Human Hepatocellular Carcinoma Xenografts Research Articles

In vivo study of effects of artesunate nanoliposomes on human hepatocellular carcinoma xenografts in nude mice

To investigate the effect of artesunate nanoliposomes on cultured cells in vitro and hepatocellular carcinoma xenografts in BALB/c-nu mice. Fluorescence polarization was applied for measurement of mitochondrial membrane fluidities; inhibition test of tumor cell proliferation in vitro was performed and nude mice xenograft model from human hepatocellular carcinoma (HCC) was established. Cytotoxicity of these compounds was evaluated by MTT assay on hepatocellular carcinoma xenografts in nude mice. Anisotropy (r-value) of blank nanoliposomes didn’t change, it had no statistically significance between the blank nanoliposomes group and the control group, it indicated that artesunate had no obvious effect on L-O2 human normal liver cells. IC50 values of artesunate nanoliposomes and artesunate API (active pharmaceutical ingredient) against HepG-2 cells were 15.997 and 19.706 μg/ml; IC50 values of the same drugs against L-O2 normal human liver cells were 100.23 and 105.54 μg/ml, respectively. Tumor growth inhibitory effect of artesunate nanoliposomes was 32.7%, and artesunate API was 20.5%, respectively. HepG-2 cells treated with artesunate nanoliposomes showed dose-dependent apoptosis. The antitumor effect of artesunate nanoliposomes on human hepatoma HepG2 cells were stronger than that of artesunate API at the same concentration.

Ultrasound Exposure Improves the Targeted Therapy Effects of Galactosylated Docetaxel Nanoparticles on Hepatocellular Carcinoma Xenografts

PurposeThe distribution of targeted nanoparticles in tumor tissue is affected by a combination of various factors such as the physicochemical properties of the nanoparticles, tumor hemoperfusion and tumor vascular permeability. In this study, the impact of the biological effects of ultrasound on nanoparticle targeting to liver carcinoma was explored.MethodsThe copolymer MePEG-PLGA was used to prepare the galactosylated docetaxel nanoparticles (GDN), and the physical and chemical properties as well as the acute toxicity were then assayed. The impact of ultrasound exposure (UE) on tumor hemoperfusion was observed by contrast-enhanced ultrasonography (CEUS), and the distribution of docetaxel in tumors and liver were detected by high performance liquid chromatography (HPLC). In the GDN combined with UE treatment group, the mice were injected intravenously with GDN, followed by ultrasound exposure on the human hepatocellular carcinoma xenografts. Twenty-eight days post-administration, the tumor growth inhibition rate was calculated, and the expression of Survivin and Ki67 in tumor tissues were determined by immunohistochemistry assay and quantitative real-time PCR.ResultsThe mean size of prepared liver-targeting nanoparticles GDN was 209.3 nm, and the encapsulation efficiency was 72.28%. The median lethal dose of GDN was detected as 219.5 mg/kg which was about four times higher than that of docetaxel. After ultrasound exposure, the tumor peak - base intensity difference value, examined by CEUS, increased significantly. The drug content in the tumor was 1.96 times higher than in the GDN treated control. In vivo, GDN intravenous injection combined with ultrasound exposure therapy achieved the best anti-tumor effect with a tumor growth inhibition rate of 74.2%, and the expression of Survivin and Ki67 were significantly decreased as well.ConclusionUltrasound exposure can improve targeting nanoparticles accumulation in the tumor, and achieve a synergism antitumor effect on the hepatocellular carcinoma xenografts.

Genistein Reinforces the Inhibitory Effect of Cisplatin on Liver Cancer Recurrence and Metastasis after Curative Hepatectomy

