Radiosensitivity Of Hepatocellular Carcinoma Cells Research Articles

KDM4C silencing inhibits cell migration and enhances radiosensitivity by inducing CXCL2 transcription in hepatocellular carcinoma

KDM4C, which is a histone lysine demethylase, has been proposed to participate in the malignant transformation and progression of several types of cancer. However, its roles in hepatocellular carcinoma (HCC) remain poorly understood. Here, we find that KDM4C protein expression is increased in HCC and promotes HCC cell growth, proliferation and migration. Furthermore, we provide evidence that depletion of KDM4C leads to a defective G2/M checkpoint, increases radiation-induced DNA damage, impairs DNA repair and enhances radiosensitivity in HCC cells. Using RNA sequencing, we identify that the chemokine CXCL2 is a downstream effector of KDM4C. KDM4C knockdown increases the binding of H3K36me3 to the promoter of CXCL2, thus upregulating CXCL2 expression and promoting CXCL2 secretion in HCC cells. Importantly, the observed effects of KDM4C depletion in HCC cells can be partially rescued by CXCL2 silencing. Thus, our findings reveal that KDM4C is involved in cell migration and radiosensitivity by modulating CXCL2 transcription, indicating that KDM4C may be a potential therapeutic target in HCC.

The Expression of Connexin 26 Regulates the Radiosensitivity of Hepatocellular Carcinoma Cells through a Mitogen-Activated Protein Kinases Signal Pathway.

Connexin 26 (Cx26) is a protein that constitutes a gap junction and is widely expressed in the liver. Abnormal expression of Cx26 is one of the important mechanisms of liver cancer, and is closely related to the transmission of radiation damage signals between cells. In the present study, we investigated the radiosensitivity of hepatocellular carcinoma (HCC) cells HepG2, with low expression of Cx26, and SK-hep-1, with high expression of Cx26 after X-ray irradiation. The cell survival, micronucleus formation and protein expressions of the mitogen-activated protein kinases (MAPK) signaling pathway were detected. The expression level of Cx26 could affect the radiosensitivity of liver cancer cells by affecting the phosphorylation of p38 and ERK proteins and regulating the expression of downstream NF-κB. Cell lines with knock-out and overexpression of Cx26 were also built to confirm the findings. Our results suggested that Cx26 might play an important role in the radiosensitivity of liver cancer and could be a potential target for clinical radiotherapy of liver cancer.

Epirubicin Enhances the Anti-Cancer Effects of Radioactive 125I Seeds in Hepatocellular Carcinoma via Downregulation of the JAK/STAT1 Pathway.

The application and promotion of 125I seed implantation technology have increased the safety and effectiveness of the clinical treatment of advanced hepatocellular carcinoma (HCC). Epirubicin (EPI) is a traditional anthracycline chemotherapy agent that has minimal side effects and has been widely used in the clinical treatment of HCC. We hypothesized that EPI would enhance the anti-cancer effects of 125I seeds via the JAK/STAT1 signaling pathway. Thus, we aimed to investigate whether EPI could enhance the radiosensitivity of HCC cells to 125I and determine the underlying molecular mechanism. This basic study was conducted in an animal laboratory at Shandong University. BALB/C male nude mice were used, and all animals were fed and treated according to the standards of the Institutional Animal Care and Use Committee of Shandong University. Both in vitro and in vivo models of 125I irradiation of HCC cells were created. The anti-cancer effects of 125I and the role of EPI in promoting these effects were evaluated using flow cytometry for apoptosis and cell cycle, CCK-8 assay for EPI drug cytotoxicity, and transwell assays for migration and invasion. The potential mediating effect of the JAK/STAT1 pathway was assessed using an isobaric tag for relative and absolute quantitation analysis to identify differentially expressed proteins after 125I treatment. Transfection of HCC cells with STAT1-RNAi were performed to determine the effect of STAT1 downregulation on 125I and EPI treatment effects. The radiosensitivity concentration of EPI promoted 125I-induced anti-cancer effects, including apoptosis, anti-proliferation, and inhibition of migration and invasion. These effects were mediated via the JAK/STAT1 pathway. Downregulation of STAT1 compromised measured anti-cancer effects, which were both confirmed in the in vivo and in vitro models. EPI can promote 125I-induced anti-cancer effects in HCC. The JAK/STAT1 pathway may be a potential target for 125I seed implantation in the treatment of HCC.

