Subcutaneous Hepatocellular Carcinoma Research Articles

Sensitization of lidocaine on hepatoma H22-bearing mice treated by mitomycin

Objective To observe the sensitization of lidocaine on subcutaneous hepatoma H22-bearing mice and abdominal cavity H22 tumor-bearing mice treated by mitomycin. Methods According to the random number table method, the mice were divided into subcutaneous tumor-bearing group and abdominal cavity tumor-bearing group, with 15 mice in each group. The mice in the two groups were further divided into three subgroups: model group, mitomycin group, mitomycin + lidocaine group, with 5 mice in each subgroup. The day before the intraperitoneal injection, the density of H22 cells obtained from peritoneal culture of one mouse was adjusted to 5×106/ml. Subcutaneous tumor-bearing group mice were injected H22 cells into the right armpit, and abdominal cavity tumor-bearing group mice were injected H22 cells into the abdominal cavity, 0.2 ml per mouse. Intraperitoneal injection was given after inoculation for 24 h (the experiment day 1), followed by intraperitoneal injection on day 5 and 9. Univariate ANOVA analysis and t test were used to analyze the solid tumor weight and tumor inhibition rate on the 11th day of subcutaneous tumor-bearing mice, and the survival time and life extension rate within 60 days of abdominal cavity tumor-bearing mice. Results The solid tumor weight of subcutaneous tumor-bearing mice model group, mitomycin group and mitomycin + lidocaine group were (3.77±1.02) g, (1.67±0.28) g, (0.74±0.19) g, respectively, and the differences in the three groups were statistically different (F = 31.753, P < 0.01); compared with the subcutaneous model group, the subcutaneous solid tumor weights of mitomycin group and mitomycin + lidocaine group were decreased and the differences were both statistically different (t = 2.10, P < 0.01; t = 3.04, P < 0.01); the subcutaneous solid tumor weight of mitomycin + lidocaine group was lower than that of mitomycin group (t = 0.93, P = 0.034). The tumor inhibition rate of mitomycin group and mitomycin + lidocaine group reached 55.70% and 80.37% respectively. The survival time of abdominal cavity tumor-bearing mice in model group, mitomycin group and mitomycin + lidocaine group was (16.80±0.84) d, (28.80±6.30) d, (40.40±12.86) d, respectively, and the differences in the three groups were statistically different (F = 10.155, P = 0.003); compared with the abdominal cavity tumor-bearing mice model group, the survival time of mice in mitomycin group and mitomycin + lidocaine group was prolonged (t = 12.00, P = 0.041; t = 23.60, P = 0.001), and it was found that survival time in mitomycin + lidocaine group was longer than that in mitomycin group (t = 11.60, P = 0.047). The life extension rate of mitomycin group and mitomycin + lidocaine group reached 71.43% and 140.48% respectively. Conclusion Lidocaine can increase the sensitization of mitomycin on hepatoma H22-bearing mice. Key words: Liver neoplasms; Lidocaine; Mitomycin; Sensitivity effect; Mice; Disease models, animal; Cells, H22

GRP78-targeted ferritin nanocaged ultra-high dose of doxorubicin for hepatocellular carcinoma therapy.

Hepatocellular carcinoma (HCC) remains one of the leading causes of cancer deaths, primarily due to its high incidence of recurrence and metastasis. Considerable efforts have therefore been undertaken to develop effective therapies; however, effective anti-HCC therapies rely on identification of suitable biomarkers, few of which are currently available for drug targeting.Methods: GRP78 was identified as the membrane receptor of HCC-targeted peptide SP94 by immunoprecipitation and mass spectrum analysis. To develop an effective anti-HCC drug nanocarrier, we first displayed GRP78-targeted peptide SP94 onto the exterior surface of Pyrococcus furiosus ferritin Fn (HccFn) by genetic engineering approach, and then loaded doxorubicin (Dox) into the cavities of HccFn via urea-based disassembly/reassembly method, thereby constructing a drug nanocarrier called HccFn-Dox.Results: We demonstrated that HccFn nanocage encapsulated ultra-high dose of Dox (up to 400 molecules Dox/protein nanocage). In vivo animal experiments showed that Dox encapsulated in HccFn-Dox was selectively delivered into HCC tumor cells, and effectively killed subcutaneous and lung metastatic HCC tumors. In addition, HccFn-Dox significantly reduced drug exposure to healthy organs and improved the maximum tolerated dose by six-fold compared with free Dox.Conclusion: In conclusion, our findings clearly demonstrate that GRP78 is an effective biomarker for HCC therapy, and GRP78-targeted HccFn nanocage is effective in delivering anti-HCC drug without damage to healthy tissue.

Enhanced anti-tumor efficacy of checkpoint inhibitors in combination with the histone deacetylase inhibitor Belinostat in a murine hepatocellular carcinoma model.

