Ionizing Radiation Potentiates Dihydroartemisinin-Induced Apoptosis of A549 Cells via a Caspase-8-Dependent Pathway

This report is designed to explore the molecular mechanism by which dihydroartemisinin (DHA) and ionizing radiation (IR) induce apoptosis in human lung adenocarcinoma A549 cells. DHA treatment induced a concentration- and time-dependent reactive oxygen species (ROS)-mediated cell death with typical apoptotic characteristics such as breakdown of mitochondrial membrane potential (Δψ_m), caspases activation, DNA fragmentation and phosphatidylserine (PS) externalization. Inhibition of caspase-8 or -9 significantly blocked DHA-induced decrease of cell viability and activation of caspase-3, suggesting the dominant roles of caspase-8 and -9 in DHA-induced apoptosis. Silencing of proapoptotic protein Bax but not Bak significantly inhibited DHA-induced apoptosis in which Bax but not Bak was activated. In contrast to DHA treatment, low-dose (2 or 4 Gy) IR induced a long-playing generation of ROS. Interestingly, IR treatment for 24 h induced G2/M cell cycle arrest that disappeared at 36 h after treatment. More importantly, IR synergistically potentiated DHA-induced generation of ROS, activation of caspase-8 and -3, irreparable G₂/M arrest and apoptosis, but did not enhance DHA-induced loss of Δψ_m and activation of caspase-9. Taken together, our results strongly demonstrate the remarkable synergistic efficacy of combination treatment with DHA and low-dose IR for A549 cells in which IR potentiates DHA-induced apoptosis largely by enhancing the caspase-8-mediated extrinsic pathway.     

Ionizing radiation can overcome resistance to TRAIL in TRAIL-resistant cancer cells

Although the majority of cancer cells are killed by TRAIL (tumor necrosis factor-related apoptosis-inducing ligand treatment), certain types show resistance to it. Ionizing radiation also induces cell death in cancer cells and may share common intracellular pathways with TRAIL leading to apoptosis. In the present study, we examined whether ionizing radiation could overcome TRAIL resistance in the variant Jurkat clones. We first selected TRAIL-resistant or -sensitive Jurkat clones and examined cross-responsiveness of the clones between TRAIL and radiation. Treatment with gamma-radiation induced significant apoptosis in all the clones, indicating that there seemed to be no cross-resistance between TRAIL and radiation. Combined treatment of radiation with TRAIL synergistically enhanced killing of TRAIL-resistant cells, compared to TRAIL or radiation alone. Apoptosis induced by combined treatment of TRAIL and radiation in TRAIL-resistant cells was associated with cleavage of caspase-8 and the proapoptotic Bid protein, resulting in the activation of caspase-9 and caspase-3. No changes in the expressions of TRAIL receptors (DR4 and DR5) and Bcl-2 or Bax were found after treatment. The caspase inhibitor z-VAD-fmk completely counteracted the synergistic cell killing induced by combined treatment of TRAIL and gamma-radiation. These results demonstrated that ionizing radiation in combination with TRAIL could overcome resistance to TRAIL in TRAIL-resistant cells through TRAIL receptor-independent synergistic activation of the cascades of the caspase-8 pathway, suggesting a potential clinical application of combination treatment of TRAIL and ionizing radiation to TRAIL-resistant cancer cells.     

A conserved Asn in TM7 of the thyrotropin receptor is a common requirement for activation by both mutations and its natural agonist

