Inhibitory Effects of Mild Hyperthermia plus Docetaxel Therapy on ER(+/–) Breast Cancer Cells and Action Mechanisms

Summary： The purpose of this study was to verify that a combination of mild hyperthermia and do- cetaxel chemotherapy produces synergistic antitumor effects and to explore the action mechanisms of this treatment approach. The effects of docetaxel on the proliferation of cells from the estrogen receptor （ER）-positive human breast cancer cell line MCF-7 and the ER-negative human breast cancer cell line MDA-MB-453 were examined by 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay, and effective experimental concentrations of docetaxel were determined. The effects of mild hy- perthermia plus docetaxel therapy on apoptosis rate in the MCF-7 and MDA-MB-453 human breast cancer cell lines were analyzed by using flow cytometry with Annexin-V fluorescein isothiocyanate （FITC）/propidium iodide （PI） staining. The effects of these combined treatments on cell cycle progres- sion in the MCF-7 and MDA-MB-453 human breast cancer cell lines were examined by using flow cy- tometry. The effects of these combined treatments on the expression of apoptosis-related proteins and proteins in the mitogen-activated protein kinase （MAPK） pathways were analyzed by using Western blotting. The effects of these combined treatments on the expression of the heat shock protein 70 （HSP70） and the multi-drug resistance （MDR） gene product P-glycoprotein （Pgp） were examined by using Western blotting. The results showed that the half-maximal inhibitory concentration （IC50） of do- cetaxel for MCF-7 and MDA-MB-453 cells was 19.57±1.12 and 21.64±2.31 gmol/L respectively. Mild hyperthermia with docetaxel therapy could increase apoptosis rate in the MCF-7 and MDA-MB-453 cells. Apoptosis rate in MCF-7 and MDA-MB-453 cells was increased from （23.66±3.59）% and （18.51±3.17）% in docetaxel treatment group to （47.12±6.73）% and （55.16±7.42）% in mild hyperthermia plus docetaxel group, indicating that the mild hyperthermia and docetaxel therapeutic approaches exhib- ited significant synergistic antitumor effects. Treatments of mild hyperthermia plus docetaxel induced G2/M cell cycle arrest in the MCF-7 and MDA-MB-453 cells. Western blotting demonstrated that pro- teins in the MAPK pathway were expressed at higher levels in docetaxel-treated cells following mild hypothermia than those in cells treated with docetaxel alone. As compared with blank control group, cells from the mild hyperthermia plus docetaxel group exhibited significantly decreased B-cell lym- phoma 2 （Bcl-2） protein expression but slightly increased Bcl-2-associated X protein （Bax） expression. Western blotting results revealed that HSP70 and Pgp expression levels were significantly increased following mild hypothermia. It was concluded that treatments of mild hyperthermia plus docetaxel in- hibited the proliferation of human breast cancer cells, promoted apoptosis of breast cancer cells, and produced synergistic antitumor effects.

A Dual-radiolabel Marker Quantifies Decrease in HT29 Xenograft Hypoxia Induced by Mild Temperature Hyperthermia

Purpose: In this project, we developed novel methods to quantify changes in tumor hypoxia following a mild tempera-ture hyperthermia (MTH) treatment in rat HT29 human colon adenocarcinoma xenograft. Materials and Methods: An exogenous hypoxia marker (IAZGP) was labeled with two radioisotopes of iodine (131I and 123I, respectively) to form two distinct tracers. The two tracers were injected into HT29-bearing nude rats 4-hour before and immediately following 41.5℃, 45-minute mild hyperthermia treatment. The distributions of the two hypoxia tracers were obtained by performing digital autoradiography on tumor sections, and image processing resulted in quantitative information at 50 μm pixel size. Results: Following the hyperthermia treatment, there was a remarkable decrease in hypoxia tracer binding. The average whole tumor hypoxia tracer targeted fraction in five animals changed from 30.3% ± 9.7% to 13.0% ± 5.3% after the hyperthermia treatment (P = 0.001). Detailed pixelby-pixel analysis of the image data revealed a decline in hypoxia tracer uptake after hyperthermia in most regions. However, there was concomitant emergence of some new regions of hypoxia identified by increased tracer uptake. In the control group, the overall hypoxia tracer targeted fraction remained almost constant, with some hypoxic tracer redistribution (putative acute hypoxia) observed. Conclusion: Reoxygenation occurred in the rat HT29 xenograft following MTH treatment. This was evident with preponderance of decreased hypoxia specific tracer uptake on tumor sections. Our methodology might be a useful tool in hypoxia study.