Immune checkpoint inhibitors are currently tested in different combinations in patients with advanced hepatocellular carcinoma (HCC). Nivolumab, an anti-PD-1 agent, has gained approval in the second-line setting in the USA. Epigenetic drugs have immune-mediated antitumor effects that may improve the activity of immunotherapy agents. Our aim was to study the therapeutic efficacy of checkpoint inhibitors (anti-CTLA-4 and anti-PD-1 antibodies) in combination with the histone deacetylase inhibitor (HDACi) Belinostat. In a subcutaneous Hepa129 murine HCC model, we demonstrated that Belinostat improves the antitumor activity of anti-CTLA-4 but not of anti-PD-1 therapy. This effect correlated with enhanced IFN-γ production by antitumor T-cells and a decrease in regulatory T-cells. Moreover, the combination induced early upregulation of PD-L1 on tumor antigen-presenting cells and late expression of PD-1 on tumor-infiltrating effector T-cells, suggesting the suitability of PD-1 blockade. Indeed, Belinostat combined with the simultaneous blockade of CTLA-4 and PD-1 led to complete tumor rejection. These results provide a rationale for testing Belinostat in combination with checkpoint inhibitors to enhance their therapeutic activity in patients with HCC.

Phosphorothioate-Modified AP613-1 Specifically Targets GPC3 when Used for Hepatocellular Carcinoma Cell Imaging

Glypican-3 (GPC3), the cellular membrane proteoglycan, has been established as a tumor biomarker for early diagnosis of hepatocellular carcinoma (HCC). GPC3 is highly expressed in more than 70% HCC tissues detected by antibody-based histopathological systems. Recently, aptamers, a short single-strand DNA or RNA generated from systematic evolution of ligands by exponential enrichment (SELEX), were reported as potential alternatives in tumor-targeted imaging and diagnosis. In this study, a total of 19 GPC3-bound aptamers were successfully screened by capillary electrophoresis (CE)-SELEX technology. After truncated, AP613-1 was confirmed to specifically target GPC3 with a dissociation constant (KD) of 59.85 nM. When modified with a phosphorothioate linkage, APS613-1 targeted GPC3 with a KD of 15.48 nM and could be used as a specific probe in living Huh7 and PLC/PRF/5 imaging, GPC3-positive cell lines, but not in L02 or A549, two GPC3-negative cell lines. More importantly, Alexa Fluor 750-conjugated APS613-1 could be used as a fluorescent probe to subcutaneous HCC imaging in xenograft nude mice. Our results indicated that modified AP613-1, especially APS613-1, was a potential agent in GPC3-positive tumor imaging for HCC early diagnosis.

Study of inhibition of hepatocellular carcinoma proliferation after down-regulating Claspin

Objective To explore the relationship between the expression of Claspin in liver cancer and the clinical features of patients and the roles of Claspin in the proliferation of hepatocellular carcinoma (HCC). Methods Using the TCGA public database, analyze the expression level of Claspin mRNA in 374 cases hepatocarcinoma tissues and 50 cases adjacent tissues, and its survival and clinical features. Real-time quantitative polymerase chain reaction (Real-time PCR) and Western blotting were used to detect the expression of Claspin mRNA and protein in hepatoma cell lines Hep3B cells. Methyl thiazol tetrazolium (MTT) method and colony formation assay were used to understand the changes of proliferation of Hep3B cells after Claspin down-regulation. The stable cell line screened out by G418 was inoculate to establish the subcutaneous xenograft tumor in nude mice, 3 groups and 6 mice each group. The tumor growth curve was plotting and histological examination was performed. Results Claspin mRNA expression was higher in HCC tissues (5.643±1.599) than in normal liver tissues (2.752±1.674) (t=11.941, P=0.000), the OS time and PFS time of the low expression Claspin mRNA patients was high than high expression patients [ (3.659±0.355) years vs (2.289±0.357) years, Log Rank=15.993, P=0.000; (6.072±0.426) years vs (4.771±0.462) years, Log Rank=11.830, P=0.001]. The level of Claspin mRNA expression was related to the pathological TNM stage (χ2=6.149, P=0.013)、grade of liver cancer (χ2=8.563, P=0.003) and AFP level (χ2=11.202, P=0.001), and was also an independent predictor of prognosis (R=0.579, P=0.009). Compared with the blank control group, the down-regulation of Claspin group decreased the proliferation ability at 72 h [(1.316±2.471)%, t=4.823, P=0.000] and the relative colony formation cells [(28.000±3.152)%, t=39.540, P=0.000]. As for the subcutaneous hepatoma xenograft in nude mice, the down-regulation group had significantly slower tumor growth than the blank group and negative control group[(144.300±15.340) mm3 vs. (377.000±20.280) mm3/(363.300±26.980) mm3,t=22.410 and 17.280, both P=0.000], the down-regulation group had significantly lower Claspin expression than he blank group (t=8.367, P=0.000) and negative control group (t=9.303, P=0.000). Conclusion The expression of Claspin is significant high in HCC and also an independent predictor of poor prognosis, down-regulation of Claspin can inhibit the proliferation of liver cancer cells and xenograft in nude mice. Key words: Claspin; Hepatocellular carcinoma; Gene expression; Proliferation

MicroRNA-140-3p enhances the sensitivity of hepatocellular carcinoma cells to sorafenib by targeting pregnenolone X receptor.