The high recurrence rate after hepatic resection in hepatocellular carcinoma (HCC) is a major obstacle to improving prognosis. The objective of the present study was to explore the function of genistein, a soy-derived isoflavone, in enhancing the inhibitory effect of cisplatin on HCC cell proliferation and on tumor recurrence and metastasis in nude mice after curative hepatectomy. Proliferation of human HCC cells (HCCLM3) was detected by 3-(4, 5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay. Synergistic effects of genistein and cisplatin were evaluated with the median-effect formula. Nude mice bearing human HCC xenografts underwent tumour resection (hepatectomy) 10 days post implantation, then received intraperitoneal administration of genistein or cisplatin alone or the combination of the two drugs. 33 days after surgery, recurrent tumours and pulmonary metastasis were evaluated individually. MMP-2 level in recurrent tumours was detected by immunohistochemistry and real-time PCR; MMP-2 expression in HCCLM3 was detected by immunocytochemistry. Genistein and cisplatin both suppressed the growth and proliferation of HCCLM3 cells. The two drugs exhibited synergistic effects even at relatively low concentrations. In vivo, mice in the combined genistein and cisplatin group had a smaller volume of liver recurrent tumors and fewer pulmonary metastatic foci compared with single drug treated groups. Cisplatin upregulated the expression of MMP-2 in both recurrent tumours and HCCLM3, while genistein abolished cisplatin-induced MMP-2 expression. Genistein reinforced the inhibitory effect of cisplatin on HCC cell proliferation and tumour recurrence and metastasis after curative hepatectomy in nude mice, possibly through mitigation of cisplatin-induced MMP-2 upregulation.

Nuclear α1-Antichymotrypsin Promotes Chromatin Condensation and Inhibits Proliferation of Human Hepatocellular Carcinoma Cells

α1-Antichymotrypsin (α1-ACT), a member of the serpin family (SERPINA3), is an acute-phase protein secreted by hepatocytes in response to cytokines such as oncostatin M. α1-ACT is a protease inhibitor thought to limit tissue damage produced by excessive inflammation-associated proteolysis. However, α1-ACT also is detected in the nuclei of cells, where its activities are unknown. Expression of α1-ACT is down-regulated in human hepatocellular carcinoma (HCC) tissues and cells; we examined its roles in liver regeneration and HCC proliferation. We measured levels of α1-ACT messenger RNA in human HCC samples and healthy liver tissue. We reduced levels of α1-ACT using targeted RNA interference in human HCC (HepG2) and mouse hepatocyte (AML12) cell lines, and overexpressed α1-ACT from lentiviral vectors in Huh7 (HCC) cells and adeno-associated viral vectors in livers of mice. We assessed proliferation, differentiation, and chromatin compaction in cultured cells, and liver regeneration and tumor formation in mice. Reducing levels of α1-ACT promoted proliferation of HCC cells in vitro. Oncostatin M up-regulated α1-ACT expression and nuclear translocation, which inhibited HCC cell proliferation and activated differentiation of mouse hepatocytes. We identified amino acids required for α1-ACT nuclear localization, and found that α1-ACT inhibits cell-cycle progression and anchorage-independent proliferation of HCC cells. HCC cells that overexpressed α1-ACT formed smaller tumors in mice than HCC cells that did not express the protein. α1-ACT was observed to self-associate and polymerize in the nuclei of cells; nuclear α1-ACT strongly bound chromatin to promote a condensed state that could prevent cell proliferation. α1-ACT localizes to the nuclei of hepatic cells to control chromatin condensation and proliferation. Overexpression of α1-ACT slows the growth of HCC xenograft tumors in nude mice.

Ethyl acetate extract of Squilla oratoria suppresses growth of HepG2 cells by inducing S phase arrest

Objective: The oceans and seas are a rich source of organisms from which anti-cancer drugs can be isolated and developed. Marine organisms have been screened in our laboratory, and organic solvent extracts of Squilla oratoria (ESO) have been shown to possess cytostatic effects on cancer cell lines of diverse origins. To explore the underlying mechanisms, the growth inhibition by ESO was investigated in the present study. Methods: Human hepatocellular carcinoma (HCC) derived cells (HepG2) were used. The cells were challenged with ESO, cell cycle profile was assayed, and level of proliferating cell nuclear antigen (PCNA) expression and that of cyclin D1 and cyclin A were evaluated with flow cytomtery. The in vivo antitumor effect of ESO was tested in nude mouse xenografts. PCNA expression was evaluated immunohistochemically in nude mouse xenograft tissues. Results: With the increase in dose of injected ESO, expression of PCNA by human HCC xenografts increased. ESO inhibited the growth of human HCC HepG2 cells both in vitro and in vivo. The effect was correlated with arrest of the cell cycle in S phase. Expression of PCNA, which is a cell-cycle regulator that promotes S phase entry, was elevated in both cell lines and xenografts whereas that of cyclins that promote M phase entry was down-regulated by exposure to ESO. Conclusion: Growth inhibition was explained by arrest of the cell cycle in S phase and down-regulation of molecules that promote cells to enter S phase.

