Sublethal Irradiation Promotes Migration and Invasiveness of Prostate Cancer PC-3 Cells：Implications for Radiotherapy of Human Prostate Cancer

OBJECTIVE To study the changes in the matrix metalloproteinases-2 and 9 （MMP2, MMP9） induced by ^60Co γ-ray external irradiation of human prostate cancer PC-3 cells. METHODS Human prostate cancer PC-3 cells were irradiated with different doses of ^60Co γ-rays. Cell migration and invasiveness were evaluated and the expression of MMP2, and MMP9 was investigated by RT-PCR, Western blotting and flow cytometry（FCM）. RESULTS Irradiation enchances invasive protential at the doses of 1,3 and 5 Gy,whereas it significantly inhibits cell migration. CONCLUSION The different doses of ^60Co γ-ray external irradiation for prostate cancer may have different effects through the changes of MMP2, and MMP9 expression.     

Study on the DNA damage induced by coal tar pitch fume extracts in rat alveolar macrophage and it‘s mechanism

The carcinogenic mechanism of coal tar pitch (CTP) as a recognized carcinogen has been studying. It is widely believed that the carcinogenicity of CTP is based on the genotoxicity of CTP. In the process of carcinogenesis caused by extrinsic chemical substance, the DNA damage mainly occurred in the initiation phase. UP to now, the most sensitive detecting endpoint for DNA damage is to detect DNA single strand breaks. The single cell gel electrophoresis has been rapidly becoming a widely used analytical procedure during the last few years, which can detect DNA strand breaks. The method is a fast, relatively inexpensive, easy to perform, non-radioactive, and very sensitive method. This method suits to different tests in vitro or in vivo. Virtually any eukaryotic cell, which could be made into single cell suspensions, can be processed for analysis of DNA damage using the single cell gel electrophoresis. The aim of the study is to investigate the role of DNA damage induced by CTP fume in rat AM, to examine the changes of ROS, MDA and SOD, and to explore the mechanism of DNA damage by CTP fume. The present study is in favor of studying the mechanism of mutagenesis and carcinogenesis induced by CTP. Method: The healthy male Wistar rats were anesthetized intraperitoneally with 40 mg pentobarbital sodium per kilogram of body weight. The animals were exanguinated by excising femoral, and collected the rat alveolar macrophage by Joseph's method. The concentration of AM had been regulated to 1.5×106 cell/ml. AMs, which had been cultured in 24-well culture plate, were divided into 4 groups. These cells were exposured to 5.0 μg/ml extracts of coal tar pitch fume, and contacted with 500 μM, 1 000 μM, and 2 000 μM of GSH respectively. These cells were divided into 4 groups. After incubation 24 hours, the indexes that had been used above were measured. Results: ①The DNA strand breaks induced by coal tar pitch fume extracts: After undergoing electrophoresis, the ‘comet’ cells are seldom in control group, but the ‘comet’ cells increased significantly in experimental groups. When the concentration of CTP fume extracts increased from 0 to 10.0 μg/ml, the incidences of ‘comet’ cell increased from 6.0% to 92.0%. There were significant differences in all groups (P<0.001). There was a dose-response relationship between the concentrations of CTP fume extracts and the incidences of ‘comet’ cell. There were correlation relationship between the grade of DNA damage and the concentration of CTP fume extracts (P<0.001). ②The level of ROS induced by CTP fume extracts in AM: After incubation 24 h, the concentration of ROS increased. The difference between control group and experimental groups were significant (P<0.001). ③ The changes of MDA and SOD in AM induced by CTP fume extracts: As the concentration of coal tar pitch fume extracts increased, the concentration of MDA increased too. The differences between experimental groups and control group were significant. But the difference of SOD activity in all groups weren’t significant. ④The role of GSH interfere: GSH could protect the DNA of AM from DNA-damage induced by CTP fume extracts. With the increasing of the concentration of GSH, the incidences of ‘comet’ cell decreased from 77.0% to 16.0%. The differences of incidences of ‘comet’ cell in all groups were different significantly. There was a dose-response relationship between the concentrations of GSH and the incidences of ‘comet’ cell. There was correlation relationship between the grade of DNA damage and the concentration of GSH (P<0.001). GSH could inhibit the increasing of ROS and MDA. The differences of SOD activity in all groups weren't significant (P>0.05). Conclusion: ①Coal tar pitch fume extracts can induce DNA single strand breaks in rat alveolar macrophage; the single cell gel electrophoresis appears to be suitable to detect DNA strand breaks in rat alveolar macrophage. ②Coal tar pitch fume extracts can induce the rat alveolar macrophage to produce reactive oxygen species (ROS), and can induce lipid peroxidation: the DNA strand breaks induced by coal tar pitch fume extracts in AM are associated with the producing of ROS. ③GSH can inhibit coal tar pitch fume extracts-induced DNA damage in rat alveola r macrophage.     

