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 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.

Radiosensitization effect of overexpression of adenovirus-mediated SIRT6 on A549 non-small cell lung cancer cells.

OBJECTIVE: To explore the radiosensitization effect of overexpression of silent information regulator 6 (SIRT6) on A549 non-small cell lung cancer (NSCLC) cells. METHODS: Adenovirus vector Ad-SIRT6 causing overexpression of SIRT6 was established. Western blotting and MTT assay were adopted to detect the level of SIRT6 protein and the inhibitory rate of A549 cell proliferation after different concentrations of adenovirus transduction (0, 25, 100, 200, and 400 pfu/cell) for 24 h. Control group, Ad-null group and Ad-SIRT6 group were designed in this experiment and virus concentration of the latter two groups was 200 pfu/cell. Colony formation assays were employed to test survival fraction (SF) of the 3 groups after 0, 2, 4, 6, 8, 10 X-ray irradiation. Flow cytometry was used to detect the status of cell cycle of 3 groups after 48 h of 4 Gy X-ray irradiation and Western blotting was used to determine the expression of apoptosis-related genes of 3 groups after 48 h of 4 Gy X-ray irradiation. RESULTS: In the range of 25 ~ 400 pfu/cell, the inhibitory rate of A549 cell proliferation increased as adenovirus concentration raised. The inhibitory rates under the concentrations of 0, 25, 100, 200, and 400 pfu/cell were 0%, 4.23 ± 0.34%, 12.7 ± 2.57%, 22.6 ± 3.38%, 32.2 ± 3.22%, 38.7 ± 4.09% and 47.8 ± 5.58% and there were significantly differences among groups (P < .05). SF in Ad-SIRT6 group was lower than Ad-null and control groups after 4 ~ 10 Gy X-ray irradiation (P < 0.05) and the sensitization enhancement ratio (SER) was 1.35 when compared with control group. Moreover, after 48 h of 4 Gy X-ray irradiation, there appeared a significant increase in G1-phase cell proportion, up-regulated expression of the level of apoptosis-promoting genes (Bax and Cleaved caspase-3), but a obvious decline in S-phase and G2-phase cell proportion and a significant decrease of the level of apoptosis- inhibiting gene (Bal-2) in the Ad-SIRT6 group (P<0.05). CONCLUSION: The over-expression of adenovirus-mediated SIRT6, which has radiosensitization effect on A549 cells of NSCLC, can inhibit the proliferation of A549 cells and cause G0/G1 phase retardation as well as induce apoptosis of cells.

Injury in Minipig Parotid Glands following Fractionated Exposure to 30 Gy of Ionizing Radiation.

OBJECTIVE: To explore the effects of 30 Gy of (60)Co γ-rays on apoptosis and reactive oxygen species (ROS) levels in minipig parotid cells as a possible mechanism for radiation-induced parotid injury. STUDY DESIGN: Experimental study. SETTING: Department of Radiotherapy, First Affiliated Hospital, Guangxi Medical University, Nanning, China. SUBJECTS AND METHODS: Forty male minipigs were divided into control and irradiated groups. Terminal deoxynucleotidyl transferase deoxyuridine triphosphate nick-end labeling was used for detecting apoptosis in the parotid cells, immunohistochemistry, and western blots were used to test expression of the B-cell lymphoma 2 (Bcl-2) and BCL2-associated X (Bax) proteins, and reverse transcription polymerase chain reaction was used to analyze the expression of Bcl-2, Bax, p53, and caspase-3 messenger ribonucleic acid. An enzyme-linked immunosorbent assay was used to detect ROS levels in the parotid tissue. RESULTS: At each time point, the apoptotic rates in the irradiated group were higher than those in the control group. Furthermore, the ROS and expression levels of Bax, p53, and caspase-3 messenger ribonucleic acid and proteins gradually increased and were higher than those in the control group. Conversely, the expression of Bcl-2 was decreased in the irradiated group (P < .05). CONCLUSIONS: Ionizing radiation induces the production of ROS and promotes changes in the expression of several apoptotic proteins, which increases apoptosis and likely contributes to the mechanism of radiation-induced parotid injury.

Radiation/paclitaxel treatment of p53-abnormal non-small cell lung cancer xenograft tumor and associated mechanism.

BACKGROUND: Mutations in key tumor suppressor genes such as tumor protein 53 (TP53) and phosphatase and tensin homolog deleted on chromosome ten (PTEN) are the main genetic alterations in cancers. TP53 mutations have been found in most patients with non-small cell lung cancer (NSCLC), whereas PTEN mutations are rarely found in lung cancer, though most NSCLCs lack PTEN protein synthesis. However, the signaling involved in radio- and chemotherapy of NSCLC with wild-type PTEN and nonfunctional p53 is not clearly understood. METHODS: In this study, we established a xenograft tumor model with H358 NSCLC cells expressing wild-type PTEN, but nonfunctional p53. Protein expression and phosphorylation of PTEN and its downstream signal molecules in NSCLC tissues were detected by Western blot. RESULTS: We demonstrated that radiation and paclitaxel alone inhibited tumor growth, but a combined therapy of radiation and paclitaxel was more effective in inhibiting NSCLC tumor growth. Interestingly, both radiation and paclitaxel significantly increased PTEN protein expression and phosphorylation. Further identification of the affected PTEN downstream molecules showed that Akt phosphorylation at Ser(473) and Thr(308) residues was significantly decreased, whereas Bax and cleaved caspase-3 levels were significantly increased in tumor tissues treated with both radiation and paclitaxel. The combined treatment was more effective than either treatment alone in regulating the studied molecules. We also found that paclitaxel, but not radiation, inhibited phosphoinositide 3-kinase (PI3K) activity. CONCLUSIONS: Our study suggested that a PTEN-PI3K-Akt-Bax signaling cascade is involved in the therapeutic effect of combined radiation/paclitaxel treatment in NSCLC without p53 expression. Our study also suggested that PTEN is an ideal target in tumors with wild-type PTEN and a lack of functional p53.