Quantification in Molecular Ultrasound Imaging

The purpose of this study was to quantitatively assess the contrast kinetics of vascular endothelial growth factor receptor 2 (VEGFR2)-targeted microbubbles (BR55; Bracco Suisse, Geneva, Switzerland) compared to clinically used microbubbles (SonoVue; Bracco SpA, Milan, Italy) in both normal liver and human hepatocellular carcinoma xenograft tumors in mice. Microbubbles were injected intravenously into healthy mice (n = 5) and mice bearing hepatocellular carcinoma xenograft tumors (n = 10). Cine loops of contrast enhancement in normal liver and the tumors were acquired for 10 minutes. Quantitative perfusion parameters were derived by fitting time-intensity curves using a dedicated mathematical model combining a bolus function and a ramp function. Immunohistochemical examinations were also performed for normal liver and tumor specimens to determine the level of VEGFR2 expression. The peak contrast enhancement observed in normal liver with BR55 was comparable to that with SonoVue, whereas a significant difference was observed in latephase enhancement at 10 minutes (ramp slope: P < .01). In the tumor model, SonoVue was rapidly cleared from the circulation, without any noticeable binding in the tumor, whereas BR55 showed a gradual decline, resulting in a longer wash-out period (mean transit time: P < .01). Immunohistochemical examinations showed that intratumoral vascular endothelial cells had sparse and weak VEGFR2 expression, whereas the sinusoidal capillaries in normal liver had much more diffuse and much stronger expression. Our results suggest that BR55 accurately reflects the VEGFR2 status in human hepatocellular carcinoma xenograft tumors. We showed that quantification applied to molecular ultrasound imaging may provide an objective method for measuring the degree of microbubble binding.

Combination of acyclic retinoid with branched‐chain amino acids inhibits xenograft growth of human hepatoma cells in nude mice

Combination chemoprevention is a promising strategy to improve the prognosis of hepatocellular carcinoma (HCC). A malfunction of retinoid X receptor-α (RXR-α) due to phosphorylation by Ras/mitogen-activated protein kinase is closely associated with liver carcinogenesis and acyclic retinoid (ACR) can prevent HCC development by inhibiting RXR-α phosphorylation. The present study examined the possible combined effects of ACR plus branched-chain amino acids (BCAA), which can also prevent the development of HCC in obese patients with liver cirrhosis, in human HCC xenografts in nude mice. This study examined the effects of the combination of ACR plus BCAA on the growth of Huh7 human HCC xenografts in nude mice. The effects of the combination on the phosphorylation of RXR-α, extracellular signal-regulated kinase (ERK), Akt and insulin-like growth factor-1 receptor (IGF-1R) proteins, and on the expression levels of retinoic acid receptor-β (RAR-β) and p21(CIP1) mRNA, were also examined by western blot and real-time reverse transcription polymerase chain reaction analyses, respectively. The combined treatment with ACR plus BCAA significantly inhibited the growth of Huh7 xenografts. The combination of these agents caused a marked inhibition of the phosphorylation of RXR-α, ERK, Akt and IGF-1R proteins in the xenografts. In addition, the expression levels of RAR-β and p21(CIP1) mRNA significantly increased by these agents. The combination of ACR and BCAA restores the function of RXR-α by inhibiting its phosphorylation and increasing the level of RAR-β, a heterodimeric partner for RXR-α, and thus suppresses the growth of HCC xenografts. Therefore, this combination might be an effective regimen for the treatment and, probably, chemoprevention of HCC.

