Liraglutide activates nature killer cell-mediated antitumor responses by inhibiting IL-6/STAT3 signaling in hepatocellular carcinoma

Inflammatory IL-6/STAT3 signaling is constitutively activated in diverse cancers and is associated with malignant cell proliferation, invasion and escape of antitumor immunosurveillance. Liraglutide, a glucagon-like peptide-1 (GLP-1) analog, is commonly used to treat insulin-resistant diabetes. In this study, for the first time, we showed that liraglutide remarkably improved the antitumor immune responses in hepatocellular carcinoma (HCC). Furthermore, we showed that the antitumor activity was mediated by nature killer cells (NKs) but not CD8⁺ T cells. Finally, we showed that liraglutide enhanced NK-mediated cytotoxicity by suppressing the IL-6/STAT3 signaling pathway in HCC cells. Our findings unveil a novel therapeutic role of liraglutide by manipulating the innate immunity in cancer therapy.     

The combination of RAD001 and NVP-BEZ235 exerts synergistic anticancer activity against non-small cell lung cancer in vitro and in vivo

The phosphoinositide 3-kinase (PI3K)-mammalian target of rapamycin (mTOR) signaling axis has emerged as a novel target for cancer therapy. Agents that inhibit PI3K, mTOR or both are currently under development. The mTOR allosteric inhibitor, RAD001, and the PI3K/mTOR dual kinase inhibitor, BEZ235, are examples of these agents. We were interested in developing strategies to enhance mTOR-targeted caner therapy. In this study, we found that BEZ235 alone effectively inhibited the growth of rapamycin-resistant cancer cells. Interestingly, the combination of sub-optimal concentrations of RAD001 and BEZ235 exerted synergistic inhibition of the growth of human lung cancer cells along with induction of apoptosis and G1 arrest. Furthermore, the combination was also more effective than either agent alone in inhibiting the growth of lung cancer xenografts in mice. The combination showed enhanced effects on inhibiting mTOR signaling and reducing the expression of c-Myc and cyclin D1. Taken together, our results suggest that the combination of RAD001 and BEZ235 is a novel strategy for cancer therapy.     

IL-6 -174G/C polymorphism and IL-6 serum levels in patients with liver cirrhosis and hepatocellular carcinoma

Recently, a link between high levels of circulating IL-6 and hepatocellular carcinoma (HCC) has been proposed. In addition, single nucleotide polymorphisms (SNPs) in the promoter region of the IL-6 gene have been reported to be related to several inflammatory-related conditions, including cancer. The purpose of this article is: (1) to evaluate the frequencies of SNPs in the IL-6 promoter region at position -174 and IL-6 serum levels in a group of patients with HCC and underlying liver cirrhosis (LC), and compare them with a group of LC patients without HCC; (2) to determine whether a possible correlation exists between the allelic variations, IL-6 serum levels, and the risk of developing HCC. The study included 105 HCC and 95 LC patients. Genomic DNA was isolated using commercially available kits. DNA samples were typed for relevant SNPs of the IL-6 promoter region (-174?G>C, G allele being associated with higher levels of the cytokine). The Restriction Fragment Length Polymorphism (RFLP-PCR) method was used to type the SNPs. IL-6 serum levels were determined using an ultrasensitive commercially available ELISA kit. IL-6 serum levels were higher in G/G compared to C/C genotypes only in HCC (z=2; p=0.04) and G/G versus G/C (z=1.8; p<0.03). IL-6 serum levels in G carriers (G/G+G/C) were higher in HCC 4.8 (0.2-17.5) versus LC patients 2.2 (0.07-11.5) (z=2.8; p=0.004). There was no difference for genotype C/C. IL-6 serum levels in HCC correlated with G carriers (G/G+G/C) (ρ=0.25, p=0.05). A positive correlation was also found between sIL-6 levels and some parameters of liver function both in LC and in HCC patients.     

