fingers and homeoboxes 2 (ZHX2), ZBTB20, alpha-fetoprotein (AFP) and glypican 3 (GPC3) transcripts in the regenerating liver tissue of a 70% hepatectomy rodent model were monitored by real-time PCR analysis at different time points. Knockdown of ZBTB20 was performed to characterize its regulatory function. RESULTS: A negatively regulating relationship between ZHX2, ZBTB20 and AFP, GPC3 was revealed from 24 to 72 hours after 70% hepatectomy. ZBTB20 appears to negatively regulate AFP and GPC3 transcription since the knockdown of ZBTB20 promoted the proliferation of hepatocytes and the expression of AFP and GPC3. CONCLUSION: In addition to AFP, GPC3 and ZHX2, ZBTB20 is a new regulator in liver regeneration and the decrease of ZBTB20 expression following 70% hepatectomy promotes AFP and GPC3 expression.

[Comprehensive measures for improving the radical resection rate and safety of Bismuth-Corlette type III hilar cholangiocarcinoma].

OBJECTIVE: To investigate the comprehensive measures for improving radical resection rate and safety of Bismuth-Corlette type III hilar cholangiocarcinoma. METHODS: The clinical data of 15 patients with Bismuth-Corlette type III hilar cholangiocarcinoma who performed radical resection from June 2009 to December 2011 was analyzed retrospectively. There were 11 male and 4 female patients, aged from 45 to 74 years (mean 59 years). The preoperative evaluation were conducted by using magnetic resonance cholangiopancreatography (MRCP), dual source spiral CT combined with IQQA-Liver CT Imaging Analysis System providing three-dimensional reconstruction of tumor, bile duct, hepatic artery and portal vein, which could help to chose the appropriate treatment modality. All patients were treated with selective hemi-hepatic vascular control of removal liver, hemi-hepatectomy combined with whole caudate lobe resection and regional lymphadenectomy. The merits of each evaluation methods and measures of surgical treatment were analyzed thoroughly. RESULTS: The preoperative evaluation modalities including the dual source spiral CT combined with IQQA-Liver CT Imaging Analysis System could clearly show the involvement of bile duct, hepatic artery and portal vein invaded by the tumor. The satisfactory postoperative recovery could be achieved by the remnant liver volume of > 40% after the hemi-hepatectomy combined with whole caudate lobe resection through the selective preoperative biliary drainage and hemihepatic vascular control. The mean complication was transient aggravated liver dysfunction. There was no death reported during perioperative period in the group. CONCLUSIONS: The preoperative imaging evaluation modalities including MRCP, dual source spiral CT combined with IQQA-Liver CT Imaging Analysis System could do favor for the preoperative evaluation of invasion degree of hilar cholangiocarcinoma and the selection of appropriate surgical treatment. Hemi-hepatectomy combined with whole caudate lobe resection and regional lymphadenectomy could be an alternative management of Bismuth-Corlette type III hilar cholangiocarcinoma.

Prognostic roles of cross-talk between peritumoral hepatocytes and stromal cells in hepatocellular carcinoma involving peritumoral VEGF-C, VEGFR-1 and VEGFR-3.