Sinomenine hydrochloride inhibits human hepatocellular carcinoma cell growth in vitro and in vivo: Involvement of cell cycle arrest and apoptosis induction

Hepatocellular carcinoma (HCC) is one of the most common malignancies worldwide. However, therapies against HCC to date have not been completely effective. Sinomenine hydrochloride (SH), an anti‑arthritis drug applied in clinical practice, has been reported to have in vitro anti‑neoplastic activity in various cancer cells. Whether SH inhibits HCC remains unknown. For this purpose, in this study, MTT assay was used to determine cell growth. Flow cytometry, Hoechst staining, DNA fragmentation, western blot analysis, immunohistochemisty and TUNEL staining were performed to investigate the mechanisms involved. The in vivo activity of SH was determined using a mouse xenograft model. SH inhibited the growth of various types of human HCC cells in vitro. We found that SH promoted cell cycle arrest in the G1 phase and sub‑G1 formation, associated with the increased p21/WAF1/Cip1 expression. Additionally, SH induced caspase‑dependent apoptosis, which involved the disruption of mitochondrial membrane potential, the increased release of cytochrome c and Omi/HtrA2 from the mitochondria into the cytoplasm, the downregulation of Bcl‑2 and the upregulation of Bax, the activation of a caspase cascade (caspase‑8, -10, -9 and -3) and PARP, as well as the decreased expression of survivin. The SH‑suppressed growth of human HCC xenografts in vivo occurred due to the decrease in proliferation and the induction of apoptosis, implicating the activation of caspase‑3, the upregulation of p21 and the downregulation of survivin. These findings suggest that SH exhibits anticancer efficacy in vitro and in vivo involving cell cycle and caspase‑dependent apoptosis and may serve as a potential drug candidate against HCC.

Combination therapy of radiofrequency ablation and bevacizumab monitored with power Doppler ultrasound in a murine model of hepatocellular carcinoma

Purpose: The purpose of this study was to monitor tumour blood flow with power Doppler ultrasound following antiangiogenic therapy with bevacizumab in order to optimally time the application of radiofrequency (RF) ablation to increase ablation diameter.Materials and methods: Athymic nude mice bearing human hepatocellular carcinoma xenografts were treated with bevacizumab and imaged daily with power Doppler ultrasound to quantify tumour blood flow. Mice were treated with RF ablation alone or in combination with bevacizumab at the optimal time, as determined by ultrasound. Ablation diameter was measured with histology and tumour microvascular density was calculated with immunohistochemistry. A computational thermal model of RF ablation was used to estimate ablation volume.Results: A maximum reduction of 27.8 ± 8.6% in tumour blood flow occurred on day 2 following antiangiogenic therapy, while control tumours increased 29.3 ± 17.1% (p < 0.05). Tumour microvascular density was similarly reduced by 45.1 ± 5.9% on day 2 following antiangiogenic therapy. Histology demonstrated a 13.6 ± 5.6% increase in ablation diameter (40 ± 21% increase in volume) consistent with a computational model.Conclusion: Quantitative power Doppler ultrasound is a useful biomarker to monitor tumour blood flow following antiangiogenic treatment and to guide the application of RF ablation as a drug plus device combination therapy.

Human Solid Tumor Xenografts in Immunodeficient Mice Are Vulnerable to Lymphomagenesis Associated with Epstein-Barr Virus

Xenografting primary human solid tumor tissue into immunodeficient mice is a widely used tool in studies of human cancer biology; however, care must be taken to prove that the tumors obtained recapitulate parent tissue. We xenografted primary human hepatocellular carcinoma (HCC) tumor fragments or bulk tumor cell suspensions into immunodeficient mice. We unexpectedly observed that 11 of 21 xenografts generated from 16 independent patient samples resembled lymphoid neoplasms rather than HCC. Immunohistochemistry and flow cytometry analyses revealed that the lymphoid neoplasms were comprised of cells expressing human CD45 and CD19/20, consistent with human B lymphocytes. In situ hybridization was strongly positive for Epstein-Barr virus (EBV) encoded RNA. Genomic analysis revealed unique monoclonal or oligoclonal immunoglobulin heavy chain gene rearrangements in each B-cell neoplasm. These data demonstrate that the lymphoid neoplasms were EBV-associated human B-cell lymphomas. Analogous to EBV-associated lymphoproliferative disorders in immunocompromised humans, the human lymphomas in these HCC xenografts likely developed from reactivation of latent EBV in intratumoral passenger B lymphocytes following their xenotransplantation into immunodeficient recipient mice. Given the high prevalence of latent EBV infection in humans and the universal presence of B lymphocytes in solid tumors, this potentially confounding process represents an important pitfall of human solid tumor xenografting. This phenomenon can be recognized and avoided by routine phenotyping of primary tumors and xenografts with human leukocyte markers, and provides a compelling biological rationale for exclusion of these cells from human solid tumor xenotransplantation assays.

