Dexiao Yuan

Sorafenib potentiates irradiation effect in hepatocellular carcinoma in vitro and in vivo

The multikinase-inhibition action of sorafenib provides strong rationales for its combination use with radiotherapy. We investigated the in vitro and in vivo effect of sorafenib combined with irradiation on hepatocellular carcinoma (HCC). Sorafenib enhanced radiosensitivity of human HCC cell lines in a schedule-dependent manner. Sorafenib selectively inhibited radiation-induced activation of vascular endothelial growth factor receptor-2 (VEGFR2) and downstream extracellular signal-regulated kinase (ERK) pathway, induced DNA damage and suppressed DNA repair capacity, decreased radiation-activated NF-κB and increased radiation-induced apoptosis. In xenograft experiments, combination treatment produced marked tumor growth delay in both concurrent and sequential schedules. These results suggest that sorafenib could potentiate irradiation effect in HCC, which warrants further investigation for its potential clinical applications.

Reciprocal bystander effect between α-irradiated macrophage and hepatocyte is mediated by cAMP through a membrane signaling pathway.

Irradiated cells can induce biological effects on vicinal non-irradiated bystander cells, meanwhile the bystander cells may rescue the irradiated cells through a feedback signal stress. To elucidate the nature of this reciprocal effect, we examined the interaction between α-irradiated human macrophage cells U937 and its bystander HL-7702 hepatocyte cells using a cell co-culture system. Results showed that after 6h of cell co-culture, mitochondria depolarization corresponding to apoptosis was significantly induced in the HL-7702 cells, but the formation of micronuclei in the irradiated U937 cells was markedly decreased compared to that without cell co-culture treatment. This reciprocal effect was not observed when the cell membrane signaling pathway was blocked by filipin that inhibited cAMP transmission from bystander cells to irradiated cells. After treatment of cells with exogenous cAMP, forskolin (an activator of cAMP) or KH-7 (an inhibitor of cAMP), respectively, it was confirmed that cAMP communication from bystander cells to targeted cells could mitigate radiation damage in U739 cells, and this cAMP insufficiency in the bystander cells contributed to the enhancement of bystander apoptosis. Moreover, the bystander apoptosis in HL-7702 cells was aggravated by cAMP inhibition but it could not be evoked when p53 of HL-7702 cells was knocked down no matter of forskolin and KH-7 treatment. In conclusion, this study disclosed that cAMP could be released from bystander HL-7702 cells and compensated to α-irradiated U937 cells through a membrane signaling pathway and this cAMP communication played a profound role in regulating the reciprocal bystander effects.

Extracellular miR-1246 promotes lung cancer cell proliferation and enhances radioresistance by directly targeting DR5

MiRNAs in the circulation have been demonstrated to be a type of signaling molecule involved in intercellular communication but little is known about their role in regulating radiosensitivity. This study aims to investigate the effects of extracellular miRNAs induced by ionizing radiation (IR) on cell proliferation and radiosensitivity. The miRNAs in the conditioned medium (CM) from irradiated and non-irradiated A549 lung cancer cells were compared using a microarray assay and the profiles of 21 miRNAs up and down-regulated by radiation were confirmed by qRT-PCR. One of these miRNAs, miR-1246, was especially abundant outside the cells and had a much higher level compared with that inside of cells. The expressions of miR-1246 in both A549 and H446 cells increased along with irradiation dose and the time post-irradiation. By labeling exosomes and miR-1246 with different fluorescence dyes, it was found that the extracellular miR-1246 could shuttle from its donor cells to other recipient cells by a non-exosome associated pathway. Moreover, the treatments of cells with miR-1246 mimic or its antisense inhibitor showed that the extracellular miR-1246 could enhance the proliferation and radioresistance of lung cancer cells. A luciferase reporter-gene transfer experiment demonstrated that the death receptor 5 (DR5) was the direct target of miR-1246, and the kinetics of DR5 expression was opposite to that of miR-1246 in the irradiated cells. Our results show that the oncogene-like extracellular miR-1246 could act as a signaling messenger between irradiated and non-irradiated cells, more importantly, it contributes to cell radioresistance by directly suppressing the DR5 gene.

SirT1 regulates radiosensitivity of hepatoma cells differently under normoxic and hypoxic conditions.