Effect of Tirapazamine and Mild Temperature Hyperthermia on the Recovery from Radiation-Induced Damage in Pimonidazole-Unlabeled Quiescent Tumor Cell Population

The aim in this study is to examine the effect of tirapazamine (TPZ) and mild temperature hyperthermia (MTH) on the repair of radiation-induced damage in pimonidazole-unlabeled quiescent (Q) tumor cells. Labeling of proliferating (P) cells in C57BL/6J mice bearing EL4 tumors was achieved by continuous administration of 5-bromo-2-deoxyuridine (BrdU). Tumors were irradiated with γ-rays at 1 h after the administration of pimonidazole followed by TPZ treatment or MTH. Twenty-four hours later, assessment of the responses of Q and total (= P + Q) cells were based on the frequencies of micronucleation and apoptosis using immunofluorescence staining for BrdU. The response of the pimonidazole-unlabeled tumor cell fractions was assessed by means of apoptosis frequency using immunofluorescence staining for pimonidazole. With γ-rays only, the pimonidazole-unlabeled cell fraction showed significantly enhanced radio-sensitivity compared with the whole cell fraction more remarkably in Q cells than total cells. However, a significantly greater decrease in radio-sensitivity in the pimonidazole-unlabeled than the whole cell fraction, evaluated using a delayed assay, was more clearly observed in Q cells than total cells. Post-irradiation MTH more remarkably repressed the decrease in radio-sensitivity in the Q cell than the total cells. Post-irradiation TPZ administration produced a large radio-sensitizing effect on both total and Q cells, especially on Q cells. On the other hand, in pimonidazole-unlabeled cell fractions in both total and Q cells, TPZ suppressed the reduction in sensitivity due to delayed assay much more efficiently than MTH, whereas no radio-sensitizing effect was produced. Not only through suppressing the recovery from radiation-induced damage but also through radio-sensitizing effect, post-irradiation TPZ administration is very useful for repressing the increase in the difference in radio-sensitivity due to the delayed assay not only between total and Q tumor cells but also between the pimonidazole-unlabeled and the whole cell fractions within the total and Q tumor cells.

低温热疗对肺腺癌细胞放射敏感性的影响

目的:研究低温热疗(≤42℃)对肺腺癌细胞株SPC A 1的放射增敏作用及其对生长周期的影响。 方法: 集落形成率实验观察低温热疗的放射增敏作用;流式细胞仪观察细胞生长周期的变化。 结果:单纯放射时细胞的 Do、Dq值分别为1.693、2.453Gy,放射合并41.5℃加热时Do、Dq值分别为1.390、1.426Gy,热增敏比为1.218。放 射及热放射后细胞周期出现了再分布。未处理组流式细胞仪检测到S期细胞比例为14.81%,照射6Gy后48、72h 分别为18.80%和31.91%;照射后立即41.5℃加热1h,48、72h的S期细胞分别为5.89%和9.08%。 结论:低 温热疗对肺腺癌细胞系有放射增敏作用,低温热疗能去除放射引起的细胞S期阻滞,其机制可能与加热抑制了肿 瘤细胞对放射所致亚致死损伤和潜在致死损伤的修复能力有关。

脑温对局灶脑缺血再灌注损伤氨基酸含量的影响

目的 研究轻度高温、亚低温对局灶脑缺血 EAA和 IAA的影响。方法 建立大鼠可复流 MCA闭塞模型 ,诱导目标脑温 ,用 HPL C荧光法检测脑组织 Glu、Asp、Gly、GABA含量。结果 轻度高温组 Glu、Asp、Gly明显增高 ,GABA短暂增高后下降 ;亚低温组 Glu、Asp、Gly降低 ,GABA持续增高。结论 轻度高温促进 [Glu]×[Gly]/ [GABA]比值增加 ,在持续增加 Glu“兴奋毒性”方面起重要作用 ;亚低温使 [Glu]× [Gly]/ [GABA]比值下降 ,在降低 Glu“兴奋毒性”方面起重要作用。轻度高温不利于 ;亚低温有利于“抑制性保护”

