Hyperthermia inhibits homologous recombination repair and sensitizes cells to ionizing radiation in a time‐ and temperature‐dependent manner

Hyperthermia has long been known as a radio‐sensitizing agent that displays anti‐tumor effects, and has been developed as a therapeutic application. The mechanisms of hyperthermia‐induced radio‐sensitization are highly associated with inhibition of DNA repair. Our investigations aimed to show how hyperthermia inactivate homologous recombination repair in the process of sensitizing cells to ionizing radiation by using a series of DNA repair deficient Chinese Hamster cells. Significant differences in cellular toxicity attributable to hyperthermia at and above 42.5°C were observed. In wild‐type and non‐homologous end joining repair mutants, cells in late S phase showed double the amount heat‐induced radio‐sensitization effects of G1‐phase cells. Both radiation‐induced DNA double strand breaks and chromatin damage resulting from hyperthermia exposure was measured to be approximately two times higher in G2‐phase cells than G0/G1 cells. Additionally, G2‐phase cells took approximately two times as long to repair DNA damage over time than G0/G1‐phase cells. To supplement these findings, radiation‐induced Rad51 foci formations at DNA double strand break sites were observed to gradually dissociate in response to the temperature and time of hyperthermia exposure. Dissociated Rad51 proteins subsequently re‐formed foci at damage sites with time, and occurred in a trend also related to temperature and time of hyperthermia exposure. These findings suggest Rad51's dissociation and subsequent reformation at DNA double strand break sites in response to varying hyperthermia conditions plays an important role in hyperthermia‐induced radio‐sensitization. J. Cell. Physiol. 228: 1473–1481, 2013. © 2012 Wiley Periodicals, Inc.     

Gossypol effects on endothelial cells and tumor blood flow

Isomers (-, +) of the antitumor agent gossypol (G) were studied for their ability to reduce tumor ATP and blood flow in rats bearing subcutaneously implanted pancreatic tumors. A 50% reduction in tumor ATP/Pi within ih of a single injection of -G was associated with a 60% decline in tumor blood flow. To determine if these changes in tumor physiology could be due to a direct drug effect on tumor endothelium, G isomers were compared for their ability to alter protein (125I-BSA) permeability and metabolic (32P) labelling of cultured endothelial cells. Treatments for ih produced no endothelial cell leakage, but 24h exposures to either -G (5 microM) or +G (50 microM) produced complete permeability of the monolayers to 125I-BSA. In contrast, 0.5-I.Oh exposures to -G (4 microM) produced 2 to 3-fold increases in phosphorylated 27 kDa heat-shock protein, hsp-27. Hsp-27 phosphoprotein isoforms were differentially labelled following -G and +G exposures with the phosphorylation profile of -G appearing most similar to that of oxyradical producing agents known to induce hsp-27 and injure endothelial cells. We postulate that the tumor ischemic effects of -G are mediated by endothelial response to oxyradical production in a mechanism similar to that of tissue ischemia-reperfusion injury.     

Synergistic antiproliferative effect of delta 12-prostaglandin J2 (delta 12-PGJ2) and hyperthermia on human esophageal cancer cell lines

delta 12-PGJ2, one of the cyclopentenone prostaglandins and the ultimate metabolite of prostaglandin D2, has been reported to have potent antiproliferative activity on various tumor cells in vitro and in vivo. In this study, the combined effect of delta 12-PGJ2 and hyperthermia on six established cell lines of human esophageal carcinoma (SGF series) was analyzed by an in vitro assay, and the degree of apoptosis induced by this combination was examined to clarify the mechanism of supra-additive effects. In five SGF cell lines, except SGF-7 cells, combination therapy with delta 12-PGJ2 and hyperthermia showed synergistic antiproliferative effects. The supra-additive combined effect of delta 12-PGJ2 and hyperthermia on esophageal cancer cells is attributed to the synergistic induction of apoptosis. delta 12-PGJ2 induced G1 accumulation and apoptosis was induced by delta 12-PGJ2 from G1 phase. Hyperthermia induced G1 accumulation and apoptosis was induced by hyperthermia during all cell phases. Both augmented G1 arrest followed by G1 phase-selective induction of apoptosis and increased apoptotic induction without cell-cycle specificity are responsible for the synergism of combined treatment with delta 12-PGJ2 and hyperthermia.     