IL-6, a risk factor for hepatocellular carcinoma: FLLL32 inhibits IL-6-Induced STAT3 phosphorylation in human hepatocellular cancer cells

Hepatocellular carcinoma (HCC) is one of the most common human cancers and the patients' five-year survival rate is very low. Growing evidence indicates that interleukin-6 is a risk factor for HCC. High serum IL-6 may promote HCC development in hepatitis B patients. Therefore, IL-6 could be considered a HCC biomarker and blockade of IL-6 pathway may be a promising therapeutic alternative for HCC. STAT3 is major pathway to mediate signal from IL-6 to the nucleus, where different genes associated with proliferation and apoptosis are regulated. We previous reported that IL-6 induces cell survival upon drug treatment in HCC cells and inhibition of IL-6/STAT3 pathway using anti-IL-6 antibody or STAT3 small-molecule inhibitor LLL12 reduces this effect. Here we summarized the recent studies of IL-6 in HCC and showed another STAT3 small-molecule FLLL32 also blocked IL-6-induced STAT3 activation in HCC cells. FLLL32 is a novel curcumin analogue, which has been described to suppress the constitutive activation of STAT3 in pancreatic and breast cancer cells in vitro and vivo. In this study, we demonstrated that FLLL32 blocked IL-6-induced STAT3 phosphorylation and nuclear translocation.     

MicroRNA200a enhances antitumor effects in combination with doxorubicin in hepatocellular carcinoma

Hepatocellular carcinoma (HCC) is often treated with doxorubicin. MicroRNAs have been shown to have important regulatory roles in cancer and serve as a target in chemoresistance. In this study, we investigated the effects of specific microRNA-200a (miR-200a) on HCC tumor cell growth and effect of doxorubicin-mediated cytotoxicity. Our results show miR-200a is downregulated in human HCC and HCC tumor cell lines. Increasing miR-200a expression inhibited HCC growth and synergized with the antitumor effects of doxorubicin. Inhibiting endogenous miR-200a promoted tumor growth and chemotherapeutic resistance. Increasing miR-200a expression inhibited tumor metabolism (ATP production, mitochondrial respiration, glycolysis), while inhibition of endogenous miR-200a reversed these effects. MiR-200a expression also increased autophagy and synergized with doxorubicin-mediated cytotoxicity. This study identifies a novel role of miR-200a in potentiating doxorubicin-mediated therapeutic effects in HCC.     

APOBEC3B and IL-6 form a positive feedback loop in hepatocellular carcinoma cells

APOBEC3 protein families, a DNA cytidine deaminase, were up-regulated in multiple tumors. However, the relationship between Hepatocellular carcinoma(HCC) and APOBEC3B(A3B) remains unknown. It has been confirmed that interleukin-6(IL-6)has significant impacts on oncogenesis of HCC. Here, we reported that the expression of IL-6 was substantially up-regulated by A3 B in HepG2 cells. A3 B induced IL-6 expression through relocating HuR to enhance the IL-6 mRNA stability. Further analysis indicated that IL-6 also increased the expression of A3 B through JAK1/STAT3 signaling pathway, which formed a positive feedback to maintain the continuous expression of A3 B and IL-6, and thereby promoted the prolonged non-resolving inflammation. Collectively, these findings suggest that A3 B is essential for oncogenesis of HCC, and is a potential target for preventive intervention.     

Synergistic antitumor effects of rhein and doxorubicin in hepatocellular carcinoma cells

The aim of this study was to investigate the synergistic antitumor activity of rhein and doxorubicin (DOX) and to elucidate the underlying mechanisms in hepatocellular SMMC-7721 and HepG2 cells. Cell growth curves, caspase-3 activity, and intracellular DOX accumulation were observed using an IncuCyte real-time video imaging system. Combination index was used to calculate synergistic potential of rhein and DOX. Cell apoptosis was detected by the Annexin V-FITC/PI apoptosis kit. Lactate dehydrogenase and adenosine triphosphate (ATP) levels were assessed using an assay kit. Oxygen consumption rates (OCR) and extracellular acidification rates were assessed by the Seahorse XFe96 Extracellular Flux Analyzer. Mitochondrial inner membrane potential (ΔΨm) was monitored with JC-1 fluorescence. Western blot analysis was used to detect the level of P-glycoprotein. Synergistic antiproliferative and proapoptotic effects were exerted by the combination of rhein at 10 μM and DOX at 2 μM in SMMC-7721 and HepG2 cells. Rhein could influenced the accumulation of DOX in both cells, which was associated with remarkably decreased mitochondrial energy metabolism and ATP levels. Rhein could reduce ΔΨm in both cells. mPTP, opener atractyloside (ATR) could accelerate the loss of ΔΨm, and further suppress the OCR induced by rhein. In contrast, the mPTP blocker cyclosporin A (Cs A) inhibited the loss of ΔΨm and the OCR induced by rhein. Our data indicate that a decline in mitochondrial energy metabolism was responsible for the synergistic antitumor effects of rhein and DOX in hepatocellular carcinoma cells. Reduction of ΔΨm and opening of mPTP inhibited the exchange of ATP/adenosine diphosphate between mitochondrial matrix and cytoplasm is the important mechanism.     