The Monoclonal Antibody CH12 Enhances the Sorafenib-Mediated Growth Inhibition of Hepatocellular Carcinoma Xenografts Expressing Epidermal Growth Factor Receptor Variant III

The multikinase inhibitor sorafenib is the first oral agent to show activity against human hepatocellular carcinoma (HCC). Although the clinical application of sorafenib has shown good tolerability in the studied populations, it also causes multiple human dose-limiting toxicities. Thus, there is a strong need to reduce the overall dose of sorafenib. We have reported that the epidermal growth factor receptor variant III (EGFRvIII) expression can decrease the sensitivity of HCC cells to chemotherapeutic drugs. Therefore, we sought to explore whether EGFRvIII can affect the sensitivity of HCC cells to sorafenib. In this study, we observed that EGFRvIII expression significantly decreased the sensitivity of HCC cells to sorafenib. To enhance the antitumor effect and reduce the overall dose of sorafenib, we evaluated the combined effects of CH12, a monoclonal antibody against EGFRvIII, and sorafenib on the growth of HCC cells expressing EGFRvIII in vitro and in vivo. The results showed that, when CH12 was combined with sorafenib, the tumor growth suppression effect was significantly increased, and the concentration of sorafenib required for growth inhibition was substantially reduced. Mechanistically, the combination could more noticeably downregulate the phosphorylation of constitutively active extracellular signal-regulated kinase (ERK), Akt (Thr308), and signal transducer and activator of transcription 3 (STAT3) than sorafenib alone. Collectively, these findings demonstrate that CH12 interacts additively with sorafenib to strongly inhibit the tumor growth of HCC xenografts expressing EGFRvIII by enhancing the sorafenib-mediated inhibition of the MEK/ERK, phosphoinositide 3-kinase/AKT, and STAT3 pathways.

The role of regulatory B cells during the progression of human hepatocellular carcinoma (162.39)

Abstract Y SHAO1, CX LI1, YY MA1, XB LIU1, CY CHU2, CC LING1, WH Yeung1, KW Lee3, ST FAN1,2, CM LO1,2, K MAN1,2 1.Department of Surgery 2.Centre for Cancer Research 3. Department of Pathology, The University of Hong Kong, Hong Kong Human regulatory B cells (hBregs) have been shown to play an important role in autoimmune diseases, but their function during human cancer progression remains elusive. In this study, we aim to unveil the underlying mechanism of hBregs regulating human hepatocellular carcinoma (HCC) growth. B cells in HCC liver tumor were examined by immunohistochemistry. We found a tendency that the amount of B cells increased with HCC progression (p=0.001), and it’s in the marginal region of HCC increased (P&amp;lt;0.001) more dramatically. In vitro, human HCC cell line MHCC-97L cells were cocultured with hBregs. hBregs promoted HCC cells proliferation (P=0.011) and migration (p=0.006) and decreased HCC cells apoptosis (p=0.078). HCC cells also promoted hBregs proliferation and migration (p=0.003). hBregs could interact with HCC cells directly through CD40-CD154 signaling. Tail vein injection of hBreg cells into SCID mice increased the size of HCC xenograft tumor (4 weeks, p=0.0221). In vivo imagining showed that hBreg cells could migrate into tumor. Our findings indicate that hBregs could promote HCC progression.

Celastrol Suppresses Growth and Induces Apoptosis of Human Hepatocellular Carcinoma through the Modulation of STAT3/JAK2 Signaling Cascade In Vitro and In Vivo