重型颅脑损伤的亚低温治疗体会

目的研究亚低温治疗对重型颅脑损伤患者疗效及预后的影响。方法回顾性分析50例重型颅脑损伤患者,随机分为亚低温组(n=25)及常规治疗组(n=25)。亚低温组接受32℃～35℃低温治疗,常规治疗组除亚低温治疗外,其余治疗与亚低温组相同。比较两组的颅内压(ICP)、并发症及预后的差异。结果亚低温治疗组伤后ICP显著低于常规治疗组(P<0.01)。应激性溃疡、肝肾功能异常和外伤性癫痫的发生率显著低于常规治疗组(P<0.01)。预后显著好于常规治疗组(P<0.05)。结论亚低温治疗能显著降低重型颅脑损伤患者的高颅压,减少并发症,改善预后和提高生活质量。

浅低温不停跳体外循环方法在重症搭桥手术中的应用

目的:总结重症患者在浅低温不停跳体外循环(extracorporeal circulation,ECC)下行冠状动脉旁路移植手术的效果。方法:2010年1月至2011年5月对34例择期行冠状动脉旁路移植术(coro-nary artery bypass grafting surgery,CABG)的重症患者(左主干病变、左心室舒张末期内径>60 mm、左心室射血分数<40%、术前Euroscore评分>6分、术前心源性休克、急性心肌梗死)施行浅低温ECC下不停跳CABG,术前均存在不稳定型心绞痛。其中10例(28%)合并糖尿病。3例(44%)患者术前应用了主动脉内球囊反搏(intra-aortic ballon pump,IABP)。分析总结重症CABG患者体外循环管理经验。结果:平均ECC时间68 min(42～123 min),机械通气时间平均18 h(6～84 h),ICU滞留时间平均2 d(14h～14 d),住院时间平均22 d(10～34 d)。12例患者术后应用IABP。无与ECC相关的神经系统并发症,肾功能不全6例(18%),并应用肾替代疗法。死亡2例(6%)。结论:对严重心功能不全或血管条件不好的重症CABG患者,采用浅低温不停跳ECC是一种安全可靠的手术方法。

不同灸法对胃经不同部位温度影响

目的:观察温和灸和隔姜灸足三里后胃经不同部位(足三里、腹部和足背)温度的变化,进一步研究艾灸的温热效应,为临床上选择适宜的灸法提供实验依据。方法:随机将30名大学生分为两组(温和灸组和隔姜灸组),用红外成像仪分别记录足三里、腹部和足背的灸前、灸后15、20、25、30 min,及停灸后的35、40 min时的温度。比较不同灸法在不同时段对足三里局部及胃经循经远端温度的影响。结果:两组在不同时段对足三里局部温度影响不同(P<0.05),对胃经远端循经部位温度影响也不同(P<0.001)。结论:不同灸法对胃经不同部位的温度有影响。施灸时和撤灸后,温和灸组足三里处平均温度高于隔姜灸组;且不同灸法在不同时段对胃经循经部位的温度影响不同。

动脉内亚低温对大鼠缺血再灌注损伤的脑保护研究

目的探讨血管内低温对于缺血再灌注损伤的脑保护作用。方法实验分A组:单纯缺血再灌注组;B组:20℃冷盐水灌注组;C组:10℃冷盐水灌注组。各组分别检测脑梗塞体积、各项生理指标、脑组织水含量、脑温和肛温;计算48h存活率和神经功能缺失评分。结果灌注20℃冷盐水可迅速将梗塞区皮质温度由(36.3±0.9)℃降至(34.4±0.8)℃,纹状体温度由(37.0±0.8)℃降至(35.1±0.8)℃,低温可持续1h以上;灌注10℃冷盐水可将梗塞区皮质温度由(36.1±0.2)℃降至(31.7±1.9)℃,纹状体温度由(36.6±0.4)℃降至(32.3±1.8)℃,低温可持续4h以上。与A组相比,B组和C组神经功能缺失评分有明显好转,脑梗塞体积显著减小,48h存活率明显增加(P<0.01)。结论局部低温灌注治疗可靠、有效,能明显缩小缺血后梗死体积,提高大鼠存活率,有较高的临床应用价值。