Intratumoral hypoxic cells are more resistant to radiotherapy due to a reduction in lifespan of DNA‐damaging free radicals and augmentation of post‐irradiation molecular restoration. SirT1, a member of the mammalian sirtuin family, deacetylates various transcription factors to trigger cell defense and survival in response to stresses and DNA damage. In this study, we provide new evidence indicating that overexpression of SirT1 in hepatoma HepG2 cells allowed the cells to become much more resistant to irradiation under hypoxia than under normoxia. When SirT1 was knocked down in both HepG2 and SK‐Hep‐1 cells, the radiosensitivity was increased, especially under hypoxia. But this enhanced radiosensitivity in SirT1‐deficient cells was extensively decreased by infecting cells with c‐Myc siRNA. Furthermore, the expression of c‐Myc protein and its acetylation were increased in the SirT1 knockdown cells and these increments under hypoxic conditions were much more notable than under normoxia. In addition, c‐Myc interference significantly suppressed phosphorylated p53 protein expression after irradiation, especially under hypoxic conditions. The current findings indicate that SirT1 confers a higher radioresistance in hypoxic cells than in normoxic cells due to the decreased levels of c‐Myc protein and its acetylation, and that a c‐Myc‐dependent radiation‐induced phosphorylated p53 may be involved. SirT1 could serve as a novel target of radiation damage and thus as a potential strategy to advance the efficiency of radiotherapy in hepatoma entities. (Cancer Sci 2012; 103: 1238–1244)

Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway contributes to the proliferation of hepatoma cells.

Hydrogen sulfide (H2S)/cystathionine γ-lyase (CSE) pathway has been demonstrated to play vital roles in physiology and pathophysiology. However, its role in tumor cell proliferation remains largely unclear. Here we found that CSE over-expressed in hepatoma HepG2 and PLC/PRF/5 cells. Inhibition of endogenous H2S/CSE pathway drastically decreased the proliferation of HepG2 and PLC/PRF/5 cells, and it also enhanced ROS production and mitochondrial disruption, pronounced DNA damage and increased apoptosis. Moreover, this increase of apoptosis was associated with the activation of p53 and p21 accompanied by a decreased ratio of Bcl-2/Bax and up-regulation of phosphorylated c-Jun N-terminal kinase (JNK) and caspase-3 activity. In addition, the negative regulation of cell proliferation by inhibition of H2S/CSE system correlated with the blockage of cell mitogenic and survival signal transduction of epidermal growth factor receptor (EGFR) via down-regulating the extracellular-signal-regulated kinase 1/2 (ERK1/2) activation. These results demonstrate that H2S/CSE and its downstream pathway contribute to the proliferation of hepatoma cells, and inhibition of this pathway strongly suppress the excessive growth of hepatoma cells by stimulating mitochondrial apoptosis and suppressing cell growth signal transduction.

Radiation Enhances the Invasiveness of Irradiated and Nonirradiated Bystander Hepatoma Cells Through a VEGF-MMP2 Pathway Initiated by p53

Recent evidence has shown that irradiation can promote the invasiveness of hepatocellular carcinoma cells and have an impact on the invasive behavior of nonirradiated surrounding cancer cells, which may enhance overall tumor aggressiveness. However, the role of the TP53 tumor suppressor gene in the invasion of irradiated hepatoma cells and their nonirradiated bystanders remain largely unknown. In the present study, we found that irradiation increased the invasiveness of human hepatoma HepG2 cells, and pretreatment of the cells with SU1498 (an inhibitor of vascular endothelial growth factor receptor 2, VEGFR2) and GM6001 (an inhibitor of matrix metalloproteinases 2, MMP2) demonstrated that radiation-enhanced invasiveness is associated with the interplay between MMP2 and VEGF signaling. In addition, while radiation-induced expression and phosphorylation of p53, inhibition of p53 function with pifithrin-α or transfection of cells with p53 siRNA significantly reduced the activation of both MMP2 and VEGF and resulted in a reduction of radiation-induced invasiveness. Interestingly, we also found that the invasiveness of the nonirradiated bystander cells was also elevated after co-culturing with irradiated cells and that bystander invasive potential was regulated paracrine in a manner by MMP2 and VEGF from the irradiated cells through a p53-dependent mechanism. Taken together, our data demonstrate that radiation-induced up-regulation of p53 is responsible for the promotion of VEGF-MMP2 pathway involved in the enhancement of invasiveness of both irradiated and bystander hepatoma cells.

Dose response of micronuclei induced by combination radiation of α-particles and γ-rays in human lymphoblast cells

Combination radiation is a real situation of both nuclear accident exposure and space radiation environment, but its biological dosimetry is still not established. This study investigated the dose-response of micronuclei (MN) induction in lymphocyte by irradiating HMy2.CIR lymphoblast cells with α-particles, γ-rays, and their combinations. Results showed that the dose-response of MN induced by γ-rays was well-fitted with the linear-quadratic model. But for α-particle irradiation, the MN induction had a biphasic phenomenon containing a low dose hypersensitivity characteristic and its dose response could be well-stimulated with a state vector model where radiation-induced bystander effect (RIBE) was involved. For the combination exposure, the dose response of MN was similar to that of α-irradiation. However, the yield of MN was closely related to the sequence of irradiations. When the cells were irradiated with α-particles at first and then γ-rays, a synergistic effect of MN induction was observed. But when the cells were irradiated with γ-rays followed by α-particles, an antagonistic effect of MN was observed in the low dose range although this combination radiation also yielded a synergistic effect at high doses. When the interval between two irradiations was extended to 4h, a cross-adaptive response against the other irradiation was induced by a low dose of γ-rays but not α-particles.