BackgroundPregnane X receptor (PXR), which is a member of the nuclear receptor protein family (nuclear receptor subfamily 1 group I member 2 [NR 1I2]), mediates the drug-resistance in the hepatocellular carcinoma (HCC) via enhancing the expression of drug-resistance-related genes which accelerate the clearance of antitumor drugs, eg, sorafenib. However, there are few reports on miRNA targeting PXR participating in the epigenetic regulation of PXR in HCC cells.Materials and methodsTargetScan 7.2, an online method, was used to predict the miRNAs potentially targeting PXR. The expression of PXR and PXR downstream genes was detected by quantitative real-time PCR (qPCR) and Western blot. The clearance of sorafenib in HCC cells was monitored by liquid chromatograph-mass spectrometer/mass spectrometer (LC-MS/MS). The effects of miRNA on sorafenib’s efficacy were examined by in vitro methods, eg, MTT, and in vivo methods, eg, subcutaneous or intrahepatic tumor model.ResultsBy virtual screening, we identified that miR-140-3p possibly targets PXR and then confirmed that the overexpression of miR-140-3p via lentiviral particles inhibited the expression of PXR in HCC cells. The downregulation of PXR’s expression by miR-140-3p led to the reduction of PXR downstream genes’ expression, which finally resulted in the decelerating clearance of sorafenib in HCC cells and enhanced the sensitivity of HCC cells to sorafenib. The effect of miR-140-3p could not modulate the expression of mutated PXR and the effect of miR-140-3p could also be inhibited by miR-140-3p’s inhibitor. Moreover, miR-140-3p enhanced the anti-tumor effect of sorafenib in both the subcutaneous and intrahepatic HCC tumor models.ConclusionOur study suggests that targeting PXR by miR-140-3p is a promising strategy for enhancing sorafenib’s efficacy during HCC treatment.

Inhibition of cyclin-dependent kinase 7 suppresses human hepatocellular carcinoma by inducing apoptosis.

Increasing evidence has shownthat THZ1, a covalent cyclin-dependent kinase 7 (CDK7) inhibitor, exhibits therapeutic effects in various tumors. However, the possible effect of THZ1 on hepatocellular carcinoma (HCC) remains unknown. Our study was to investigate the roles of THZ1 in HCC cells and in subcutaneous HCC modeland illustrate the molecular mechanisms. The phosphorylation levels of Ser2, Ser5, and Ser7 within RNA polymerase II (RNAPII) C-terminal domain (CTD) and the expression levels of Ki67, Mcl-1, survivin, XIAP, and p53 in HCC cells under different conditions were examined by Western blot analysis. Cell growth and apoptosis were assessed via 3-(4,5-dimethylthiazolyl-2)-2, 5-diphenyltetrazolium bromide (MTT) assay and flow cytometry analysis, respectively. Tumor volume was assessed in HCC mice with THZ1 or vehicle treatment and immunohistochemical (IHC) analysis was conducted on excised tumors. THZ1 significantly inhibited the phosphorylation of Ser2, Ser5, and Ser7 within RNAPII-CTD in the dose-dependent and irreversible manner. MTT assay and flow cytometry analysis showed that THZ1 inhibited HCC cell proliferation and induced apoptosis, respectively. Western blot analysis indicated THZ1 significantly upregulated p53 expression and downregulated the expressions of Mcl-1, survivin, XIAP, and Ki67. THZ1 suppressed tumor growth in Hep3B xenografted mice in a time-dependent manner. IHC analysis indicated that tumors in THZ1 group had less Ki67+ cells and more cleaved caspase-3+ cells than those in vehicle group. THZ1 exhibited anti-HCC effects through irreversibly inhibiting CDK7 activity, decreasing RNAPII-CTD phosphorylation, inducing p53 expression and inhibiting antiapoptotic gene expressions, which subsequently induced apoptosis and inhibited proliferation of HCC cells.

Combination of radiation and interleukin 12 eradicates large orthotopic hepatocellular carcinoma through immunomodulation of tumor microenvironment

ABSTRACTImmunotherapies have shown promising results in certain cancer patients. For hepatocellular carcinoma (HCC), the multiplicity of an immunotolerant microenvironment within both the tumor, and the liver per se may limit the efficacy of cancer immunotherapies. Since radiation induces immunogenic cell death and inflammatory reactions within the tumor microenvironment, we hypothesized that a combination therapy of radiation and lasting local immunostimulating agents, achieved by intratumoral injection of an adenoviral vector encoding interleukin 12, may reverse the immunotolerant microenvironment within a well-established orthotopic HCC toward a state favorable for inducing antitumor immunities. Our data showed that radiation and IL-12 combination therapy (RT/IL-12) led to dramatic tumor regression in animals bearing large subcutaneous or orthotopic HCC, induced systemic effect against distant tumor, and significantly prolonged survival. Radiation monotherapy induced tumor regression at early times but afterwards most tumors regained exponential growth, while IL-12 monotherapy only delayed tumor growth. Mechanistic studies revealed that RT/IL-12 increased expression of MHC class II and co-stimulatory molecules CD40 and CD86 on tumor-infiltrating dendritic cells, suggesting an improvement of their antigen presentation activity. RT/IL-12 also significantly reduced accumulation of tumor-infiltrating myeloid-derived suppressor cells (MDSCs) and impaired their suppressive functions by reducing production of reactive oxygen species. Accordingly, tumor-infiltrating CD8+ T cells and NK cells were significantly activated toward the antitumor phenotype, as revealed by increased expression of CD107a and TNF-α. Together, our data showed that RT/IL-12 treatment could reset the intratumoral immunotolerant state and stimulate activation of antitumor cellular immunity that is capable of eliminating large established HCC tumors.