高温、亚低温对大鼠脑缺血再灌注损伤的作用及其机制研究

目的 :研究轻度高温、亚低温对大鼠脑缺血再灌注损伤组织兴奋性氨基酸 (EAA )与氧自由基的相互关系及病理损伤程度的影响。方法 :6 0只 Wistar大鼠按不同脑温条件随机分为生化组 (n =2 8)和病理组 (n =32 ) ,采用改良 Nagasawa局灶脑缺血再灌注模型 ,观察脑缺血再灌注损伤组织谷氨酸 (Glu)、超氧化物歧化酶 (SOD)、丙二醛 (MDA )的变化及光镜、电镜下的病理变化。结果 :轻度高温明显加重常温脑缺血再灌注损伤组织 Glu、MDA的升高 (P <0 .0 1)及 SOD的下降 (P <0 .0 5 ) ,加重常温脑缺血再灌注组织病理损伤程度 ;亚低温的作用则相反。结论 :轻度高温可能通过同时促进 EAA合成、释放和氧自由基生成系统活化 ,造成大鼠脑缺血再灌注组织损伤加重 ;亚低温可能通过同时抑制 EAA合成、释放和氧自由基生成系统活化 ,减轻大鼠脑缺血再灌注组织损伤程度 ,对大鼠脑缺血再灌注损伤组织起保护作用。

脑温变化对大鼠局灶脑缺血再灌注组织兴奋性氨基酸的影响

目的 研究轻度高温、亚低温对局灶脑缺血组织兴奋性氨基酸 (EAA)的影响。方法 建立大鼠大脑中动脉缺血再灌注线栓模型 ,诱导目标脑温 ,用HPLC荧光法检测脑组织谷氨酸 (Glu)、天冬氨酸 (Asp)、甘氨酸 (Gly)含量。结果 轻度高温组Glu、Asp、Gly明显增高 ;亚低温组则明显降低。结论 轻度高温促进EAA增高 ,在持续增加Glu“兴奋毒性”方面起重要作用 ;亚低温抑制EAA增高 ,在降低Glu“兴奋毒性”方面起重要作用。

体温与卒中的关系

体温是影响脑缺血预后的一个重要因素。发热通过多种机制可直接或间接与卒中有关。文章回顾 了体温与脑缺血的关系以及体温对预后的影响,探讨了其可能的机制。

局灶脑缺血再灌注脑温对抑制性氨基酸的影响

目的 研究轻度高温、亚低温对局灶脑缺血抑制性氨基酸的影响。 方法 建立大鼠大脑中动脉缺血再灌注模型 ,诱导目标脑温 ,用 HPL C荧光法检测脑组织 GABA含量。 结果 轻度高温组GABA短暂增高后下降 ,亚低温组 GABA持续增高。 结论 轻度高温使脑内 GABA水平下调 ,在加重缺血神经元损伤方面起着不容忽视的作用 :亚低温使 GABA水平持续上调 ,在保护缺血神经元方面起重要作用。

脑温不同对大鼠缺血脑组织c-fos和bcl-2表达的影响

目的 :研究脑缺血后不同脑温对相关基因表达的影响 ,探讨亚低温脑保护作用的机制。方法 :Sprague -Dawley(SD)大鼠 ,随机分为常温 (37- 38℃ )脑缺血、亚低温 (31- 32℃ )脑缺血、高温 (41- 4 2℃ )脑缺血和假手术组 ,每组 12只动物。脑缺血动物模型采用改良的Pulsinelli四动脉阻断法。脑组织中Fos和bcl- 2蛋白的检测采用链菌素亲生物素 -过氧化酶连接法 (SP)免疫组织化学技术。结果 :常温脑缺血再灌注可诱导Fos蛋白的表达 ,亚低温可使Fos蛋白的表达提早而增多 ,高温却使脑缺血时Fos蛋白的表达减少。常温脑缺血再灌注可诱导大脑皮质bcl- 2蛋白表达 ;亚低温增加bcl- 2蛋白表达 ;高温减少bcl- 2蛋白表达。结论 :脑缺血后即时开始亚低温可增加Fos和bcl- 2蛋白的表达 ,是亚低温脑保护作用的机制 ;

