Enhancement Of Radiation-induced Apoptosis Research Articles

Withaferin A enhances radiation-induced apoptosis in Caki cells through induction of reactive oxygen species, Bcl-2 downregulation and Akt inhibition

Withaferin A (Wit A), a natural compound derived from the medicinal plant Withania somnifera, has been reported for the anti-tumor effects, including the inhibition of tumor cell growth, metastasis and angiogenesis. In this study, we investigated the effect of Wit A on radiation-induced apoptosis in human renal cancer cells (Caki cells). Our results showed that, compared with Wit A or radiation alone, the combination of both resulted in a significant enhancement of apoptosis, showing the increase in the cleavage of caspase-3 and PARP as well as sub-G1 cell population. In addition, reactive oxygen species (ROS) generation was correlated with the enhancement of radiation-induced apoptosis by Wit A. Wit A downregulated Bcl-2 protein levels and ectopic expression of Bcl-2 in Caki cells attenuated the apoptosis induced by Wit A plus radiation. Taken together, these results indicate that Wit A enhanced radiation-induced apoptosis in Caki cells through ROS generation, down-regulation of Bcl-2 and Akt dephosphorylation. Thus, our study shows that Wit A may be used as an effective radiosensitizer in cancer therapy.

Disruption of Sag/Rbx2/Roc2 induces radiosensitization by increasing ROS levels and blocking NF-κB activation in mouse embryonic stem cells

SAG (sensitive to apoptosis gene; also known as RBX2 or ROC2) is a dual-function protein with antioxidant activity when acting alone or E3 ligase activity when complexed with other components of SCF (Skp1, cullins, F-box proteins) E3 ubiquitin ligases. SAG acts as a survival protein to inhibit apoptosis induced by a variety of stresses. Our recent work showed that SAG siRNA silencing sensitized cancer cells to radiation but the mechanism responsible remains elusive. Here we report that complete elimination of Sag expression via a gene-trapping strategy significantly sensitized mouse embryonic stem (ES) cells to radiation, with a sensitizing enhancement rate of 1.5–1.6. Radiosensitization was associated with increased steady-state levels of intracellular ROS (including superoxide) 24 h after irradiation as well as enhancement of radiation-induced apoptosis. Furthermore, Sag elimination abrogated IκBα degradation leading to inhibition of NF-κB activation. Further detailed analysis revealed that IκBα is a direct substrate of SAG-SCF β-TrCP E3 ubiquitin ligase. Taken together, these results support the hypothesis that Sag elimination via gene disruption sensitizes ES cells to radiation-induced cell killing by mechanisms that involve increased steady-state levels of ROS and decreased activation of NF-κB.

Enhancement of radiation-induced apoptosis by Podophyllum hexandrum.

The aqueous extract of Podophyllum hexandrum (RP-1), which has been recently reported to manifest radioprotective and anti-tumour properties, has been investigated for its mode of action. RP-1, under in-vitro conditions dose-dependently chelated metal ions, inhibited radiation or metal ion-induced hydroxyl radicals and lipid peroxidation and scavenged superoxide anions. Intraperitoneal administration of RP-1 to mice pre-irradiation (10 Gy) induced more DNA fragmentation and lipid peroxidation in thymocytes maximally at 4 and 8 h, respectively, in comparison with RP-1 treatment or irradiation. Flow-cytometric quantification of sub-diploid peak, oligonucleosomal cleavage assay (ladder) and depletion of total thiols also corroborated the ability of RP-1 to enhance radiation-induced apoptosis. RP-1 in presence of 100 microM CuSO(4) induced strand breaks in plasmid DNA and addition of metal chelators (EDTA and deferoxamine) inhibited the strand scission. Treatment with a major constituent of RP-1, podophyllin, did not cause strand breaks, but isolated constituents of RP-1, quercetin or podophyllotoxin, induced strand breaks. Depending on its concentration in the milieu, RP-1 acted as a pro- or antioxidant modifying the radiation-induced apoptosis and therefore could be exploited for cancer management.