Cumulative evidences(s) have established that the constitutive activation of STAT3 plays a pivotal role in the proliferation, survival, metastasis, and angiogenesis and thus can contribute directly to the pathogenesis of hepatocellular carcinoma (HCC). Thus, novel agents that can inhibit STAT3 activation have potential for both prevention and treatment of HCCs. The effect of celastrol on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was investigated. The in vivo effect of celastrol on the growth of human HCC xenograft tumors in athymic nu/nu mice was also examined. We observed that celastrol inhibited both constitutive and inducible STAT3 activation, and the suppression was mediated through the inhibition of activation of upstream kinases c-Src, as well as Janus-activated kinase-1 and -2. Vanadate treatment reversed the celastrol-induced modulation of STAT3, suggesting the involvement of a tyrosine phosphatase. The inhibition of STAT3 activation by celastrol led to the suppression of various gene products involved in proliferation, survival, and angiogenesis. Celastrol also inhibited the proliferation and induced apoptosis in HCC cells. Finally, when administered intraperitoneally, celastrol inhibited STAT3 activation in tumor tissues and the growth of human HCC xenograft tumors in athymic nu/nu mice without any side effects. Overall, our results suggest for the first time that celastrol exerts its antiproliferative and proapoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.

Iron Deprivation Suppresses Hepatocellular Carcinoma Growth in Experimental Studies

Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death, and iron overload is a significant risk factor in the development of HCC. In this study, we investigated the potential application of depriving iron by a novel iron chelator, thiosemicarbazone-24 (TSC24), in HCC treatment. Two HCC cell lines and HFE knockout (HFE(-/-)) mice were used to determine iron chelation efficiency of TSC24. The anticancer effects of TSC24 on HCC were analyzed in vitro and in athymic xenograft mouse models. Treatment with TSC24 significantly decreased the cellular iron concentration in hepatoma cells and the serum iron concentration in HFE(-/-) mice by blocking iron uptake and interfering with normal regulation of iron levels. Moreover, the viability of HCC cell lines was reduced by TSC24. Confirming the mechanism of the agent, this decrease in viability could be partially rescued by addition of exogenous iron. TSC24 also suppressed tumor growth in athymic mice bearing human HCC xenografts in a concentration-dependent manner, without apparent toxicity in parallel with a decrease in the serum iron level. Further studies revealed that TSC24 efficiently triggered cell-cycle arrest and apoptosis in Hep3B and HepG2 cell lines. TSC24 is a potent iron chelator that suppresses human HCC tumor growth by disrupting iron homeostasis, reducing available iron, and triggering cell-cycle arrest and apoptosis, without apparent host toxicity at effective doses. Thus, TSC24 shows great potential for the treatment of HCC.

Positron Emission Tomography of Human Hepatocellular Carcinoma Xenografts in Mice Using Copper (II)-64 Chloride as a Tracer with Copper (II)-64 Chloride

The aim of this study was to assess copper metabolism of human hepatocellular carcinoma (HCC) with positron emission tomographic (PET) imaging using copper (II)-64 chloride ((64)CuCl(2)) as a tracer. PET imaging of athymic mice (n = 5) bearing extrahepatic HCC xenografts was performed 24 hours after the intravenous injection of (64)CuCl(2), followed by exvivo tissue radioactivity assay. Expression of human copper transporter 1 (hCTR1) in HCC cells and tissues was examined by real-time reverse transcription polymerase chain reaction and immunohistochemistry analysis, respectively. The extrahepatic HCC xenografts in mice with increased uptake of (64)Cu radionuclide were visualized on the micro-PET images obtained 24 hours after the intravenous injection of (64)CuCl(2). PET quantitative analysis revealed increased (64)Cu radioactivity in tumor tissues (2.7 ± 0.6 %ID/g) compared to that in the soft tissue of the left shoulder opposite to the tumor site (0.6 ± 0.2 %ID/g) and the brain (0.7 ± 0.1 %ID/g) but lower than that of the liver (16.6 ± 1.3 %ID/g). Expression of hCTR1 in the HCC cells and xenograft tumor tissues was demonstrated by real-time reverse transcription polymerase chain reaction and immunohistochemistry analysis, respectively. The expression level of hCTR1 in the Hep3B HCC xenograft tissues was lower than that detected in the normal hepatic tissues and the tissue samples of well-differentiated primary HCC. Variable expression of hCTR1 was detected in the tissue samples of moderately differentiated primary HCC. Extrahepatic human HCC xenografts in mice could be localized with (64)CuCl(2) PET imaging, which might be useful for the localization and quantitative assessment of copper metabolism in extrahepatic metastases of HCC in humans.

