Study of the radiosensitization of docetaxel in human esophageal squamous carcinoma ECA109 cell line.

The aim of this study was to determine the radio sensitization of docetaxel in human esophageal squamous carcinoma ECA109 cell line by observing the effects of docetaxel in ECA109 cell proliferation, cell cycle distribution, apoptosis rate and related protein expression. Docetaxel inhibits the proliferation in ECA109 cell line in a dose-dependent and time-dependent manner, and 1nM was chosen for radio sensitization according to the inhibition curves. The D0 and SF2 values of ECA109 cells were 3.00Gy and 0.95, respectively, and of docetaxel (1nM) with irradiation group were 2.54Gy and 0.88. G0/G1 decreased (P<0.05), G2/M phase saw a spike (P<0.05) in the docetaxel with radiation group at 12h, 24h and 48h, while the apoptotic index witnessed a surge at 24h and 48h (P<0.05). The docetaxel with radiation group obtained a higher expression of p21 and bax protein than the docetaxel group and the radiation group (P<0.05), and a higher ratio of bcl-2/bax than the others (P<0.05). Docetaxel could inhibit the proliferation in ECA109 cell line. p21, bax, bcl-2 and other related proteins can regulate cell cycle phase distribution and induce cell apoptosis, thereby increasing the radiosensitivity effect of docetaxel in ECA109 cell line.

The Evaluation of Survivin and Bcl-2 Expression on the Medical Radiation Doses for Neural Tube Defect Development.

AIM: To investigate the effects of different radiation doses on the development of the neural tube defect in chick embryos using computed tomography (CT), and assess its correlation with survivin and Bcl-2 expressions. MATERIAL AND METHODS: A total of 150 chicken eggs were used and grouped into five categories. In Group 1 (n=30), the embryos were not exposed to radiation. In Group 2 (n=30), the embryos were irradiated using lung cancer screening chest CT protocol. In Groups 3 and 4 (n=30 each), the abdominopelvic and adult routine head CT protocols, respectively, were used to irradiate the embryos. In Group 5 (n=30), the embryos were irradiated using adult brain perfusion CT protocol. Subsequently, the embryos were examined under a stereomicroscope to assess the presence of neural tube developmental abnormalities. Moreover, immunohistochemical staining was performed to determine the survivin and Bcl-2 expression levels. RESULTS: The risk of developing neural tube defect increased with the amount of exposed radiation. Moreover, no significant correlation was observed between the survivin and Bcl-2 expression levels and the radiation dose. CONCLUSION: Overall, the results of this study indicate that the radiation from CT may cause neural tube defect in chicken embryos.

The Effect of Indocyanine Green Antimicrobial Photothermal/Photodynamic Therapy on the Expression of BCL-2 and BAX Messenger RNA Levels in Human Gingival Fibroblast Cells.

BACKGROUND: Antimicrobial photothermal/photodynamic therapy (PTT/PDT) with indocyanine green (ICG) is an adjuvant therapeutic approach in the treatment of periodontitis. To explore whether PTT/PDT with ICG causes cell death by apoptosis in human gingival fibroblast (HGF) cells, BAX and BCL-2 genes expression as key events for apoptosis were evaluated in this study. MATERIALS AND METHODS: HGF cells were treated with: 1) different concentrations (500-2000 µg/mL) of ICG alone, 2) Diode laser irradiation alone with a fluency of 39.06 J/cm2; 3) PTT/PDT combined different concentrations (500-2000 µg/mL) of ICG with an 808 nm diode laser with a fluency of 39.06 J/cm2, and 4) controls (untreated cells). After that, BAX and BCL-2 messenger RNA levels were evaluated by real-time quantitative reverse transcription PCR. RESULTS: PTT/PDT with 500 µg/mL of ICG caused significant increases in the expression of the BAX gene, with an 8.5-fold increase, which was approximately 7- and 8.5-fold higher than PTT/PDT with ICG for 1500 and 2000 µg/mL of ICG, respectively, indicating induction of apoptosis in HGF cells. ICG (in different test concentrations), diode laser, and PTT/PDT with ICG (1500 and 2000 µg/mL of ICG) treatment displayed insignificant increases in expression levels of BAX (all p>0.05). Our experiments showed an insignificant increase (1.1-1.6-fold) in the expression of BCL-2 after ICG, diode laser, and PTT/PDT with ICG treatment (all p>0.05). CONCLUSIONS: This study suggests that various concentration of ICG can be the diverse expression of BAX responses to PTT/PDT on HGF cells.