The antitumor effect of bleomycin combined with bestatin against Ehrlich ascites carcinoma in mice

The effect of bleomycin against Ehrlich ascites carcinoma transplanted subcutaneously to mice used in combination with bestatin was investigated. Male Balb/c mice weighting approximately 20 g and bred in our laboratories were used in this study. Each mouse was injected in its left lateral abdominal region subcutaneously with 7 X 10(6) tumor cells in 0.2 ml of ascites fluid. The mice were divided into four groups: control, bestatin alone (5 mg/kg intraperitoneally on Days 9-14), bleomycin alone (10 mg/kg intraperitoneally on Days 7 and 8), and bestatin plus bleomycin. Our results show that bestatin enhances the antitumor effect of bleomycin against Ehrlich ascites carcinoma as measured by the increased survival rates. Being an agent of very low toxicity, bestatin should be considered as a part of the chemoimmunotherapy protocol.     

HBV感染对肝癌组织MMP-2和PTEN表达的影响

目的探讨肝癌组织MMP-2(基质金属蛋白酶-2)和PTEN(张力蛋白和辅助蛋白同源、第10号染色体丢失的磷酸酶基因)蛋白表达与临床病理参数的关系以及HBV感染对两者表达的影响。方法用免疫组织化学(S-P)链霉菌抗生物素过氧化物酶法检测21例肝癌组织和10例癌旁组织MMP-2和PTEN蛋白表达。结果MMP-2在肝癌组织阳性表达率为69.0%,明显高于癌旁组织的40.0%(P<0.05),在高中分化和低分化组分别为31.6%和50.0%(P>0.05),门脉癌栓转移和未转移组分别为100%和21.1%(P<0.01)。在血清HBsAg阳性和阴性组MMP-2表达阳性率分别为66.7%和33.3%(P<0.05);PTEN蛋白在肝癌组织表达缺失率为32.5%,明显高于癌旁组织的10.0%(P<0.05),在高中分化和低分化组缺失率分别为47.4%和50.0%(P>0.05),门脉癌栓转移与未转移组分别为50.0%和26.3%(P<0.05),在血清HBsAg阳性和阴性组PTEN表达缺失率分别为11.1%和0(P>0.05)。结论HBV感染对肝癌细胞基质金属蛋白酶的分泌起上调作用,有利于癌细胞的浸润和转移;HBV感染能使PTEN蛋白表达缺失增加。     

在HBV感染的肝癌组织中Fas/FasL表达的作用

目的探讨Fas／FasL（Fas配体）在原发性肝细胞肝癌（HCC）组织中的表达及其与乙肝病毒（HBV）感染的关系。方法用免疫组化S-P法检测21例肝癌组织及其10例癌旁组织Fas／FasL的表达。结果在肝癌和癌旁组织中Fas表达的阳性率分别为52．38％和80．00％（P〈0．05）,FasL分别为66．67％和40．00％（P〈0．05）。在HBsAg阳性和阴性组Fas表达的阳性率分别为50．00％和66．67％（P〉0．05）;FasL分别为61．11％和100％（P〈0．05）。肝癌组织中Fas、FasL表达与性别、年龄、肿瘤大小无关,与癌栓转移呈负相关,FasL与分化程度有关。结论肝癌细胞能下调Fas及上调FasL的表达而使凋亡受阻,HBV感染能抑制肝癌组织FasL的表达,可能是HBV在HCC的发牛及发展讨稃中的致痛机制。     

Celastrol exerts synergistic effects with PHA-665752 and inhibits tumor growth of c-Met-deficient hepatocellular carcinoma in vivo

PHA665752 (PHA), a selective small molecule c-Met Inhibitor, potently inhibited HGF-stimulated and constitutive c-Met phosphorylation, as well as HGF and c-Met-driven phenotypes of a variety of tumor cells including hepatocellular carcinoma cells. However, these effects were impaired in c-Met-deficient cancer cells. In the present study, we investigated the potential anti-human c-Met-deficient hepatocellular carcinoma effects of Celastrol, a novel triterpene, and its combination with PHA. Human hepatocellular carcinoma cells BEL-7402 (c-Met-positive) and Huh7 (c-Met-deficient) were treated with different dose of PHA with or without equal dose of Celastrol, and cell growth, cell cycle and apoptosis were evaluated, respectively, by MTT assay, flow cytometry and Caspase3/7 activity. Nude mice bearing Huh7 xenografts were used to assess the in vivo anti-tumor activity. Our results showed that Celastrol at high concentration (>1.0 μM) induced G2/M arrest and apoptosis with the activation of Caspase3/7 in Huh7 cells whereas at low concentration (<1.0 μM) had no obvious effects. Low concentration Celastrol presented significant combined effects with PHA on Huh7 cells and Huh7 xenografts in terms of growth inhibition, migration inhibition and apoptosis induction. These results suggest that Celastrol and its combination with PHA present the therapeutic potential on c-Met-deficient hepatocellular carcinoma, and deserve further preclinical and clinical studies.     