Arsenic trioxide (As2O3) inhibits cell growth and induces apoptosis in certain types of cancer cells including acute promyelocytic leukemia, prostate and ovarian carcinomas, but its effect on response of tumor cells to ionizing radiation has never been explored before. Here we demonstrate that As2O3 can sensitize human cervical cancer cells to ionizing radiation both in vitro and in vivo. As2O3 in combination with ionizing radiation have a synergistic effect in decreasing clonogenic survival and in the regression of established human cervical tumor xenografts. Pretreatment of the cells with As2O3 also synergistically enhanced radiation-induced apoptosis. Apoptosis of the cells by combined treatment of As2O3 and radiation was associated with reactive oxygen species generation and loss of mitochondrial membrane potential, resulting in the activation of caspase-9 and caspase-3. The combined treatment also resulted in an increased G2/M cell cycle distribution at the concentration of As2O3 which did not alter cell cycle when applied alone. These results indicate that As2O3 can synergistically enhance radiosensitivity of human cervix carcinoma cells in vitro and in vivo, suggesting a potential clinical applicability of combination treatment of As2O3 and ionizing radiation in cancer therapies.     

Futile caspase-8 activation during the apoptotic cell death induced by DNA damaging agents in human B-lymphoblasts

Caspase-8 plays an essential role in apoptosis induced by Fas activation. Moreover, caspase-8 can be processed also in response to exposure to genotoxic agents. To decipher the role of caspase-8 in DNA damaging agent (DDA)-induced apoptosis as well as the pathway(s) leading to its activation in response to genotoxic stress, we investigated caspase-8 processing induced by ionizing radiation (IR) or mitomycin C (MMC) treatment in human B-lymphoblasts. Altogether, our observations establish that caspase-8 is actively processed in both receptor-mediated and DDA-induced cell death. However, while Fas-dependent apoptosis absolutely required caspase-8 activity, it is not necessary for completion of the apoptotic program induced by IR and MMC. Experiments performed to understand the molecular pathway(s) of the caspase-8 activation after DDA demonstrated that for both IR and MMC, the Fas/Fas-L interaction is dispensable. Data obtained from caspase inhibitors and from lymphoblasts carrying mutations in ATM and FANCC proteins, involved in DDA response, clearly showed that distinct mechanisms are responsible for caspase-8 activation by IR and MMC in B-lymphoblasts. IR-dependent processing of caspase-8 involves ATM, mitochondrial collapse, FANCC, and caspase-3 activation. Caspase-8 activation by MMC evokes the mitochondrial pathways involving FANCC but not ATM. Collectively, our data indicate that caspase-8 activation is essentially a bystander effect and not a major determinant of the behavior of DDA-exposed cells.     

Loss of the Fanconi anemia group C protein activity results in an inability to activate caspase-3 after ionizing radiation

Fanconi anemia (FA) is a human genetic disease featuring cancer predisposition, genetic instability and DNA damage hypersensitivity. Although abnormalities in DNA repair and cell cycle checkpoint have been proposed as the underlying defect in this syndrome, these hypotheses did not provide full explanations of the complex phenotype. Although not exclusive of such possibilities, alterations in the control of apoptosis might account for the pleiotropic phenotype of this syndrome. We and others have previously reported a deregulation of the apoptotic response to mitomycin C, suggesting that the products of the Fanconi anemia group C protein (FANCC) contribute to the regulation of apoptosis. To explore the functional importance of the apoptotic alterations in FA we analyzed biochemical steps of the execution phase of apoptosis stimulated by another DNA damaging agent, the gamma-ray using FA cell lines derived from complementation group C (FA-C) independent patients. It is shown that the poly(ADP-ribose) polymerase, a target of caspase-3, is not cleaved in FA-C after ionizing radiation (IR). Moreover, caspase-3 is not processed in its active form and, its activity is not increased by IR in FA-C cells compared to normal cells. Altogether, these results demonstrate that loss of the FANCC activity results in a deficiency of the IR-induced apoptosis which is due to an inability to activate caspase-3. Our work suggests that apoptosis signaling induced by mitomycin C and IR is subject to common regulation involving the FANCC protein.     

