Human Hepatocellular Carcinoma Xenograft Tumors Research Articles

Effect of UV-curable and injectable dendrimer matrix on tumor pharmacokinetics and survival in a hepatocellular carcinoma mouse model using interstitial photodynamic therapy.

e15598 Background: Intratumoral drug delivery in cancer treatment has shown to increase drug accumulation in tumors and decrease adverse effects of systemic therapies. The active agent is typically required to stay in contact with the tumor and thus attachment directly into the tumor or to the surrounding tissue is warranted. In addition, a long-acting release of the active drug is vital. Dendrimers are nano-sized, radially symmetric molecules with homogeneous and monodisperse structures, which can used to encapsulate drug molecules for controlled release with the aid of external stimuli at physiological pH. Methods: We prepared a dendrimeric drug delivery matrix based on a PEG-core. The model drug (5-fluorouracil or doxorubicin) was covalently bonded to the core. The release of the drug was observed up to 29 days in vitro. The outer shell of the matrix was partially modified to obtain a fast UV-curing and subsequent attachment to the tumor tissue utilizing interstitial photodynamic therapy. We compared the pharmacokinetics, tissue distribution and antitumor efficacy of conventional systemic therapy and the intratumoral therapy in xenograft-bearing mice. In addition, we monitored blood parameters and body weight of the animals for adverse effects. Results: The dendrimer matrix caused significantly greater inhibition of human hepatocellular carcinoma xenograft tumor growth when compared with systemic therapy, without increasing toxicity. The matrix enabled a significant increase in drug accumulation in tumors, and markedly extended the survival of mice compared with systemic treatments. A larger decrease in blood parameters as well as spleen weight was observed in the systemic therapy group especially when using 5-fluorouracil. Conclusions: Our study shows that the intratumoral treatment strategy based on a novel UV-curable dendrimer hydrogel matrix enhances antitumor activity by improving pharmacokinetics and drug accumulation and increases efficacy in comparison to conventional systemic therapy. These results highlight the potential use of dendrimer hydrogels in the treatment of hepatocellular carcinoma.

Inhibitory effect of migration-inducing gene-7-shRNA recombinant retrovirus combined with endostatin on growth and metastasis of hepatoma xenograft

Objective: To investigate the inhibitory effect of migration-inducing gene-7(Mig-7)interfered with retrovirus-mediated RNA(shRNA)combined with recombinant human endostatin(ES)on the growth and metastasis of subcutaneous xenograft of human hepatoma cells in nude mice. Methods: Two Mig-7-mRNA oligonucleotide sequences(Mig-7-shRNA-1 and Mig-7-shRNA-2)and one sequence as a negative control(Mig-7-shRNA-N)were designed. The specific Mig-7-shRNA recombinant retrovirus expression vector plasmid was constructed and used for the transfection of human hepatoma MHCC-97H cells with high expression of Mig-7. The subcutaneous xenograft tumor model of human hepatocellular carcinoma(HCC)in nude mice was established, and according to the condition of transfection and administration, the nude mice were divided into pSIREN-M1 group, pSIREN-MN group, ES group, and pSIREN-M1+ES group. The xenograft tumor volume, mass, and metastasis were compared between groups. Immunohistochemistry was used to observe the formation of vasculogenic mimicry(VM)in xenograft tumor and the difference in tumor microvascular density(MVD), and Western blot was used to measure the expression of Mig-7 and vascular endothelial growth factor(VEGF)in each group. A one-way analysis of variance was used for comparison between groups, and the Fisher's exact test was used for comparison of continuous data between groups. Results: Compared with the pSIREN-MN group, the pSIREN-M1 group had significantly lower xenograft tumor volume, mass, and metastasis rate, Mig-7 expression, and formation of VM(P < 0.05), as well as significantly higher VEGF expression and MVD(P < 0.05). Compared with the pSIREN-MN group, the ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, VEGF expression, and MVD(P < 0.05), as well as significantly higher Mig-7 expression and formation of VM(P < 0.05). Compared with the pSIREN-M1 group and the ES group, the pSIREN-M1+ES group had significantly lower xenograft tumor volume, mass, and metastasis rate, Mig-7 expression, formation of VM, VEGF expression, and MVD(P < 0.05). Conclusion: Mig-7-shRNA recombinant retrovirus combined with ES has a better inhibitory effect on the growth and metastasis of HCC xenograft tumor than Mig-7-shRNA recombinant retrovirus or ES alone. The anti-tumor angiogenesis therapy alone, which targets vascular endothelial cells in vivo, has a limited effect, since it may promote the formation of VM.

Histone acetyl transferase GCN5 promotes human hepatocellular carcinoma progression by enhancing AIB1 expression.

BackgroundGeneral control non-depressible 5 (GCN5) is a crucial catalytic component of a transcriptional regulatory complex that plays important roles in cellular functions from cell cycle regulation to DNA damage repair. Although GCN5 has recently been implicated in certain oncogenic roles, its role in liver cancer progression remains vague.ResultsIn this study, we report that GCN5 was overexpressed in 17 (54.8 %) of 31 human hepatocellular carcinoma (HCC) specimens. Down-regulation of GCN5 inhibited HCC cell proliferation and xenograft tumor formation. GCN5 knockdown decreased the protein levels of the proliferation marker proliferating cell nuclear antigen (PCNA) and amplified in breast cancer 1 (AIB1), but increased the protein levels of cell cycle inhibitor p21Cip1/Waf1 in HepG2 cells. GCN5 regulated AIB1 expression, at least in part, by cooperating with E2F1 to enhance AIB1 transcription. Consistently, GCN5 expression was positively correlated with AIB1 expression in human HCC specimens in two GEO profile datasets.ConclusionSince AIB1 plays a promoting role in HCC progression, our results propose that GCN5 promotes HCC progression at least partially by regulating AIB1 expression. This study implicates that GCN5 might be a potential molecular target for HCC diagnosis and treatment.

