Hepatocellular Carcinoma Growth In Mice Research Articles

Cell division cyclin 25C knockdown inhibits hepatocellular carcinoma development by inducing endoplasmic reticulum stress.

Cell division cyclin 25C (CDC25C) is a protein that plays a critical role in the cell cycle, specifically in the transition from the G2 phase to the M phase. Recent research has shown that CDC25C could be a potential therapeutic target for cancers, particularly for hepatocellular carcinoma (HCC). However, the specific regulatory mechanisms underlying the role of CDC25C in HCC tumorigenesis and development remain incompletely understood. To explore the impact of CDC25C on cell proliferation and apoptosis, as well as its regulatory mechanisms in HCC development. Hepa1-6 and B16 cells were transduced with a lentiviral vector containing shRNA interference sequences (LV-CDC25C shRNA) to knock down CDC25C. Subsequently, a xenograft mouse model was established by subcutaneously injecting transduced Hepa1-6 cells into C57BL/6 mice to assess the effects of CDC25C knockdown on HCC development in vivo. Cell proliferation and migration were evaluated using a Cell Counting Kit-8 cell proliferation assays and wound healing assays, respectively. The expression of endoplasmic reticulum (ER) stress-related molecules (glucose-regulated protein 78, X-box binding protein-1, and C/EBP homologous protein) was measured in both cells and subcutaneous xenografts using quantitative real-time PCR (qRT-PCR) and western blotting. Additionally, apoptosis was investigated using flow cytometry, qRT-PCR, and western blotting. CDC25C was stably suppressed in Hepa1-6 and B16 cells through LV-CDC25C shRNA transduction. A xenograft model with CDC25C knockdown was successfully established and that downregulation of CDC25C expression significantly inhibited HCC growth in mice. CDC25C knockdown not only inhibited cell proliferation and migration but also significantly increased the ER stress response, ultimately promoting ER stress-induced apoptosis in HCC cells. The regulatory mechanism of CDC25C in HCC development may involve the activation of ER stress and the ER stress-induced apoptosis signaling pathway.

A CRISPR-Cas9 library screening identifies CARM1 as a critical inhibitor of ferroptosis in hepatocellular carcinoma cells

Ferroptosis is an iron-catalyzed form of regulated cell death that results from the accumulation of lipid peroxidation products and reactive oxygen species to a lethal content. However, the transcriptional regulation of ferroptosis is not well understood. Sorafenib, a standard drug for hepatocellular carcinoma (HCC), induces ferroptosis in HCC cells. In this study, we conducted a CRISPR-Cas9 library screening targeting epigenetic factors and identified coactivator-associated arginine methyltransferase 1 (CARM1) as a critical inhibitor of ferroptosis. CARM1 depletion intensified Sorafenib-induced ferroptosis, resulting in decreased cell viability, reduced cellular glutathione level, increased lipid peroxidation, and altered mitochondrial crista structure. Additionally, we investigated a CARM1 inhibitor (CARM1i) as a potential ferroptosis inducer. Combining the CARM1i with Sorafenib enhanced the induction of ferroptosis. Notably, both CARM1 knockdown and CARM1i showed cooperative effects with Sorafenib in inhibiting HCC growth in mice. The underlying mechanism involves CARM1-catalyzed H3R26me2a on the promoter of glutathione peroxidase 4, leading to its transcriptional activation and subsequent ferroptosis inhibition. Furthermore, Sorafenib treatment induced the transcription of CARM1 through the MDM2-p53 axis. In summary, our findings establish CARM1 as a critical ferroptosis inhibitor and highlight the potential of CARM1is as novel ferroptosis inducers, providing promising therapeutic strategies for HCC treatment.

Nanodrug removes physical barrier to promote T-cell infiltration for enhanced cancer immunotherapy.