Characterization of herpes simplex virus 1 strains as platforms for the development of oncolytic viruses against liver cancer

Diverse oncolytic viruses (OV) are being designed for the treatment of cancer. The characteristics of the parental virus strains may influence the properties of these agents. To characterize two herpes simplex virus 1 strains (HSV-1 17syn(+) and HFEM) as platforms for virotherapy against liver cancer. The luciferase reporter gene was introduced in the intergenic region 20 locus of both HSV-1 strains, giving rise to the Cgal-Luc and H6-Luc viruses. Their properties were studied in hepatocellular carcinoma (HCC) cells in vitro. Biodistribution was monitored by bioluminescence imaging (BLI) in athymic mice and immune-competent Balb/c mice. Immunogenicity was studied by MHC-tetramer staining, in vivo killing assays and determination of specific antibody production. Intratumoural transgene expression and oncolytic effect were studied in HuH-7 xenografts. The H6-Luc virus displayed a syncytial phenotype and showed higher cytolytic effect on some HCC cells. Upon intravenous or intrahepatic injection in mice, both viruses showed a transient transduction of the liver with rapid relocalization of bioluminescence in adrenal glands, spinal cord, uterus and ovaries. No significant differences were observed in the immunogenicity of these viruses. Local intratumoural administration caused progressive increase in transgene expression during the first 5 days and persisted for at least 2 weeks. H6-Luc achieved faster amplification of transgene expression and stronger inhibition of tumour growth than Cgal-Luc, although toxicity of these non-attenuated viruses should be reduced to obtain a therapeutic effect. The syncytial H6-Luc virus has a strong oncolytic potential on human HCC xenografts and could be the basis for potent OV.

Highly specific off-target binding identified and eliminated during the humanization of an antibody against FGF receptor 4

Off-target binding can significantly affect the pharmacokinetics (PK), tissue distribution, efficacy and toxicity of a therapeutic antibody. Herein we describe the development of a humanized anti- fibroblast growth factor receptor 4 (FGFR4) antibody as a potential therapeutic for hepatocellular carcinoma (HCC). A chimeric anti FGFR4 monoclonal antibody (chLD1) was previously shown to block ligand binding and to inhibit FGFR4 mediated signaling as well as tumor growth in vivo. A humanized version of chLD1, hLD1.vB, had similar binding affinity and in vitro blocking activity, but it exhibited rapid clearance, poor target tissue biodistribution and limited efficacy when compared to chLD1 in a HUH7 human HCC xenograft mouse model. These problems were traced to instability of the molecule in rodent serum. Size exclusion high performance liquid chromatography, immunoprecipitation and mass spectral sequencing identified a specific interaction between hLD1.vB and mouse complement component 3 (C3). A PK study in C3 knock-out mice further confirmed this specific interaction. Subsequently, an affinity-matured variant derived from hLD1.vB (hLD1.v22), specifically selected for its lack of binding to mouse C3 was demonstrated to have a PK profile and in vivo efficacy similar to that of chLD1 in mice. Although reports of non-specific off-target binding have been observed for other antibodies, this represents the first report identifying a specific off-target interaction that affected disposition and biological activity. Screens developed to identify general non-specific interactions are likely to miss the rare and highly specific cross-reactivity identified in this study, thus highlighting the importance of animal models as a proxy for avoiding unexpected clinical outcomes.

Inhibitory effect of pyrrolidine dithiocarbamate combined with matrine on the growth of human hepatocellular carcinoma xenografts