Circ-LARP1B knockdown restrains the tumorigenicity and enhances radiosensitivity by regulating miR-578/IGF1R axis in hepatocellular carcinoma

Introduction and ObjectivesCircular RNA La Ribonucleoprotein 1B (circ-LARP1B) was reported to serve as an oncogene in many types of cancers. Radiotherapy (RT) is an important element of the multimodal treatment concept in malignancies. Here, this work aimed to investigate the role of circ-LARP1B in the tumorigenesis and radiosensitivity of hepatocellular carcinoma (HCC). Patients or Materials and MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of genes and proteins. In vitro experiments were conducted using cell counting Kit-8 (CCK-8), colony formation, EDU, transwell, and tube formation assays, respectively. Dual-luciferase reporter assay was employed to identify the target relationship between miR-578 and circ-LARP1B or IGF1R (insulin-like growth factor 1 receptor). In vivo assay was performed using murine xenograft model. ResultsCirc-LARP1B was highly expressed in HCC tissues and cells, and high expression of circ-LARP1B was closely associated with poor prognosis. Functional experiments demonstrated that circ-LARP1B silencing impaired cell proliferation, invasion, angiogenesis and reduced radioresistance in vitro. Mechanistically, circ-LARP1B could competitively bind with miR-578 to relieve the repression of miR-578 on the expression of its target gene IGF1R. Further rescue assay confirmed that miR-578 inhibition reversed the inhibitory effects of circ-LARP1B knockdown on HCC cell malignant phenotypes and radioresistance. Moreover, miR-578 overexpression restrained tumorigenicity and enhanced radiosensitivity in HCC cells, which were attenuated by IGF1R up-regulation. Besides that, circ-LARP1B knockdown impeded tumor growth and enhanced irradiation sensitivity in HCC in vivo. ConclusionsCirc-LARP1B knockdown restrained HCC tumorigenicity and enhanced radiosensitivity by regulating miR-578/IGF1R axis, providing a new target for the treatment of HCC.

Verbascoside enhances radiosensitivity of hepatocellular carcinoma cells through regulating miR-101-3p/Wee1 axis.

Verbascoside is a kind of phenylpropanoid glycoside derived from multiple medicinal plants, exerting anti-tumor effects in diverse human malignancies. However, the function of Verbascoside on the radiosensitivity of hepatocellular carcinoma (HCC) cells remains unknown. Human Huh7 and HepG2 cell lines were treated with Verbascosideis, and cell viability was detected with cell counting kit-8 (CCK-8) assay. Quantitative real-time polymerase chain reaction (qRT-PCR) was applied to detect miR-101-3p expression, and Western blot was used to quantify the expression of WEE1 G2 checkpoint kinase (WEE1). Then, CCK-8 and flow cytometry assays were used to detect the proliferation and apoptosis of HCC cells after Verbascoside and X-ray combined treatment, and the expressions of WEE1 and apoptosis-related proteins Bax and Bcl-2 were detected by Western blot. Verbascoside could improve the radiosensitivity of HCC cells in a dose-dependent manner. Verbascoside increased the expression of miR-101-3p but reduced WEE1 expression in HCC cells. Additionally, WEE1 was identified as a target of miR-101-3p. MiR-101-3p inhibition or WEE1 overexpression could reverse the effect of Verbascoside on the viability and apoptosis of HCC cells. Verbascoside increases the radiosensitivity of hepatocellular carcinoma cells via modulating miR-101-3p/WEE1 axis.

MiR-122 radiosensitize hepatocellular carcinoma cells by suppressing cyclin G1

Purpose Emerging evidence has shown that radiotherapy is an effective treatment for hepatocellular carcinoma (HCC), Micro(mi)RNAs are involved in regulating radiosensitivity in many cancers. MiR-122 accounts for approximately 70% of all cloned miRNAs in the liver, but there are few reports about whether it is involved in regulating of radiosensitivity in HCC cells. Materials and Methods HCC cells (HepG2 and Huh7) overexpressing miR-122 were constructed by transfecting them with lentiviral-miR-122. Then, their proliferation ability was analyzed by the MTT, and colony formation assays and a xenograft tumor model was used to detect their radiosensitivity. The expression of cyclin G1 mRNA and protein was detected by the quantitative real-time polymerase chain reaction and western blotting, respectively. Results Overexpression of miR-122 inhibited the proliferation of, and radiosensitized HCC cells. Cyclin G1 mRNA and protein level were suppressed in HepG2 tumors overexpression miR-122. Conclusion MiR-122 may be useful as a potential radiosensitizer for HCC, and its mechanism is related to the regulation of cyclin G1.