Suppression of BRD4 inhibits human hepatocellular carcinoma by repressing MYC and enhancing BIM expression.

Bromodomain 4 (BRD4) is an epigenetic regulator that, when inhibited, has anti-cancer effects. In this study, we investigated whether BRD4 could be a target for treatment of human hepatocellular carcinoma (HCC). We show that BRD4 is over-expressed in HCC tissues. Suppression of BRD4, either by siRNA or using JQ1, a pharmaceutical BRD4 inhibitor, reduced cell growth and induced apoptosis in HCC cell lines while also slowing HCC xenograft tumor growth in mice. JQ1 treatment induced G1 cell cycle arrest by repressing MYC expression, which led to the up-regulation of CDKN1B (P27). JQ1 also de-repressed expression of the pro-apoptotic BCL2L11 (BIM). Moreover, siRNA knockdown of BIM attenuated JQ1-triggered apoptosis in HCC cells, suggesting an essential role for BIM in mediating JQ1 anti-HCC activity.

Inhibitory effect of biosynthetic nanoscale peptide Melittin on hepatocellular carcinoma, driven by survivin promoter.

Hepatic resection and orthotopic liver transplantation are the only potentially curative treatments for hepatocellular carcinoma (HCC) but are indicated only in a minority of patients. Biosynthetic nanoscale peptide Melittin (Mel) is postulated to disrupt microbial phospholipid membranes by formation of stable or transient pores. Survivin, a member of the inhibitor of apoptosis family, is transcriptionally upregulated in most malignant tissues but not in normal tissues. It has been reported that the survivin promoter activity is tumor-specific and makes it a good candidate for construction of gene therapy vectors. In the present study, a non-viral vector (pSURV-Mel), encoding Mel gene, was developed to evaluate its anti-tumor effect in HCC cell lines and in vivo in a mouse model of human HCC xenograft tumor. Our results showed that the survivin promoter is specifically activated in tumor cells, and the pSURV-Mel plasmid expressed Mel selectively in tumor cells and also induced cytotoxicity. Moreover, intratumoral Injection of pSURV-Mel significantly suppressed the growth of xenograft tumors. Mechanistically, pSURV-Mel induced cell death by an apoptosis-dependent pathway. All taken together, this study elucidates a relatively safe, highly effective and cancer specific gene therapy strategy for HCC. The mechanisms of non-viral vector-induced cell death which were revealed by this work will shed light on the construction of more powerful vectors for cancer therapy.

Glutaminase 2 negatively regulates the PI3K/AKT signaling and shows tumor suppression activity in human hepatocellular carcinoma.

The tumor suppressor p53 and its signaling pathway play a critical role in tumor prevention. As a direct p53 target gene, the role of glutaminase 2 (GLS2) in tumorigenesis is unclear. In this study, we found that GLS2 expression is significantly decreased in majority of human hepatocellular carcinoma (HCC). Restoration of GLS2 expression in HCC cells inhibits the anchorage-independent growth of cells and reduces the growth of HCC xenograft tumors. Interestingly, we found that GLS2 negatively regulates the PI3K/AKT signaling, which is frequently activated in HCC. Blocking the PI3K/AKT signaling in HCC cells largely abolishes the inhibitory effect of GLS2 on the anchorage-independent cell growth and xenograft tumor growth. The GLS2 promoter is hypermethylated in majority of HCC samples. CpG methylation of GLS2 promoter inhibits GLS2 transcription, whereas reducing the methylation of GLS2 promoter induces GLS2 expression. Taken together, our results demonstrate that GLS2 plays an important role in tumor suppression in HCC, and the negative regulation of PI3K/AKT signaling contributes greatly to this function of GLS2. Furthermore, hypermethylation of GLS2 promoter is an important mechanism contributing to the decreased GLS2 expression in HCC.

Inhibition of STAT3 dimerization and acetylation by garcinol suppresses the growth of human hepatocellular carcinoma in vitro and in vivo

BackgroundConstitutive activation of signal transducer and activator of transcription 3 (STAT3) has been linked with proliferation, survival, invasion and angiogenesis of a variety of human cancer cells, including hepatocellular carcinoma (HCC). Thus, novel agents that can suppress STAT3 activation have potential for both prevention and treatment of HCC. Here we report, garcinol, a polyisoprenylated benzophenone, could suppress STAT3 activation in HCC cell lines and in xenografted tumor of HCC in nude mice model.Experimental designDifferent HCC cell lines have been treated with garcinol and the inhibition of STAT3 activation, dimerization and acetylation have been checked by immunoblotting, immuno-fluorescence, and DNA binding assays. Xenografted tumor model has been generated in nude mice using HCC cell line and effect of garcinol in the inhibition of tumor growth has been investigated.ResultsGarcinol could inhibit both constitutive and interleukin (IL-6) inducible STAT3 activation in HCC cells. Computational modeling showed that garcinol could bind to the SH2 domain of STAT3 and suppress its dimerization in vitro. Being an acetyltransferase inhibitor, garcinol also inhibits STAT3 acetylation and thus impairs its DNA binding ability. The inhibition of STAT3 activation by garcinol led to the suppression of expression of various genes involved in proliferation, survival, and angiogenesis. It also suppressed proliferation and induced substantial apoptosis in HCC cells. Remarkably, garcinol inhibited the growth of human HCC xenograft tumors in athymic nu/nu mice, through the inhibition of STAT3 activation.ConclusionOverall, our results suggest that garcinol exerts its anti-proliferative and pro-apoptotic effects through suppression of STAT3 signaling in HCC both in vitro and in vivo.

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.

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.

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.

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.

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.