CELL TRANSFORMATION IN VITRO BY -RAY COMBINED WITH EP

Malignant transformation of syrian hamsterembryo(SHE) cells in vitro was induced bylow-dose of Co-60 γ-ray combined withestradiol valerate and hydroxyprogesteronecaproate (EP).The transformation was notobserved in the groups that the γ-ray or EPwas given alone.SHE cells were seeded in     

贝伐单抗联合放射方案的实验研究

Objective To evaluate the impact of the hypoxia induced by bevacizumab on the antitumor effect in combining with irradiation in CNI-H441 xenografts in mice. Methods Bevacizumab of 5 mg/kg mouse for groups of control, bevacizumab alone, irradiation alone, earlier combination (EC), and later combination (LC) were initially injected peritoneally. Single irradiation of 14 Gy (122Sc γ-ray) was given at the 4th hour for the group of irradiation alone, 24th hour for EC group, and 72nd hour for LC group after the initial injection. Tumor hypoxia, micro vessels density and permeability of tumor vasculature,pathological responses, apoptosis, and tumor growth delay curve were evaluated after using bevacizumab and/or irradiation. Results Although it was lower than the control at the 24 hr after using bevacizumab (3. 1 × 106: 6.1 × 106 ;t = - 1.73 ,P > 0. 05), the HIF-1α rapidly increased to 3 - 4 times and 2 - 3 times of the control at day 3 (7.4 × 106: 20. 4 × 106; t = 2. 36, P < 0. 05) and lasted until day 10, which was consistent with the changes of tumor function vessels count. The count of residual micro vessel density count in LC group was higher than that in groups of EC and irradiation at day 3 after irradiation (9. 33: 3. 17;t =- 2. 43, P < 0. 05). The apoptotic count of tumor cells was lower in LC group than that in EC group (23.33: 43.83; t= 2.54, P< 0.05, at day 3 after radiation). Tumor growth delay time of LC groupwas shorter than that of EC groups (10. 5 days vs. 23. 0 days , t = 2. 67 , P < 0. 05) . Conclusions Hypoxia level induced by bevacizumab decreases the antitumor effect in later combination of bevacizumab and irradiaion. It shows a time window that determines whether the combination of bevacizumab and irradiation will be benefit or diverse.     

贝伐单抗联合放射方案的实验研究

Objective To evaluate the impact of the hypoxia induced by bevacizumab on the antitumor effect in combining with irradiation in CNI-H441 xenografts in mice. Methods Bevacizumab of 5 mg/kg mouse for groups of control, bevacizumab alone, irradiation alone, earlier combination (EC), and later combination (LC) were initially injected peritoneally. Single irradiation of 14 Gy (122Sc γ-ray) was given at the 4th hour for the group of irradiation alone, 24th hour for EC group, and 72nd hour for LC group after the initial injection. Tumor hypoxia, micro vessels density and permeability of tumor vasculature,pathological responses, apoptosis, and tumor growth delay curve were evaluated after using bevacizumab and/or irradiation. Results Although it was lower than the control at the 24 hr after using bevacizumab (3. 1 × 106: 6.1 × 106 ;t = - 1.73 ,P > 0. 05), the HIF-1α rapidly increased to 3 - 4 times and 2 - 3 times of the control at day 3 (7.4 × 106: 20. 4 × 106; t = 2. 36, P < 0. 05) and lasted until day 10, which was consistent with the changes of tumor function vessels count. The count of residual micro vessel density count in LC group was higher than that in groups of EC and irradiation at day 3 after irradiation (9. 33: 3. 17;t =- 2. 43, P < 0. 05). The apoptotic count of tumor cells was lower in LC group than that in EC group (23.33: 43.83; t= 2.54, P< 0.05, at day 3 after radiation). Tumor growth delay time of LC groupwas shorter than that of EC groups (10. 5 days vs. 23. 0 days , t = 2. 67 , P < 0. 05) . Conclusions Hypoxia level induced by bevacizumab decreases the antitumor effect in later combination of bevacizumab and irradiaion. It shows a time window that determines whether the combination of bevacizumab and irradiation will be benefit or diverse.     