[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.

Bcl-XL protein is markedly decreased in UVB-irradiated basal cell carcinoma cell lines through proteasome-mediated degradation.

There is considerable evidence that the excessive ultraviolet radiation B (UVB) from sunlight is implicated in skin damage, ultimately inducing the death of keratinocytes. The UVB-induced apoptotic pathways are tightly regulated by the balance between pro-apoptotic and anti-apoptotic molecules. Among them, modulations of Bcl2 family proteins are important to decide the fate of UVB-irradiated cells. If the apoptotic pathway does not work properly, the damaged cells have a chance to transform into a carcinoma, such as basal cell carcinoma or squamous cell carcinoma of the skin. To develop a strategy of inducing apoptosis of skin cancer cells, the current study was performed to investigate the apoptotic pathway, especially focused on Bcl2 family proteins, in curcumin or UVB-treated basal cell carcinoma cell lines. Our data showed that the decreased proliferation rates and apoptotic DNA laddering were clearly observed in UVB irradiation, but not markedly observed in curcumin treatment. The decreased expression of Bcl-XL, which is involved in protection of apoptosis, was also clearly observed in UVB-irradiated cells without markedly changing mRNA levels. However, the expression of Bax or Bcl2 were not markedly changed by UVB-irradiation. The decreased expression of Bcl-XL protein after UVB treatment was partially restored in the presence of MG132, which is an inhibitor of proteasome, implying that the down-regulation of Bcl-XL may be regulated by the proteasome-mediated degradation. Our data demonstrated that the expression of Bcl-XL protein was decreased by proteasome-mediated degradation prior to change of mRNA level in UVB-induced apoptotic basal cell carcinoma cell lines, thereby these results will offer fundamental information to develop a strategy of inducing apoptosis of skin cancer cells.

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.

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.

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.

Dissection of transcriptional and non-transcriptional p53 activities in the response to genotoxic stress.

Following genotoxic stress, p53 either rescues a damaged cell or promotes its elimination. The parameters determining a specific outcome of the p53 response are largely unknown. In mouse fibroblasts treated with different irradiation schemes, we monitored transcriptional and non-transcriptional p53 activities and identified determinants that initiate an anti- or a pro-apoptotic p53 response within the context of p53-independent stress signaling. The primary, transcription-mediated p53 response in these cells is anti-apoptotic, while induction of p53-dependent apoptosis requires an additional, transcription-independent p53 activity, provided by high intracellular levels of activated p53. High intracellular levels of p53 were selectively generated after apoptosis-inducing high-dose UV-irradiation, and correlated with a strongly delayed upregulation of Mdm2. Following high-dose UV-irradiation, p53 accumulated in the cytoplasm and led to activation of the pro-apoptotic protein Bax. As p53-dependent Bax-activation is transcription-independent, we postulated that certain transcription-deficient mutant p53 proteins might also exert this activity. Indeed we found an endogenous, transcription-inactive mutant p53 that upon genotoxic stress induced Bax-activation in vivo. Our results demonstrate the impact and in vivo relevance of non-transcriptional mechanisms for wild-type and mutant p53-mediated apoptosis.

Pulsating electromagnetic field induces apoptosis of rat's bowel Cajal's cells.

UNLABELLED: We previously have shown that pulsating electromagnetic field (PEMF) reduce expression of interstitial cells of Cajal (ICCs) in the rat gastrointestinal tract. Aim of present study was to determine whether diminished expression of ICCs in the rat's bowel after PEMF exposure was related to apoptosis and to PEMF dose. METHODS: rats were divided into two groups (n= 32). First group (n = 16) was exposed to four rising doses of PEMF from one dose 12.5 x 10(3) A(2) x h/m(2) to four doses 50 x 10(3) A(2) x h/m(2). Second group (n = 16) served as a control. Tissue samples of the rat duodenum and colon from exposed to PEMF and control animals were fixed and paraffin embedded and cryostat frozen. The tangential paraffin bowel sections were stained with anti c-Kit antibody. C-Kit positive cells were assessed by image analysis. Apoptosis detection in rat's tissues was performed with rabbit polyclonal anti-Bax antibody. RESULTS: the surface of c-Kit immunopositive cells decreased in the duodenum and colon of rats stimulated with PEMF in a dose dependent manner with increase in expression of pro-apoptotic Bax protein in c-Kit immunopositive myenteric cells. The apoptosis - inducing action of PEMF on the c-Kit immunoreactivity of Cajal's cells suggests a possible therapeutical implications in diseases associated with overactive smooth muscles dysfunction. Pulsating electromagnetic field (PEMF) induced changed immunoreactivity in rat's myenteric Cajal cells. C-Kit diminished reactivity of ICCs was proved to be caused by triggering of apoptotic pathwa in ICCs upon PEMF stimulation. PEMF generated apoptosis was dependant on applied dose of PEMF and detected by immunostaining with antibody against proapoptic protein Bax.