盐霉素增加鼻咽癌CNE-2细胞放疗敏感性的实验研究

目的:探讨盐霉素（Sal）对鼻咽癌CNE-2细胞的放疗增敏作用及其可能的机制。方法:CCK8测定不同浓度盐霉素、不同剂量放疗及联合应用对CNE-2细胞增殖的抑制作用;平板克隆实验观察盐霉素联合放疗后细胞的克隆形成率;Hoechst33258染色观察细胞凋亡;流式细胞术检测细胞凋亡率及细胞周期的变化情况。结果:CCK8结果显示,盐霉素和放疗对鼻咽癌CNE-2细胞的生长有显著的抑制作用,呈浓度和剂量依赖性,且联合组抑制作用强于单纯药物组或放疗组。平板克隆实验显示盐霉素联合放疗后能显著降低细胞的克隆形成率;Hoechst33258染色显示盐霉素联合放疗治疗后细胞凋亡现象更明显;细胞流式术检测显示联合组较单纯药物组或放疗组凋亡率增加,盐霉素对放疗具有增敏作用。药物组和放疗组均能使G2/M期细胞比例增加,两者联合效果更明显。结论:盐霉素能增加人鼻咽癌CNE-2细胞的放疗敏感性,其作用机制可能与周期阻滞及其凋亡诱导相关。     

circRNA在消化道肿瘤中的研究进展

circRNA是广泛存在的内源性非编码RNA,通过反式剪接使3'端和5'端共价结合形成闭合的环状结构,具有高度稳定性、生物进化保守性和组织表达特异性。circRNA具有miRNA “海绵作用”、调控亲本基因表达、提高转录水平及翻译蛋白质等潜在功能。在消化道肿瘤中,circRNA主要发挥miRNA “海绵作用”,从而影响癌细胞的增殖、分化、凋亡及侵袭。另外,circRNA在肿瘤中特异性表达,且表达水平与癌旁组织有显著差异。因此,高度保守、稳定的circRNA序列可能成为消化道肿瘤早期诊断及预后标志物。本文将从circRNA的概况以及与各消化道肿瘤（食管癌、胃癌、结直肠、肝癌等）的相关性进行综述。      

表没食子儿茶素没食子酸酯（EGCG）对鼻咽癌细胞CNE1、CNE2放射增敏作用的初步研究

目的:研究表没食子儿茶素没食子酸酯（EGCG）对鼻咽癌细胞CNE1、CEN2的放射增敏作用及其可能机制。方法:体外培养CNE1、CNE2细胞,CCK-8实验得到EGCG IC20值50μg/ml,即为实验浓度。实验分为4组,对照组:含有和其他组相等浓度的DMSO溶解剂;药物组:DMSO溶解的EGCG;照射组:X线照射组;实验组:EGCG联合X线照射组。体外培养CNE1、CNE2细胞至对数生长期,药物组及实验组分别给予药物即EGCG处理,培养24h后进行相应剂量的X线照射后收集细胞。采用克隆形成实验检测不同射线剂量（0、2、4、6、8、10Gy）处理后各组的克隆形成率、细胞存活率及放射增敏比（SER）,流式细胞分析法检测各组细胞的凋亡情况及细胞周期,Western bolt检测Akt蛋白的表达,RT-PCR检测Akt mRNA的表达。结果:平板克隆实验:照射组及实验组随着X线剂量的逐步增加,克隆形成率随之下降,细胞存活分数下降,呈现剂量依赖效应（P＜0.05）;相同剂量下,实验组比照射组克隆形成率低,实验组Do、Dq明显低于单纯照射组（P＜0.05）,CNE1细胞株SER为1.4962,CNE2细胞株SER为1.1846,说明EGCG具有放射增敏作用（P＜0.05）。流式细胞分析:各实验组与对照组相比,G2/M期百分比升高（P＜0.05）,表明存在G2/M期阻滞,单纯射线组比单纯药物组对G2/M期的阻滞作用更明显,二者联合的实验组表现为协同作用。实时荧光定量实验:各实验组与对照组相比,Akt mRNA表达下降（P＜0.05）,实验组比单纯照射组及单纯药物组更明显。Western blot实验:各实验组与对照组相比,Akt蛋白表达均有下降（P＜0.05）,实验组比单纯照射组及单纯药物组更明显。结论:EGCG对鼻咽癌细胞CNE1、CNE2具有放射增敏作用,其机制可能是通过抑制细胞增殖、促进细胞凋亡、抑制细胞周期、抑制射线诱导的Akt活化而产生。     