Immune Responses of Dendritic Cells Loaded with Antigens from Apoptotic Cholangiocarcinoma Cells Caused by γ-Irradation

Objective:To investigate the induction cytotoxic T cells(CTLs) with antitumor activity and therapeutic efficacy after dendritic cells(DCs) acquired antigen from apoptotic cholangiocarcinoma cells caused by γ-irradiation. Methods:DCs from peripheral blood mononuclear cells (PBMC) that maintain the antigen capturing and processing capacity charateristic of immature cells have been established in vitro, using granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4). Then, in cholangiocarcinoma cells apoptosis was induced by γ-irradiation. The experimental groups were as follows:(1)coculture of DCs and apoptotic cancer cells and T cells;(2)coculture of DCs and necrotic cancer cells and T cells;(3)coculture of DCs, cultured cancer cell and T cells. They are cocultured for 7 days.DCs and T cells were riched, isolated and their antitumor response was tested. Results:The cells had typical dendritic morphology, expressed high levels of CDla and B7, acquired antigen from apoptotic cells caused by γ-irradiation and induced an increased T cell stimulatory capacity in mixed lymphocyte reactions (MLR). Conclusion:DCs obtained from PBMCs using GM-CSF and IL-4 can efficiently present antigen derived from apoptotic cells caused by γ-irradiation and efficiently induce T cells.This strategy, therefore, may present an effective approach to transduce DCs with antigen.     

Influence of PPAR -γligand on radiation - induced PAI - 1 and TGF -βmRNA in fibroblast cells

Objective To observe the influence of peroxisome proliferator activated receptor-γligand (PPAR-γ, pioglatazone) on expression of PAI-1 and TGF-β mRNA and proliferation in fibroblast cells before and after X-ray radiation, and to study the effect of PPAR-γon normal cells during radiation induced fibrosis process. Methods RT-PCR method was used to measure PPAR-γgene expression in L929 cells.After X-ray irradiation of 10 Gy,4 Gy or 2 Gy, the expressions of PAI-1 and TGF-β mRNA in mouse lung fibroblast cells (L929) were measured using RT-PCR. After X-ray irradiation and pioglatazone treatment,the influence of pioglatazone on PAI-1 and TGF-β was measured using RT-PCR method. MTT method was used to test cell proliferation after the treatment of irradiation and pioglatazone. Results PPAR-γ mRNA expression was observed in L929 cells. Expression of PAI-1 and TGF-β mRNA reached the highest level 483.40,P =0. 090) ). At 48 h after the treatment of pioglatazone and 10 Gy radiation, pioglatazone decreased 0. 36, 0. 34 and 0. 32( F = 3.90, P = 0. 040) ). The inhibitory effect was significantly increased when L9292. 50,P =0. 005)). Conclusions X-ray irradiation can increase the expression of PAI-1 and TGF-β in L929 cells. Pioglatazone can decrease the expression of radiation-induced PAI-1 and TGF-β, and restrain the fibroblast proliferation.     

Effects of Imbalance of Apoptosis and Proliferation on Large Bowel Carcinogenesis in Mice