The dense extracellular matrix (ECM) is a key barrier to tumor infiltration of cytotoxic T lymphocytes (CTLs), which greatly compromises T cell-dependent immunotherapy of hepatocellular carcinoma (HCC). Herein, hyaluronidase (HAase), IL-12, and anti-PD-L1 antibody (αPD-L1) were co-delivered using a pH and MMP-2 dual-sensitive polymer/calcium phosphate (CaP) hybrid nanocarrier. The dissolution of CaP triggered by tumor acidity facilitated the release of IL-12 and HAase responsible for ECM digestion, enhancing the tumor infiltration and proliferation of CTLs. Furthermore, the in situ-released αPD-L1 inside tumor, as triggered by an overexpressed MMP-2, prevented the tumor cell from escaping the killing effects of CTLs. Such combination strategy induced a robust antitumor immunity for efficiently suppressing HCC growth in mice. Additionally, tumor acidity-sheddable polyethylene glycol (PEG) coating enhanced the tumor accumulation of nanocarrier and reduced the immune-related adverse events (irAEs) induced by on-target off-tumor αPD-L1. This dual-sensitive nanodrug demonstrates an effective immunotherapy paradigm for other dense ECM-characterized solid tumors.

Cyclin-Dependent Kinase 9 Inhibition as a Potential Treatment for Hepatocellular Carcinoma

Purpose: Composite cyclin-dependent kinase (CDK) inhibition has shown potential as a treatment for hepatocellular carcinoma (HCC) in preclinical studies. We tested whether the specific inhibition of CDK9 was effective against HCC. Methods: The effects of two specific CDK9 inhibitors, BAY1143572 and AZD4573, in HCC cell lines were examined. We tested the in vivo efficacy of CDK9 inhibition in mouse xenograft models of HuH7 human HCC cells and in an orthotopic model of BNL mouse HCC cells. Overexpression and knockdown of CDK9 were performed to confirm the efficacy of CDK9 inhibition. Results: CDK9 inhibitors exhibited potent antiproliferative activities in HCC cells regardless of the levels of c-myc expression while inhibiting the downstream signals of CDK9, such as the phosphorylation of RNA polymerase II. These 2 CDK9 inhibitors induced apoptosis in HCC cells and reduced the expression of antiapoptotic proteins such as myeloid cell leukemia-1 and survivin. In the xenograft studies, mice receiving either CDK9 inhibitor exhibited significantly slower tumor growth than did the mice receiving vehicles. In the orthotopic model, the HCC growth in mice receiving a CDK9 inhibitor also tended to be slower than that in the control group. Overexpression of CDK9 in HuH7 cells reduced the efficacy of both CDK9 inhibitors. Knockdown of CDK9 expression reduced the proliferative activities of HCC cells. Conclusion: We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.

Cyclin dependent kinase 9 inhibition as a potential treatment for hepatocellular carcinoma.

425 Background: Composite cyclin dependent kinase (CDK) inhibition has shown potential as a treatment for hepatocellular carcinoma (HCC) in preclinical studies. We tested whether the specific inhibition of CDK9 was effective against HCC. Methods: The effects of two specific CDK9 inhibitors, BAY1143572 and AZD4573, on cell viability and apoptosis in HCC cell lines were examined. We tested the in vivo efficacy of CDK9 inhibition in mouse xenograft models of HuH7 human HCC cells and in an orthotopic model of BNL mouse HCC cells. Overexpression and knockdown of CDK9 were performed to confirm the efficacy of CDK9 inhibition. Results: Both BAY1143572 and AZD4573 exhibited potent antiproliferative activities in HCC cell lines including HuH7, HLE, and HepG2, regardless of the levels of c-myc expression while inhibiting the downstream signals of CDK9, such as the phosphorylation of RNA polymerase II. These 2 CDK9 inhibitors induced apoptosis in HCC cells and reduced the expression of antiapoptotic proteins such as myeloid cell leukemia-1, survivin, BimEL, and the X-linked inhibitor of apoptosis protein. Reduction of CDK9 expression through siRNA knockdown also reduced the phosphorylation of RNA polymerase II and the colony formations of HuH7 and HLE cells. In the xenograft studies, mice receiving either CDK9 inhibitor exhibited a significantly lower tumor growth rate than did the mice receiving vehicles. In the orthotopic model, the HCC growth in mice receiving BAY1143572 also tended to be slower than that in the control group. Overexpression of CDK9 in HuH7 cells reduced the efficacy of both CDK9 inhibitors. Conclusions: We demonstrated the in vitro and in vivo activity of CDK9 inhibition on multiple HCC cell lines. Our data support further clinical development of CDK9 inhibitors as a treatment for HCC.

Irradiated whole-cell vaccine suppresses hepatocellular carcinoma growth in mice via Th9 cells.