Thermal sensitivity of endothelial cells

Experimental work indicates that one of the mechanisms of tumor control by hyperthermia may be damage to blood vessels, resulting in decreased blood flow to the neoplasms. Among the various elements of the microvasculature, endothelial cells are the most important possible targets of thermal injury. Furthermore, neoplasms have a significantly higher proportion of proliferating endothelial cells than do normal tissues. Thus it is necessary to establish the thermal sensitivity of endothelial cells and to explore possible differences in response between resting and proliferating endothelium. We studied the in vitro thermal sensitivity of murine and human capillary endothelial cells compared to human fibroblasts by following cell survival and growth recovery. Nonstimulated endothelial cells are more sensitive than fibroblasts. Their sensitivity is dose dependent within the range of 42 to 45 degrees C/30 min. Stimulation to proliferate by endothelial cell growth factor (ECGF) renders these cells even more sensitive. Morphologic studies confirm these thermal effects in endothelial cells and fibroblasts. These findings support a direct effect of hyperthermia on endothelial cells, which appears to be more severe in proliferating cells. This may explain the reduced blood flow in heated tumors and may indicate a valuable therapeutic gain for hyperthermia.     

沙利度胺通过诱导G1阻滞和凋亡抑制血管内皮细胞增殖

沙利度胺是一种抗血管生成药物,临床上用于治疗多种肿瘤,但其抗肿瘤血管生成机制尚不十分清楚. 本文采用MTT法观察沙利度胺对体外培养的血管内皮细胞增殖的影响. 结果发现,沙利度胺能够抑制血管内皮细胞的增殖,其IC50为16.47 μg/mL;然后采用Hoechst染色和流式细胞仪检测细胞凋亡和细胞周期,发现沙利度胺能够诱导内皮细胞凋亡,并干扰细胞的周期,出现G0/G1期阻滞. 最后,通过Western印迹方法分析沙利度胺对血管内皮细胞Bcl-2蛋白表达的影响,发现抗凋亡的Bcl-2蛋白表达水平随沙利度胺浓度增大而降低. 初步结果提示,沙利度胺可能通过阻遏抗凋亡分子Bcl-2表达,激活诱导G1期阻滞的信号通路而抑制内皮细胞新生,从而抑制肿瘤生长. 诱导内皮细胞凋亡及G1期阻滞的具体分子机制正在研究中.     

Combined use of hyperthermia and irradiation cause antiproliferative activity and cell death to human esophageal cell carcinoma cells--mainly cell cycle examination

In clinically hyperthermia and irradiation therapy for malignant neoplasms are known that they have antiproliferative activity and cell death (including apoptosis) inducing activity. However not only mechanisms of cell death induction but treatment effects of them still have been unclear. In this time we showed that cell cycles from G0/G1 phase to S-G2/M phase were delayed by hyperthermia and G2/M phase accumulation were caused immediately by irradiation. And we also demonstrated that the combination treatments of hyperthermia and irradiation induced synergistic antiproliferative effects and strong effects of cell death to human esophageal carcinoma cell lines. Although treatments of hyperthermia and irradiation were mild individually, combination treatment of hyperthermia and irradiation were useful for esophageal carcinoma treatment.     