To investigate the relationship between activation of nuclear factor-K-gene binding (NF-κB) and apoptosis induced by matrine(MT) in transplanted tumor of human hepatocellular carcinoma in nude mouse. Tumors were established by injection of hepatocellular carcinoma cell line HepG2 into the back of nude mice. The mice were divided randomly into four groups: Control group, MT group (35 mg/kg), PDTC group (120 mg/kg) and Combination group: PDTC + MT group (120 mg/kg + 35 mg/kg), the reagents were injected peritoneally. The tumor growth curve of nude mice bearing transplanted tumor were observed and the inhibition ratios were evaluated. Apoptosis of carcinoma cells was analyzed by TUNEL. The DNA-binding activity of NF-κB was determined by electrophoretic mobility shift assay (EMSA). Expression of bcl-2 and bax in carcinoma tissue were detected by immunohistochemical method. NF-κB mRNA, bcl-2 mRNA and bax mRNA in carcinoma tissue were detected by RT-PCR. Pyrrolidine dithiocarbamate (PDTC) could enhance the inhibition of matrine on carcinoma proliferation (P < 0.05). The apoptosis and activation of NF-κB in carcinoma cells could be induced by matrine. PDTC significantly suppressed NF-κB activation induced by matrine in carcinoma cells from 93.64 ± 2.95 to 65.78 ± 5.65 (F = 124.754, P < 0.01). Meanwhile, PDTC increased the apoptosis induced by matrine from 55.9% ± 2.8% to 74.3% ± 4.8% (P < 0.05).A positive correlation observed between the expressions of NF-κB and of bcl-2 (Pearson correlation coefficient = 0.983, P < 0.01). Matrine could induce apoptosis and activation of NF-κB in transplanted tumor. PDTC could increase apoptosis in hepatocellular carcinoma cells might be due to the suppression of NF-κB activation and the enhancement of bcl-2 expression.

Suppression of Signal Transducer and Activator of Transcription 3 Activation by Butein Inhibits Growth of Human Hepatocellular Carcinoma In Vivo

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide and the third cause of global cancer mortality. Increasing evidence suggest that STAT3 is a critical mediator of oncogenic signaling in HCC and controls the expression of several genes involved in proliferation, survival, metastasis, and angiogenesis. Thus, the novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. The effect of butein on STAT3 activation, associated protein kinases, STAT3-regulated gene products, cellular proliferation, and apoptosis was investigated. The in vivo effect of butein on the growth of human HCC xenograft tumors in male athymic nu/nu mice was also examined. We tested an agent, butein, for its ability to suppress STAT3 activation in HCC cells and nude mice model along with prospectively testing the hypothesis of STAT3 inhibition in a virtual predictive functional proteomics tumor pathway technology platform. We found that butein inhibited both constitutive and inducible STAT3 activation in HCC cells. The suppression was mediated through the inhibition of activation of upstream kinases c-Src and Janus-activated kinase 2. Butein inhibited proliferation and significantly potentiated the apoptotic effects of paclitaxel and doxorubicin in HCC cells. When administered intraperitoneally, butein inhibited the growth of human HCC xenograft tumors in male athymic nu/nu mice. Overall, cumulative results from experimental and predictive studies suggest that butein exerts its antiproliferative and proapoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.

Growth Suppression of Human Hepatocellular Carcinoma Xenografts by a Monoclonal Antibody CH12 Directed to Epidermal Growth Factor Receptor Variant III

Human hepatocellular carcinoma (HCC) is considered difficult to cure because it is resistant to radio- and chemotherapy and has a high recurrence rate after curative liver resection. Epidermal growth factor receptor variant III (EGFRvIII) has been reported to express in HCC tissues and cell lines. This article describes the efficacy of an anti-EGFRvIII monoclonal antibody (mAb CH12) in the treatment of HCC xenografts with EGFRvIII expression and the underlying mechanism of EGFRvIII as an oncogene in HCC. The results demonstrated that CH12 bound preferentially to EGFRvIII with a dissociation constant (K(d)) of 1.346 nm/liter. In addition, CH12 induces strong antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in Huh7-EGFRvIII (with exogenous expression of EGFRvIII) and SMMC-7721 (with endogenous expression of EGFRvIII) cells. Notably, CH12 significantly inhibited the growth of Huh7-EGFRvIII and SMMC-7721 xenografts in vivo with a growth inhibition ratio much higher than C225, a U. S. Food and Drug Administration-approved anti-EGFR antibody. Treatment of the two HCC xenografts with CH12 significantly suppressed tumor proliferation and angiogenesis. Mechanistically, in vivo treatment with CH12 reduced the phosphorylation of constitutively active EGFRvIII, Akt, and ERK. Down-regulation of the apoptotic protectors Bcl-x(L), Bcl-2, and the cell cycle regulator cyclin D1, as well as up-regulation of the cell-cycle inhibitor p27, were also observed after in vivo CH12 treatment. Collectively, these results indicate that the monoclonal antibody CH12 is a promising therapeutic agent for HCC with EGFRvIII expression.