Reverse resistance to radiation in KYSE-150R esophageal carcinoma cell after epidermal growth factor receptor signal pathway inhibition by cetuximab

Background and purpose The purpose of our study is to examine the capacity of cetuximab to reverse radiation resistance and investigate molecular mechanisms in human radiation-resistant esophageal carcinoma cell line KYSE-150R. Materials and methods The radioresistant cell line KYSE-150R was established by using fractionated irradiation (FIR). The KYSE-150R cell line was exposed to radiation, treatment with cetuximab, and combined treatment. Cell cycle distribution and apoptosis were analyzed using flow cytometry. Radiation survival was analyzed using clonogenic assays. RT 2 profiler™ PCR array was performed to analyze EGF/PDGF signaling pathway genes. Results The established esophageal carcinoma cell line KYSE-150R showed higher radioresistance than parental cell line. Cetuximab could reverse the radiation resistance of KYSE-150R cells. Cell cycle analysis showed that combination with radiation and cetuximab resulted in the accumulation of cells in G1 and G2/M phases, with the reduction of cells within the S phase. Cetuximab enhanced the apoptosis induced by radiation. RT 2 profiler™ array showed that some intracellular signaling genes deriving from EGF/PDGF signaling pathway regulated by cetuximab. Conclusions Irradiation combined with EGFR blocked by cetuximab may reverse the resistance to radiation in radioresistant esophageal carcinoma cell. The mechanisms may include cell cycle perturbation and enhancement of radiation-induced apoptosis. Further studies are needed to evaluate the role of cetuximab in combination with radiotherapy in the management of esophageal carcinoma.

Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair

We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59 CJ-OH (API; 1-5 micromol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G(1) cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the gamma-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.

Radiosensitization of EMT6 mammary carcinoma cells by 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide

Background and purpose Previously, we have reported that 2-benzoyl-3-phenyl-6,7-dichloroquinoxaline 1,4-dioxide (DCQ) is a radiosensitizer. Here, we investigate the mechanism of radiosensitization. Materials and methods EMT6 cells were treated with DCQ for 4 h prior to ionizing radiation (IR). Flow cytometry, clonogenic assay, TUNEL, and Western blotting were performed to assess the effect of treatment on cells. Results Propidium iodide staining of EMT6 cells treated with IR ± DCQ revealed high numbers of cells with decreased DNA, consistent with an apoptotic response. TUNEL assay revealed apoptosis was 4%, 38%, and 49% 24 h after treatment with IR alone, DCQ alone, and DCQ + IR, respectively. Clonogenic assays revealed that the survival of irradiated EMT6 cells was significantly reduced by DCQ treatment. DCQ treatment abrogated the radiation-induced expression of p21 and p53. The increased apoptosis observed in DCQ + IR-treated cells was correlated to suppression of radiation-induced phosphorylation of Akt and the expression of Bcl-X L. DCQ also caused the phosphorylation of mitogen-activated protein kinases Erk and Jnk. Conclusions The radiosensitization effect of DCQ occurs through enhancement of radiation-induced apoptosis, which correlates to the inhibition of p-Akt kinase and Bcl-X L and the activation of Erk and Jnk kinases, but appears independent of p53 induction or modulation of Bax/Bcl-2 gene expression. These data suggest DCQ should be tested as a radiosensitizer in vivo and has potential in the treatment of human solid tumors.

Enhancement of radiation-induced apoptosis in raji cell line by Bcl-2 antisense oligodeoxynucleotide

Objective: To investigate whether the Bcl-2 antisense oligonucleotide (ASODN) may enhance radiation-induced apoptosis in Raji cell line. Methods: Cell surviving fraction was determined using the trypan blue dye exclusion assay. The expression level of Bcl-2 protein was assayed by immunofluorescence using fluoresce isothiocyanate label. Apoptosis was detected by Giemsa staining and flow cytomertric cell cycle analysis. Results: It was found that Bcl-2 ASODN combined with radiation had significantly reduced the number of viable cells (P<0.05). There was no difference on cell survival between mismatch Bcl-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone. Bcl-2 ASODN combined with radiation could significantly inhibit expression of Bcl-2 protein in Raji cells (P<0.05). Cells treated with Bcl-2 ASODN combined with radiation at 72 h displayed classic apoptotic changes. Apoptosis rates of Raji cells treated with Bcl-2 oligodeoxynucleotide/radiation combination and radiation-treated cells alone, respectively. Conclusion: Bcl-2 antisense oligonucleotide can enhance radiation-induced apoptosis in Raji cell line.