Thymoquinone Enhances the Effect of Gamma Knife in B16-F10 Melanoma Through Inhibition of Phosphorylated STAT3.

BACKGROUND: Patients with brain metastasis from melanoma have a dismal prognosis with poor survival time. Gamma Knife (GK) is an effective treatment to control brain metastasis from melanoma. Thymoquinone (TQ) has emerged as a potential therapeutic option due to its antiproliferative effects on various cancers. The purpose of the study was to assess the effect of GK on B16-F10 melanoma cells in vitro and intracerebral melanoma in vivo, and its synergistic effect in combination with TQ. METHODS: The effects of GK and combination treatment of GK and TQ were studied on B16-F10 melanoma cells by evaluating cytotoxicity with an adenosine triphosphate assay, apoptosis by acridine orange staining, and genotoxicity by comet assay. Western blot analysis was performed to investigate the expression of STAT3, p-STAT3 (Tyr705), JAK2, p-JAK2, caspase-3, Bax, Bcl-2, survivin, and ß-actin. Expression of inflammatory cytokines was assessed by enzyme-linked immunosorbent assay. GK alone and in combination with TQ was assessed in an established intracerebral melanoma tumor in mice. RESULTS: The effects of GK on cytotoxicity, genotoxicity, and apoptosis were enhanced by TQ in B16-F10 melanoma cells. GK induced apoptosis through inhibition of p-STAT3 expression, which in turn regulated pro- and antiapoptotic proteins such as caspase-3, Bax, Bcl-2, and survivin. Adding TQ to GK irradiation further enhanced this apoptotic effect of GK irradiation. GK was shown to reduce the levels of tumor-related inflammatory cytokines in B16-F10 melanoma cells. This effect was more pronounced when TQ was added to GK irradiation. GK with 15 Gy increased the survival of mice with intracerebral melanoma compared with untreated mice. However, despite the additive effect of TQ in addition to GK irradiation on B16-F10 melanoma cells in vitro, TQ did not add any significant survival benefit to GK treatment in mice with intracerebral melanoma. CONCLUSIONS: Our findings suggest that TQ would be a potential therapeutic agent in addition to GK to enhance the antitumor effect of irradiation. Further studies are required to support our findings.

Long-term exposure to 835 MHz RF-EMF induces hyperactivity, autophagy and demyelination in the cortical neurons of mice.

Radiofrequency electromagnetic field (RF-EMF) is used globally in conjunction with mobile communications. There are public concerns of the perceived deleterious biological consequences of RF-EMF exposure. This study assessed neuronal effects of RF-EMF on the cerebral cortex of the mouse brain as a proxy for cranial exposure during mobile phone use. C57BL/6 mice were exposed to 835 MHz RF-EMF at a specific absorption rate (SAR) of 4.0 W/kg for 5 hours/day during 12 weeks. The aim was to examine activation of autophagy pathway in the cerebral cortex, a brain region that is located relatively externally. Induction of autophagy genes and production of proteins including LC3B-II and Beclin1 were increased and accumulation of autolysosome was observed in neuronal cell bodies. However, proapoptotic factor Bax was down-regulted in the cerebral cortex. Importantly, we found that RF-EMF exposure led to myelin sheath damage and mice displayed hyperactivity-like behaviour. The data suggest that autophagy may act as a protective pathway for the neuronal cell bodies in the cerebral cortex during radiofrequency exposure. The observations that neuronal cell bodies remained structurally stable but demyelination was induced in cortical neurons following prolonged RF-EMF suggests a potential cause of neurological or neurobehavioural disorders.

On the mechanism of cellular toxicity in breast cancer by ionizing radiation and chemotherapeutic drugs.

Breast cancer is the second leading cause of cancer mortality and the most frequent cancer found in women around the globe. The development of breast cancer is a multistep and complicated process that includes the development of ductal and lobular cells into atypical hyperplasia, carcinoma in situ, and invasive carcinoma, with an ability to metastasize. The efficacy of radiotherapy in breast cancer seems to be reduced because of a frequently observed lack of cellular sensitivity to apoptosis. Both Bcl-2 and p53 are linked to apoptosis pathways and are known to play a role in the outcome of radiotherapy. Resistance of tumor cells to therapeutic drugs and the undesirable cytotoxicity of normal cells are frequently observed in treatment outcomes in clinics. Research is, therefore, needed to develop strategies for improving the protocols of chemotherapy and radiotherapy in patients with breast cancer. This review focuses on understanding the molecular mechanisms of enhanced tumor cell killing by the combined action of certain anticancer drugs together with gamma radiation in vitro, with possible implications for practical applications in clinics.