Abstract 2415: HEY1 counteracts hypoxia-induced oxidative stress via transcriptionally repressing PINK1 in hepatocellular carcinoma

Abstract Background and Objective: Excessive accumulation of oxidative stress/reactive oxygen species (ROS) can be harmful to cancer cells. Hypoxia or O2 deprivation, which is commonly found in hepatocellular carcinoma (HCC), is a crucial factor that contributes to elevated ROS level in HCC cells as hypoxia causes inefficient transfer of electrons in the mitochondria. To survive, HCC cells need to devise strategies to counteract and balance hypoxia-induced oxidative stress. While it is known that hypoxia inducible factors (HIFs) are essential to metabolic reprogramming in HCC cells under hypoxia, there are significant gaps in knowledge about underlying mechanisms and transcriptional targets of HIFs. Experimental Procedures: Gene profiling of HCC cell lines (exposed to 20% and 1% O2) was analyzed by transcriptome sequencing to identify novel candidate responsible for counteracting hypoxia-induced oxidative stress. ShRNA-mediated gene silencing and gene activation by CRISPR-dCas9 system were used to modify transcriptional expression of HEY1 for different functional assays. Transmission electron microscopy was used to visualize the mitochondrial structure. Orthotopic and subcutaneous HCC implantation models were used to evaluate the role of HEY1 in HCC progression. Transcriptome sequencing and ChIP assay were performed to identify novel transcriptional targets of HEY1. Results: We showed that transcriptional repressor HEY1 was induced under hypoxia and directly regulated by HIF-1α. Overexpression of HEY1 was associated with poor overall survival in HCC patients. Importantly, we identified PINK1 as a novel repression target of HEY1. PINK1 is known to protect cells against mitochondrial dysfunction. We demonstrated that HEY1 actively repressed PINK1 and downregulation of PINK1 led to loss of mitochondrial mass and impaired mitochondrial cristae formation, subsequently decreasing intracellular ROS level. Downregulation of PINK also associated with poor overall survival and decrease-free in HCC patients. Genetic ablation of HEY1 in HCC cells profoundly reduced tumor growth and lung metastasis while genetic ablation of PINK1 in HCC cells reversely promoted HCC growth. Strikingly, HEY1 and PINK1 expressions reversely correlated in human HCC tissues. Conclusion: This study unprecedentedly identifies an upstream regulatory mechanism of PINK1, which controls the oxidative stress in HCC cells. It also reveals a novel molecular mechanism by which ablation of HEY1 leads to elevation of oxidative stress, making HCC cells more vulnerable. Targeting HEY1 represents an attractive therapeutic approach against HCC. Citation Format: David Kung-Chun Chiu, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Aki Pui-Wah Tse, Dicky Cheuk-Ting Law, Vincent Wai-Hin Yuen, Larry Lai Wei, Hui-Yu Koh, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. HEY1 counteracts hypoxia-induced oxidative stress via transcriptionally repressing PINK1 in hepatocellular carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2415.

CD8+ T cells mediate the antitumor activity of frankincense and myrrh in hepatocellular carcinoma

BackgroundTumor-promoting inflammation is an emerging hallmark of cancer, which participates in both cancer progression and immune escape. Hepatocellular carcinoma (HCC) is a typical inflammation-related cancer with an extremely poor prognosis. Frankincense and myrrh are anti-inflammation agents commonly used in clinic. The purpose of this study is to investigate whether extract of frankincense and myrrh (FM) downregulates inflammatory microenvironment of HCC and thereby restores antitumor immune responses.MethodsThe water-decocting FM was obtained and quantified. HCC cell lines HCCLM3 and Hepa1-6 were used to evaluate the efficacy of FM targeting NF-κB and STAT3 signaling with western blot and qRT-PCR analysis. CD8+NKG2D+ cells were derived from human peripheral blood and were used for evaluation of immune cells-mediated inflammation and oncolysis on HCCLM3 cells. The antitumor efficacy of FM was investigated both in immune compromised and immune competent mice bearing subcutaneous HCC. Mice received daily oral gavage of FM at 60 mg/kg. Immune activity within tumor microenvironment (TME) was assessed by ELISpot assay and flow cytometry, respectively. Depletion of CD8+ T cells or NK cells was achieved by intraperitoneal injection of respective neutralizing antibody.ResultsFM significantly inhibited the activation of NF-κB and STAT3 signaling in HCC cells induced by cytokines (TNF-α or IL-6) and in co-culture system with CD8+NKG2D+ cells. Furthermore, FM sensitized HCC cells to CD8+NKG2D+ cells-mediated oncolysis. In HCC-bearing mice, FM at a non-toxic dose failed to reduce tumor growth in immune compromised mice, whereas it significantly inhibited tumor growth and prolonged life span in immune competent mice. While the number of IFN-γ-producing cells within TME was increased in mice treated with FM, the infiltration of CD8+ T cells and NK cells was not increased. Finally, we identified that depletion of CD8+ T cells rather than NK cells abrogated the antitumor activity of FM.ConclusionsOur results show for the first time that CD8+ T cells mediate the antitumor activity of FM at a non-toxic dose. This may provide new insights to this ancient mysterious prescription in cancer therapy, which offers a novel and practical therapeutic strategy and the possibilities of combined immunotherapy for HCC as well as other inflammation-related cancers in clinic.