OBJECTIVE To observe the pattern of changes in the proliferation and apoptosis at different stages of large bowel carcinoma in mice, and to explore the effects of the imbalance of apoptosis and proliferation at different stages of large-intestine carcinogenesis.METHODS An experimental animal model for large intestine carcinogenesis of KUNMING-strain mice was used. The carcinomas were induced by subcuteneous injection of dimethylhydrazine (DMH) and the distribution and density changes of proliferating and apoptotic cells observed through multistages toward cancer formation. The animals were killed in groups at the 12th, 18th, 24th,and 32nd weeks of carcinoma induction. The apoptotic and proliferating cells were labeled separately using TUNEL and PCNA immunohistochemical staining methodsRF, RESULTS In the normal mouse mucosa, all the apoptotic cells were situated in the superficial layers, however, the proliferating cells were situated in the basement layers, and the amount of both were small. In the early stage of carcinoma induction, the proliferation and the apoptotic cells slightly increased in amount, but there were no obvious changes in their ratio. In the medium stage, the densities of both distinctly increased, but there were no obvious changes in the ratio. In the late stage, the densities of the proliferating and the apoptotic cells in the non-carcinoma mucosa were higher than those at other stages. The proliferating cells in the dysplastic mucosa increased progressively with the increasing degree of the lesions. Although the apoptotic cells increased, their changes did not occur with the degree of the lesions. Their ratio showed a decreasing tendency with the degree of the lesions.CONCLUSIONS (①The presence of an imbalance between cell proliferation and apoptosis was confirmed in the course of large intestine carcinogenesis in a mouse model. ②In the early stage of carcinoma induction both proliferation and apoptosis were at a low level; in the medium stage, they were both at a high level; and in the late stage (that is in carcinoma), proliferation was at a very high level, while apoptosis was at a low level. ③The proliferating cells increased progressively with the degree of dysplasia. There were no obvious changes in the apoptotic cells and their ratio to the proliferating cell sshowed a progressively increasing tendency. ④In the stage of cancer formation, the most essential change was the excessive decrease in the ratio of apoptosis to proliferation. These results support the hypothesis of “Cell Selective Proliferation“, which was raised by authors previously in a study on human large bowel carcinoma.     

Antiproliferative effects of zinc-citrate compound on hormone refractory prostate cancer

Objective: To investigate the antiproliferative effects of zinc‐citrate compound on hormone refractory prostate cancer (HRPC). Methods: HRPC cell line (DU145) and normal prostate cell line (RWPE-1) were treated with zinc, citrate and zinc-citrate compound at different time intervals and concentrations to investigate the effect of zinc-citrate compound. Mitochondrial (m)-aconitase activity was determined using aconitase assay. DNA laddering analysis was performed to investigate apoptosis of DU145 cells. Molecular mechanism of apoptosis was investigated by Western blot analysis of P53, P21 waf1 , Bcl-2, Bcl-xL and Bax, and also caspase-3 activity analysis. Results: Treatment with zinc-citrate compound resulted in a time-and dose-dependent decrease in cell number of DU145 cells in comparison with RWPE-1. M-aconitase activity was significantly decreased. DNA laddering analysis indicated apoptosis of DU145 cells. Zinc-citrate compound increased the expression of P21 waf1 and P53, and reduced the expression of Bcl-2 and Bcl-xL proteins but induced the expression of Bax protein. Zinc-citrate compound induced apoptosis of DU145 cells by activation of the caspase-3 pathway. Conclusion: Zinc-citrate compound can induce apoptotic cell death in DU145, by caspase-3 activation through up-regulation of apoptotic proteins and down-regulation of antiapoptotic proteins.     

In Vitro Anti-irradiation Effects of a Polypeptide from Chlamys Farreri

OBJECTIVE To investigate the anti-irradiation effects of a polypeptide from Chlamys farreri(PCF) on γ-ray induced damage to mouse thymocytes.METHODS Thymocytes were randomly divided into 6 groups including an untreated control, a model group, groups treated with 5, 2.5 and 1.25 mg PCF/ml and a 0.1% vitamin C group. Cell viability, morphology, nucleic acid and enzymatic changes of the cells 3 h after 3 Gy γ-ray irradiation at a dose rate of 0.6 Gy/min were determined.RESULTS γ-Ray irradiation decreased the cell viability and SOD activity and increased MDA levels and the apoptotic number of cells. PCF increased cell viability and SOD activity and decreased MDA levels and the apoptotic number of the cells in a dose-dependant manner.CONCLUSION PCF pretreatment can attenuate cytotoxicity, inhibit apoptosis, reduce the level of MDA and maintainthe activity of SOD.Therefore PCF has anti-irradiation effects on γ-ray induced damage of mouse thymocytes.     