亚低温联合高压氧治疗脑出血术后临床效果观察

目的探讨亚低温联合高压氧(HBO)治疗脑出血术后的临床效果。方法选择66例脑出血术后患者随机分为亚低温联合HBO治疗组和对照组各33例;对照组接受常规综合治疗,治疗组在此基础上行早期亚低温联合HBO治疗。观察两组患者术后1周颅内压(ICP)、入出院时临床格拉斯哥昏迷量表(GCS)评分、动脉血氧分压、血氧饱和度以及治疗后6个月格拉斯哥结局量表(Glasgow Outcome Scale,GOS)评定预后的情况,比较两组临床疗效。结果治疗组患者术后1周ICP显著低于对照组(P<0.01),动脉血氧分压、血氧饱度明显提高(P<0.01),GCS评分和GOS分级明显优于对照组(P均<0.01)。结论脑出血术后患者经亚低温联合HBO治疗能明显提高疗效和改善患者的预后,减少后遗症的发生,提高生活质量。

热化疗对人乳腺癌细胞株MCF-7凋亡的影响

目的探讨亚高温热化疗的体外抗肿瘤作用。方法 (1)采用MTT法(四甲基偶氮唑盐比色法)观察紫杉醇对人乳腺癌细胞(MCF-7)生长的影响,筛选出有效实验浓度。(2)将体外培养的MCF-7细胞分为空白对照组、化疗组(紫杉醇)、热疗组(聚束微波加热)及热化疗组(聚束微波加热联合紫杉醇),各组MCF-7细胞分别经相应干预后,采用流式细胞术(Annexin V-FITC/PI)检测上述各组MCF-7细胞凋亡情况。结果 (1)热疗后48 h,41℃热疗组细胞凋亡率高于空白对照组、40℃、40.5℃热疗组及热疗后24 h组(P<0.05)。(2)5μg/m L化疗干预48 h、10μg/m L化疗干预24 h细胞凋亡率均分别高于其他各组(P<0.05)。(3)5μg/m L热化疗组干预24 h、48 h后细胞凋亡率均随着热疗温度的升高而升高(P<0.05);10μg/m L热化疗组干预24 h后细胞凋亡率随着热疗温度的升高而升高,干预48 h后细胞凋亡率随着热疗温度的升高而降低。(4)细胞凋亡率的顺序为:5μg/m L热化疗组48 h>10μg/m L热化疗组24 h>10μg/m L化疗组24 h>5μg/m L化疗组48 h>41℃热疗组>空白对照组。结论在体外,一定温度的热疗可杀伤肿瘤细胞,热疗联合化疗协同杀伤肿瘤细胞作用更明显,但并非化疗药物浓度越高越有效,温度越高越有效。

41℃及43℃温热疗联合聚乙二醇化脂质体阿霉素治疗兔VX2肿瘤的疗效对比研究

目的探讨41℃及43℃温热疗(MH)触发聚乙二醇化脂质体阿霉素(PLD)释放阿霉素治疗兔VX2肿瘤的疗效差异。方法 12只VX2移植瘤兔随机分为2组:PLD+41℃MH组(n=6)和PLD+43℃MH组(n=6)。为达到完全的肿瘤缓解,进行多次高剂量耳缘静脉推注给药(5 mg/kg,每周1次,共治疗3次),使用嵌入式超声温热治疗仪对肿瘤部位进行30 min局部MH。结果在观察终点,PLD+41℃MH组血管形成指数(VI)、瘤体质量、外周血谷草转氨酶(GOT)、乳酸脱氢酶(LDH)、肌酸激酶(CPK)值均明显低于PLD+43℃MH组。结论同43℃下30 min MH相比,41℃下30 min MH联合PLD可以明显提高抗肿瘤疗效,降低系统毒性。