A new apatinib microcrystal formulation enhances the effect of radiofrequency ablation treatment on hepatocellular carcinoma.

IntroductionRadiofrequency ablation (RFA) is the foremost treatment option for advanced hepatocellular carcinoma (HCC), however, rapid and aggressive recurrence of HCC often occurs after RFA due to epithelial–mesenchymal transition process. Although combination of RFA with sorafenib, a molecular targeted agent, could attenuate the recurrence of HCC, application of this molecular targeted agent poses a heavy medical burden and oral administration of sorafenib also brings severe side effects.Materials and methodsIn this study, we prepared an apatinib microcrystal formulation (Apa-MS) that sustainably releases apatinib, a novel molecular targeted agent, for advanced HCC treatment. We injected apatinib solution or Apa-MS into subcutaneous HCC tumors.ResultsIt was found that Apa-MS exhibited slow apatinib release in vivo and in turn inhibited the epithelial–mesenchymal transition of HCC cells for extended time. Moreover, in rodent HCC model, Apa-MS enhanced the antitumor effect of RFA treatment.ConclusionBased on these results, we conclude that Apa-MS, a slow releasing system of apatinib, allows apatinib to remain effective in tumor tissues for a long time and could enhance the antitumor effect of RFA on HCC.

Hepatitis transactivator protein X promotes extracellular matrix modification through HIF/LOX pathway in liver cancer

Hepatocellular carcinoma (HCC), accounting for 90% of primary liver cancer, is a lethal malignancy that is tightly associated with chronic hepatitis B virus (HBV) infection. HBV encodes a viral onco-protein, transactivator protein X (HBx), which interacts with proteins of hepatocytes to promote oncogenesis. Our current study focused on the interaction of HBx with a transcription factor, hypoxia-inducible factor-1α (HIF-1α), which is stabilized by low O2 condition (hypoxia) and is found to be frequently overexpressed in HCC intra-tumorally due to poor blood perfusion. Here, we showed that overexpression of HBx by tetracycline-inducible systems further stabilized HIF-1α under hypoxia in HBV-negative HCC cell lines. Reversely, knockdown of HBx reduced HIF-1α protein stabilization under hypoxia in HBV-positive HCC cell lines. More intriguingly, overexpression of HBx elevated the mRNA and protein expression of a family of HIF-1α target genes, the lysyl oxidase (LOX) family in HCC. The LOX family members function to cross-link collagen in the extracellular matrix (ECM) to promote cancer progression and metastasis. By analyzing the collagens under scanning electron microscope, we found that collagen fibers were significantly smaller in size when incubated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells in vitro. Transwell invasion assay further revealed that less cells were able to invade through the matrigel which was pre-treated with conditioned medium from HBx knockdown HCC cells as compared to control HCC cells. Orthotopic and subcutaneous HCC models further showed that knockdown of HBx in HCC cells reduced collagen crosslinking and stiffness in vivo and repressed HCC growth and metastasis. Taken together, our in vitro and in vivo studies showed the HBx remodeled the ECM through HIF-1α/LOX pathway to promote HCC metastasis.

Synergistic anti-hepatoma effect of bufalin combined with sorafenib via mediating the tumor vascular microenvironment by targeting mTOR/VEGF signaling.