Silencing the Expression of Cyclin G1 Enhances the Radiosensitivity of Hepatocellular Carcinoma In Vitro and In Vivo by Inducing Apoptosis

Radiotherapy plays an important role in the treatment of hepatocellular carcinoma (HCC). Cyclin G1 is a novel member of the cyclin family, and it is abnormally expressed in HCC. In this study we investigated the role of cyclin G1 in the radiotherapy of HCC cells. The expression of cyclin G1 was silenced by transfection of cyclin G1-siRNA into HepG2 cells and Huh7 cells, and the expression of cyclin G1 mRNA and protein was measured by qRT-PCR and Western blot analysis. The proliferation was analyzed using MTT assay, and the radiosensitivity of HCC cells was detected using colony formation assay and a xenograft tumor model. The expression of apoptosis-related proteins (Bcl-2 and Bax) was detected by Western blot analysis, and caspase-3 was detected using fluorimetry. The expression of cyclin G1 mRNA and protein in HepG2/Huh7-cyclin G1-siRNA cells was found to be significantly decreased compared to that in HepG2/Huh7 cells. Silencing the expression of cyclin G1 inhibited the proliferation of HCC cells and enhanced radiosensitivity in HCC cells in vitro and in vivo. Knockdown of cyclin G1 expression significantly decreased Bcl-2 expression, and increased Bax expression and caspase-3 activity in HCC cells. Silencing of cyclin G1 expression enhances the radiosensitivity of HCC cells in vitro and in vivo. The mechanism for this may be related to the regulation of apoptosis-related proteins.

CLTRN, Regulated by NRF1/RAN/DLD Protein Complex, Enhances Radiation Sensitivity of Hepatocellular Carcinoma Cells Through Ferroptosis Pathway

Radiation therapy is a viable treatment option for patients with unresectable hepatocellular carcinoma (HCC). However, radiation resistance and adverse effects are issues that needs to be addressed. Herein, for the first time, we investigated the ability of collectrin (CLTRN) to enhance radiosensitivity in patients with HCC. Transcriptome sequencing technology (RNA-seq technology) was used to analyze the transcription-level changes in the genes in HepG2 cells before and after x-ray irradiation. Combining the results with the HCC tissue RNA-seq data, we determined the ultimate target gene through bioinformatics analysis and cellular verification. A series of cellular and molecular biology techniques were applied invitro and invivo to confirm whether CLTRN can enhance radiosensitivity in HCC cells. Subsequently, the downstream action mechanism, the upstream transcription factor, and the interaction proteins of CLTRN were determined. First, we confirmed that CLTRN is the target gene for radiation therapy and verified the association between CLTRN and radiosensitivity. Invivo and invitro experiments were performed. Investigation of the gene regulatory mechanism revealed that the genes analyzed at the transcriptome level after CLTRN overexpression were mostly enriched in the glutathione metabolic pathway. As glutathione metabolism forms a vital link in ferroptosis, we surmised that CLTRN is associated with ferroptosis. This was confirmed through detection of cellular iron, determination of reactive oxygen species levels, use of transmission electron microscopy, and monitoring of ferroptosis-related protein indicators. Lastly, we investigated whether nuclear respiratory factor 1 is the upstream transcription factor of CLTRN and whether dihydrolipoamide dehydrogenase and members of the RAS oncogene family are its interacting proteins. CLTRN is a vital regulator of radiation sensitivity and could serve as a novel therapeutic target or prognostic marker in HCC treatment.

Anti-GPC3 Antibody-Conjugated BEZ235 Loaded Polymeric Nanoparticles (Ab-BEZ235-NP) Enhances Radiosensitivity in Hepatocellular Carcinoma Cells by Inhibition of DNA Double-Strand Break Repair.