Candidate tumour suppressor Fau regulates apoptosis in human cells: an essential role for Bcl-G.

FAU, which encodes a ubiquitin-like protein (termed FUBI) with ribosomal protein S30 as a carboxy-terminal extension, has recently been identified as a pro-apoptotic regulatory gene. This activity may be mediated by Bcl-G (a pro-apoptotic member of the Bcl-2 family) which can be covalently modified by FUBI. FAU gene expression has been shown to be down-regulated in human breast, prostate and ovarian tumours, and this down-regulation is strongly associated with poor prognosis in breast cancer. We demonstrate here that ectopic FAU expression increases basal apoptosis in human T-cell lines and 293T/17 cells, whereas it has only a transient stimulatory effect on ultraviolet-C (UVC)-induced apoptosis. Conversely, siRNA-mediated silencing of FAU gene expression has no effect on basal apoptosis, but attenuates UV-induced apoptosis. Importantly, prior knockdown of Bcl-G expression ablates the stimulation of basal apoptosis by FAU, consistent with an essential downstream role for Bcl-G, itself a candidate tumour suppressor, in mediating the apoptosis regulatory role of FAU. In 293T/17 cells, Bcl-G knockdown also attenuates UV-induced apoptosis, so that Bcl-G may constitute a common factor in the pathways by which both FAU and UV-irradiation induce apoptosis. UV irradiation increases Bcl-G mRNA levels, providing an explanation for the transient nature of the effect of ectopic FAU expression on UV-induced apoptosis. Since failure of apoptosis is fundamental to the development of many cancers, the pro-apoptotic activity of the Fau/Bcl-G pathway offers an attractive explanation for the putative tumour suppressor role of FAU.

Beta-lapachone suppresses radiation-induced activation of nuclear factor-kappaB.

Anticancer effects of beta-lapachone (beta-lap) are due to generation of ROS and metabolic catastrophes as a result of NAD(P)H:quinone oxidoreductase (NQO1)-mediated futile cycling between the oxidized and reduced forms of beta-lap. It has been shown that NQO1 is also essential for the TNF-induced activation of NF-kappaB and that beta-lap suppresses the TNF-induced NF-kappaB activation. We investigated whether or not NQO1 is involved and beta-lap suppresses the radiation-induced NF-kappaB activation using A549 human lung cancer cells and NQO1-knock down A549 cells (shNQO1 A549 cells). Irradiation with 4 Gy markedly increased the DNA binding activity of NF-kappaB in A549 cells, but not in the shNQO1 A549 cells, thus demonstrating that NQO1 plays a pivotal role in irradiation-induced NF-kappaB activation. Treatment with 10 micronM beta-lap for 4 h almost completely abrogated the radiation-induced increase in NF-kappaB activation and the transcription of NF-kappaB target genes such as bcl2, gadd45beta and cyclinD1. Moreover, beta-lap markedly suppressed the activation of IkappaB kinase gamma (IKKgamma) and the subsequent phosphorylation of IkappaBalpha, thereby inhibiting NF-kappaB activation. It is concluded that beta-lap suppresses the radiation-induced activation of NF-kappaB by interrupting the involvement of NQO1 in the activation of NF-kappaB, thereby inhibiting the transcription of survival signals. The radiosensitization caused by beta-lap may, in part, be attributed to beta-lap-induced suppression of NF-kappaB activation.

Changes of apoptosis, p53, and bcl-2 by irradiation in poorly differentiated thyroid carcinoma cell lines: a prognostic marker for the prospect of therapeutic success?