Sorafenib inhibits tumor growth primarily by inhibiting vessel formation, however, its efficacy requires improvement, therefore, the development of strategies which augment its antiangiogenic effect are of primary concern. Bufalin inhibits tumor cell proliferation and metastasis, and induces apoptosis. In our previous study, it was demonstrated that the antiangiogenic effect of sorafenib was improved by bufalin in human umbilical vein endothelial cells(HUVECs). However, whether bufalin synergizes with sorafenib by affecting the tumor vascular microenvironment remains to be elucidated. In the present study, it was found that hepatocellular carcinoma(HCC) cell proliferation was inhibited by either bufalin or sorafenib following incubation for 24h, and the inhibition was enhanced upon treatment with a combination of the two. Conditioned medium (CM), comprising supernatant from HCC cells was collected from each of the treatment groups. The migration and tubule formation were suppressed the most in the combination-CM treated HUVECs. The secretion of vascular endothelial growth factor(VEGF) was decreased in HCC cells treated with the combination-CM, as determined by an angiogenesis array, enzyme-linked immunosorbent assay(ELISA) and western blot analysis. The inhibition of tube formation in HUVECs treated with the combination-CM was reversed by incubation with VEGF. The invivo experiments demonstrated that subcutaneous HCC cell tumors from mice treated with the combination treatment expressed the lowest levels of VEGF, as evidenced by immunohistochemistry and ELISA. Additionally, the level of phosphorylated mechanistic target of rapamycin(mTOR) was reduced in HUVECs pretreated with the phosphoinositide3-kinase inhibitor PI103. Furthermore, the migration of HCC cells and HUVEC tube formation were attenuated by PI103 pretreatment. In conclusion, the results revealed a synergistic anti-hepatoma effect of bufalin combined with sorafenib via affecting the tumor vascular microenvironment by targeting mTOR/VEGF signaling.

CTLA-4 blockade induces indoleamine 2,3-dioxygenase where treatment with 1- methyl-d-tryptophan displays greater treatment effect in tumors with high ido expression

Background: As hepatocellular carcinoma (HCC) traditionally occurs in chronically inflamed livers, these tumors are often immunogenic and therefore potential targets for immunotherapy. CTLA-4 blockade has been utilized in two trails in HCC with promising results. However, not all patients have the same response to therapy by a largely unknown mechanism. Indoleamine 2,3- dioxygenase (IDO) with its two forms, IDO1 and IDO2, is part of kynurenine pathway for the metabolism of the essential amino acid tryptophan. It has been demonstrated that IDO acts as an immunosuppressive enzyme altering the tumor microenvironment and is upregulated in inflammatory states with the most potent inducer being IFN-γ. High levels of IDO in tumors including HCC have been associated with a poor prognosis. This study aimed at identifying IDO as a potential mechanism of immune escape from CTLA-4 directed therapy. Methods: The drug 1-methyl-D-tryptophan (1-D-MT), which has been shown to inhibit IDO2 activity, was utilized alone or in combination with a CTLA-4 blocking antibody in treatment of mice with subcutaneous and orthotropic HCC. Induction of IDO RNA in tumor cells was evaluated by quantitative polymerase chain reaction (qPCR). Results: Combination therapy with 1-D-MT and an anti-CTLA-4 blocking antibody produces a greater anti-tumor effect in both a subcutaneous and orthotropic model than either drug alone. Additionally, tumors with high IDO induction showed a better response to combination treatment. Mice injected with anti-CTLA-4 antibody showed higher IDO2 expression in tumors. Conclusion: These results provide evidence that IDO expression induces an additional effect of immunosuppression in the tumor microenvironment, especially after anti-CTLA-4 therapy, and support a combination therapy with IDO inhibitors for a successful immunotherapy treatment.

Pharmacological or transcriptional inhibition of both HDAC1 and 2 leads to cell cycle blockage and apoptosis via p21Waf1/Cip1 and p19INK4d upregulation in hepatocellular carcinoma.

Histone deacetylases (HDACs) are commonly dysregulated in cancer and represent promising therapeutic targets. However, global HDAC inhibitors have shown limited efficacy in the treatment of solid tumours, including hepatocellular carcinoma (HCC). In this study, we investigated the therapeutic effect of selectively inhibiting HDAC1 and 2 in HCC. HDAC1 inhibitor Tacedinaline (CI994), HDAC2 inhibitor Santacruzamate A (CAY10683), HDAC1/2 common inhibitor Romidepsin (FK228) and global HDAC inhibitor Vorinostat (SAHA) were used to treat HCC cells. Cell cycle, apoptosis and the protein levels of CDKs and CDKNs were performed to evaluate HCC cell growth. Inhibition of HDAC1/2 by RNAi was further investigated. Combined inhibition of HDAC1/2 led to HCC cell morphology changes, growth inhibition, cell cycle blockage and apoptosis in vitro and suppressed the growth of subcutaneous HCC xenograft tumours in vivo. p21Waf1/Cip1 and p19INK4d , which play roles in cell cycle blockage and apoptosis induction, were upregulated. Inhibition of HDAC1/2 by siRNA further demonstrated that HDAC1 and 2 cooperate in blocking the cell cycle and inducing apoptosis via p19INK4d and p21Waf1/Cip1 upregulation. Finally, H3K18, H3K56 and H4K12 in the p19INK4d and p21Waf1/Cip1 promoter regions were found to be targets of HDAC1/2. Pharmacological or transcriptional inhibition of HDAC1/2 increases p19INK4d and p21Waf1/Cip1 expression, decreases CDK expression and arrests HCC growth. These results indicated a potential pharmacological mechanism of selective HDAC1/2 inhibitors in HCC therapy.