低氧对肺动脉内皮细胞PDGF—B链释放及平滑肌细胞生长的影响

应用蛋白ｄｏｔｂｌｏｔ技术检测了低氧内皮细胞条件培养液（ＨＥＣＣＭ）和常氧内皮细胞条件培养液（ＮＥＣＣＭ）内ＰＤＧＦ相对含量,并利用［３Ｈ］－ＴｄＲ掺入法和流式细胞术观察了ＨＥＣＣＭ和ＮＥＣＣＭ及加入特异ＰＤＧＦ抗体对肺动脉平滑肌细胞（ＰＡＳＭＣ）生长的影响。结果表明,ＨＥＣＣＭ中的ＰＤＧＦ含量明显高于ＮＥＣＣＭ;ＨＥＣＣＭ能明显增强ＰＡＳＭＣ内ＤＮＡ合成,促进ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期;当预先加入ＰＤＧＦ－Ｂ链抗体时,则会明显地抑制ＨＥＣＣＭ对ＰＡＳＭＣ的ＤＮＡ合成,阻止ＰＡＳＭＣ从Ｇｏ／Ｇ１期进入Ｓ期。结果提示,低氧时ＰＡＳＭＣ增殖与肺动脉内皮细胞分泌释放ＰＤＧＦ增加有关     

Endothelial cell subpopulations in vitro: cell volume, cell cycle, and radiosensitivity

Vascular endothelial cells (EC) are important clinical targets of radiation and other forms of free radical/oxidant stresses. In this study, we found that the extent of endothelial damage may be determined by the different cytotoxic responses of EC subpopulations. The following characteristics of EC subpopulations were examined: 1) cell volume; 2) cell cycle position; and 3) cytotoxic indexes for both acute cell survival and proliferative capacity after irradiation (137Cs, gamma, 0-10 Gy). EC cultured from bovine aortas were separated by centrifugal elutriation into subpopulations of different cell volumes. Through flow cytometry, we found that cell volume was related to the cell cycle phase distribution. The smallest EC were distributed in G1 phase and the larger cells were distributed in either early S, middle S, or late S + G2M phases. Cell cycle phase at the time of irradiation was not associated with acute cell loss. However, distribution in the cell cycle did relate to cell survival based on proliferative capacity (P less than 0.01). The order of increasing radioresistance was cells in G1 (D0 = 110 cGy), early S (135 cGy), middle S (145 cGy), and late S + G2M phases (180 cGy). These findings 1) suggest an age-related response to radiation in a nonmalignant differentiated cell type and 2) demonstrate EC subpopulations in culture.     

Inhibition of endothelial cell proliferation by platelet factor-4 involves a unique action on S phase progression

Modulation of endothelial cell proliferation and cell cycle progression by the "chemokine" platelet factor-4 (PF-4) was investigated. PF-4 inhibited DNA synthesis, as well as proliferation of endothelial cells derived from large and small blood vessels. Inhibition by PF-4 was independent of the type and the concentration of stimuli used for the induction of endothelial cell proliferation. Inhibition of cell growth by PF-4 was reversible. The effects of PF-4 were antagonized by heparin. Cell cycle analysis using [3H]thymidine pulse labeling during traverse of synchronous cells from G0/G1 to S phase revealed that addition of PF-4 during G1 phase completely abolished the entry of cells into S phase. In addition, PF-4 also inhibited DNA synthesis in cells that were already in S phase. In exponentially growing cells, addition of PF-4 resulted in an accumulation of > 70% of the cells in early S phase, as determined by FACS (Becton-Dickinson Immunocytometry Systems, Mountain View, CA). In cells synchronized in S phase by hydroxyurea and then released, addition of PF-4 promptly blocked further progression of DNA synthesis. These results demonstrate that in G0/G1-arrested cells, PF-4 inhibited entry of endothelial cells into S phase. More strikingly, our studies have revealed a unique mode of endothelial cell growth inhibition whereby PF-4 effectively blocked cell cycle progression during S phase.     