The enhancement of radiation-induced apoptosis by a survivin mutant adenovirus vector

The enhancement of radiation-induced apoptosis by a survivin mutant adenovirus vector

Enhancement of Radiation-induced Apoptosis by 6-Formylpterin

Radiation-induced apoptosis and its possible enhancement in the presence of 6-formylpterin (6-FP), a metabolite of folic acid, were examined in human myelomonocytic lymphoma U937 cells. When cells were treated with 6-FP at a nontoxic concentration of 300 μM, and then exposed to X-rays at a dose of 10 Gy, significant enhancement of radiation-induced apoptosis as determined by nuclear morphological change, phosphatidylserine (PS) externalization and DNA fragmentation were observed. Flow cytometry for the detection of intracellular hydrogen peroxide (H2O2) revealed that 6-FP increased the formation of intracellular H2O2, which further increased when the cells were irradiated. Decrease of mitochondria trans-membrane potential (MMP), release of cytochrome c from mitochondria, and activation of caspase-3 were enhanced after the combined treatment. Remarkable activation of protein kinase C δ (PKC δ) and its translocation from cytosol to mitochondria were detected in combined treatment. Increase of intracellular Ca2+ concentrations ([Ca2+]i) was also observed, however, neither calpain I nor calpain II could inhibit the apoptosis. In addition, c-Jun NH2-terminal kinase ( JNK) activation was not enhanced in the combined treatment. A protein involved in a caspase-independent apoptosis pathway, apoptosis inducing factor (AIF), remained unchanged even 3 h after treatment. These results indicate that intracellular H2O2 generated by 6-FP enhances radiation-induced apoptosis via the mitochondria-mediated caspase-dependent pathway, with the active involvement of PKC δ.

Enhancement of radiation-induced apoptosis by preirradiation with low-dose X-rays in human leukemia MOLT-4 cells.

The effects of low-dose preirradiation on the process of radiation-induced cell death were investigated in human leukemic MOLT-4 cells. By 0.2 Gy of X-rays given 12 h prior to a challenge dose of 5 Gy, the process of apoptosis was accelerated. The acceleration was associated with a certain increase in caspase 3 activity, a disruption of the mitochondrial transmembrane potential, and an accumulation of p53 proteins. This finding is in contrast to the radiation adaptive responses in which a small dose of preirradiation would induce certain radiation resistance and decrease the cell death after irradiation with higher doses.

Head and neck cancer as a clinical model for molecular targeting of therapy: combining EGFR blockade with radiation

Purpose/Objective: The primary purpose of this presentation is to develop the concept that molecular blockade of specific growth factor receptors and signal transduction pathways in combination with radiation will prove a valuable cancer therapeutic strategy. More specifically, the rationale for molecular blockade of the epidermal growth factor receptor (EGFR) system in combination with ionizing radiation for epithelial tumors, such as squamous cell carcinomas (SCCs) of the head and neck (H&N), is described. Methods and Materials: Preclinical experimentation with in vitro and in vivo model systems regarding the capacity of EGFR blockade, using the monoclonal antibody C225, to modulate SCC tumor growth behavior and response to radiation is presented. The rationale for new clinical trials that are currently exploring this concept are presented. Results: Blockade of the EGFR system in SCC cell lines with C225 induces G 1 cell cycle arrest with an associated decrease in the S-phase fraction. Inhibition of tumor cell proliferation is readily measured following C225 exposure and the corresponding alterations in expression of key regulators of the G 1-S cell cycle phase transition are identified. Exposure of SCCs to C225 in culture enhances radiosensitivity following single-dose radiation exposure. Profound augmentation of the in vivo radiation response of SCC tumor xenografts in athymic mice is similarly demonstrated following systemic administration of C225. Preliminary studies are presented regarding potential underlying mechanisms of action for this enhanced tumor response to the combination of C225 and radiation including: (a) proliferative growth inhibition, (b) enhancement of radiation-induced apoptosis, (c) inhibition of damage repair, and (d) downregulation of tumor angiogenic response. Preliminary observations from the Phase III multicenter clinical trial examining C225 plus radiation therapy for advanced H&N cancer patients are provided. Conclusion: Molecular inhibition of the EGFR signal transduction system in combination with radiation represents a promising investigational area in cancer therapeutics. Epithelial tumors that are rich in their expression of EGFR (e.g., SCC of the H&N) hold special promise for receptor blockade approaches. More broadly, the ultimate therapeutic effect of selected molecular agents which block specific growth factor receptors and signaling pathways may be enhanced when delivered in combination with radiation.

Inhibition of the mitogen activated protein kinase pathway potentiates radiation-induced cell killing via cell cycle arrest at the G2/M transition and independently of increased signaling by the JNK/c-Jun pathway.