BACKGROUND: Poorly differentiated thyroid carcinoma (PDTC) has an unfavorable prognosis. Surgical management is the principal treatment approach. In addition, radioiodine treatment and external beam radiotherapy (EBRT) are given to reduce the risk of local relapse. Despite aggressive therapy, the response to treatment tends to become increasingly poorer over time. The objective of this study was to investigate the induction of apoptosis by EBRT as a function of p53 and bcl-2 protein levels in PDTC. The predictive value of these molecules with respect to treatment efficacy was evaluated. MATERIALS AND METHODS: Two different cell lines of PDTC (FTC-133 and ML-1) were irradiated with a dose of 30 Gy. Apoptotic cells were quantified using terminal deoxynucleotidyltransferase-dUTP nick-end labeling staining without irradiation, 48 and 96 hours after irradiation. The protein levels of p53 and bcl-2 were measured simultaneously using flow cytometry and western blotting. The cell cycle distribution was determined. RESULTS: Untreated FTC-133 cells showed a high rate of apoptosis, a high protein level of p53, and a low bcl-2 protein level. Forty-eight hours after irradiation, a slight reduction in apoptotic cells was observed in conjunction with an increase in bcl-2 and p53 protein levels. The slightly reduced fraction of apoptotic cells remained at the same level up to 96 hours after irradiation, whereas the p53 protein level was further downregulated. The cell cycle distribution showed a significant G2/M arrest after 48 hours and recovery 96 hours after irradiation. ML-1 cells did not show any detectable p53 levels and revealed a low rate of apoptosis which significantly increased 48 hours after irradiation. Ninety-six hours after irradiation, a decrease in apoptosis was detectable. The protein level of bcl-2 increased significantly within 48 hours and decreased 96 hours after irradiation. The cell cycle distribution showed a G2/M arrest after 48 hours without a recovery 96 hours after irradiation. CONCLUSIONS: The p53 and bcl-2 expression profiles and the observed apoptotic rates of FTC-133 and ML-1 under irradiation are consistent with a more aggressive FTC-133 phenotype. Alterations in p53- and bcl-2 protein levels yield predictive information for EBRT efficacy.

Adenovirus-mediated siRNA targeting Mcl-1 gene increases radiosensitivity of pancreatic carcinoma cells in vitro and in vivo.

BACKGROUND: Myeloid cell leukemia-1 (Mcl-1), an anti-apoptotic member of the B cell lymphoma/leukemia-2 (Bcl-2) family, has been shown to be involved in apoptosis and the cell cycle. Mcl-1 is overexpressed in many malignancies, including pancreatic cancer. The aim of this study was to investigate the effect of siRNA targeted against Mcl-1 on the radiosensitivity of human pancreatic carcinoma cells. METHODS: In 3 pancreatic cancer cell lines, the expression of Mcl-1 mRNA was determined by RT-PCR assay, and the dose-dependent cytotoxicity of radiation was also assessed. Furthermore, the effects of adenovirus-mediated siRNA targeted against Mcl-1 on radiosensitivity of PANC-1 cells both in vitro and in vivo were evaluated. RESULTS: Pancreatic cancer cells showed various levels of Mcl-1 mRNA that were correlated with the radiosensitivity of tumor cells. AdU6/shMcl-1 significantly downregulated the expression of Mcl-1 gene in PANC-1 cells, the most radioresistant cell line. Furthermore, Mcl-1 downregulation could significantly enhance radiosensitivity of PANC-1 cells in vitro and in vivo. The mechanism might be correlated with apoptosis enhancement by activating celluar caspase-3. CONCLUSION: Mcl-1 might be a therapeutic target for radiosensitization of pancreatic carcinoma cells. Adenovirus-mediated siRNA targeting of Mcl-1 could enhance the radiosensitivity of pancreatic cancer cells both in vitro and in vivo, and thus might be a potential strategy for ameliorating cancer.

[Experimental study on apoptosis of gastric cancer cell line MKN45 induced by continuous irradiation from iodine-125 seeds].

OBJECTIVE: To investigate the apoptotic effect of iodine-125 seeds on gastric cancer cell line MKN45 and its changes, including the changes of cellular morphosis, early apoptotic rate and Bcl-2/Bax expression, and to provide further evidence for the clinical application of iodine-125 seeds on gastric cancer. METHODS: Human gastric cancer cell line MKN45 in treatment group was continuously exposed to discoid radiative sources made by seven iodine-125 seeds, while the control group not treated. 72 hours later, the gastric cancer cells in each group were collected, and observed respectively under light and electron microscope. Apoptotic rate were detected with DAPI staining. Early apoptotic rate was detected by flow cytometry (FCM). And Bcl-2/Bax expression was detected by RT-PCR. RESULTS: 1) Typical morphologic changes of apoptotic cells were observed under light and electronic microscope in treatment group. 2) Apoptotic rate detected with DAPI staining in treatment group was higher than that in control group (p<0.01). 3) Early apoptotic rate in treatment group was higher than that in control group (P<0.01). 4) Increased intracellular Bax expression level detected by RT-PCR in treatment group was observed (P<0.01). 5) Bax expression level in treatment group was increased obviously, with the decrese of Bcl-2 expression by semi-quantitative RT-PCR detection. The ratio of Bcl-2 to Bax in treatment group was lower than that in control group. CONCLUSION: Continous irradiation by iodine-125 seeds can induce apoptosis of human gastric cancer cell line MKN45. The decreasing ratio of Bcl-2 to Bax may be one of the mechanism of human gastric cancer cell line MKN45's apoptosis.