Inhibition of Ephrin B3-mediated survival signaling contributes to increased cell death response of non-small cell lung carcinoma cells after combined treatment with ionizing radiation and PKC 412

Radiation therapy is frequently used to treat non-small cell lung cancers (NSCLCs). We have previously shown that a combination of ionizing radiation (IR) and the staurosporine analog PKC 412, but not Ro 31–8220, increases cell death in NSCLC cells. To identify genes involved in the enhancement of cell death, a total gene profiling in response to co-administration of (i) PKC 412 with IR, or (ii) Ro 31–8220 with IR was implemented. These combined treatments caused upregulation of 140 and 179 genes and downregulation of 253 and 425 genes, respectively. Certain genes were selected and verified by real-time quantitative PCR and, of these genes, robust suppression of Ephrin B3 expression was suggested as a possible cell death-inducing mechanism of combined treatment with IR and PKC 412. Indeed, silencing of Ephrin B3 using siRNA in NSCLC cells resulted in a major alteration of their morphology with an elongated phenotype, decreased proliferation and increased cell death signaling. Moreover, silencing of Ephrin B3 in combination with IR caused a decrease in IR-mediated G₂-arrest, induced cellular senescence, inhibited MAPK ERK and p38 phosphorylation, and caused an upregulation of p27^kip1 expression. Finally, silencing of Ephrin B3 in combination with IR sensitized U-1810 cells to IR-induced apoptosis. In conclusion, we identify and describe Ephrin B3 as a putative signaling molecule involved in the response of NSCLC cells to combined treatment with PKC 412 and ionizing radiation.     

Gene expression profiles of normal human fibroblasts after exposure to ionizing radiation: a comparative study of low and high doses

Several types of cellular responses to ionizing radiation, such as the adaptive response or the bystander effect, suggest that low-dose radiation may possess characteristics that distinguish it from its high-dose counterpart. Accumulated evidence also implies that the biological effects of low-dose and high-dose ionizing radiation are not linearly distributed. We have investigated, for the first time, global gene expression changes induced by ionizing radiation at doses as low as 2 cGy and have compared this to expression changes at 4 Gy. We applied cDNA microarray analyses to G1-arrested normal human skin fibroblasts subjected to X irradiation. Our data suggest that both qualitative and quantitative differences exist between gene expression profiles induced by 2 cGy and 4 Gy. The predominant functional groups responding to low-dose radiation are those involved in cell-cell signaling, signal transduction, development and DNA damage responses. At high dose, the responding genes are involved in apoptosis and cell proliferation. Interestingly, several genes, such as cytoskeleton components ANLN and KRT15 and cell-cell signaling genes GRAP2 and GPR51, were found to respond to low-dose radiation but not to high-dose radiation. Pathways that are specifically activated by low-dose radiation were also evident. These quantitative and qualitative differences in gene expression changes may help explain the non-linear correlation of biological effects of ionizing radiation from low dose to high dose.     

HBXIP,a binding protein of HBx,regulates maintenance of the G2/M phase checkpoint induced by DNA damage and enhances sensitivity to doxorubicin-induced cytotoxicity

To maintain the integrity of the genome, cells need to detect and repair DNA damage before they complete cell division. Hepatitis B x-interacting protein (HBXIP), a binding protein of HBx (Hepatitis B virus × protein), is aberrantly overexpressed in human cancer cells and show to promote cell proliferation and inhibit apoptosis. The present study is designed to investigate the role of HBXIP on the DNA damage response. Our results show that HBXIP acts as an important regulator of G2/M checkpoint in response to DNA damage. HBXIP knockdown increases phospho-histone H2AX expression and foci formation after treatment with ionizing radiation (IR). HBXIP regulates the ATM-Chk2 pathway following DNA damage. Depletion of HBXIP abrogates IR-induced G2/M cell cycle checkpoints, accompanying decrease the expression of phospho-Cdc25C, phospho-Cdc2 (Tyr15) and p27. We also show that downregulation of HBXIP expression sensitizes cancer cells to chemotherapy, as evidenced by an increase in apoptosis and cleavage of caspase-3 and caspase-9. Our data suggest that HBXIP can function as a mediator protein for DNA damage response signals to activate the G2/M checkpoint to maintain genome integrity and prevent cell death.     