BACKGROUND: Peritumoral liver tissue could play a potential role in hepatocellular carcinoma (HCC) progression and patient survival via angiogenesis- and lymphangiogensis-related factors. The prognostic role of these factors in hepatocytes and stromal cells in HCC patients after curative resection remains to be explored. METHODS: Tumor tissue and surrounding peritumoral tissue were obtained from 145 resected HCC patients without lymph node metastasis (LNM) and 37 resected HCC patients with LNM. Tissue microarrays were constructed from duplicate cores of tumor tissue and surrounding peritumoral tissue from each resected specimen. Immunohistochemistry and real-time polymerase chain reaction were used to evaluate the expression of vascular endothelial growth factor-A (VEGF-A), VEGF-C, VEGF receptor-1(VEGFR-1), VEGFR-2, and VEGFR-3. Macrophage infiltration was determined by CD68 staining. Correlations between the expression of these factors and overall survival (OS) and time to recurrence (TTR) were studied. RESULTS: The peritumoral expression of VEGF-A, VEGF-C, VEGFR-1, VEGFR-2, and VEGFR-3 were significantly higher than expression of these factors in tumors. VEGFR-1 was mostly located in peritumoral macrophages, while VEGF-C and VEGFR-3 were mostly located in peritumoral hepatocytes. HCC with high peritumoral co-expression of VEGF-C, VEGFR-1, and VEGFR-3 was associated with higher peritumoral distribution of macrophages (0.87%±0.26% versus 0.45%±0.20%), LNM (32.4% versus 12.0%), shorter TTR (10.2 months versus 34.5 months), and poor prognosis (19.4 months versus 49.3 months). CONCLUSION: Expression of VEGF-C, VEGFR-1, and VEGFR-3 in peritumoral liver tissue is associated with a unique type of HCC that has a poorer outcome after hepatectomy.

Antiangiogenic therapy promoted metastasis of hepatocellular carcinoma by suppressing host-derived interleukin-12b in mouse models.

Antiangiogenic therapy, specially sorafenib, has become the standard of care for patients with advanced hepatocellular carcinoma (HCC), however, the improvement in survival time is not satisfactory. Previous studies have found that, in some circumstances, antiangiogenic therapy promoted tumor metastasis and the mechanistic studies were mainly focus on cancer-cell-autonomous manners. In two experimental metastasis models with tail-vein injection with hepatoma cells and an orthotopic HCC mouse model, we found that pretreatment with two vascular endothelial growth factor receptor (VEGFR) inhibitors, sunitinib and sorafenib, facilitated tumor cell survival in blood stream and promoted lung metastasis from tumors that were subsequently incubated after drug discontinuation, indicating that host response joined into the pro-metastatic effects. An antibody microarray identified that interleukin (IL)-12b was decreased in the peripheral blood of the mice treated with the two VEGFR inhibitors. IL-12b suppression in macrophages and dendritic cells from host organs was found to play a crucial role in treatment-induced metastasis. Supplement with recombinant mouse IL-12b or restoration of IL-12b expression in the host by zoledronic acid, which was previously reported to enhance IL-12 expression in vitro and in vivo, alleviated the metastasis-promoting effects of sunitinib and sorafenib. These studies suggest that host response to VEGFR inhibitors facilitates HCC metastasis and restoration of IL-12b expression could translate into clinical benefits.

Direct transformation of lung microenvironment by interferon-α treatment counteracts growth of lung metastasis of hepatocellular carcinoma.

BACKGROUND: Interferon (IFN)-α is effective in inhibiting tumor growth and metastasis of hepatocellular carcinoma (HCC). However, the biologic mechanisms of IFN-α treatment in lung metastasis are not yet clear. METHODS: The effect of IFN-α treatment was studied by using an orthotopic xenograft model and measuring tumor size and lung metastasis. Pretreatment with IFN-α before implantation of tumor was done to explore the effect of IFN-α on lung tissues. Cytokines and macrophages were measured by immunohistochemistry and/or PCR assay, using human origin or mouse origin primers to differentiate the sources. Circulating tumor cells (CTCs) were also assayed by flow cytometry. RESULTS: IFN-α treatment did not decrease the number of CTCs (0.075% ± 0.020% versus 0.063%±0.018%, P = 0.574, IFN-α-treated versus control groups), but did decrease the number and size of lung metastasis (number: 1.75 ± 1.0 versus 28.0 ± 6.3, P = 0.008; size [pixels]: 116.8 ± 72.2 versus 5226.4 ± 1355.7, P = 0.020), and inhibited macrophage infiltration (0.20% ± 0.04% versus 1.36% ± 0.21%, P = 0.0058) and alteration of matrix metalloproteinase (MMP)-9 expression (mean integrated optical density (IOD): 5.1 ± 1.7 versus 21.9 ± 0.4, P<0.000) in the lung, which was independent of the primary tumor. CONCLUSION: IFN-α inhibited lung metastasis by directly modulating the lung microenvironment.