姜黄素对人鼻咽癌CNE-2Z细胞的放射增敏作用及其作用机制

目的:研究姜黄素(curcumin)对人鼻咽癌CNE-2Z细胞放射增敏作用并初步探讨其作用机制。方法:应用MTT法检测姜黄素药物毒性,用克隆形成实验观察其对放射敏感性的影响,用流式细胞仪(FCM)技术分析对CNE-2Z细胞周期分布和姜黄素联合辐射引起的细胞周期阻滞的影响。结果:不同浓度的姜黄素作用于CNE-2Z细胞后,其细胞毒性呈剂量依赖性,在较低浓度(10μmol/L)时,即可降低放射后CNE-2Z细胞的克隆形成率,其放射增敏比为1.52±0.25。姜黄素以及姜黄素联合辐射作用CNE-2Z细胞24h后,细胞周期主要阻滞在辐射敏感时相G2/M期。结论:姜黄素对CNE-2Z细胞有放射增敏作用,其机制可能与其引起的细胞周期阻滞等因素有关。     

circRNA在肿瘤中的研究进展

环状RNA（circRNA）是3'-端与5'-端共价结合生成的闭合环状RNA分子,在肿瘤中可作为原癌基因和抑癌基因,通过海绵吸附miRNA、结合RNA结合蛋白、结合酶与相应底物、调控转录以及翻译多肽或蛋白等机制发挥重要的生物作用。circRNA通过circRNA-miRNA-mRNA轴影响多种肿瘤的发生与发展,具有肿瘤诊断和预后标志物的潜力,成为继miRNA及长链非编码RNA（lncRNA）之后又一个研究热点。     

Curcumin enhances the effects of 5-fluorouracil and oxaliplatin in mediating growth inhibition of colon cancer cells by modulating EGFR and IGF-1R

Curcumin (diferuloylmethane), which has been shown to inhibit growth of transformed cells, has no discernible toxicity and achieves high levels in colonic mucosa. 5-fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but with limited success. The present investigation was, therefore, undertaken to examine whether curcumin in combination with conventional chemotherapeutic agent(s)/regimen will be a superior therapeutic strategy for colorectal cancer. Indeed, results of our in vitro studies demonstrated that curcumin together with FOLFOX produced a significantly greater inhibition (p < 0.01) of growth and stimulated apoptosis (p < 0.001) of colon cancer HCT-116 and HT-29 cells than that caused by curcumin, 5-FU, curcumin + 5-FU or FOLFOX. These changes were associated with decreased expression and activation (tyrosine phosphorylation) of EGFR, HER-2, HER-3 (72-100%) and IGF-1R (67%) as well as their downstream effectors such as Akt and cycloxygenase-2 (51-97%). Furthermore, while these agents produced a 2-3-fold increase in the expression of IGF-binding protein-3 (IGFBP-3), curcumin together with FOLFOX caused a 5-fold increase in the same, when compared to controls. This in turn led to increased sequestration of IGF by IGFBP-3 rendering IGF-1 unavailable for binding to and activation of IGF-1R. We conclude that the superior effects of the combination therapy of curcumin and FOLFOX are due to attenuation of EGFRs and IGF-1R signaling pathways. We also suggest that inclusion of curcumin to the conventional chemotherapeutic agent(s)/regimen could be an effective therapeutic strategy for colorectal cancer.     