Co-activation of ERK, NF-kappaB, and GADD45beta in response to ionizing radiation

NF-kappaB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45beta is a primary cell cycle element responsive to NF-kappaB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-kappaB, ERK and GADD45beta are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-kappaB transactivation and GADD45beta expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-kappaB. Transfection of dominant negative mutant IkappaB that blocks NF-kappaB nuclear translocation, inhibited ERK activity and GADD45beta expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45beta strikingly blocked IR-induced NF-kappaB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-kappaB, ERK, and GADD45beta are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.     

A critical role for AKT activation in protecting cells from ionizing radiation-induced apoptosis and the regulation of acinus gene expression

Although AKT activation leads to the activation of various pathways related to cell survival, the roles of AKT in modulating cellular responses induced by ionizing radiation in normal human cells remain unclear. Here we show that low-dose radiation of 0.05 Gy did not affect cell death, but high-dose radiation (> 0.2 Gy) induced apoptosis through the activation of caspases and acinus cleavage. Ionizing radiation induced acinus phosphorylation via AKT activation. Thus, we examined the effect of AKT activation on radiation-induced cell death using CCD-18Lu cells transduced with a retroviral vector expressing constitutively active AKT (CA-AKT). The overexpression of CA-AKT rendered the cells resistant to ionizing radiation and prevented the proteolytic cleavage of acinus via phosphorylation. In addition, overexpression of CA-AKT resulted in the upregulation of acinus expression by activation of the NF-κB pathway. On the other hand, suppression of endogenous AKT expression by siRNA resulted in the reduction of acinus expression and enhanced the radiation-induced apoptosis in both CCD-18Lu and IM-9 cells. Our results suggest that AKT activation inhibits cell death during radiation-induced apoptosis through the regulation of phosphorylation and expression of acinus. The AKT/NF-κB/acinus pathway functions as one of the important regulatory mechanisms required for modulating ionizing radiation sensitivity.     

Trifluoperazine stimulates ionizing radiation induced cell killing through inhibition of DNA repair

The effect of trifluoperazine (TFP), a phenothiazine derivative antipsychotic drug, on ionizing radiation (IR) induced cell killing through inhibition of DNA repair was investigated in human cell lines. In clonogenic survival assay, TFP augmented IR induced cell killing. Also, TFP enhanced micronucleus formation in irradiated human lymphocytes. The effect of TFP and other known DNA repair inhibitors like wortmannin and caffeine, on irradiated cells, was compared by MTT assay. On the other hand, TFP failed to increase the toxicity induced by H2O2. Repair of DNA double strand breaks induced by IR was markedly inhibited by TFP, as determined by field inversion gel electrophoresis (FIGE). Further, TFP increased radiation induced apoptosis, which was accompanied by enhanced G2/M arrest. Thus, our results strongly suggest that TFP inhibits repair of DNA damage induced by IR, which significantly implicates the possibility of using TFP as an adjuvant to radiotherapy.     

ATM blocks tunicamycin-induced endoplasmic reticulum stress

Endoplasmic reticulum stress (ER-stress) is associated with ataxia telangiectasia mutated (ATM) gene. We present here conclusive data showing that ATM blocks ER-stress induced by tunicamycin or ionizing radiation (IR). X-box protein-1 (XBP-1) splicing, GRP78 expression and caspase-12 activation were increased by tunicamycin or IR in Atm-deficient AT5BIVA fibroblasts. Activation of caspase-12 and caspase-3 by tunicamycin was significantly reduced in cells transfected with wild-type Atm (AT5BIVA/wtATM). Atm knockdown by siRNA, however, noticeably elevated ER-stress and chemosensitivity to tunicamycin. In summary, we present substantial data demonstrating that ATM blocks the ER stress signaling associated with cancer cell proliferation.     