The cell cycle dependence of thermotolerance. II. CHO cells heated at 45.0 degrees C

This report extends our investigations of the cell cycle dependence of the expression of thermotolerance to include tolerance expressed by Chinese hamster ovary (CHO) cells exposed to 45.0 degrees C hyperthermia. We examined the response of asynchronous cells following exposure at 45.0 degrees C. A maximum in thermotolerance under these conditions was reached approximately 12 hr after a 15-min exposure to 45.0 degrees C hyperthermia and progressively decreased thereafter. Cells were delayed in S and G2 phase for 24 hr, after which time cell growth resumed. We then characterized the response of CHO cell populations synchronized in G1 or early or late S phase. We observed that the expression of tolerance depended on the position of cells in the cell cycle and was modulated by changes in the sensitivity of cells as they progressed through the cell cycle subsequent to the tolerance induction dose. We measured the variation in the sensitivity of these cells to 45.0 degrees C hyperthermia throughout the cell cycle and found substantial changes as cells progressed through S phase. Cells in early S phase were the most sensitive to heat at this temperature, and as these cells progressed through S phase, they became progressively more resistant. In addition, G1 cells were delayed for approximately 15 to 18 hr by a 15-min, 45.0 degrees C heat pulse, whereas S-phase cells were delayed to a lesser extent. The data presented in this report suggest that the induction of thermotolerance is relatively non-cell-cycle specific, but the magnitude of expression of tolerance depends on the position of cells in the cell cycle at the time of the subsequent challenge heat dose.     

Magnolol suppresses proliferation of cultured human colon and liver cancer cells by inhibiting DNA synthesis and activating apoptosis

Magnolol, a hydroxylated biphenyl compound isolated from the Chinese herb Hou p'u of Magnolia officinalis, has been reported to have anti-cancer activity. In the present study, magnolol at very low concentrations of 3-10 microM inhibited DNA synthesis and decreased cell number in cultured human cancer cells (COLO-205 and Hep-G2) in a dose-dependent manner, but not in human untransformed cells such as keratinocytes, fibroblasts, and human umbilical vein endothelial cells (HUVEC). Magnolol was not cytotoxic at these concentrations and this indicates that it may have an inhibitory effect on cell proliferation in the subcultured cancer cell lines. [(3)H] thymidine incorporation and flow cytometry analyses revealed that magnolol treatment decreased DNA synthesis and arrested the cells at the G0/G1 phase of the cell cycle. Moreover, the magnolol-induced cell cycle arrest occurred when the cyclin-CDK system was inhibited, just as p21 protein expression was augmented. When magnolol concentration was increased to 100 microM, apoptosis was observed in COLO-205 and Hep-G2 cells, but not in cultured human fibroblasts and HUVEC. COLO-205 cells implanted subcutaneously in nude mice formed solid tumors; subsequent daily i.p.-injections of magnolol led to profound regression of these tumors of up to 85%. In these tumors, an increase in the expression of p21 protein level and the occurrence of apoptosis were observed. These findings demonstrate for the first time that magnolol can inhibit the proliferation of tumor cells in vitro and in vivo.     

Cell cycle-dependent changes in tissue transglutaminase mRNA levels in bovine endothelial cells.

The pattern of transglutaminase gene expression through the cell cycle was examined by Northern blot analysis using cultured bovine endothelial cells and a cDNA probe. When the cells reached confluency or were arrested in G0/G1 phase by nutrition deprivation, transglutaminase mRNA rose to a very high level; S- and M-phase extracts showed high and low levels, respectively. Subcellular localization studies by sucrose gradient centrifugation and immunostaining demonstrated that the majority of transglutaminase is present in cytosols throughout the cycle. The cell cycle-dependent changes in the transglutaminase mRNA levels strongly support the implicated involvement of the enzyme in cell growth, differentiation, and senescence.     