Combination of fractionated irradiation with anti‐VEGF expressing vaccinia virus therapy enhances tumor control by simultaneous radiosensitization of tumor associated endothelium

Oncolytic viruses are currently in clinical trials for a variety of tumors, including high grade gliomas. A characteristic feature of high grade gliomas is their high vascularity and treatment approaches targeting tumor endothelium are under investigation, including bevacizumab. The aim of this study was to improve oncolytic viral therapy by combining it with ionizing radiation and to radiosensitize tumor vasculature through a viral encoded anti‐angiogenic payload. Here, we show how vaccinia virus‐mediated expression of a single‐chain antibody targeting VEGF resulted in radiosensitization of the tumor‐associated vasculature. Cell culture experiments demonstrated that purified vaccinia virus encoded antibody targeting VEGF reversed VEGF‐induced radioresistance specifically in endothelial cells but not tumor cells. In a subcutaneous model of U‐87 glioma, systemically administered oncolytic vaccinia virus expressing anti‐VEGF antibody (GLV‐1h164) in combination with fractionated irradiation resulted in enhanced tumor growth inhibition when compared to nonanti‐VEGF expressing oncolytic virus (GLV‐1h68) and irradiation. Irradiation of tumor xenografts resulted in an increase in VACV replication of both GLV‐1h68 and GLV‐1h164. However, GLV‐1h164 in combination with irradiation resulted in a drastic decrease in intratumoral VEGF levels and tumor vessel numbers in comparison to GLV‐1h68 and irradiation. These findings demonstrate the incorporation of an oncolytic virus expressing an anti‐VEGF antibody (GLV‐1h164) into a fractionated radiation scheme to target tumor cells by enhanced VACV replication in irradiated tumors as well as to radiosensitize tumor endothelium which results in enhanced efficacy of combination therapy of human glioma xenografts.     

Curcumin enhances the anticancer effects of trichostatin a in breast cancer cells

Breast cancer patients with HER‐2 positive or estrogen receptor negative tumors have a poor prognosis because these tumors are aggressive and respond poorly to standard therapies. Histone deacetylase (HDAC) inhibitors have been shown to decreased cell survival, which suggests that HDAC inhibitors may be developed for preventing and treating breast cancer. Curcumin has anti‐inflammatory and proapoptotic effects in cancer cells. We determined whether the HDAC inhibitor, Tricostatin A (TSA) in combination with curcumin would produce greater antiproliferative and apoptotic effects than either agent alone. Increasing the concentration of curcumin from 10 to 20 µM enhanced the growth inhibitory effects of the combination in SkBr3 and 435eB breast cancer cells, which was accompanied by decreased viability along with decreased phosphorylation of ERK and Akt. The decreased cell viability observed in SkBr3 cells when curcumin was combined with TSA led to a G0/G1 cell cycle arrest and increased p21 and p27, and decreased Cyclin D1 protein expression. The combination induced cleavage of caspase 3 and poly(ADP‐ribose) polymerase‐1, suggesting that cell death occurred by apoptosis. There were no changes in protein expression of Bcl2, Bax, or Bcl‐xL and decreased expression of p53. The combination increased protein expression of phosphorylated JNK and phosphorylated p38. Pharmacological inhibition of JNK, but not p38, attenuated the decreased viability induced by the curcumin and TSA combination. We conclude that p53 independent apoptosis induced by combining curcumin and TSA involves JNK activation. These findings provide a rationale for exploring the potential benefits of the combination of curcumin with TSA for treatment of breast cancer. © 2012 Wiley Periodicals, Inc.     