To assess AB-BEZ235-NP potential as a radio-sensitizer in hepatocellular carcinoma models. By comparing hepatocellular carcinoma cell with simple radiation or combined AB-BEZ235-NP therapy, the HCC apoptosis and self-repair level have significant differences in mortality rates and cell migration abilities. Cell proliferation and DNA damage increased by pretreatment with AB-BEZ235-NP after irradiation; further studies on the repair pathway indicated that AB-BEZ235-NP inhibited the important pathway of DSB repair. Our results further show that AB-BEZ235-NP significantly inhibits the phosphorylation of the canonical protein, γ-H2AX, in the NHEJ DSB repair pathway and Serine Protein Kinase (SPK) ATM, and TP53-Binding Protein one. More importantly, AB-BEZ235-NP increased the mount of mean γ-H2AX Foci in irradiated cells, indicating that AB-BEZ235-NP can selectively inhibit DSB repair in HCC cells. Therefore, these results clearly eludicate that treatment with AB-BEZ235-NP is a potential promising therapy which can increase the radiosensitivity to HCC.

Effect of CCR4 on sorafenib radiosensitivity and tumorigenesis of hepatocellular carcinoma cells in nude mice

Objective To investigate the effect of CC chemokine receptor 4(CCR4) on sorafenib radiosensitivity and tumorigenesis of hepatocellular carcinoma cells in nude mouse models. Methods Western blot was used to detect the expression of CCR4 in hepatocellular carcinoma cell line. Lentivirus was utilized to construct PLC/PRF/5 and SMMC-7721 cell lines stably overexpressing and silencing CCR4, which were verified by Western blot. The influence of CCR4 on the radiosensitivity of hepatocellular carcinoma cells was assessed by plate clone formation assay. The effect of CCR4 on the tumorigenesis in hepatocellular carcinoma cells in vivo was evaluated by tumorigenesis assay in nude mice. Results CCR4 was highly expressed in highly-metastatic hepatocellular carcinoma cells and lowly expressed in hepatocellular carcinoma cells with low metastases. The PLC/PRF/5 and SMMC-7721 cells, which stably overexpressed and silenced CCR4, were successfully established. Overexpression of CCR4 reversed the inhibitory effect of sorafenib radiotherapy on PLC/PEF/5, whereas knockdown of CCR4 could increase the radiosensitivity of SMMC-7721 to sorafenib. Overexpressing CCR4 could promote the tumorigenicity of PLC/PEF/5, whereas knockdown of CCR4 could inhibit the tumorigenicity of SMMC-7721 in nude mice. Conclusion CCR4 overexpression significantly reduces the radiosensitivity of PLC/PRF/5 and increases the tumorigenicity in nude mice, whereas knockdown of CCR4 considerably increases the chemosensitivity and radiosensitivity of SMMC-7721 and suppresses the tumorigenicity in nude mice. Key words: Chemokine receptor; Sorafenib; irradiation; Hepatocellular carcinoma cell line; Nude mice

Metformin enhances radiosensitivity in hepatocellular carcinoma by inhibition of specificity protein 1 and epithelial-to-mesenchymal transition.

Radiotherapy becomes more and more important in hepatocellular carcinoma (HCC) due to the development of technology, especially in unresectable cases. Metformin has a synergistic benefit with radiotherapy in some cancers, but remains unclear in HCC. This study aims to investigate the effect of metformin on radiosensitivity of HCC cells and the roles of specificity protein 1 (Sp1) as a target of metformin. The SMMC-7721 cell line was exposed to various doses of γ-ray irradiation (0, 2, 4, 6, and 8 Gy) and with or without different concentrations of metformin (0, 1, 5, 10, and 20 mM) to measure the radiosensitivity using MTT assay. Flow cytometry was used to determine cell cycle by propidium iodide (PI) staining and apoptosis by Hoechst 33342/PI staining and Annexin V-FITC/PI staining. Real-time polymerase chain reaction and Western blotting were performed to analyze the Sp1 mRNA and protein expressions of Sp1 and epithelial-to-mesenchymal transition (EMT) marker E-cadherin and Vimentin. The invasion capability was measured by the Boyden chamber assay. In SMMC-7721 cells exposed to irradiation, metformin reduced proliferation and survival cells at various concentrations (0, 1, 5, 10, and 20 mM) and induced cell cycle arrest, apoptosis, and inhibited invasion. In SMMC-7721 cells with irradiation, the mRNA and protein expressions of Sp1 were significantly decreased by metformin as well as a selective Sp1 inhibitor. Metformin attenuated transforming growth factor-β1 induced decrease of E-cadherin and increase of Vimentin proteins. Metformin demonstrated enhanced radiosensitivity and inhibition of EMT in HCC cells. Sp1 might be a target of metformin in radiosensitization.