Liver cancer is one of the most common malignant tumors with no available satisfactory treatment. The aim of the present study was to investigate the anti-tumor effect of an irradiated hepatocellular carcinoma (HCC) whole-cell vaccine and its underlying mechanisms. Hepa1-6 and H22 HCC cell lines were irradiated in preparation for whole-cell vaccine production. Subsequently, two HCC tumor-bearing mouse models were created by injecting these Hepa1-6 and H22 cells into the abdominal skin of C57BL/6 and ICR mice, respectively. The mice were immunized with the corresponding whole-cell vaccine the next day, and then once a week until the end of the experimental period. Tumor growth, blood T helper (Th)9 cells and plasma interleukin (IL)-9 levels were monitored during the immunization period. Th9 cells were also induced by in vitro co-culture of the whole-cell vaccine with lymphocytes from the spleen and lymph nodes of the corresponding mice. Alterations of gene expression in transcription factor (TF) were determined by reverse transcription-quantitative PCR, and Th9 cells were detected using flow cytometry. The whole-cell vaccine effectively suppressed HCC tumor growth, as indicated by slower tumor growth and a smaller tumor size in the immunized group compared with the control. The percentage of blood Th9 cells and the concentration of plasma IL-9 were significantly increased in the immunized group. The whole-cell vaccine also induced Th9 cell differentiation and upregulated the expression of TFs PU.1, interferon regulatory factor 4 and basic leucine zipper transcriptional factor ATF-like. These results suggest that the irradiated HCC whole-cell vaccine inhibited tumor growth by increasing Th9 cell numbers in HCC mice

Tumor methionine metabolism drives T-cell exhaustion in hepatocellular carcinoma

T-cell exhaustion denotes a hypofunctional state of T lymphocytes commonly found in cancer, but how tumor cells drive T-cell exhaustion remains elusive. Here, we find T-cell exhaustion linked to overall survival in 675 hepatocellular carcinoma (HCC) patients with diverse ethnicities and etiologies. Integrative omics analyses uncover oncogenic reprograming of HCC methionine recycling with elevated 5-methylthioadenosine (MTA) and S-adenosylmethionine (SAM) to be tightly linked to T-cell exhaustion. SAM and MTA induce T-cell dysfunction in vitro. Moreover, CRISPR-Cas9-mediated deletion of MAT2A, a key SAM producing enzyme, results in an inhibition of T-cell dysfunction and HCC growth in mice. Thus, reprogramming of tumor methionine metabolism may be a viable therapeutic strategy to improve HCC immunity.

MET Inhibitors Promote Liver Tumor Evasion of the Immune Response by Stabilizing PDL1

Inhibitors of MET have not produced satisfactory outcomes in trials of patients with liver cancer. We investigated the mechanisms of liver tumor resistance to MET inhibitors in mice. We tested the effects of MET inhibitors tivantinib and capmatinib in the mouse hepatocellular carcinoma (HCC) cell line HCA-1 and in immune-competent and immunodeficient mice with subcutaneous tumors grown from this cell line. Tumors were collected from mice and tumor cells were analyzed by time-of-flight mass cytometry. We used short hairpin RNAs to weaken expression of MET in Hep3B, SK-HEP-1, HA59T, and HA22T liver cancer cell lines and analyzed cells by immunoblot, immunofluorescence, and immunoprecipitation assays. Mass spectrometry was used to assess interactions between MET and glycogen synthase kinase 3β (GSK3B), and GSK3B phosphorylation, in liver cancer cell lines. C57/BL6 mice with orthotopic tumors grown from Hep1-6 cells were given combinations of capmatinib or tivantinib and antibodies against programmed celldeath 1 (PDCD1; also called PD1); tumors were collected andanalyzed by immunofluorescence. We analyzed 268 HCCsamples in a tissue microarray by immunohistochemistry. Exposure of liver cancer cell lines to MET inhibitors increased their expression of PD ligand 1 (PDL1) and inactivated cocultured T cells. MET phosphorylated and activated GSK3B at tyrosine 56, which decreased the expression of PDL1 by liver cancer cells. In orthotopic tumors grown in immune-competent mice, MET inhibitors decreased the antitumor activity of T cells. However, addition of anti-PD1 decreased orthotopic tumor growth and prolonged survival of mice compared with anti-PD1 or MET inhibitors alone. Tissue microarray analysis of HCC samples showed an inverse correlation between levels of MET and PDL1 and a positive correlation between levels of MET and phosphorylated GSK3B. In studies of liver cancer cell lines and mice with orthotopic tumors, MET mediated phosphorylation and activated GSK3B, leading to decreased expression of PDL1. Combined with a MET inhibitor, anti-PD1 and anti-PDL1 produced additive effect to slow growth of HCCs in mice.