Xanthogranulomatous cholecystitis: a clinicopathological study of its association with gallbladder carcinoma.

OBJECTIVES: To determine the distribution of macrophages (MΦ) in both xanthogranulomatous cholecystitis (XGC) and gallbladder carcinoma (GBC) and to analyze the association between XGC and GBC. METHODS: From January 2009 to June 2011, 110 patients with gallbladder diseases, including 35 with GBC, 45 with XGC and 30 with chronic cholecystitis (CC), were enrolled. Immunohistochemistry stain and real-time polymerase chain reaction using oncogenes (BCL-2, c-Myc) and anti-oncogene genes (p53, p21) were performed, serum expressions of tumor marker (CA19-9, CA724 and CA242) were also conducted. MΦ were used to determine their potential role in the carcinogenesis of GBC. RESULTS: BCL-2 and c-Myc expressions gradually increased among CC, XGC and GBC (P = 0.032 and P = 0.020, respectively); while p53 and p21 were similar in the three groups (P = 0.167 and P = 0.122, respectively). Serum BCL-2 and c-Myc were significantly correlated with their tissue levels; in terms of serum tumor markers, which gradually increased among CC, XGC and GBC, however, CA242 and CA724 were both negative in XGC but positive in GBC. Furthermore, gradually increasing MΦ counts were observed among CC, XGC and GBC groups; c-Myc and CA724 were independent predictors for the differentiation of XGC and GBC. CONCLUSIONS: XGC is an uncommon inflammatory condition distinct from CC and may be associated with the precancerous nature of GBC for its upregulated oncogenes and MΦ biology. c-Myc and CA724 were independent predictors for the differentiation of XGC and GBC.

Diagnostic and prognostic role of laparoscopic staging for gallbladder carcinoma.

BACKGROUND: The aim of this study was to evaluate the diagnostic and prognostic role of staging laparoscopy in gallbladder carcinoma (GBC). METHODS: From January 2007 through December 2010, 79 GBC patients without evidence of metastatic disease on preoperative imaging underwent staging laparoscopy. Peritoneal and liver metastases were assessed by a single surgeon in a systematic manner. Resection rate, safety, and survival analysis were compared between the laparoscopy group and no laparoscopy group. RESULTS: Disseminated disease was detected in 27 patients and no further surgery was performed; the overall accuracy for detecting unresectable disease was 67.5% (27/40), with 39 (75%) and 27 (51.9%) receiving resection and curative resection. In 203 GBC patients undergoing laparotomy, 90 (44.3%) and 53 (26.1%) patients received resection and curative resection; therefore, the resection rate and curative resection rate were significantly much higher in the laparoscopy group (p < 0.000). CONCLUSIONS: Staging laparoscopy in GBC is sensitive in detecting disseminated disease and increases the curative resection rate, shortens the recovery time, and has no negative implications on overall survival; therefore, we suggest the routine use of staging laparoscopy in patients with GBC without evidence of disseminated disease on preoperative imaging.

Up-regulation of platelet-derived growth factor-A is responsible for the failure of re-initiated interferon alpha treatment in hepatocellular carcinoma.