A phenanthroline derivative enhances radiosensitivity of hepatocellular carcinoma cells by inducing mitochondria-dependent apoptosis

Combining radiosensitizers with ionizing radiation (IR) is an effective strategy to increase the radiation therapeutic effect for hepatocellular carcinoma (HCC) patients. A phenanthroline derivative, 2-phenyl-imidazo [4, 5 f] [1, 10] phenanthroline (L02), had been synthesized. This study investigated the radiosensitization and mechanisms of L02 combined with IR against HCC. The radiosensitization of L02 combined with IR was evaluated by the sensitivity enhancement ratio (SER) and the isobolographic analysis. The toxicity of L02 and cisplatin were compared by the zebrafish model. The cell cycle and apoptosis were examined by flow cytometry. DNA damage was measured by comet assay and the expressions of apoptosis related proteins were analyzed by western blotting. L02 was effective in sensitizing HCC to IR. The SERs in HepG2 and BEL7402 were 1.41 and 1.28, respectively. The sensitization of L02 was comparable with cisplatin. L02 treatment with IR had synergistic anti-tumor effect. L02 enhanced the percentage of IR induced apoptosis cells. L02 increased comet tail in comet assay when combined with IR. L02 sensitized HCC to IR by the activation of P53 signaling, the decrease in Bcl-2, up-regulation of cytochrome c and the subsequent activation of caspase-3. L02 sensitizes HCC to IR, mostly likely by inhibiting cell proliferation, inducing DNA damage and mitochondria-dependent apoptosis. L02 may be a novel radiosensitizer for HCC.

A20 enhances the radiosensitivity of hepatocellular carcinoma cells to 60Co-γ ionizing radiation.

The radioresistance of hepatocellular carcinoma (HCC) cells is a critical obstacle for effectively applying radiotherapy (RT) in HCC treatment. NF-κB, an important transcription factor, can influence critical cell fate decisions by promoting cell survival or anti-apoptosis in response to cell-stress, e.g. chemotherapies or ionizing radiation (IR). A20, also named as tumor necrosis factor α induced protein 3 (TNFAIP3), is a dominant negative regulator of NF-κB pathway and its functions in HCC are largely unknown. The present work aimed to reveal the role of A20 plays in affecting the radiosensitivity of HCC cells. Higher expression of A20 was detected in hepatic non-tumor cell line or clinical specimens compared with HCC cell lines or clinical specimens. A20 decreased the expression of proteins mediating cellular stress/injury response or epithelial-mesenchymal transition (EMT) process. Overexpression of A20 via adenovirus enhanced the effect of 60Co-γ ionizing radiation (IR) on HCC cells’ injury, e.g. G2/M arrest or DNA double strands break (DSB). Moreover, A20 also enhanced the in vitro or in vivo survival inhibiting of HCC cells induced by IR. These results reveal the roles of A20 in HCC radiosensitization and overexpression of A20 would be a novel strategy for HCC radiotherapy.

Abstract 5190: Oncoprotein SET determines the radiosensitivity of hepatocellular carcinoma cell and antagonizing SET augments the effects of radiotherapy via reactivating PP2A-mediated Akt downregulation