Effect of 6mT static magnetic field on the bcl-2, bax, p53 and hsp70 expression in freshly isolated and in vitro aged human lymphocytes.

An increasing number of evidence indicates that static magnetic fields (SMFs) are capable of altering apoptosis, mainly through modulation of Ca(2+) influx. Here we present data that suggest apoptotic-related gene expression as an alternative pathway, through which exposure to 6milliTesla (mT) SMF can interfere with apoptosis. Exposure to 6mT SMF affects the apoptotic rate (spontaneous and drug-induced) and [Ca(2+)](i) in isolated human lymphocytes; the aged cells are more susceptible to exposure than fresh ones. The exposure to 6mT exerted a protective effect on chemical or physical-induced apoptosis, irrespective of the age of the cells. The investigation of the gene expression of bcl-2, bax, p53 and hsp70 in freshly isolated and in culture-aged human lymphocytes indicates that these genes are modulated by SMF exposure in the experimental conditions used, in a gene-, age- and time-dependent manner. The exposure of isolated lymphocytes to SMF for up to 24h modulated increased bax and p53 and decreased hsp70, and bcl-2. The amount of increment and/or decrement of the proteins varied for each gene examined and was independent of the apoptotic inducers. Finally, the same stress applied to freshly isolated or aged lymphocytes resulted in different modulation of bcl-2, bax and hsp70.

Promotion of PDT efficacy by a Bcl-2 antagonist.

Photodynamic therapy (PDT) directed against the endoplasmic reticulum (ER) is also known to target antiapoptotic Bcl-2 family proteins. This effect is associated with the initiation of both apoptosis, a cell death pathway, and autophagy, an organelle recycling system that can lead to survival or cell death. In this study, we examined the ability of the Bcl-2 antagonist HA14-1 to promote the photodynamic efficacy of PDT directed at the ER. At concentrations that independently caused only a small loss of viability, HA14-1 markedly enhanced the proapoptotic and phototoxic effects of ER photodamage. These results provide additional evidence that the antiapoptotic properties of Bcl-2 constitute an important determinant of photokilling, and demonstrate that synergistic effects can result when PDT is coupled with pharmacologic suppression of Bcl-2 function.

Expression of survivin and p16(INK4a)/Cdk6/pRB proteins and induction of apoptosis in response to radiation and cisplatin in meningioma cells.

Although meningiomas represent the most common class of tumors of the central nervous system, the molecular events underlying their genesis and development are still not well defined, and therapeutic approaches based on the genetics of these tumors are currently lacking. In the present study we have used the immunoblotting technique to show that the p16(INK4A), Cdk6 and pRB proteins are differentially expressed in primary meningioma cells with 20-, 30- and 36-fold difference between the lowest and the highest levels of each protein, respectively. In addition, we present evidence that the level of the anti-apoptosis survivin protein is high in these benign tumors. Moreover, the annexin V-associated flow cytometry technique was used to show that 60% of meningioma cell cultures underwent apoptosis in response to both gamma-rays and cisplatin, and 50% of these cells exhibited significant sensitivity to hydroxyurea. These agents triggered apoptosis through the mitochondrial pathway, by increasing the Bax/Bcl-2 ratio. Interestingly, the induction of apoptosis following radiation and cisplatin was significant in all cells that expressed low levels of p16(INK4A), Cdk6 and pRB proteins. These data shed more light on the molecular biology of meningioma cells and suggest that survivin and proteins of the RB pathway could play a determinant role in the development and the treatment of meningiomas.

Bcl-2 small hairpin RNAs enhance radiation-induced apoptosis in A549 cells.