Mechanism of antitumor effect on mouse hepatocellular carcinoma by intratumoral injection of OK-432, a streptococcal preparation

Intratumoral (i.t.) injection of OK-432, a streptococcal preparation, into implanted tumors of mouse hepatocellular carcinoma (MIH-2) showed antitumor effect including tumor eradication. Intraperitoneal administration of same dose OK-432 did not exhibit tumor suppressive effect. In vitro cytotoxic test suggested that direct cytotoxic effect of OK-432 was not associated with antitumor activity by i.t.-OK-432 treatment. It was also found that Toll-like receptor 4 signaling was not involved in i.t.-OK-432 treatment. Three mice out of five, which had shown tumor eradication by i.t.-OK-432 treatment did not reject re-challenge of MIH-2 cells. Splenocytes from i.t.-OK-432 treated mice did not produce IFN-gamma by stimulation with MIH-2 cells in vitro, but produced abundant IFN-gamma by stimulation with OK-432. Immunofluorescence microscopy demonstrated that CD4+T cells, but not CD8+T cells, infiltrated to i.t.-OK-432 treated tumor tissue produced IFN-gamma. Tumor-infiltrating CD4+T cells from i.t.-OK-432 treated tumor tissue produced IFN-gamma by in vitro stimulation with OK-432 higher than those from untreated tumor tissue. IFN-gamma directly induced apoptosis of MIH-2 cells in vitro. Collectively, i.t.-OK-432 treatment induced priming of CD4+T cells to antigenecity of OK-432, and repetitive i.t.-OK-432 treatment induced IFN-gamma production from OK-432-sensitized CD4+T cells in tumor site, leading to apoptosis of MIH-2 cells susceptible to IFN-gamma.     

Cellular mechanisms of the antitumor activity of recombinant IL-6 in mice.

The systemic administration of human rIL-6 to mice resulted in the regression of established, 3-day pulmonary micrometastases from two weakly immunogenic tumors, but not from a nonimmunogenic tumor, in the absence of observable toxicity. Although IL-6 alone failed to have a significant therapeutic impact on advanced, 10-day pulmonary macrometastases from weakly immunogenic tumors, substantial cure rates of mice could be achieved when this cytokine was combined with cyclophosphamide. Histologic analysis of the lungs of mice receiving IL-6 revealed infiltration with lymphoid cells during the regression of pulmonary nodules from a weakly immunogenic tumor. IL-6-mediated tumor regression could be abrogated after selective in vivo depletion of either CD4 or CD8 T cell subsets by the systemic administration of specific mAb. In vivo generation of tumor-specific CTL, but not of lymphokine-activated killer cells, was detected in the lungs of IL-6-treated mice during regression of pulmonary metastases. Collectively, these findings demonstrate a role for IL-6 in the treatment of established solid tumors that have the capacity to elicit T cell responses in the host. Differences in host cellular mechanisms involved in tumor regression mediated by immunotherapy using IL-6 vs IL-2 are discussed.     

A synergistic interaction of IL-6 and IL-1 mediates the thymocyte-stimulating activity produced by recombinant IL-1-stimulated fibroblasts

We characterized the ability of normal human lung fibroblasts to elaborate thymocyte-stimulating activity, spontaneously, and in response to rIL-1. Supernatants from unstimulated fibroblasts did not contain thymocyte-stimulating activity, whereas supernatants from fibroblasts incubated with rIL-1 alpha or rIL-1 beta contained more thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. This heightened thymocyte-stimulating activity was mediated by fibroblast-derived IL-6 inasmuch as it was neutralized by anti-serum against human rIL-6, and rIL-1-stimulated fibroblasts to accumulate messenger RNA for IL-6 and produce soluble IL-6 protein. However, IL-6 alone could not account for the intensity of this effect because rIL-6 only weakly stimulated thymocyte proliferation. In addition, antisera against the rIL-1 moiety that was used to prepare the supernatant had different effects on supernatants that contained and did not contain active IL-6. In the presence of IL-6 these antisera caused a greater decrease in thymocyte-stimulating activity than could be accounted for by passively transferred rIL-1 alone. When the IL-6 was neutralized the remaining thymocyte-stimulating activity could be quantitatively accounted for and neutralized by antisera against the rIL-1 that was passively transferred. Furthermore, rIL-6 and rIL-1 (alpha or beta) synergized in stimulating thymocyte proliferation. Thus, rIL-1 stimulates fibroblasts to produce a thymocyte-stimulating activity that is largely mediated by a synergistic interaction of fibroblast-derived IL-6 and IL-1. These findings suggest that fibroblast production of IL-6 may mediate or amplify some of the tissue effects of IL-1. In addition they suggest that biologic effects previously attributed to IL-1 may be due to IL-6 alone or the concerted action of IL-1 and IL-6.     