The ability of low dose ionizing radiation (2 Gy) to modulate the activities of the mitogen activated protein kinase (MAPK) and c-Jun NH2-terminal kinase (JNK1) cascades in human monocytic leukemia (U937/pREP4) cells and in cells over-expressing dominant negative c-Jun (TAM67) (U937/TAM67) was investigated. Radiation exposure caused prolonged ( approximately 1 h) MAPK activations in U937 cells. In contrast, low dose irradiation weakly modulated JNK1 activity in these cells. Inhibition of the MAPK pathway by use of the specific MEK1/2 inhibitor (10 microM PD98059) in both U937/pREP4 and U937/TAM67 cells prior to radiation exposure permitted strong prolonged radiation-induced activations of JNK1. Expression of TAM67 decreased the ability of radiation to cause apoptosis compared to control transfected cells. However, combined MEK1/2 inhibition and radiation exposure in both cell types caused a large decrease in suspension culture growth and a large increase in apoptosis, when compared to either treatment alone. Reduced proliferation after combined irradiation and PD98059 treatment in both cell types correlated with prolonged cell cycle arrest in G2/M phase. Prolonged growth arrest was abolished when MEK1/2 inhibitor was removed 6 h following irradiation, which was associated with a reduction in apoptosis. The ability of MEK1/2 inhibition to cause prolonged G2/M growth arrest was reduced in U937 cells stably transfected with a p21Cip-1/WAF1 antisense construct (U937/p21AS). This data correlated with an enhancement of radiation-induced apoptosis and a reduced ability of MEK1/2 inhibition to potentiate apoptosis. Collectively our data demonstrate that inhibition of MEK1/2 function increases the radiation sensitivity of U937 cells, independently of c-Jun function, and decreases the ability of these cells to recover from the radiation-induced G2/M cell cycle checkpoint arrest. In addition, our data also demonstrate that the ability of MEK1/2 inhibition to potentiate radiation-induced cell death in U937 cells in part requires an ability of cells to express low levels of p21Cip-1/WAF1.

DNA damage-associated cell cycle and cell death control is differentially modulated by caffeine in clones with p53 mutations.

Caffeine is known to potentiate the cytotoxic effects of DNA damaging agents and increases the sensitivity of p53-deficient cells to X-irradiation (X-IR). We have analyzed the cell cycle and cell death control after X-IR in the absence or presence of caffeine in hematological cell lines with various configurations of the p53 gene; EBV-immortalized lymphoblastoid cells with heterozygous p53 mutation (wt/mt), human leukemia cell lines HL60 and KOPM28 with no and mutant p53 expression, respectively. These cell lines display an impaired G0/G1 checkpoint and G2 delay following X-IR, and resistance to apoptosis, which are in accordance with findings previously reported. When irradiated in combination with caffeine, all these cell lines overrode the G2 delay and accumulated at G0/G1. The cell cycle modifications in these cell lines correlated with the increase in radiation-induced p34Cdc2 kinase activity by caffeine. These cell cycle control modifications by caffeine, however, were not associated with enhancement of radiation-induced apoptosis or reduction of clonogenic growth activity in these cell lines. These results suggest that the cytocidal effect of caffeine may need to be verified independently of its cell cycle regulatory activities at least in some cases with p53 mutation.

Modulation of radiation-induced apoptosis by thiolamines

. Exposure to the thiolamine radioprotector N (2-mercaptoethyl)-1,3-propanediamine (WR-1065) induced apoptosis in the mouse TB8.3 hybridoma after a 60-min (LD50 =4.5 mm) or during a 20-h (LD50=0.15 mm) exposure. In contrast, a 20-h exposure to 17 mm l-cysteine or 10 mm cysteamine was required to induce 50% apoptosis within 20 h. Apoptosis was not induced by either a 60-min or 20-h exposure to 10 mm of the thiazolidine prodrugs ribose-cysteine (RibCys) or ribosecysteamine (RibCyst). Thiolamine-induced apoptosis appeared to be a p53-independent process since it was induced by WR-1065 exposure in human HL60 cells. Exposure to WR-1065 (4 mm for 15 min) or cysteine (10 mm for 60 min) before and during irradiation protected cells against the induction of both DNA double-strand breaks and apoptosis, while exposure to RibCys (10 mm for 3 h) did not. Treatment with either WR-1065, cysteine, RibCys or RibCyst for 60 min beginning 60 min after irradiation did not affect the level of radiation-induced apoptosis. In contrast, treatment with either cysteine, cysteamine or RibCys for 20 h beginning 60 min after irradiation enhanced radiationinduced apoptosis. Similar experiments could not be conducted with WR-1065 because of its extreme toxicity. Our results indicate that thiolamine enhancement of radiation-induced apoptosis is not involved in their previously reported capacity to reduce radiation-induced mutations.