BACKGROUND: Postoperative interferon-α(IFN-α) treatment delays hepatocellular carcinoma(HCC) recurrence and prolongs patient survival, and may thus be an effective form of adjuvant therapy. However, clinical observations found that HCC recurs in some patients within 8 months of IFN-α treatment being discontinued. We investigated whether HCC regrowth appears after IFN-α is discontinued, whether re-initiated IFN-α is effective, and the underlying mechanisms of IFN-α treatment. METHODS: The human HCC nude mouse model LCI-D20 was used to study the effects of IFN-α treatment, discontinued IFN-α treatment, and re-initiated IFN-α treatment on tumor growth. Tumor weight, microvessel density(MVD), serum vascular endothelial growth factor (VEGF), and tumor cell apoptosis were analyzed. Angiogenesis-related factors were studied using cDNA microarray in different tumor samples and confirmed using reverse transcription-polymerase chain reaction(RT-PCR) and Western blotting assays. Finally, imatinib was added with re-initiated IFN-α treatment to improve efficacy. RESULTS: IFN-α (1.5 × 107 U/kg/day for 20 days) suppressed HCC growth by 60.3% and decreased MVD by 52.2% compared with the control. However, tumor regrowth occurred after IFN-α was discontinued, and re-initiated IFN-α treatment was not effective for inhibiting tumor growth or reducing MVD compared with a saline-treated group. cDNA microarray showed VEGF was down-regulated while platelet-derived growth factor-A (PDGF-A) was up-regulated when IFN-α treatment was re-initiated. These findings were further confirmed with RT-PCR and Western blotting assay. The combination of imatinib with re-initiated IFN-α reduced HCC weight by 30.7% and decreased MVD by 31.1% compared with IFN-α treatment only (P=0.003 and 0.015, respectively). CONCLUSION: Tumor regrowth occurred after IFN-α treatment was discontinued. Re-initiated IFN-α treatment was not effective and was associated with up-regulation of PDGF-A, while the VEGF remained suppressed. The combination of a PDGF-receptor inhibitor with IFN-α improved the effect of the re-initiated treatment.

Sorafenib down-regulates expression of HTATIP2 to promote invasiveness and metastasis of orthotopic hepatocellular carcinoma tumors in mice.

BACKGROUND & AIMS: Antiangiogenic agents can sometimes promote tumor invasiveness and metastasis, but little is known about the effects of the antiangiogenic drug sorafenib on progression of hepatocellular carcinoma (HCC). METHODS: Sorafenib was administered orally (30 mg · kg(-1) · day(-1)) to mice with orthotopic tumors grown from HCC-LM3, SMMC7721, or HepG2 cells. We analyzed survival times of mice, along with tumor growth, metastasis within liver and to lung, and induction of the epithelial-mesenchymal transition. Polymerase chain reaction arrays were used to determine the effects of sorafenib on gene expression patterns in HCC cells. We analyzed regulation of HIV-1 Tat interactive protein 2 (HTATIP2) by sorafenib and compared levels of this protein in tumor samples from 75 patients with HCC (21 who received sorafenib after resection and 54 who did not). RESULTS: Sorafenib promoted invasiveness and the metastatic potential of orthotopic tumors grown from SMMC7721 and HCC-LM3 cells but not from HepG2 cells. In gene expression analysis, HTATIP2 was down-regulated by sorafenib. HCC-LM3 cells that expressed small hairpin RNAs against HTATIP2 (knockdown) formed less invasive tumors in mice following administration of sorafenib than HCC-LM3 without HTATIP2 knockdown. Alternatively, HepG2 cells that expressed transgenic HTATIP2 formed more invasive tumors in mice following administration of sorafenib. Sorafenib induced the epithelial-mesenchymal transition in HCC cell lines, which was associated with expression of HTATIP2. Sorafenib regulated expression of HTATIP2 via Jun-activated kinase (JAK) and signal transducer and activator of transcription (STAT)3 signaling. Sorafenib therapy prolonged recurrence-free survival in patients who expressed lower levels of HTATIP2 compared with higher levels. CONCLUSIONS: Sorafenib promotes invasiveness and the metastatic potential of orthotopic tumors from HCC cells in mice, down-regulating expression of HTATIP2 via JAK-STAT3 signaling.

Antiangiogenic effects of pazopanib in xenograft hepatocellular carcinoma models: evaluation by quantitative contrast-enhanced ultrasonography.