Role of DNA methylation in long-term low-dose γ-rays induced adaptive response in human B lymphoblast cells

Abstract Purpose: With widespread use of ionizing radiation, more attention has been attracted to low-dose radiation (LDR); however, the mechanisms of long-term LDR-induced bio-effects are unclear. Here, we applied human B lymphoblast cell line HMy2.CIR to monitor the effects of long-term LDR and the potential involvement of DNA methylation. Materials and methods: HMy2.CIR cells were irradiated with 0.032 Gy γ-rays three times per week for 1–4 weeks. Some of these primed cells were further challenged with 2 Gy γ-rays. Cell proliferation, micronuclei formation, gene expression of DNA methyltransferases (DNMT), levels of global genomic DNA methylation and protein expression of methyl CpG binding protein 2 (MeCP2) and heterochromatin protein-1 (HP1) were measured. Results: Long-term LDR enhanced cell proliferation and clonogenicity and triggered a cellular adaptive response (AR). Furthermore, global genomic DNA methylation was increased in HMy2.CIR cells after long-term LDR, accompanied with an increase of gene expression of DNMT1 and protein expression of MeCP2 and HP1. After treatment with 5-aza-2′-deoxycytidine (5-aza-dC), a DNA methyltransferase inhibitor, the long-term LDR-induced global genomic DNA hypermethylation was decreased and the AR was eliminated. Conclusion: Global genomic DNA hypermethylation accompanied with increases of DNMT1 and MeCP2 expression and heterochromatin formation might be involved in long-term LDR-induced adaptive response.

Topotecan‐Loaded Mesoporous Silica Nanoparticles for Reversing Multi‐Drug Resistance by Synergetic Chemoradiotherapy

Multi-drug resistance (MDR) has become a major challenge for the further improvement of chemotherapy. Thus, more effective strategies for further enhancing the treatment against cancer by overcoming MDR are warranted. In this study, by the encapsulation of the radiosensitizing drug TPT into mesoporous silica nanoparticles (MSNs), the combined use of drug-delivered chemotherapy and high-energy X-ray induced radiotherapy could produce synergetic chemoradiotherapeutic effects to kill multi-drug resistant cells through significant DNA damage, thus leading to an efficient circumvention of MDR. We hope that this synergetic dual-mode treatment strategy may achieve higher oncolytic efficacy and find use in future clinical anti-MDR applications.

Cadmium-Induced Adaptive Response in Cells of Chinese Hamster Ovary Cell Lines with Varying DNA Repair Capacity

Abstract Pan, Y., Yuan, D., Zhang, J., Xu, P., Chen, H. and Shao, C. Cadmium-Induced Adaptive Response in Cells of Chinese Hamster Ovary Cell Lines with Varying DNA Repair Capacity. Radiat. Res. 171, 446–453 (2009). The combined exposure to environmental toxicants such as heavy metals and radiation is an important research area in health protection. The aim of this study was to investigate the role of DNA repair and the phosphatidylinositol 3-kinase (PI3K) family in the cadmium-induced adaptive response to toxicants or radiation. Using cells of three Chinese hamster ovary (CHO) cell lines with different capacities to repair DNA damage, we found that pre-exposure to cadmium at a nonlethal concentration could induce an adaptive response to a subsequent challenge with cadmium or radiation in all the cell lines. The magnitude of the adaptive response in adapted cells was dependent on several factors, including DNA repair capacity, the priming dose of cadmium, and the challenging dose of cadmium or radiation. When the cells were challenged with 50 μM CdCl2, the adaptive response was less evident in XRCC1-defective EM-C11 cells than in cells of the other two cell lines. Moreover, treatment of cells with wortmannin or KU-55933 eliminated the adaptive response in all the cell lines. Our data suggest that the triggering of a cadmium-induced adaptive response was independent of DNA repair capacity. Single-strand break (SSB) repair or base excision repair (BER) rather than double-strand break (DSB) repair was mainly involved in the adaptive response. This response of cells to a further challenge with cadmium or radiation may be mediated through the ataxia telangiectasia mutated (ATM) pathway.