血必净注射液对人鼻咽癌细胞生物学行为的影响

目的 目前临床应用血必净注射液治疗急性放射性炎症反应取得了良好的疗效,但血必净注射液是否会影响肿瘤的生长和发展,此问题目前罕见报道.本实验探讨了血必净注射液对人鼻咽癌HNE-2细胞生物学行为的影响,为临床应用奠定基础.方法 应用不同浓度(0、0.25、0.50、0.75、1.00、1.25 mg/mL)血必净注射液刺激鼻咽癌细胞HNE-2,四甲基偶氮唑蓝[3-(4,5-dimethylthiazol-yl)-2,5-diphenyl tetrazolium bromide,MTT]实验检测鼻咽癌细胞的生长增殖;应用流式细胞仪检测细胞凋亡及细胞周期;应用Transwell实验及蛋白质印迹法检测检测血必净注射液对鼻咽癌细胞侵袭、转移的影响及上皮间质转化(epithelial-mesenchymal transition,EMT)相关蛋白的表达.在裸鼠移植瘤模型中观察对照组、血必净组、放疗组、放疗+血必净组荷瘤鼠肿瘤生长曲线.结果 MTT实验显示,小剂量血必净刺激鼻咽癌细胞生长,但随浓度的增加,血必净注射液对鼻咽癌细胞增殖的抑制作用逐渐增强.空白对照组吸光度值为1.012±0.069,血必净0.25 mg/mL组为1.034±0.061,0.50 mg/mL组为0.974±0.077,0.75 mg/mL组为0.806±0.103,1.00 mg/mL组为0.656±0.098,1.25 mg/mL组为0.626±0.017,F=28.353,P＜0.001.凋亡实验显示,血必净处理20 h,空白对照组细胞凋亡率为(3.97±0.13)％,血必净0.25 mg/mL组为(8.79±0.36)％,0.50 mg/mL组为(16.07±0.52)％,0.75 mg/mL组为(25.60±0.57)％,1.0 mg/mL组为(26.93±0.74)％,1.25 mg/mL组为(30.84±0.69)％,F=1 167.35,P＜0.001;血必净处理40 h,空白对照组细胞凋亡率为(5.58±0.31)％,血必净0.25 mg/mL组为(7.42±0.23)％,0.50 mg/mL组为(9.30±0.08)％,0.75 mg/mL组为(10.02±0.39)％,1.00 mg/mL组为(12.6±0.31)％,1.25 mg/mL组为(13.65±0.35)％,F=311.28,P＜0.001.随着血必净浓度增大,细胞凋亡率增加.鼻咽癌荷瘤动物模型实验显示,血必净注射液可以抑制鼻咽癌移植瘤的生长,与放疗有协同作用.单独应用血必净注射液对鼻咽癌细胞的细胞周期无明显影响,可抑制EMT相关蛋白的表达和细胞的运动.Transwell实验结果显示,空白对照组细胞穿孔数为120.50±8.35,血必净0.25 mg/mL组为94.5±2.08,0.50 mg/mL组为85.00±2.58,0.75 mg/mL组为77.25±2.21,1.00 mg/mL组为74.00±4.83,1.25 mg/mL组为61.75±1.71,F=88.873,P＜0.001.结论 血必净注射液对人鼻咽癌细胞的细胞周期无明显影响,但对细胞增殖和移植瘤生长,以及细胞运动有一定抑制作用,具有潜在的放疗增敏作用.     

Dose-rate dependence of heat radiosensitization

The dose rate dependence of heat radiosensitization was studied using rat astrocytoma cells in culture and a clinically relevant protocol of heat dose and heat radiation sequence. Cells were treated with a minimally toxic heat dose of 43°C for 30 minutes, after which they were irradiated with varying doses of radiation at dose rates ranging from 0.567 to 300 cGy/min. This heat dose substantially reduced the extrapolation number ($\text{n}\text{?}$), but had little effect on D_o of the radiation survival curve at dose rates of 50 cGy/min or greater. At dose rates less than 10 cGy/min, 43°C for 30 min had little a on $\text{n}\text{?}$ and only for the lowest dose rate studied (0.567 cGy/min) was there a significant reduction in D_o (60%). The thermal enhancement ratio did not vary inversely with radiation dose rate over the dose rate range studied but, instead, was maximal at the two dose rate extremes (0.567 and 300 cGy/min). These data demonstrate that a clinically relevant heat dose enhances very low dose rate, as well as high dose rate, ionizing radiation, but suggest that little benefit is to be gained from using dose rates intermediate between conventional radiotherapeutic high dose rates or dose rates representative of interstitial implants.     