BACKGROUND: Antiangiogenesis is a promising therapy for advanced hepatocellular carcinoma (HCC), but the effects are difficult to be evaluated. Pazopanib (GW786034B) is a pan-vascular endothelial growth factor receptor inhibitor, the antitumor effects or antiangiogenic effects haven't been investigated in HCC. METHODS: In vitro direct effects of pazopanib on human HCC cell lines and endothelial cells were evaluated. In vivo antitumor effects were evaluated in three xenograft nude mice models. In the subcutaneous HCCLM3 model, intratumoral blood perfusion was detected by contrast-enhanced ultrasonography (CEUS), and serial quantitative parameters were profiled from the time-intensity curves of ultrasonograms. RESULTS: In vitro proliferation of various HCC cell lines were not inhibited by pazopanib. Pazopanib inhibited migration and invasion and induced apoptosis significantly in two HCC cell lines, HCCLM3 and PLC/PRF/5. Proliferation, migration, and tubule formation of human umbilical vein endothelial cells were inhibited by pazopanib in a dose-dependent manner. In vivo tumor growth was significantly inhibited by pazopanib in HCCLM3, HepG2, and PLC/PRF/5 xenograft models. Various intratumoral perfusion parameters changed over time, and the signal intensity was significantly impaired in the treated tumors before the treatment efficacy on tumor size could be observed. Mean transit time of the contrast media in hotspot areas of the tumors was reversely correlated with intratumoral microvessel density. CONCLUSIONS: Antitumor effects of pazopanib in HCC xenografts may owe to its antiangiogenic effects, and the in vivo antiangiogenic effects could be evaluated by quantitative CEUS.

Bevacizumab enhances chemosensitivity of hepatocellular carcinoma to adriamycin related to inhibition of survivin expression.

PURPOSE: In recent years, anti-angiogenesis drugs have shown promising clinical effects against many tumors, particularly in combination with chemotherapy. Although the combination has become a standard of care for many tumors, the mechanisms of the chemosensitizing activity of anti-angiogenic drugs are not fully understood. Here, we sought to determine if anti-angiogenesis drug bevacizumab could enhance the chemosensitivity of HCC by inhibition of survivin. METHODS: After treatment of human umbilical vein endothelial cells (HUVECs) and hepatocellular carcinoma (HCC) cell line PLC/PRF/5 (PLC) with bevacizumab or/and adriamycin, the direct effects were examined by survival assays, and the expression of Akt, Phospho-Akt and survivin were evaluated by western blot. Tumor growth was observed in a human HCC xenograft nude mouse model treated with different drugs, and the expression of PCNA, CD31 and survivin in tumor tissues were evaluated by means of immunohistochemistry. RESULTS: Bevacizumab enhanced the chemosensitivity of HCC by inhibiting the VEGF-PI3 K/Akt-survivin signaling cascade in endothelial cells. The combination of bevacizumab with adriamycin therapy resulted in better outcomes compared with monotherapy in hepatocellular carcinoma xenografts; bevacizumab significantly inhibited tumor angiogenesis and growth. In addition, bevacizumab reduced survivin expression in tumor tissues, including tumor vascular endothelial cells in vivo, although it did not inhibit survivin expression in tumor cells in vitro. CONCLUSION: These results implicate the bevacizumab-increased efficacy of adriamycin via an inhibition of survivin expression in malignant cells as well as tumor vasculature cells, which provides other insights into the mechanism of enhanced efficacy by combination of VEGF blocker and chemotherapeutic agents.

Expression and prognostic significance of placental growth factor in hepatocellular carcinoma and peritumoral liver tissue.

Vascular endothelial growth factor-targeted therapy is a promising treatment for hepatocellular carcinoma (HCC), but its clinical benefit is often accompanied by acquired resistance. In animal studies, antiplacental growth factor therapy is effective with less resistance. The role of placental growth factor (PlGF) in the progression of HCC is not clear. In our study, we used immunohistochemistry in tissue microarrays to investigate PlGF expression in tumor and peritumoral liver tissues from 105 patients with HCC. Intratumoral and peritumoral PlGF mRNA expression was analyzed in another cohort of 37 patients. Peritumoral PlGF expression was significantly higher than intratumoral PlGF expression (p < 0.001). Intratumoral PlGF expression was not associated with patients' overall survival (OS) or time to recurrence (TTR). However, peritumoral PlGF expression, which was associated with tumor size, presence of intrahepatic metastasis, TNM stage and Barcelona Clinic Liver Cancer stage, was an independent risk factor for OS (p = 0.026) and TTR (p = 0.041). The prognostic value of peritumoral PlGF expression was further validated in a validation cohort (n = 394). We inferred that the elevation of PlGF in peritumoral liver might be induced by hypoxia. We found that peritumoral PlGF expression was associated with hypoxia-inducible factor-1α (p = 0.017). PlGF expression was elevated in L02, a hepatic cell line, under hypoxic conditions in vitro. These findings indicate that high peritumoral PlGF expression is associated with tumor recurrence and survival after resection of HCC. PlGF could be a target of adjuvant therapy and deserves further investigations.