Inhibition of mitochondrial respiration overcomes hepatocellular carcinoma chemoresistance

The clinical management of advanced hepatocellular carcinoma (HCC) is challenging due to its resistance to chemotherapy. In our work, we demonstrate that an antiparasitic drug atovaquone at clinically relevant concentrations is active against chemoresistant HCC. We show that atovaquone inhibits proliferation and induces apoptosis in not only HCC parental cells but also cells exposed to long time culture of chemotherapeutic agents. Consistently, the combination of atovaquone with cisplatin or doxorubicin achieved remarkably greater efficacy than single drug alone. Mechanistically, atovaquone overcomes HCC chemoresistance via supressing mitochondrial respiration and inducing oxidative stress. Atovaquone but not cisplatin or doxorubicin is ineffective in mitochondrial respiration-deficient ρ0, confirming mitochondria as a specific upstream target of atovaquone. Interestingly, we show that prolonged exposure of HCC cells to chemotherapeutic agents induces higher level of mitochondrial respiration, suggesting that tumors which develop chemoresistance after chemotherapy might be more dependent on mitochondrial respiration than primary tumors and explaining the sensitivity of chemoresistant HCC cells to atovaquone. We further show that atovaquone at tolerable does significantly inhibits chemoresistant HCC growth in mice throughout the duration of treatment. In line with in vitro data, we observe the increased oxidative stress in atovaquone-treated tumors. Our findings highlight the dependency of chemoresistant HCC on mitochondrial respiration and demonstrate that atovaquone is a potential drug to overcome HCC chemoresistance.

Non-invasive quantitative imaging of hepatocellular carcinoma growth in mice by micro-CT using liver-targeted iodinated nano-emulsions

Hepatocellular carcinoma (HCC) is the only cancer for which non-invasive diagnosis is recognized by international guidelines. Contrast agent free ultrasound imaging, computed tomography (CT) and/or magnetic resonance imaging are techniques used for early detection and confirmation. Clinical evidence depicts that CT is 30% less precise as compared to MRI for detection of small tumors. In our work, we have reported some novel tools that can enhance the sensitivity and precision of CT applied to preclinical research (micro-CT). Our system, containing non-toxic nano-droplets loaded with iodine has high contrasting properties, liver and hepatocyte specificity and strong liver persistence. Micro-CT was performed on HCC model implanted in nude mice by intrahepatic injection. Contrast agent was administrated intravenously. This method allows an unprecedented high precision of detection, quantitative measurement of tumor volume and quantitative follow-up of the tumor development.

Chemical or genetic Pin1 inhibition exerts potent anticancer activity against hepatocellular carcinoma by blocking multiple cancer-driving pathways

Hepatocellular carcinoma (HCC) is one of the most prevalent and malignant cancers with high inter- and intra-tumor heterogeneity. A central common signaling mechanism in cancer is proline-directed phosphorylation, which is further regulated by the unique proline isomerase Pin1. Pin1 is prevalently overexpressed in human cancers including ~70% of HCC, and promotes tumorigenesis by activating multiple cancer-driving pathways. However, it was challenging to evaluate the significance of targeting Pin1 in cancer treatment until the recent identification of all-trans retinoic acid (ATRA) as a Pin1 inhibitor. Here we systematically investigate functions of Pin1 and its inhibitor ATRA in the development and treatment of HCC. Pin1 knockdown potently inhibited HCC cell proliferation and tumor growth in mice. ATRA-induced Pin1 degradation inhibited the growth of HCC cells, although at a higher IC50 as compared with breast cancer cells, likely due to more active ATRA metabolism in liver cells. Indeed, inhibition of ATRA metabolism enhanced the sensitivity of HCC cells to ATRA. Moreover, slow-releasing ATRA potently and dose-dependently inhibited HCC growth in mice. Finally, chemical or genetic Pin1 ablation blocked multiple cancer-driving pathways simultaneously in HCC cells. Thus, targeting Pin1 offers a promising therapeutic approach to simultaneously stop multiple cancer-driving pathways in HCC.

