In vitro cellular radiosensitivity of human malignant melanoma

The agar diffusion chamber assay was used to construct in vitro radiation cell survival curves for two human malignant melanoma xenografts. Both tumors had survival curves of shoulder-exponential shape with large shoulders (Dq 2.38, 2.92 Gy). Comparison with previous studies of 4 human tumor xenografts in the same system suggests that large shoulders are more common in melanomas than other tumor types. This supports the case for large fraction irradiation in the clinical therapy of melanoma.     

Silencing of R-Spondin1 increases radiosensitivity of glioma cells

Although radiation therapy is the most effective postoperative adjuvant treatment, it does not substantially improve the long-term outcomes of glioma patients because of the characteristic radioresistance of glioma. We found that R-Spondin1 (Rspo1) expression was elevated in high-grade gliomas and was associated with worse overall survival and disease-free survival. Rspo1 expression was also associated with reduced survival rates in glioma patients after treatment with radiotherapy and temozolomide (RT-TMZ). Importantly, Rspo1 was dramatically upregulated after radiation treatment in patients with glioma. Rspo1 silencing by shRNA potentiated glioma cell death upon radiation treatment. In a xenograft nude mouse model, combining radiation and silencing of Rspo1 potentiated tumor growth inhibition. Thus, combining radiotherapy with silencing of Rspo1 is a potential therapeutic approach.     

干扰GTPBP4基因对食管癌EC9706细胞放射敏感性影响

目的:探讨沉默GTPBP4基因对人食管癌EC9706细胞系放射敏感性影响。方法:通过GEO数据库分析食管癌患者组织中GTPBP4的表达情况。利用重组质粒载体介导的RNA干扰(RNAi)技术转染食管癌EC9706细胞系,使细胞中GTPBP4基因沉默,观察GTPBP4基因沉默对EC9706细胞增殖、凋亡及放射敏感性影响。通...     

shRNA沉默Sp1对胶质瘤细胞放射敏感性的影响

目的 探讨下调Sp1基因表达对胶质瘤细胞放射敏感性的影响。方法 设计并合成编码shRNA的寡核苷酸序列,克隆入LV3（H1/GFP&Puro）载体中构建重组体。然后重组慢病毒分别感染U251和U87细胞,嘌呤霉素筛选得到稳定转染细胞株。荧光定量RT-PCR和蛋白质印迹法分别检测细胞中Sp1 mRNA和蛋白表达水平;克隆存活法检测细胞存活;流式法检测细胞周期;免疫荧光法检测DNA损伤程度。结果 两株胶质瘤细胞在感染72 h后荧光显微镜观察Sp1慢病毒载体均高表达,RT-PCR和蛋白质印迹法结果显示转染后shRNA-U87和shRNA-U251细胞Sp1 mRMA和蛋白表达量均明显降低（P<0.01）,Sp1敲除率达90%以上。shRNA-U251和shRNA-U87细胞在10%细胞存活水平下的放射增敏比（SER）分别为1.39和1.18;两株Sp1敲除组细胞经辐照后G₂/M期比率及γ-H2AX foci数目与对照组相比均极显著增大。结论 shRNA沉默Sp1增大了X线诱导的G₂/M期阻滞,加重了DNA双链断裂程度,提高了胶质瘤细胞的放射敏感性。     

shRNA沉默Sp1对胶质瘤细胞放射敏感性的影响北大核心CSCD

目的探讨下调Sp1基因表达对胶质瘤细胞放射敏感性的影响。方法设计并合成编码shRNA的寡核苷酸序列,克隆入LV3(H1/GFP&Puro)载体中构建重组体。然后重组慢病毒分别感染U251和U87细胞,嘌呤霉素筛选得到稳定转染细胞株。荧光定量RT-PCR和蛋白质印迹法分别检测细胞中Sp1 mRNA和蛋白表达水平;克隆存活法检测细胞存活;流式法检测细胞周期;免疫荧光法检测DNA损伤程度。结果两株胶质瘤细胞在感染72 h后荧光显微镜观察Sp1慢病毒载体均高表达,RT-PCR和蛋白质印迹法结果显示转染后shRNA-U87和shRNA-U251细胞Sp1 mRMA和蛋白表达量均明显降低(P<0.01),Sp1敲除率达90%以上。shRNA-U251和shRNA-U87细胞在10%细胞存活水平下的放射增敏比(SER)分别为1.39和1.18;两株Sp1敲除组细胞经辐照后G2/M期比率及γ-H2AX foci数目与对照组相比均极显著增大。结论shRNA沉默Sp1增大了X线诱导的G2/M期阻滞,加重了DNA双链断裂程度,提高了胶质瘤细胞的放射敏感性。     

Possible relationship between chromosome alterations and in vitro cellular radiosensitivity of human malignant melanoma

In the present study, in vitro radiation survival analysis was performed on 8 human malignant melanoma cell lines with defined karyotypes. Exponentially growing cells were irradiated to doses of 0 to 8 Gy and examined for soft agar clonogenicity. Two groups emerged from this analysis: 4 cell lines with a small shoulder (extrapolation number, n less than 2) and 4 cell lines with a large shoulder (n greater than 2). Of possible significance, 4/4 cell lines with extrapolation numbers greater than 2 demonstrated clonal structural abnormalities of chromosome 7. In contrast, 3/4 cell lines with extrapolation numbers less than 2 had no structural abnormalities of chromosome 7. These very preliminary results suggest that structural alterations of chromosome 7 may be associated with melanoma tumors with high extrapolation numbers.     