Abstract Background: SET is a novel oncoprotein with growing evidence suggesting its roles in promoting the survival of hepatocellular carcinoma (HCC) and other types of malignant disease. Notably, pre-clinical data suggesting SET overexpression in cancer cell leaded to the development of resistance to various anti-cancer medications, and antagonizing SET reversely enhanced therapeutic efficacy. Radiotherapy (RT) is a commonly used anti-cancer modality, but its role for the treatment of HCC is largely limited for the concern of endogenous radioresistance. In this report, we explored the role of SET regarding the radiosensitivity of HCC cells. Furthermore, we explored whether SET serve as a valid target to enhance RT against HCC. Method: A panel of HCC cell lines, including Hep3B, PLC5, HA22T and HA59T, were used for in vitro experiments, and the PLC5 subcutaneous xenograft mice model was used for in vivo testing. HCC cells and tumors were treated with RT and/or SET antagonist and harvested for subsequent experiments. The viability of cancer cells were assessed by sub-G1, colony and sphere formation assay. Molecular events were determined by western blot and PP2A activity. Result: Through manipulating the expression of SET in cancer cells, we showed the crucial role of SET in mediating the effects of RT in HCC cells. Using colony formation and hepatosphere formation assay, we showed that RT significantly reduced the number and size of both tumor colony and sphere formed by Hep3B and PLC5 cells. In contrast, ectopic expression of SET in Hep3B and PLC5 cells abolished the effects of RT. More importantly, SET-knockdown oppositely potentiated the RT-induced growth inhibition in HCC cells. Based on our findings, we hypothesized that targeting SET may exert potential to enhance RT for the treatment of HCC. In order to validate our hypothesis, we tested the effects of RT in combine with a novel SET antagonist, EMQA, developed and characterized in our previous works. As expected, we found that EMQA significantly potentiated the anti-HCC effects of RT in vitro and in vivo. Moreover, we found that antagonizing SET to restore PP2A-mediated p-Akt downregulation was responsible for the synergism between EMQA and RT. Conclusion Oncoprotein SET plays a critical role in affecting the radiosensitivity of HCC cells. Using SET antagonist plus RT showed promising results in pre-clinical HCC models, and yields further investigation. Citation Format: Man-Hsin Hung, Chao-Yuan Huang, Chih-Ting Shih, Min-Hsien Tsai, Yung-Jen Hsiao, Tzu-I Chao, Feng-Shu Hsieh, Chung-Wai Shiau, Kuen-Feng Chen. Oncoprotein SET determines the radiosensitivity of hepatocellular carcinoma cell and antagonizing SET augments the effects of radiotherapy via reactivating PP2A-mediated Akt downregulation [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 5190. doi:10.1158/1538-7445.AM2017-5190

MiR-26b enhances radiosensitivity of hepatocellular carcinoma cells by targeting EphA2.

Although low-dose radiotherapy (RT) that involves low collateral damage is more suitable for hepatocellular carcinoma (HCC) than traditional high-dose RT, but to achieve satisfactory therapeutic effect with low-dose RT, it is necessary to sensitize HCC cells to irradiation. This study was aimed to determine whether radiosensitivity of HCC cells can be enhanced using miR-26b by targeting erythropoietin producing human hepatocelluar A2 (EphA2). The levels of miR-26b and EphA2 expression in multiple HCC cell lines were assessed by qPCR and western blotting, respectively, and compared with those in a hepatic cell line. HCC 97H cells were transfected with miR-26b mimics, EphA2-ShRNA or EphA2 over-expression vector before exposure to low-dose irradiation. Different degrees of miR-26b down-regulation and EphA2 up-regulation were observed in all HCC cell lines, among which the HCC 97H cell line expressed the lowest level of miR-26b and highest level of EphA2. EphA2 was verified as the target of miR-26b by dual luciferase reporter assay. HCC 97H cells transfected with miR-26b mimics or EphA2-ShRNA reduced the expression of EphA2 protein, with significantly lower cell proliferation rate and cell invasion ability and higher apoptosis rate in response to low-dose irradiation than those in the non-transfected cells. These results were reversed after EphA2 was overexpressed by transfection with the EphA2 overexpression vector. Co-transfection with miR-26b mimics and EphA2 overexpression vector barely altered EphA2 expression level and cell response to low-dose irradiation. These data suggest that miR-26b enhances radiosensitivity of HCC 97H cells by targeting EphA2 protein.

Dovitinib Acts As a Novel Radiosensitizer in Hepatocellular Carcinoma by Targeting SHP-1/STAT3 Signaling

Hepatocellular carcinoma (HCC) is among the most lethal human malignancies, and curative therapy is not an option for most patients. There is growing interest in the potential benefit of combining targeted therapies with radiation therapy (RT). This study aimed to characterize the efficacy and mechanism of an investigational drug, dovitinib, used in combination with RT. HCC cell lines (PLC5, Hep3B, SK-Hep1, HA59T, and Huh-7) were treated with dovitinib, RT, or both, and apoptosis and signal transduction were analyzed. Dovitinib treatment resulted in Src homology region 2 (SH2) domain-containing phosphatase 1 (SHP-1)-mediated downregulation of p-STAT3 and promoted potent apoptosis of HCC cells. Ectopic expression of STAT3, or inhibition of SHP-1, diminished the effects of dovitinib on HCC cells. By ectopic expression and purified recombinant proteins of various mutant forms of SHP-1, the N-SH2 domain of SHP-1 was found to be required for dovitinib treatment. Overexpression of STAT3 or catalytic-dead mutant SHP-1 restored RT-induced reduction of HCC cell survival. Conversely, ectopic expression of SHP-1 or activation of SHP-1 by dovitinib enhanced the effects of RT against HCC invitro and invivo. SHP-1/STAT3 signaling is critically associated with the radiosensitivity of HCC cells. Combination therapy with RT and the SHP-1 agonist, such as dovitinib, resulted in enhanced invitro and invivo anti-HCC effects.