Silver nanocrystals sensitize magnetic-nanoparticle-mediated thermo-induced killing of cancer cells

Magnetic nanoparticles (MNPs) can heat up tumor tissues and induce killing of cancer cells under external AC magnetic field. However, magnetic nanoparticles hyperthermia (MNPH) requires high concentration of MNPs that are injected into the tumor in order to obtain clinically needed thermal dose because of the complicated heat transfer in vivo and the limited heat quality of MNPs. To cut down the dose of MNPs and enhance the effect of this Nanotherapy, we prepared silver nanoparticles (AgNPs) with different sizes and investigated the effects of these AgNPs on cancer cells in MNPH treatment. It was found that AgNPs could enhance thermo-sensitivity of glioma cells and this effect was size dependent. AgNPs could induce cell cycles arrested in G(2)/M phase and enhanced the apoptosis rate of cancer cells after hyperthermia. In glioma bearing rats model, MNPH combined with AgNPs could enhance Bax expression in cancer cells. Our results suggested that AgNPs could be a potential thermo-sensitizer and could be further developed for the design of Ag nanostructure-based thermal seeds for MNPH therapy.     

肝癌细胞与内皮细胞的粘附力学特性研究

采用细胞同步技术和微管吸吮技术,从细胞周期的角度研究不同细胞周期肝癌细胞(hepatocellularcarcinoma cells,HCC)与脐静脉内皮细胞(HUVEC)的粘附力学特性。结果表明,未同步化肝癌细胞各周期时相的细胞百分比为:G_0/G_1期,53.51％;G_2/M期,11.01％;S期,35.48％。采用胸腺嘧啶脱氧核苷、秋水仙碱顺序阻断和胸腺嘧啶脱氧核苷双阻断后释放培养的方法可分别获得G_1期和S期的肝癌细胞,其平均同步率分别为69.02％和96.50％。G_1期肝癌细胞与人脐静脉内皮细胞的粘附力比S期相应值明显降低(P<0.01),与未同步化肝癌细胞组比较也得到同样结果,而S期与未同步化肝癌细胞组的粘附力值无明显差别。肝癌细胞与脐静脉内皮细胞的粘附力随着粘附时间的变化而变化,在30～60min内迅速增长,60min之后维持在较稳定的水平,即300×10~(10)N左右。提示:在肝癌细胞与内皮细胞的粘附过程中,S期细胞可能起的作用更大;肝癌细胞和内皮细胞上粘附分子表达呈现时间效应,从而体现出粘附和去粘附的行为特征。     

Addition of both platelets and thrombin in combination accelerates tumor cells to adhere to endothelial cells In vitro

Summary Platelets and coagulation are involved in the pathogenesis of blood-borne metastases. The aim of this study is to obtain more information about the mechanisms involved in the initial adhesion of tumor cells to endothelial cells. In short term experiments with tumor cells, suspended in the medium of cultured endothelial cells, we tested whether addition of both platelets and thrombin cause more tumor cell adhesion to endothelial cells, than when either platelets or thrombin are acting alone. HeLa cells or HT29 cells, prelabeled with radioactive ⁵¹Cr, human platelets, and thrombin were added to human endothelial cell cultures. Following 15 min of shaking at 37° C, the percentage of tumor cell adhesion was calculated. The percentages of adhering tumor cells with the presence of both platelets and thrombin were greatly increased compared to controls. Addition of hirudin 2 min before thrombin lowered the adhesion percentage of tumor cells. Hirudin added immediately before and 2 min after thrombin gave only minor effects. When the endothelium was treated with superoxide dismutase, catalase, and mannitol, the adhesion of tumor cells was lowered with catalase and superoxide dismutase. The cause of tumor cell-endothelial cell interaction is probably complex. Our results show that activated platelets enhance the tumor cell adhesion, and that generation of active oxygen species may be important in the initial phase of the interaction.     