Bcl-2, a prominent member of the family of proteins, is responsible for dys-regulation of apoptosis and resistance to chemotherapy and radiotherapy. This study investigated whether small hairpin RNA (shRNA) targeting Bcl-2 could render A549 cells more susceptible to gamma radiation-induced apoptosis. Recombinant Bcl-2 shRNAs expression vector were transfected into A549 cells with Lipofectamine 2000. Transfected cells were screened in 800 mg/ml G418 screening medium, and after stable transfection, silencing was examined. Expression of the Bcl-2 protein was assayed using Western blot in A549 cells. Inhibition of cell growth was assessed by a MTT assay. Apoptosis was determined by morphological observation and flow cytometry. Expression levels of Bcl-2 protein from A549 cells decreased after stable transfection with Bcl-2 shRNAs. No differences in Bcl-2 protein levels between control shRNA group and untreated cells were noted. After stable transfection with Bcl-2 shRNAs the viability of cells was less than after stable transfection with those with control shRNAs and untransfected A549, respectively (P<0.05). Control shRNA had no significant effect on growth of cells. Radiation significantly inhibited the growth of cells stably transfected with Bcl-2 shRNA (P<0.05). No difference in survival between the cells with control shRNA and untransfected cells was noted. Using Giemsa staining, cells stably transfected with Bcl-2 shRNA combined with radiation at 48 h displayed changes of apoptosis. After treatment with radiation apoptotic rates of the A549 cells stably transfected with Bcl-2 shRNA significantly increased (P<0.05), compared with the cells with control shRNA and untransfected cells. shRNAs against the Bcl-2 mRNA increases radiation-induced apoptosis in A549 cells.

Bcl-2 is the target of a UV-inducible apoptosis switch and a node for UV signaling.

Sunlight's UVB radiation triggers cell signaling at multiple sites to induce apoptosis. The integration of these signal entry sites is not understood. Here we show that P53 and E2f1 constitute a UV-inducible apoptosis switch. At low-UV doses, wild-type cells resemble the OFF state of an siP53-treated cell, whereas at high-UV doses, the apoptosis frequency transitions to the fully ON behavior of an siE2f1-treated cell. The switch's target is Bcl-2: Rapid Bcl-2 down-regulation in response to UVB-induced DNA photoproducts is lost in P53-deficient cells, but, as for apoptosis, is restored when both P53 and its inhibited target E2f1 are absent. P53's down-regulation of Bcl-2 is mediated entirely through E2f1. Bcl-2 is also down-regulated by a separate pathway triggered by DNA photoproducts in the absence of P53 and E2f1. Four UV pathways terminating on Bcl-2 contribute to apoptosis after UVB irradiation. The apoptosis lost in p53(-/-) is completely restored by siBcl-2, implying that Bcl-2 is a rate-limiting member of this network. These results identify Bcl-2 as an integrator of several UV-induced proapoptotic signals and show that it, in turn, suppresses a direct UV-apoptosis pathway. UV-induced apoptosis requires both UV activation of the direct pathway and a separate UV disinhibition of this pathway through P53-E2f1-Bcl-2.

Enhancement of macrosphelide-induced apoptosis by mild hyperthermia.

Hyperthermia is a useful adjunct in cancer therapy as it can increase the effectiveness and decrease the toxicity of currently available cancer treatments such as chemotherapy and radiation. In the present study, we investigated whether 41 degrees C hyperthermia (mild HT) for 20 min can enhance macrosphelide (MS5)-induced apoptosis in human lymphoma U937 cells. Our results revealed that, compared with MS5 (5 microM) and mild HT alone, the combined treatment exhibited significant enhancement in apoptosis at 6 h, which was evaluated by observing morphological changes and DNA fragmentation. Marked increase in the reactive oxygen species (ROS) generation was observed immediately after the combined treatment. Significant increase in Fas externalization, caspase-8 and caspase-3 activation, and loss of mitochondrial membrane potential (MMP) was found after the combined treatment compared with MS5 and mild HT alone. Moreover, this combination can also alter the expression of apoptosis-related proteins as evident by the cleavage of Bid and down-regulation of Bcl-2 while no change in the expression of Bax was observed. Furthermore, an immediate rise in the intracellular calcium ion ([Ca(2+)]i) concentration was observed after the combined treatment, which continuously increased in a time-dependent manner. In addition, mild HT treatment alone also increases [Ca(2+)]i concentration without inducing apoptosis. Our data indicate that early increase in ROS generation is mainly responsible for the enhancement of apoptosis after the combined treatment.