Cell-type-dependent induction of eotaxin and CCR3 by ionizing radiation

Eotaxin is an eosinophil-specific C-C chemokine that is implicated in the pathogenesis of eosinophilic inflammatory diseases, such as asthma and atopic dermatitis, by acting specifically on its receptor CCR3. Using RT-PCR analysis, we show that the expression of eotaxin is upregulated upon treatment with ionizing radiation (IR) in human dermal fibroblasts, but not in the bronchial epithelial cell line A549. In contrast, the gene encoding CCR3 is markedly induced in both cell types. None of the genes coding for other CCR3 ligands are significantly induced by IR in these cell types. cDNA array analysis of irradiated versus nonirradiated A549 cells and human dermal fibroblasts confirm and extend these results, and support the observation that regulation of eotaxin/CCR3-induction by IR occurs in a selective and cell-type-dependent manner. They further suggest that the induction of signaling via eotaxin and CCR3 may be an important step leading to eosinophilia in patients with radiation exposure.     

FPTB, a novel CA-4 derivative, induces cell apoptosis of human chondrosarcoma cells through mitochondrial dysfunction and endoplasmic reticulum stress pathways

Chondrosarcoma is a malignant primary bone tumor that responds poorly to both chemotherapy and radiation therapy. The aim of this study was to elucidate the mechanism of the novel Combretastatin A-4 derivative, 2-(furanyl)-5-(pyrrolidinyl)-1-(3,4,5-trimethoxybenzyl)benzoimidazole (FPTB)-induced human chondrosarcoma cells apoptosis. FPTB induced cell apoptosis in human chondrosarcoma cell line but not primary chondrocytes. FPTB induced up-regulation of Bax and Bak, down-regulation of Bcl-2 and Bcl-XL and dysfunction of mitochondria in chondrosarcoma. FPTB also triggered endoplasmic reticulum (ER) stress, as indicated by changes in cytosol-calcium levels. We found that FPTB increased glucose-regulated proteins (GRP)78 but not GRP94 expression. In addition, treatment of cells with FPTB induced calpain expression and activity. Transfection of cells with GRP78 or calpain siRNA reduced FPTB-mediated cell apoptosis. Therefore, FPTB-induced apoptosis in chondrosarcoma cells through the mitochondria dysfunction and involves caspase-9 and caspase-3-mediated mechanism. FPTB also induced cell death mediated by increasing ER stress, GPR78 activation, and Ca(2+) release, which subsequently triggers calpain, caspase-12 and caspase-3 activity, resulting in apoptosis.     

Down-regulation of thiamine transporter THTR2 gene expression in breast cancer and its association with resistance to apoptosis

The recent molecular identification of two thiamine transporters, SLC19A2 (THTR1) and SLC19A3 (THTR2), has provided the opportunity to study thiamine transporter gene expression in human malignancies. We compared RNA levels of both THTR1 and THTR2 in a panel of human breast tumors and corresponding normal tissues. THTR2 RNA levels were down-regulated in breast cancer to 14% of the level found in corresponding normal tissues, while THTR1 levels were unchanged. Both thiamine transport genes were cloned and expressed in a breast cancer cell line to examine the impact of reconstituted thiamine transport gene expression on drug and radiation sensitivity and on resistance to apoptosis. THTR2-transfected breast cancer cells showed a 2.5-fold increase in specific THTR2 activity and a 3-fold increase in cytotoxicity against a bromoacetyl ester derivative of thiamine. Surprisingly, these cells also showed a 3-fold increase in sensitivity to doxorubicin and an increase in sensitivity to ionizing radiation, but no change in sensitivity to methotrexate or paclitaxel. TUNEL assays demonstrate an increase in apoptosis in THTR2-transfected cells exposed to doxorubicin and radiation, and Western blot analysis suggests that apoptosis associated with these cytotoxic stresses is mediated at least in part by a caspase-3-dependent pathway. Therefore, thiamine transporter THTR2 gene expression is down-regulated in breast cancer, which may contribute to resistance to apoptosis in these tumors.