MicroRNA-26b Enhances the Radiosensitivity of Hepatocellular Carcinoma Cells by Targeting EphA2.

Sensitizing hepatocellular carcinoma (HCC) cells to irradiation is important to achieve satisfactory therapeutic effect with low-dose radiotherapy. Erythropoietin-producing hepatocellular carcinoma A2 (EphA2) is a member of the Eph receptor family that constitutes the largest family of tyrosine kinase receptors. EphA2 overexpression is one of the poor prognostic factors in many progressive cancers. Importantly, EphA2 is a potential target of microRNA-26b (miR-26b), and miR-26b expression is down-regulated in several types of cancer. In this study, we measured the expression levels of miR-26b and EphA2 protein in seven human HCC cell lines by quantitative PCR and western blot analysis, respectively. Overall, lower miR-26b expression levels tended to be associated with higher EphA2 levels in HCC cell lines. Among the cell lines examined, 97H HCC cells expressed the lowest level of miR-26b and highest level of EphA2 protein. Thus, using 97H HCC cells, EphA2 mRNA was verified as the target of miR-26b by the luciferase reporter assay. Accordingly, a synthetic miR-26b, miR-26b mimics, was used to mimic the function of endogenous miR-26b. In 97H HCC cells transfected with miR-26b mimics or short-hairpin RNA targeting EphA2 mRNA, expression of EphA2 protein was reduced, which was associated with significantly lower proliferation rate and invasion ability and with higher apoptosis rate in response to low-dose irradiation, compared to control cells. In contrast, 97H HCC cells over-expressing EphA2 showed higher proliferation rate and invasion ability and lower apoptosis rate upon irradiation. These data suggest that miR-26b enhances the radiosensitivity of 97H HCC cells by targeting EphA2 protein.

Abstract 3122: The DNA-PK inhibitor NU7441 inhibits non-homologous end joining, enhances radio- and chemosensitivity and increases dependence on homologous recombination in hepatocellular carcinoma cell lines

Abstract Background DNA double-strand breaks (DSBs), the most cytotoxic lesions induced by ionizing radiation (IR) and anticancer drugs such as topoisomerase II poisons (e.g., doxorubicin), are repaired by non-homologous end joining (NHEJ) and homologous recombination (HR). DNA-dependent protein kinase (DNA-PK), which initiates NHEJ, is up-regulated in hepatocellular carcinoma (HCC) (GEO profiles), possibly contributing to anticancer therapy resistance. To assess DNA-PK as a potential therapeutic target for chemo- and radio-sensitisation in HCC we determined the effect of the DNA-PK inhibitor, NU7441, on DSB repair and cytotoxicity in HCC cells. Methods DNA-PK protein levels and activation by IR (Western blot), DSB levels (y-H2AX foci), HR (RAD51 foci), cell growth (DAPI fluorescence) and cytotoxicity (colony formation) following exposure to IR or doxorubicin was determined in a panel of 6 hepatoma cell lines (HepG2, Hep3B, Huh7, SNU-182, SNU475 and PLC/PRF/5). Results DNA-PK protein concentration and activity did not vary significantly across the panel (±24% and ±37%, respectively) but there were cell-specific sensitivities to IR and doxorubicin (e.g. HepG2 2-fold more resistant than Hep3B). NU7441 significantly sensitised all cells to both doxorubicin (average PF50 4.3±3.0) and IR (average PF50 3.9±1.1) in growth inhibition assays and significantly reduced survival (4.8 to 3.3-fold) in colony forming assays. Following exposure to IR, NU7441 significantly delayed yH2AX focus clearance in all cell lines (e.g. only 13% cleared at 4 hr compared to 50% in control), but increased RAD51 focus formation (3-fold). Conclusion While chemo- and radio-sensitivity in HCC cells was not dependent on DNA-PK expression or activity, NU7441 causes greater than 2-fold chemo- and radiosensitisation in all cells. This was accompanied by a substantial reduction in the rapid phase of DNA repair (NHEJ-dependent) and a shift to a greater reliance on the slower HR repair. DNA-PK inhibitors may have potential as chemo- and radio-sensitisors in hepatoma patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3122. doi:1538-7445.AM2012-3122