DNA vaccine encoding α-fetoprotein fused with the ornithine decarboxylase degradation signal significantly suppresses the hepatocellular carcinoma growth in mice

Hepatocellular carcinoma (HCC) is the most frequent primary malignancy of the liver in humans. The available anti-HCC therapies are ineffective, and their use has substantial limitations. DNA vaccines expressing α-fetoprotein (AFP), which is a marker of HCC, were recently found to induce the immune response against AFP in vivo. However, the vectors used as vaccines failed to ensure the necessary level of protection, and the immunization procedures were rather complex. We showed earlier that a DNA vaccine encoding HIV-1 reverse transcriptase (RT) with the C end fused with the ornithine decarboxylase (ODC) degradation signal induced a strong Th-1 immune response against RT in mice. In this work, we designed a set of novel DNA vaccines encoding AFP or AFP with the ODC degradation signal. Transfected cells demonstrated efficient expression and fast proteasomal degradation of the recombinant AFP. The antitumor immune response was stimulated in immunized animals, and a notable retardation of HCC growth was observed as a result of protective vaccination.

Oxysophoridine suppresses the growth of hepatocellular carcinoma in mice: In Vivo and cDNA microarray studies

To observe the in vivo effects of oxysophoridine on hepatocellular carcinoma in mice and to study the related mechanisms. C57BL mice were inoculated with mouse hepatoma H22 cells subcutaneously, then divided into 5 groups (14 per group), and treated with oxysophoridine (50, 100, or 150 mg/kg) or cisplatin (4 mg/kg) for 10 days. Inhibitory rate of tumor, body weight gain, and influence indices on internal organs (liver, spleen and thymus) were evaluated. The differentially expressed genes between the oxysophoridine-treated group, and the control group were analyzed using cDNA microarray and quantitative real-time PCR (qRT-PCR) experiments. Compared with the tumor weight of the control group (2.75±0.66 g), oxysophoridine significantly suppressed hepatocellular carcinoma growth in mice (P <0.01), with 0.82±0.36 g, 0.57±0.22 g, and 1.22±0.67 g for the tumor weight in the low, moderate, and high dose treatment group, respectively. The moderate dose led to the highest inhibitory rate, 79.3%. Observation of body weight gain and influence on three organs showed that compared with cisplatin, oxysophoridine produced fewer side effects in vivo. cDNA microarray and qRT-PCR showed that the most significant differentially expressed genes in the tumor samples of oxysophoridine-treated mice were mostly involved in regulating apoptosis, with the Tnfrsf11b (osteoprotegerin) gene being the most significantly affected. Oxysophoridine was a promising compound for developing drugs against hepatocellular carcinoma, and its anti-hepatoma effect was probably related to osteoprotegerin activation.

Inhibition effect of 1-methyl-tryptophan on transplant hepatocellular carcinoma growth in mice subcutaneous

Objective To investigate the inhibitory effect of 1-methyl-tryptophan (1-MT) on transplanted hepatocellular carcinoma in mice.Methods Human hepatocellular carcinoma subcutaneous tumor models were established in mice,and the mice were divided into hepG2 group,empty plasmid group,indoleamine 2,3-dioxygenase (IDO) saline group,IDO) 5-fluoropyrimidine (5-FU) group,IDO 1-MT group,and the group combining IDO 1-MT with 5-FU treatment (n=8 in each group).The tumor growth,tumor volume and pathological examination were observed and the expression of IDO in tumor tissues was determined by immunohistochemistry.Results Compared with hepG2 and the empty vector saline groups,IDO saline group had bigger tumor,faster growth,and the differences were statistically significant (P＜0.05).Compared with IDO saline group,5-FU group,1-MT group and combination treatment groups showed smaller tumor volume and weight,and the tumor inhibitory rates were 86.54％,79.95％,94.46％,respectively.There were significant differences between these groups (P＜0.05).However,there were no significant differences in tumor volumes between 5-FU group and 1-MT group (P＞0.05).HE pathological observation of ceils in each treatment group showed reduced density,increased necrotic area and significant decrease in peripheral blood alpha-fetoprotein (P＜0.05).Conclusions IDO can promote the growth of liver cancer cells involved in immune escape.1-MT can inhibit the transplanted tumor growth in mice,and therefore may enhance the chemotherapeutic efficacy. Key words: 1-methiyl tryptophan; Indoleamine 2,3-dioxygenase; Hepatocellular carcinoma; Immune therapy