Ionizing radiation induces apoptosis and inhibits neuronal differentiation in rat neural stem cells via the c-Jun NH2-terminal kinase (JNK) pathway.

A substantial number of neural stem cells (NSCs) continue to proliferate and generate neurons in the central nervous system throughout life. Ionizing radiation, an important adjuvant therapy for glioma patients, may damage NSCs and cause neuronal deficits, such as cognitive dysfunction and memory impairment. However, the precise mechanism of radiation effects on death and differentiation of NSCs remains largely unknown. Here, we found that radiation induced apoptosis in NSCs via the mitochondrial pathway, upregulating the ratio of Bax to Bcl-2 and releasing cytochrome c into the cytoplasm. Radiation also inhibited neuronal differentiation of NSCs by 50%. Of the three stress-associated mitogen-activated protein kinases (MAPKs), only c-Jun NH(2)-terminal kinase (JNK) was activated in NSCs after radiation. Interestingly, JNK inhibition by the specific inhibitor SP600125 rescued NSCs from apoptosis and improved neuronal differentiation. Furthermore, we examined whether radiation directly inhibits neuronal differentiation or not. Radiation did not affect the promoter activity of NeuroD, a basic helix-loop-helix transcription factor that regulates the expression of neuronal differentiation markers. Radiation induced more apoptosis in NeuroD-positive cells than NeuroD-negative cells. We concluded that radiation activates JNK and induces apoptosis, especially in neural progenitor cells, resulting in the inhibition of neurogenesis. Our findings raise the possibility that JNK inhibition has therapeutic potential in protecting NSCs from the adverse effects of radiation.

Amifostine has an inhibitory effect on the radiation-induced p53-branched cascade in the immature mouse ovary.

The organic thiophosphate, amifostine, is a promising pharmacological compound showing selective protection in many tissues against the toxic side-effects of radiation and cytotoxic drugs. The aim of the present study was to assess the radioprotective effects of amifostine on ovarian follicles. Three-week-old female mice, with or without pretreatment with amifostine, were irradiated with 6.42 Gy of gamma-ray. Reduced proliferation of granulosa cells was verified with BrdU staining and the incidences of follicular degeneration increased in ovarian follicles in the gamma-ray-irradiated mice compared to that of the control or amifostine-treated group. Biochemical changes caused by gamma-irradiation provoked a rise of p53 and Bax protein and a decline of the inactive form in caspase-3 and PARP protein. Caspase-3 and PARP cleaved into active peptides during apoptosis. This process was confirmed by the result of this study, which was that the amount of the stable form decreased immediately after irradiation. In the amifostine treatment group before irradiation, the increased rate of p53 and Bax was suppressed, particularly in the LDs-treated group. The relationship between PARP and caspase-3 levels showed the effect of amifostine exposure before irradiation. In conclusion, amifostine had an inhibitory effect on ovarian programmed cell death induced by gamma-ray, affecting the expression of apoptotic signaling molecules and the level of proliferation of the granulosa cells.

TRAIL sensitizes for ionizing irradiation-induced apoptosis through an entirely Bax-dependent mitochondrial cell death pathway.

The death ligand TRAIL has been suggested as a suitable biological agent for the selective induction of cell death in cancer cells. Moreover, TRAIL synergizes with DNA-damaging therapies such as chemotherapeutic drugs or ionizing irradiation (IR). Here, we show that synergy of TRAIL and IR, that is, crosssensitization between TRAIL and IR for induction of apoptosis, entirely depends on Bax proficiency in human DU145 and HCT116 carcinoma cells. DU145 prostate carcinoma cells that have lost Bax protein expression due to mutation fail to activate caspase-3 and -9 when exposed to TRAIL and IR. In contrast, TRAIL sensitized for IR-induced apoptosis and vice versa upon reconstitution of Bax expression. Notably, both DU145 and HCT116 still express significant levels of the multidomain proapoptotic Bcl-2 homolog Bak. This indicates that Bak is not sufficient to mediate crosssensitization and synergism between IR and TRAIL. These data clearly establish distinct roles for Bax and Bak in linking the TRAIL death receptor pathway to the mitochondrial apoptosis signaling cascade upon DNA damage by IR.