Mouse Monoclonal Antibodies Against Progranulin (PGRN/GEP) as Therapeutics in Preclinical Cancer Models.

The use of monoclonal antibody (mAb) has become a unique means of targeted therapy for human cancers. mAb-based therapies have shown survival benefits by applying alone or in combination with chemotherapeutics. Being a humanized biomolecule with exquisite target specificity, mAb demonstrated effects in a relatively lower dose range with limited off-target harm to the patients. Nowadays, novel targets involved in tumorigenic mechanisms and biomarkers expressed exclusively on cancer cell surface are being constantly discovered. The potential effects of their specific mAb could be investigated in the preclinical cancer model. In this chapter, we outlined our experimental procedures in determining the feasibility of novel mAb in the preclinical cancer model, with an example of progranulin (PGRN/GEP) mAb against hepatocellular carcinoma (HCC) tumor in mouse model. This chapter included the establishment of subcutaneous and orthotopic HCC tumor in mouse model, the injection of the mouse monoclonal antibody in combination with cytotoxic chemotherapeutics, the assessment of tumor development, and the analyses of the molecular changes of the tumor cells.

High mobility group box 1 promotes sorafenib resistance in HepG2 cells and in vivo

BackgroundPrimary liver cancer is a lethal malignancy with a high mortality worldwide. Currently, sorafenib is the most effective molecular-targeted drug against hepatocellular carcinoma (HCC). However, the sorafenib resistance rate is high. The molecular mechanism of this resistance has not been fully elucidated. High mobility group box 1 (HMGB1) is a multifaceted protein that plays a key role in the proliferation, apoptosis, metastasis and angiogenesis of HCC cells. In addition, HMGB1 has been suggested to contribute to chemotherapy resistance in tumours, including lung cancer, osteosarcoma, neuroblastoma, leukaemia, and colorectal cancer. This study investigated the association between HMGB1 and sorafenib resistance in HCC.MethodsHepG2 cells with HMGB1 knockdown or overexpression were generated. The efficacy of sorafenib in these cells was tested using flow cytometry and a cell counting assay. The subcellular localization of HMGB1 in HepG2 cells following sorafenib treatment was measured by western blotting and confocal microscopy. A murine subcutaneous HCC model was generated to examine the association between HMGB1 and the sensitivity of sorafenib treatment.ResultsThe HMGB1 knockdown cells exhibited a significantly higher apoptotic level and lower cell viability than the normal HMGB1 expressing cells following the sorafenib treatment. In addition, the cell viability observed in the HMGB1 overexpressing cells was higher than that observed in the control cells following the sorafenib intervention. Sorafenib had a better tumour inhibition effect in the HMGB1 knockdown group in vivo. The amount of mitochondrial HMGB1 decreased, while the amount of cytosolic HMGB1 increased following the exposure to sorafenib. Altogether, HMGB1 translocated from the mitochondria to the cytoplasm outside the mitochondria following the exposure of HepG2 cells to sorafenib.ConclusionsA novel potential role of HMGB1 in the regulation of sorafenib therapy resistance in HCC was observed. The knockdown of HMGB1 restores sensitivity to sorafenib and enhances HepG2 cell death, while HMGB1 overexpression blunts these effects. The translocation of HMGB1 from the mitochondria to the cytosol following sorafenib treatment provides new insight into sorafenib resistance in HCC.

Immature colon carcinoma transcript-1 promotes cell growth of hepatocellular carcinoma via facilitating cell cycle progression and apoptosis resistance.

Immature colon carcinoma transcript-1 (ICT1) is a newly identified oncogene, which regulates proliferation, cell cycle progression and apoptosis of cancer cells. However, the clinical significance, biological function and underlying mechanisms of ICT1 in hepatocellular carcinoma (HCC) remain poorly known. In the present study, we showed that the expression of ICT1 in HCC tissues were notably overexpressed compared to corresponding non-tumor tissues. Accordingly, the relative levels of ICT1 were upregulated in HCC cell lines compared with LO2 cells. The positive expression of ICT1 was correlated with large tumor size and advanced TNM tumor stage. Kaplan-Meier plots indicated that ICT1-positive expression in HCC patients showed a prominent shorter survival. In addition, ICT1 knockdown inhibited proliferation and cell cycle progression, and induced apoptosis in HepG2 cells. While, ICT1 overexpression showed opposite effects on these cellular processes of Hep3B cells. Invivo experiments demonstrated that ICT1 deficiency reduced the growth of subcutaneous HCC in nude mice. Notably, ICT1 knockdown reduced the levels of CDK1, cyclinB1 and Bcl-2 and increased the expression of Bax in HepG2 cells. ICT1 overexpression resulted in upregulation of CDK1, cyclinB1 and Bcl-2, and downregulation of Bax in Hep3B cells. Furthermore, microRNA-134 (miR-134) was recognized as a direct upstream regulator and inversely modulated ICT1 abundance in HCC cells. Altogether, our data support that miR-134 regulation of ICT1 facilitates malignant phenotype of HCC cells probably via cell cycle and apoptosis-associated proteins including CDK1, cyclinB1, Bcl-2 and Bax.