Inhibition of experimental HCC growth in mice by use of the kinase inhibitor DMAT

The multi-kinase-inhibitor Sorafenib has been shown to prolong survival of patients suffering from hepatocellular carcinoma (HCC). We investigated effects of the serine/threonine kinase inhibitor 2-Dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole (DMAT) on experimental HCC growth, and identified mechanisms and target kinases of DMAT. Our results show that DMAT application in vivo reduced tumor growth in a xenotransplant model by interference with tumor cell proliferation. Biochemical parameters and histology following DMAT administration revealed no alterations in liver tissue. Similar to Sorafenib, DMAT interfered with NFκB activation and Wnt-signaling. Of the kinases inhibited by DMAT at almost equimolar IC50, CK2 and PIM-3 were found to be over-expressed or more active in hepatoma cells and human HCC tissue. Knockdown of PIM-3 or CK2 by shRNA revealed that both kinases are important for hepatoma cell proliferation and survival. In conclusion, DMAT reduces HCC growth by interference with NFκB- and Wnt-signaling. PIM-3 and CK2 seem to be important target kinases. Inhibition of these kinases by application of inhibitors, e.g., DMAT, might represent a promising therapeutic approach in future HCC therapy.

AAV-mediated TRAIL gene expression driven by hTERT promoter suppressed human hepatocellular carcinoma growth in mice

A major obstacle in the development of effective recombinant adeno-associated virus (rAAV) mediated gene therapy is infection specificity and gene targeting. In the present study, we investigated whether the human telomerase reverse transcriptase (hTERT) promoter could drive tumor-specific expression of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), an apoptosis apoptosis-inducing protein with potential toxic effects on normal cells. Our data demonstrated that hTERT promoter-driven tumor-specific expression of TRAIL decreased the cellular viability of tumor cells, but not normal cells. TRAIL expression driven by hTERT promoter inhibited tumor growth significantly in vivo and combination of viral infection with 5-fluorouracil (5-Fu) suppressed tumor growth more efficiently. Intra-venous injection of virus showed that the recombinant virus was predominantly distributed in the liver, but not in other major tissues tested, and no transgene expression was detected in the liver. Furthermore, serum enzyme and liver histology analysis confirmed that liver function is unaffected by TRAIL expression, significant as the liver is frequently metastasized and scattered with tumors from other organs, which are unpractical to treat by intra-tumor injection. Together our results demonstrate that rAAV-mediated TRAIL expression is a promising strategy in gene therapy for treatment of cancer.

Suppression of Hepatocellular carcinoma growth in mice via Mycelia of Liquid‐ Cultured Phellinus Liteus, is associated with inhibition of tumor cell growth and natural killer cell activation

Phellinus Liteus is a perennial fungus which is selectively parasitic on Morus rubra. This fungus is know as the Chinese medicine, Souou, and rich with polysaccharide and polysaccharide-protein complex. The aim of this study was to determine Phellinus Liteus¡¦s anti-tumor effect in a murine model of hepatocellular carcinoma ( HCC). Eight weeks old male SCID mice were transplanted with Hep3B cells, followed by dairly Phellinus Liteus administration for 8 weeks. Phellinus Liteus administration induced a significant reduction in tumor size, improved survival rate, and was association with a significant increase in splenocytes. There was no significant difference between groups was noted in any of the other serum cytokine level. However, Phellinus Liteus administration induces a significant suppression of human HCC. This effect is mediated by induction of natural killer cell activation, along with direct inhibition of tumor growth.