Indomethacin overcomes doxorubicin resistance by decreasing intracellular content of glutathione and its conjugates with decreasing expression of γ-glutamylcysteine synthetase via promoter activity in doxorubicin-resistant leukemia cells

Drug resistance continues to be a serious problem in cancer therapy. We investigated whether indomethacin, which inhibits cyclooxygenases, is able to overcome doxorubicin resistance in K562/ADR leukemia cells. Indomethacin at 10 μM increased the cytotoxicity of doxorubicin and vincristine in K562/ADR cells. Intracellular glutathione content was elevated in K562/ADR cells. Indomethacin treatment decreased glutathione content and glutathione-conjugates in K562/ADR cells. Increased expression of γ-glutamylcysteine synthetase (γ-GCS) was observed in K562/ADR cells, but this expression was decreased by indomethacin treatment. The activity of the γ-GCS promoter from K562/ADR cells decreased after indomethacin treatment in MDA231 cells. These data strongly suggest that the cyclooxygenase inhibitor indomethacin increases the cytotoxicity of doxorubicin by decreasing the intracellular contents of glutathione and its conjugates with decreasing expression of γ-GCS by inhibiting γ-GCS promoter activity.     

THE LATE EFFECTS ON BONE MARROWS IN MICE AFTER TOTAL BODY IRRADIATION BY P(35) BE FAST NEUTRONS AND γ RAYS

Thebonemarrowisadose-limitingcellre11ewaltissueforwide-fieldirradiation,inspiteofbothearlyandlateperiodaftertheradiotherapy.Thedamageofthehernatopoieticstemcellandmicroenvironmentofthebonemarrowcanbefoundafterirradiationandthelong-termmyelosuppressioncanalsoberesuItedinwhenwide-fieldirradiationisused.It'sveryimportantforfurtherclinicaltherapytounderstandthcIateeffectsonbonemarrowsafterwide-fieldortotalbodyirradiation.Inthispaperthelateeffectsonbonemarrowsinmiceaftertotalbodyirracliationwithsi…     

Growth Inhibiton of Human Breast Cancer Cell Line MDAMB-231 by Rosiglitazone through Activation of PPARγ

Objective  To investigate the anti-proliferative effect of rosiglitazone and its relationship to peroxisome proliferator-activated receptor γ (PPARγ) in human breast cancer cell line MDA-MB-231 and evaluate the potential application value of rosiglitazone for breast cancer therapy. Methods  The cytostatic effect of rosiglitazone on MDA-MB-231 cells was measured by the MTT assay. Cell-cycle kinetics was assessed by flow cytometry. Apoptotic cells were determined by the TUNEL assay. MDA-MB-231 cells were treated with rosiglitazone or in combination with the PPARγ antagonist GW9662 to investigate the effect of rosiglitazone on cell proliferation and its relationship to PPARγ. Results  The results showed that rosiglitazone could inhibit growth of MDA-MB-231 cells in a dose- and time-dependent manner with an IC₅₀ value of 5.2 μmol/L at 24 h after the drug was added into the culture. Cell cycle analysis showed that the percentage of G₀/G₁ phase cells increased, S phase cells decreased, and cells were arrested in G1 phase with increasing concentrations of rosiglitazone. Detectable signs of apoptotic cell death caused by rosiglitazone occurred at a concentration of 100 μmol/L and the apoptotic rate was (18 ± 3)%. PPARγ selective antagonist GW9662 could partially reverse the inhibitory effect of rosiglitazone on proliferation of MDA-MB-231 cells. Conclusion  It was concluded that rosiglitazone can inhibit growth of MDA-MB-231 cells via PPARγ activation and a high concentration of rosiglitazone can also induce MDA-MB-231 cell apoptosis. These results suggest that PPARγ represents a putative molecular target for chemopreventive therapy and rosiglitazone may be effective in the treatment of breast cancer.     