The Combined Antitumor Effects of 125I Radioactive Particle Implantation and Cytokine-Induced Killer Cell Therapy on Xenograft Hepatocellular Carcinoma in a Mouse Model.

The combination of radiotherapy and immunotherapy has shown great promise in eradicating tumors. For example, 125I radioactive particle implantation and cytokine-induced killer cell therapies have demonstrated efficacy in treating hepatocellular carcinoma. However, the mechanism of this combination therapy remains unknown. In this study, we utilized cytokine-induced killer cells obtained from human peripheral blood mononuclear cells along with 125I radioactive particle implantation to treat subcutaneous hepatocellular carcinoma xenograft tumors in BALB/c nude mice. The effects of combination therapy on tumor growth, tumor cell apoptosis and proliferation, animal survival, and immune indexes were then assessed. The results indicated that 125I radioactive particle implantation combined with cytokine-induced killer cells shows a much greater antitumor therapeutic effect than either of the therapies alone when compared to control treatments. Mice treated with a combination of radiotherapy and immunotherapy displayed significantly reduced tumor growth. 125I radioactive particle implantation upregulated the expression of major histocompatibility complex (MHC) class I chain-related gene A in hepatocellular carcinoma cells and enhanced cytokine-induced killer cell–mediated apoptosis through activation of caspase-3. Furthermore, cytokine-induced killer cells supplied immune substrates to induce a strong immune response after 125I radioactive particle implantation therapy. In conclusion, 125I radioactive particle implantation combined with cytokine-induced killer cell therapy significantly inhibits the growth of human hepatocellular carcinoma cells in vivo and improves animal survival times through mutual promotion of antitumor immunity, presenting a promising therapy for hepatocellular carcinoma.

Abstract 2941: Inhibition of hypoxia-induced ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) restrains myeloid-derived suppressor cell (MDSC) accumulation and sensitizes tumors to immune checkpoint inhibition

Abstract Background and Objective: Rapidly expanding knowledge on cancer immunology has introduced promising anti-cancer therapeutic approaches which involve the activation of T cells to combat cancer cells. Accumulating studies have indicated that the efficacy of immunotherapies is critically determined by the stromal cell components in tumors. Myeloid-derived suppressor cells (MDSCs), are regarded as one of the major immune cell types that possess immunosuppressive activities against T cells which allow cancers to escape immune surveillance and become non-responsive to immune checkpoints blockade. To increase the efficacy of immunotherapy, novel strategies to target MDSC in tumors are warranted. Hypoxia, oxygen (O2) shortage, frequently occurs in tumors due to abnormal vasculature. Using hepatocellular carcinoma (HCC) as a model, we have previously observed MDSC preferentially accumulates in hypoxic regions of human HCC tissues. Here, we aim to identify hypoxia-induced therapeutic targets that are critical for MDSC accumulation in tumors. Experimental Procedures: Transcriptome sequencing in multiple HCC cell lines exposed to hypoxia and normoxia and HCC clinical specimens was performed to identify potential hypoxia-induced genes relevant to HCC development. MDSCs were isolated from HCC-bearing mice by magnetic bead sorting for different functional assays. LC-MS was performed to evaluate the level of extracellular metabolites. Flow cytometry was used to detect the frequencies of tumor-infiltrating MDSCs in orthotopic and subcutaneous HCC mouse models. Results: We showed that hypoxia, through stabilization of hypoxia-inducible factor-1 (HIF-1), induced ectoenzyme, ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2/ CD39L1), in cancer cells, causing its over-expression in HCC clinical specimens. Over-expression of ENTPD2 was found as a poor prognostic indicator for HCC patients. Mechanistically, we demonstrated that ENTPD2 converted extracellular ATP to 5’-AMP which prevents the differentiation of monocytic MDSCs to dendritic cells, therefore promoting the maintenance of MDSCs in vitro and in vivo. Therapeutically, we found that ENTPD2 inhibitor POM-1 restrained MDSC accumulation and tumor growth, substantially enhancing the efficiency and efficacy of immune checkpoints inhibitors. Conclusion: Our study reveals a novel mechanism whereby hypoxia/HIF-1 in cancer cells governs tumor-infiltrating MDSCs. Our data suggest that ENTPD2 may be a good prognostic marker and therapeutic target for cancer patients especially those receiving immune therapy. Citation Format: David Kung-Chun Chiu, Aki Pui-Wah Tse, Iris Ming-Jing Xu, Robin Kit-Ho Lai, Hui-yu Koh, Felice Ho-Ching Tsang, Larry Lai Wei, Chun-Ming Wong, Irene Oi-Lin Ng, Carmen Chak-Lui Wong. Inhibition of hypoxia-induced ectonucleoside triphosphate diphosphohydrolase 2 (ENTPD2) restrains myeloid-derived suppressor cell (MDSC) accumulation and sensitizes tumors to immune checkpoint inhibition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2941. doi:10.1158/1538-7445.AM2017-2941