Adeno-associated virus-mediated expression of apolipoprotein (a) kringles suppresses hepatocellular carcinoma growth in mice

Hepatocellular carcinoma (HCC) constitutes more than 90% of all primary liver cancers. HCC is a hypervascular tumor that develops from dedifferentiation of small avascular HCC and is therefore a good target for anti-angiogenic gene therapy. Recent studies have identified apolipoprotein(a) [apo(a)] kringles LK68 and LK8 (LKs) as having a potential antiangiogenic and anti-tumor activity, and the current study evaluates the therapeutic potential of gene therapy with recombinant adeno-associated virus carrying genes encoding LKs (rAAV-LK) in the treatment of hypervascular HCC. We generated rAAV-LK to obtain persistent transgene expression in vivo, which is essential for anti-angiogenic therapy. The rAAV-produced LKs substantially inhibited proliferation and migration of human umbilical vein endothelial cells (HUVECs) in vitro, validating their anti-angiogenic potential. Intramuscular administration of rAAV-LK gave 60% to 84% suppression (P < .05) of tumor growth in mice bearing subcutaneously transplanted HCC derived from Huh-7 and Hep3B cells, respectively. Histological and immunohistochemical analyses of HCC tumor sections showed that a single administration of rAAV-LK gave rise to persistent expression of LKs that inhibited tumor angiogenesis and triggered tumor apoptosis, and, thus, significantly suppressed tumor growth. The administration of rAAV-LK provided a significant survival benefit (P < .05), and 3 of 10 rAAV-LK-treated mice were still alive without visible tumors and without clinical symptoms 188 days after treatment. In conclusion, rAAV-LK is a potential candidate for anti-angiogenic gene therapy in the treatment of HCC.

INHIBITION OF WILMS TUMOR XENOGRAFT PROGRESSION BY HALOFUGINONE IS ACCOMPANIED BY ACTIVATION OF WT-1 GENE EXPRESSION

Wilms tumor (WT) is the most common malignant neoplasm of the urinary tract in children. Although it is curable with long-term survival, the combination of surgery, chemotherapy and often radiotherapy in some cases results in severe complications in adulthood. Therefore, novel therapeutic strategies that would decrease treatment burden and improve outcome for high risk patients are required. We evaluated the efficacy of halofuginone, an inhibitor of collagen type I synthesis and angiogenesis, to inhibit WT development in xenografts models. WTs derived from 2 patients with favorable histology at different disease stages were implanted subcutaneously or orthotopically in the kidneys of nude mice. Halofuginone was administered intraperitoneally (2 mug per mouse every other day) or given in the diet (1 part per million). Independent of disease stage, tumor location or administration route, halofuginone caused a decrease in angiogenesis that resulted in marked inhibition of tumor development. This result was accompanied by a reduction in collagen synthesis, reduced levels of hepatocyte growth factor receptor MET and increased levels of the tumor suppressor protein WT1. In culture halofuginone increased the synthesis of WT1 in the human WT cell-line SK-NEP-1 and in other cancer cell lines such as hepatocellular carcinoma and prostate cancer. In SK-NEP-1 halofuginone also lowered erb B2 levels and reduced cell proliferation. These results suggest that halofuginone is a potent inhibitor of WT progression. Because of its unique mode of action, halofuginone may decrease the treatment burden when combined with chemotherapy.

Suppression of hepatocellular carcinoma growth in mice via leptin, is associated with inhibition of tumor cell growth and natural killer cell activation

Leptin exerts potent immune modulatory properties. The aim of this study was to determine leptin's anti-tumor effect in a murine model of hepatocellular carcinoma (HCC). In vivo, athymic nude mice were transplanted with Hep3B cells, followed by daily leptin administration for 6 weeks. Leptin administration induced a significant reduction in tumor size, improved survival rate, and was associated with a significant increase in peripheral natural killer (NK) cell number. Splenocytes from leptin-treated mice featured decreased expression of CIS mRNA. SCID mice featured a similar leptin-associated tumor suppression. In contrast, NK-deficient SCID-beige mice developed larger tumors which were unresponsive to leptin. NK cells incubated in vitro with increasing doses of leptin demonstrated increased cytotoxicity and proliferation. Incubation of leptin with hepatoma cells induced a dose-dependent reduction in proliferation, suggesting a direct anti-tumor effect. Leptin induced increased mRNA expression of STAT2 and SOCS1 on HCC cell lines. Leptin administration induces a significant suppression of human HCC. This effect is mediated by induction of natural killer cell proliferation and activation, along with direct inhibition of tumor growth. Decreased NK expression of inhibitory CIS and over-expression of the antiproliferative STAT2 and SOCS1 proteins in HCC lines may underline the anti-cancerous effects of leptin.