甘氨双唑钠对ⅣB期鼻咽癌后半程放射治疗增敏的临床研究

目的:探讨甘氨双唑钠(CMNa)在ⅣB期鼻咽癌放射治疗的增敏作用和临床应用价值.方法:选择ⅣB期鼻咽癌,面颈联合野加双下颈前后对穿照射36 Gy后,颈部转移淋巴结残留≥5 cm的患者40例,分为前瞻性分析试验组(CMNa+放疗)和对照组各20例.试验组采用CMNa800 mg/m<'2>,用100 mL生理盐水稀释溶解,于30 min内静脉滴完,在60 min内进行3D-CRT放疗.对照组仅行3D-CRT放疗.结果:放疗70 Gy结束后,评定疗效,颈部转移淋巴结缓解率(CR+PR):试验组90%(18/20),对照组55%(11/20),P=0.01;原发灶(T)缓解率:试验组95%(19/20),对照组65%(13/20),P=0.02.结论:CMNa对鼻咽癌有良好的放疗增敏作用,特别是对于颈部淋巴结转移灶大的患者,近期疗效明显,且药物耐受性好.远期效果有待进一步观察.     

In vitro and in vivo radiosensitization induced by hydroxyapatite nanoparticles

Background

Previous study showed that hydroxyapatite nanoparticles (nano-HAPs) inhibited glioma growth in vitro and in vivo; and in a drug combination, they could reduce adverse reactions. We investigated the possible enhancement of radiosensitivity induced by nano-HAPs.

Methods

In vitro radiosensitization of nano-HAPs was measured using a clonogenic survival assay in human glioblastoma U251 and breast tumor brain metastatic tumor MDA-MB-231BR cells. DNA damage and repair were measured using γH2AX foci, and mitotic catastrophe was determined by immunostaining. The effect of nano-HAPs on in vivo tumor radiosensitivity was investigated in a subcutaneous and an orthotopic model.

Results

Nano-HAPs enhanced each cell line''s radiosensitivity when the exposure was 1 h before irradiation, and they had no significant effect on irradiation-induced apoptosis or on the activation of the G₂ cell cycle checkpoint. The number of γH2AX foci per cell was significantly large at 24 h after the combination modality of nano-HAPs + irradiation compared with single treatments. Mitotic catastrophe was also significantly increased at an interval of 72 h in tumor cells receiving the combined modality compared with the individual treatments. In a subcutaneous model, nano-HAPs caused a larger than additive increase in tumor growth delay. In an orthotopic model, nano-HAPs significantly reduced tumor growth and extended the prolongation of survival induced by irradiation.

Conclusions

These results show that nano-HAPs can enhance the radiosensitivity of tumor cells in vitro and in vivo through the inhibition of DNA repair, resulting in an increase in mitotic catastrophe.     

拓扑替康对鼻咽癌放射增敏机制的研究

目的:研究拓扑替康(TPT)对人鼻咽癌裸鼠移植瘤放射增敏的机制。方法:把放射增敏实验各组[RT TPT组(增敏组),TPT组(药物组),RT组(放射组),生理盐水组(对照组) ]肿瘤标本制备成单细胞悬液,用流式细胞仪分析细胞周期,凋亡指数以及凋亡相关基因p53、bcl 2、bax的表达。结果:增敏组增殖指数 ( 19. 3±1. 7 )%降低,凋亡指数增加,和药物组(25. 2±0. 3)%,放射组 (26. 1±0. 3)%,对照组 (35. 7±2. 5)%比较有统计学意义;p53,bcl 2,bax的表达,各处理组与对照组比较,差异均没有统计学意义。结论:改变细胞周期,促进凋亡可能是拓扑替康增敏作用在细胞水平的重要机制,但诱导凋亡可能不依赖p53,bcl 2,bax基因。     