Innate Anti-breast Cancer Immunity of Apoptosis-resistant Human <Emphasis Type="Italic">γδ</Emphasis>-T cells

Summary We previously identified a CD2-initiated signaling pathway which inhibits activation-induced cell death in mitogen-stimulated human γδ-T cells permitting the large-scale expansion of these cells. Here we report the innate anti-tumor activity of expanded human γδ-T cells against human breast cancer cells. Apoptosis-resistant human γδ-T cells which were expanded in vitro from cultured human peripheral blood mononuclear cells displayed lytic activity against breast cancer cell lines MDA-MB-231, MCF-7 and T-47D, but failed to kill normal human skin fibroblasts and normal human liver cells. Monoclonal antibodies (mAb) directed against the γδ-T cell receptor (TCR) or mAb directed against either the Vγ9 or the Vδ2 TCR chains were able to block γδ-T cell-mediated lysis of MDA-MB-231 cells. In addition, mAb against intercellular adhesion molecules-1 (ICAM-1/CD54) or CD18 (β subunit of ICAM-1 counter–receptor) also blocked γδ-T cell-mediated killing of MDA-MB-231 cells. Ex vivo expanded human γδ-T cells are thus able to innately recognize and kill human breast cancer cells in a γδ-TCR-dependent manner; ICAM-1 and CD18 also appear to be involved in the interactions between sensitive breast cancer cells and cytolytic γδ-T cells. As apoptosis-resistant human γδ-T cells can now readily be expanded to large numbers (clinical scale), these findings must be considered in the context of developing adoptive immunotherapy strategies to exploit γδ-T cell innate immune responses for the primary or adjuvant treatment of breast cancer.     

Lipid Peroxidation and Activity of Some Antioxidant Enzymes in Patients with Glioblastoma and Astrocytoma

The aim of this study was to evaluate the concentration of malondialdehyde—MDA (one of the lipid peroxidation products)—in blood plasma and erythrocytes and the activity of superoxide dismutase (SOD) and catalase (CAT) in red blood cells of patients with a primary brain tumour. The study was performed on 24 patients with a brain tumour (9 with glioblastoma and 15 with asrocytoma) treated in the Department and Clinic of Neurosurgery and Neurotraumatology at Ludwik Rydygier Medical University in Bydgoszcz. The control group consisted of 20 healthy volunteers. A statistically significant higher MDA concentration in erythrocytes and blood plasma and a higher activity of SOD or CAT in erythrocytes was shown in patients with a brain tumour as compared to the control group. Neither the histological type of tumour nor surgery has an effect on the tested biochemical parameters.     

In vitro effects of R-verapamil on the cytokine environment and T-lymphocyte proliferation when human T-lymphocyte activation takes place in the presence of acute myelogenous leukemia blasts

 We investigated the effects of R-verapamil on the cytokine environment and T-lymphocyte proliferation when human T-lymphocytes were activated in the presence of accessory cells containing a large population of acute myelogenous leukemia (AML) blasts (nonirradiated blasts for cytokine studies, 50 Gy irradiated blasts in proliferation studies). In the presence of AML blasts, R-verapamil inhibited interleukin 4 (IL4) and interferon-γ (IFNγ) release from polyclonal T-cell lines activated with the T-cell mitogen phytohemagglutinin (PHA). R-verapamil also inhibited both the proliferation and the release of IFNγ and IL10 by normal T-cells stimulated with allogeneic peripheral blood mononuclear cells derived from AML patients. This antiproliferative effect of R-verapamil was seen in the presence of exogenous IL2 but was not observed in the presence of exogenous IL1β or granulocyte/macrophage colony-stimulating factor GM–CSF). In addition R-verapamil inhibited the release of IL1β and tumor necrosis factor α during allogeneic stimulation. Received: 7 January 1996/Accepted: 11 April 1996     

Mechanism of action and spectrum of cell types susceptible to doxorubicin photochemotherapy

We have shown that, whereas argon ion laser irradiation alone is not cytocidal for L929 cells, it greatly increases the cytotoxicity of intracellular doxorubicin. The present study showed that light enhancement of doxorubicin cytotoxicity was not restricted to stock L929 cells, but could also be demonstrated using L929 cells selected for doxorubicin resistance and several standard cell lines that are relatively resistant to doxorubicin prior to selection. Light-enhanced cytotoxicity resulted in extensive nuclear DNA loss and was strongly inhibited by anoxia. These findings suggest that the mechanism by which light exposure enhances doxorubicin cytotoxicity involves DNA damage by intranuclear generation of reactive oxygen species. Received: 10 June 1996 / Accepted: 16 November 1996