Changes of c-fos, Malondialdehyde and Lactate in Brain Tissue after Global Cerebral Ischemia under Different Brain Temperatures

Summary： Under global cerebral ischemia, the effect of different brain temperature on cerebral ischemic injury was studied. Male Sprague-Dawley rats were divided into normothermic （37-38℃） ischemia, mild hypothermic （31 32℃） ischemia, hyperthermic （41-42℃） ischemia and sham-operated groups. Global cerebral ischemia was established using the Pulsinelli four-vessel occlusion model and brain temperature was maintained at defined level for 60 min after 20omin ischemia. The expression of c-fos protein and the levels of malondialdehyde （MDA） and lactate in brain regions were detected by immunochemistry and spectrophotometrical methods, respectively. C-fos positive neurons were found in the hippocampus and cerebral cortex after cerebral ischemia reperfusion. Mild hypothermia increased the expression of c-fos protein in both areas, whereas hyperthermia decreased the expression of c-los protein in the hippocampus at 24 h reperfusion, and the cerebral cortex at 48 h reperfusion when compared to normothermic conditions. In normothermic, mild hypothermic and hyperthermic ischemia groups, the levels of MDA and lactate in brain tissue were increased at 24, 48 and 72 h reperfusion fol- lowing 20-min ischemia as compared with the sham-operated group （P〈0.01）. The levels of MDA and lactate in mild hypothermic group were significantly lower than those in normothermic group （P〈0.01）. It is suggested that brain temperature influences the translation of the immunoreactive protein product of c-fos after global cerebral ischemia, and MDA and lactate are also affected by hypothermia and hyperthermia.

Metal-polyphenol-coordinated nanomedicines for Fe(II) catalyzed photoacoustic-imaging guided mild hyperthermia-assisted ferroustherapy against breast cancer

Ferroustherapy has gained great attention for anti-cancer treatment in recent years. Enlightened by temperature-mediated Fenton reaction in industrial waste water removal, we designed a iron-based polyphenol-coordinated nanomedicines for mild hyperthermia-assisted anti-cancer ferroustherapy. In brief, Fe-GA@BSA nanoparticles was synthesized by self-assembly and sorafenib(SRF) was loaded into Fe-GA@BSA to establish Fe-GA@BSA-SRF nanomedicines. The result nanomedicines can induce ferroptosis in cancer cells by accelerating Fenton reaction. And the photothermal effect of Fe-GA@BSA-SRF was used for mild hyperthermia-assisted ferroustherapy. The nanomedicines performs good anti-cancer therapeutic efficacy by inducing the production of ROS and inhibiting glutathione peroxidase 4(GPX4) expression in vitro and in vivo. Besides, the broad absorption of Fe-GA@BSA-SRF in near infrared region endows it with photoacoustic imaging ability. This study provides ideas about rational design on iron-based nanoparticles for anti-cancer ferroustherapy.

Mild hyperthermia-enhanced chemo-photothermal synergistic therapy using doxorubicin-loaded gold nanovesicles

Gold nanovesicles(GVs) with unique plasmonic property and large cavity hold great potential as a stimuli-responsive nanocarrier to deliver drugs for efficient tumor chemotherapy and other therapies synergistically.Herein,we developed doxorubicin-loaded gold nanovesicles(DGVs),offering infrared thermal(IRT) and photoacoustic(PA) dual-modal imaging guided mild hype rthermia-enhanced chemophotothermal cancer synergistic therapy.The DGVs are self-assembled by gold nanoparticles modified with amphiphilic copolymer in a predetermined concentration of doxorubicin through film rehydration method.Under the influence of laser excitation,the as-prepared DGVs exhibited good photothermal effect,which triggered the structural disruption of GVs and thus,allowed the efficient release of encapsulated DOX to enhance cell uptake for fluorescence imaging and tumor chemotherapy,respectively.In addition,DGVs also showed a strong PA and IRT signals in vivo.Our study demonstrated the potential of DGVs as stimuli-responsive drug delivery systems and cancer theranostics.