The emerging roles of exosomal miRNAs in nasopharyngeal carcinoma

Nasopharyngeal carcinoma (NPC) is a unique subtype of head and neck cancer that is endemic to Southern China and Southeast Asia. Due to the concealed location and intrinsic invasiveness of this disease, majority of NPC patients are diagnosed with advanced stages (III and IV) and poor prognosis. Chemoradiotherapy resistance is a major problem for NPC patients, leading to incomplete local elimination, recurrence and metastasis. Therefore, it is of great significance to seek novel biomarkers and effective therapeutic regimen for clinical management of this deadly cancer. Exosomes are tiny membrane vesicles with a lipid bilayer secreted by most cells in the body, which are widely distributed in various body fluids. They are functionally active in different physiopathological process by carrying and transmitting important signal molecules such as miRNA, mRNA, protein, lipid, etc. Exosomal miRNAs play an important role in tumorigenesis and development of NPC. They are extensively involved in NPC cell proliferation, migration, invasion, neovascularization, radiotherapy resistance and the regulation of tumor immune microenvironment through intercellular communication and control of gene expression. Moreover, exosomal miRNAs can be used as valuable biomarkers for early diagnosis and therapeutic targets of NPC.     

叶酸修饰埃洛石纳米管负载姜黄素对乳腺癌细胞及移植瘤的放射增敏作用

目的:探究叶酸修饰埃洛石纳米管负载姜黄素（HNTs-PEG-FA/Cur）对乳腺癌MCF-7细胞及4T1荷瘤鼠的靶向放射增敏作用。方法:用化学改性和物理吸附的方法合成纳米放疗增敏药物HNTs-PEG-FA/Cur,采用透射电子显微镜、傅里叶红外光谱仪和X射线光电子能谱仪进行形貌和化学组分表征,MTT法检测不同浓度纳米药物对人乳腺癌MCF-7细胞活性的影响,流式细胞术、活/死细胞荧光染色和克隆形成实验评估HNTs-PEG-FA/Cur联合放疗对MCF-7细胞的促凋亡作用和放射增敏作用。最后,通过建立乳腺癌4T1细胞小鼠移植瘤模型,评估联合治疗对小鼠肿瘤体积生长的抑制效果。结果:成功将姜黄素负载于叶酸修饰的HNTs管腔内。所制备的HNTs-PEG-FA/Cur在等效姜黄素浓度为5 μmol/L时对MCF-7细胞无明显毒性,并能有效增强X射线诱导的细胞凋亡和杀伤作用（增敏比为1.25）。体内抑瘤实验结果显示HNTs-PEG-FA/Cur联合放疗对小鼠肿瘤生长抑制率（56.75%）>单独射线组（24.16%）>单独药物组（6.67%）。结论:HNTs-PEG-FA作为姜黄素的新型纳米药物载体,能有效提高姜黄素生物利用度并且对乳腺癌具有靶向放射增敏潜力。     

甘氨双唑钠联合放射治疗中晚期鼻咽癌临床研究

目的:观察甘氨双唑钠(sodiumglyci-didazole,CMNa)在中晚期鼻咽癌放射治疗中的作用。方法:60例中晚期鼻咽癌患者随机分观察组(CMNa 放疗)和对照组(单纯放疗组)各30例。CMNa静脉滴入,每次0·75g,每周1、3和5用药,用药后1h内放疗。放疗采用常规外照射方案。结果:鼻咽原发灶CR率在观察组和对照组分别是93%和73%,两组差异有统计学意义,χ2=4·320,P=0·0377。颈淋巴结CR率两组分别是90%和63%,两组差异亦有统计学意义,χ2=5·963,P=0·0146。两组毒副反应如皮肤、黏膜、消化道反应及血白细胞下降等差异无统计学意义,P>0·05。结论:CMNa联合放射治疗可提高鼻咽癌的肿瘤局部控制率,而毒副反应不增加。