Evaluation of two modified ECF regimens in the treatment of advanced gallbladder cancer.

Gallbladder cancer is a rare disease and it is associated with a poor clinical outcome and survival. A standard therapy for it has not been established yet. The aim of this study is to evaluate efficacy and safety of two modified ECF regimens in advanced gallbladder cancer patients. Clinical data of 38 patients with advanced gallbladder cancer treated with modified ECF regimen were reviewed retrospectively. Of them, 21 patients received an epirubicin, cisplatin, and 5-FU/LV combination therapy. Seventeen patients received a chemotherapy of epirubicin, cisplatin, and capecitabine. Partial response was achieved in fourteen (36.84%) patients with a median duration of 5 months (range, 3-13 months), while stable disease was achieved in eight patients (21.05%). The median time to progression was 4.0 months (95% CI, 3.62-4.58 months). And the median overall survival was 9.8 months (95% CI, 7.26-12.34 months). Responders demonstrated better survival than non-responders (median survival time: 16 vs. 6.9 months, P = 0.008). The median survival time for epirubicin-, cisplatin- and capecitabine-treated patients was 9.2 versus 8.9 months for epirubicin-, cisplatin- and 5-FU/LV-treated patients. There was no statistical difference between both treatment groups in terms of survival time (P = 0.769). Regimen-related toxicity resulted in at least one treatment delay or dosage reduction in 63.2 and 34.2% patients, respectively. There were no chemotherapy-related deaths during the study. Modified ECF regimen with epirubicin, cisplatin and 5-FU/LV or substituting capecitabine for 5-FU/LV is still a potentially effective therapeutic chemotherapy for patients with advanced gallbladder cancer, and toxicity was manageable. There was no remarkable difference in efficacy between the two regimens.

[Somatostatin enhanced anti-tumor effect of doxorubicin on gallbladder cancer cells through the regulation of intracellular drug concentration].

OBJECTIVE: To investigate the possible mechanisms by which Somatostatin (SST) enhances the anti-tumor effect of doxorubicin (DOX) on gallbladder cancer cells. METHODS: GBC-SD cells were grouped into 4 groups: SST-treated group, DOX-treated group, SST+DOX co-treated group and control group. The concentrations of SST and DOX were 75 µg/ml and 5 µg/ml based on our previous studies. In control group, cells were cultivated with phosphate buffered saline (PBS). In experimental groups, cells were cultivated with medium and the corresponding drugs. After drug treatment, cell viability was examined by MTT assay at 6, 12, 24 and 36 h respectively. Meanwhile, intracellular concentrations of doxorubicin in each group was determined by microspectrofluorimetry; Real-time polymerase chain reaction (RT-PCR) was used to determine the expressions of MDR1 mRNA in the cells at different time points and the expressions of P-gp protein, a product of MDR1 mRNA, were determined by Western blot analysis. RESULTS: SST did not exhibit significant inhibitory effect on the proliferation of GBC-SD cells as compared to that of control group (P>0.05). SST+DOX co-treatment group and DOX showed significantly inhibitory effect on the growth of GBC-SD cells at Hour 12 post-treatment. However no statistical difference was found between SST+DOX and DOX groups. Interestingly, at Hour 24 post-treatment, SST+DOX group showed more robust inhibitory effect on GBC-SD cells as compared to DOX alone group. Moreover, SST could significantly down-regulate the expressions of MDR1 mRNA and P-gp protein. SST could increase intracellular DOX concentration. And the difference of intracellular DOX concentration between SST+DOX group and DOX group at Hour 24 was statistically significant. CONCLUSIONS: In our experiment, SST decreases the expression of MDR1 mRNA and P-gp protein so as to reduce the efflux of DOX and elevate DOX concentrations in GBC-SD cells. This eventually leads to enhanced cytotoxic effects of DOX on GBC-SD cells.