Large-scale network models of IL-1 and IL-6 signalling and their hepatocellular specification

The pro-inflammatory cytokines interleukin 1 (IL-1) and 6 (IL-6) are crucially involved in the regulation of a multitude of physiological processes, in particular coordinating the immune response upon bacterial infection and tissue injury. Both interleukins induce complex signalling cascades and trigger the production of mitogenic, pro-proliferative, anti-apoptotic, chemotactic, and pro-angiogenic factors thereby affecting the delicate balance between regeneration vs. invasive growth, tumourigenesis and metastasis. Moreover, several links to insulin resistance have been found within their associated signalling networks. Focusing on this from a systems biology perspective, we introduce comprehensive large-scale network models of IL-1 and IL-6 signalling which are based on a logical modelling approach and reflect the current biological knowledge. Theoretical network analysis enabled us to uncover general topological features and to make testable predictions on the stimulus-response behaviour of the networks. In this context, non-intuitive network-wide species dependencies as well as structures of regulatory feedback and feed-forward mechanisms could be characterised. By integrating high-throughput phosphoproteomic data from primary human hepatocytes we optimised the model structures to obtain models with high prediction accuracy for hepatocytes. Our model-based data analysis, for instance, suggested model modifications regarding (i) Akt contribution to IL-1-stimulated p38 MAPK activation and (ii) insignificant p38 MAPK activation in response to IL-6. In light of the presented results and in conjunction with the detailed model documentations, both models hold great potential for theoretical studies and practical applications.     

In vivo neutralization of human IL-6 (hIL-6) achieved by immunization of hIL-6-transgenic mice with a hIL-6 receptor antagonist

Neutralization of IL-6 represents an attractive therapeutic option in several diseases, including B cell neoplasia, osteoporosis, and autoimmunity. Therapeutic attempts in humans have shown that administration of injectable doses of a mAb to IL-6 does not provide efficient neutralization of the cytokine in vivo. Therefore, alternative approaches are needed. In this study, we evaluated whether the Ab response to human IL-6 (hIL-6) elicited by vaccination with Sant1 (a hIL-6 variant with seven amino acid substitutions) was able to fully correct in vivo the clinical and biological effects of a chronic endogenous overproduction of hIL-6 in the hIL-6-transgenic NSE/hIL-6 mice. Because of the overexpression of hIL-6, occurring since birth, with circulating levels in the nanogram per milliliter range, NSE/hIL-6 mice have a marked decrease in growth rate, associated with decrease in insulin-like growth factor I levels, and represent an animal model of the growth impairment associated with human chronic inflammatory diseases. Following immunization with Sant1, but not with hIL-6, NSE/hIL-6 mice developed high titers of polyclonal Abs to hIL-6. The Abs, acquired by transplacental transfer, effectively neutralized IL-6 activities in vivo as shown by the complete correction of the growth defect and normalization of insulin-like growth factor levels in the hIL-6-transgenic offspring. Immunization with Sant1 could therefore represent a novel and simple therapeutic approach for the specific neutralization of IL-6 in humans.     

白介素24的抗肿瘤机理

白介素24(interleukin 24,IL-24)是利用消减杂交技术,从重组的纤维母细胞干扰素和密执毒素共同作用的黑色素瘤细胞株中筛选得到的一种高表达基因。由于IL-24能选择性地诱导肿瘤细胞凋亡,而对正常细胞没有细胞毒作用,因此在肿瘤治疗研究中受到人们广泛的重视。IL-24诱导肿瘤细胞凋亡的机理涉及多种信号途径,有些信号途径目前还不十分清楚。本文就IL-24通过启动不同的信号途径,诱导广泛的肿瘤细胞凋亡的机理作一综述,从而为IL-24抗肿瘤机理研究提供一些有用的信息。