IFNgamma-inducible CXCL10/CXCR3 axis alters the sensitivity of HEp-2 cells to ionizing radiation

Radiotherapy is a conventional approach for anti-cancer treatment, killing tumor cells through damaging cellular DNA. While increasing studies have demonstrated that tumors generated the tolerance to radiation and tumor immune system was found to be correlated to radiotherapy resistance. Therefore, it is critical to identify potential immune factors associated with the efficacy of radiotherapy. Here in this study, we evaluated the sensitivities of different tumor cells to radiation and determined HEp-2 cells as the radio-resistant tumor cells for further investigation. IFNgamma as a key regulator of host immune response showed the potential to sensitize tumors to ionizing radiation (IR). Besides, IFNgamma-induced CXC chemokine ligand 10 (CXCL10) was found to be necessary for effective IR-induced killing of cultured HEp-2 cells. Increased clonogenic survival was observed in CXCL10-depleted HEp-2 cells and CXCL10-KO cells. Additionally, the loss of CXCL10 in HEp-2 cells showed less progression of the G0/G1 phase to G2/M when exposed to IR (8 Gy). Local IR (20 Gy) to nude mice bearing HEp-2 tumors significantly reduced tumor burden, while fewer effects on tumor burden in mice carrying CXCL10-KO tumors were observed. We furtherly evaluated the possible roles the chemokine receptor CXCR3 plays in mediating the sensitivity of cultured HEp-2 cells to IR. Altered expression of CXCR3 in HEp-2 cells affected IR-induced killing of HEp-2 cells. Our data suggest the IFNgamma-activated CXCL10/CXCR3 axis may contribute to the effective radiation-induced killing of HEp-2 cells in vitro.     

与转染Endostatin基因的角朊细胞共培养时内皮细胞增殖特性变化

构建含Endostatin基因的腺病毒载体,将Endostatin基因导入培养的角朊细胞,并采用套皿法共培养角朊细胞与内皮细胞,测定培养液中Endostatin含量、内皮细胞增殖周期各时相比例、内皮细胞凋亡及细胞抑制率。结果表明转染Endostatin基因的角朊细胞可有效表达并分泌Endostatin,连续培养3天后,培养液中Endostatin含量可达226ng/ml;与转基因角朊细胞共培养的内皮细胞凋亡百分数与抑制率分别为(32.7±7.1)%、(60.5±8.3)%,均显著高于对照组[(7.3±2.0)%,(13.8±1.6)%],且G0/G1期比例明显高于对照组,而S期、G2/M期比例及增殖指数显著低于对照组。因此,转染Endostatin基因角朊细胞与内皮细胞共培养时,角朊细胞可通过分泌Endostatin促进内皮细胞凋亡,并抑制其增殖。     

Stimulation of de novo synthesis of prostaglandin G/H synthase in human endothelial cells by phorbol ester

The present study was undertaken to determine the mechanism by which phorbol ester stimulates eicosanoid synthesis in endothelial cells. We observed that phorbol 12-myristate 13-acetate (PMA) actively stimulated eicosanoid synthesis over a prolonged period of time, and the stimulatory effect was abolished by cycloheximide and actinomycin D. Western blot was employed to test the hypothesis that PMA elicited sustained eicosanoid synthesis via the stimulation of de novo synthesis of prostaglandin G/H synthase (cyclooxygenase, EC 1.14.99.1). Treatment of cultured human umbilical vein endothelial cells resulted in an enhancement of the 70-kDa immunoreactive prostaglandin G/H synthase band over the control cells treated with medium alone. The enhancement was abolished by cycloheximide. Human umbilical vein endothelial cells were then metabolically labeled with L-[35S]methionine, and the effect of PMA on methionine incorporation was evaluated by immunoblotting. PMA increased the synthetic rate of prostaglandin G/H synthase over the control cells. By pulse-chase experiments, we further showed that prostaglandin G/H synthase has a rapid turnover rate (t1/2 less than 10 min) in control cells, and PMA had no effect on the enzyme turnover. Our data indicate that PMA increases the synthesis of prostaglandin G/H synthase which is required for circumventing the autoinactivation of prostaglandin G/H synthase and hence permit sustained conversion of arachidonic acid into eicosanoids.