High expression of macrophage colony-stimulating factor-1 receptor in peritumoral liver tissue is associated with poor outcome in hepatocellular carcinoma after curative resection.

BACKGROUND: Macrophage colony-stimulating factor 1 receptor (CSF-1R) expression in hepatocellular carcinoma (HCC) and its prognostic values are unclear. This study evaluated the prognostic values of the intratumoral and peritumoral expression of CSF-1R in HCC patients after curative resection. METHODS: Tissue microarrays containing material from cohort 1 (105 patients) and cohort 2 (32 patients) were constructed. Immunohistochemistry was performed and prognostic values of these and other clinicopathological data were evaluated. The CSF-1R mRNA level was assessed by quantitative real-time polymerase chain reaction in cohort 3 (52 patients). RESULTS: Both the CSF-1R density and its mRNA level were significantly higher in peritumoral liver tissue than in the corresponding tumor tissue. CSF-1R was distributed in a gradient in the long-distance peritumoral tissue microarray, with its density decreasing as the distance from the tumor margin increased. High peritumoral CSF-1R was significantly associated with more intrahepatic metastases and poorer survival. Peritumoral CSF-1R was an independent prognostic factor for both overall survival and time to recurrence and affected the incidence of early recurrence. However, intratumoral CSF-1R did not correlate with any clinicopathological feature. Peritumoral CSF-1R was also associated with both overall survival and time to recurrence in a subgroup with small HCCs (< or =5 cm). CONCLUSIONS: Peritumoral CSF-1R is associated with intrahepatic metastasis, tumor recurrence, and patient survival after hepatectomy, highlighting the critical role of the peritumoral liver milieu in HCC progression. CSF-1R may become a potential therapeutic target for postoperative adjuvant treatment.

Depletion of tumor-associated macrophages enhances the effect of sorafenib in metastatic liver cancer models by antimetastatic and antiangiogenic effects.

PURPOSE: To investigate the role of macrophages in tumor progression under sorafenib treatment and to explore whether combination of drugs that deplete macrophages improved the antitumor effect of sorafenib. EXPERIMENTAL DESIGN: Tumor growth, lung metastasis, and tumor angiogenesis were observed in HCCLM3-R and SMMC7721, two human hepatocellular carcinoma xenograft nude mouse models, when treated with sorafenib (30 mg/kg daily, n = 6 per group) or a vehicle as control. Macrophage infiltration was measured in the peripheral blood and in sorafenib-treated tumor by immunohistochemistry and flow cytometry with F4/80 antibody and CD11b antibody. The effect of macrophage depletion on tumor angiogenesis and metastasis after sorafenib treatment, using two drug target macrophages, zoledronic acid (ZA) and clodrolip, was measured in the two models of hepatocellular carcinoma. RESULTS: Although sorafenib significantly inhibited tumor growth and lung metastasis, it induced a significant increase in peripheral recruitment and intratumoral infiltration of F4/80- and CD11b-positive cells, which was accompanied with elevation of colony-stimulating factor-1, stromal-derived factor 1alpha, and vascular endothelial growth factor in the tumor and elevation of plasma colony-stimulating factor-1 and mouse vascular endothelial growth factor in peripheral blood, suggesting the role of macrophages in tumor progression under sorafenib treatment. Depletion of macrophages by clodrolip or ZA in combination with sorafenib significantly inhibited tumor progression, tumor angiogenesis, and lung metastasis compared with mice treated with sorafenib alone. ZA was more effective than clodrolip. CONCLUSIONS: Macrophages may have an important role in tumor progression under sorafenib treatment. ZA is promising when combined with sorafenib to enhance its antitumor effect.