柳氮磺吡啶通过铁死亡途径增强结直肠癌细胞放射敏感性研究

目的 探究低剂量柳氮磺吡啶（SAS）对结直肠癌细胞的放疗增敏作用。方法 CCK‐8检测不同浓度SAS对结直肠癌细胞的增殖抑制作用,采用凋亡、自噬等抑制剂联合SAS处理结直肠癌细胞,CCK‐8法比较细胞活力。采用锥虫蓝染色、平板克隆形成、细胞生长曲线等实验评价低浓度SAS联合放疗对结直肠癌细胞的体外放疗增敏作用。通过检测细胞内脂质过氧化物、铁死亡蛋白标志物（GPX4、PTGS2）等,探究低浓度SAS联合放疗对细胞铁死亡的促进作用。利用小鼠皮下移植瘤模型,明确SAS的体内放疗增敏效应。结果 高浓度SAS可诱导结直肠癌细胞凋亡与铁死亡。低浓度SAS增强结直肠癌细胞放疗敏感性,该效应可被铁死亡抑制剂（Fer‐1）阻断。低浓度SAS联合放疗显著增加细胞内脂质过氧化物含量与PTGS2表达,降低GPX4表达。皮下移植瘤模型中SAS亦显示显著的放疗增敏效应。结论 低浓度SAS通过铁死亡途径增强结直肠癌细胞放疗敏感性。     

柳氮磺吡啶通过铁死亡途径增强结直肠癌细胞放射敏感性研究

目的 探究低剂量柳氮磺吡啶（SAS）对结直肠癌细胞的放疗增敏作用。方法 CCK‐8检测不同浓度SAS对结直肠癌细胞的增殖抑制作用,采用凋亡、自噬等抑制剂联合SAS处理结直肠癌细胞,CCK‐8法比较细胞活力。采用锥虫蓝染色、平板克隆形成、细胞生长曲线等实验评价低浓度SAS联合放疗对结直肠癌细胞的体外放疗增敏作用。通过检测细胞内脂质过氧化物、铁死亡蛋白标志物（GPX4、PTGS2）等,探究低浓度SAS联合放疗对细胞铁死亡的促进作用。利用小鼠皮下移植瘤模型,明确SAS的体内放疗增敏效应。结果 高浓度SAS可诱导结直肠癌细胞凋亡与铁死亡。低浓度SAS增强结直肠癌细胞放疗敏感性,该效应可被铁死亡抑制剂（Fer‐1）阻断。低浓度SAS联合放疗显著增加细胞内脂质过氧化物含量与PTGS2表达,降低GPX4表达。皮下移植瘤模型中SAS亦显示显著的放疗增敏效应。结论 低浓度SAS通过铁死亡途径增强结直肠癌细胞放疗敏感性。     

小干扰RNA沉默Bcl-2表达增强食管癌细胞放射敏感性研究

目的 探讨Bcl-2基因的特异性小干扰RNA (siRNA)对食管癌细胞(EC9706细胞)Bcl-2蛋白表达和凋亡的影响及放射增敏作用。方法 化学合成Bcl-2基因的siRNA,通过脂质体转染法转入EC9706细胞。流式细胞仪检测转染前后Bcl-2蛋白表达及细胞凋亡,成克隆分析siRNA联合X线照射对EC9706细胞的放射敏感性影响。结果 Bcl-2 siRNA1、Bcl-2 siRNA2、Bcl-2 siRNA3转染后Bcl-2蛋白在EC9706细胞中的阳性表达率分别为25.13％、38.87％、30.55％,较对照组的84.28％明显下降(t =4.01、3.04、3.64,P均＜0.05)。Bcl-2 siRNA1转染组凋亡率为33.86％,明显高于空白对照组的5.51％(t=6.55,P＜0.01)和阴性siRNA1转染组的5.59％(t=6.54,P＜0.01);Bcl-2 siRNA1联合X线照射的细胞凋亡率为56.76％,明显高于单纯照射组的24.51％(t=3.59,P＜0.05)。成克隆分析的Bcl-2 siRNA1转染加照射组D0、Dq、SF2值均低于单纯照射组,放射增敏比为1.33(D0值之比)。结论 Bcl-2基因的特异性siRNA可明显增强EC9706细胞的放射敏感性,具有良好临床应用前景。     

SIRT1基因沉默对DLBCL细胞放射敏感性影响

目的 探究SIRT1基因沉默对DLBCL放射敏感性影响。方法 利用免疫组织化学观察SIRT1在DLBCL组织中的蛋白表达情况,蛋白印迹法检测SIRT1在DLBCL细胞系OCI-Ly3、SU-DHL-2、SU-DHL-4及人正常B细胞永生化细胞系HMy2.CIR中的表达情况。利用Lipofectamine 2000转染试剂盒将si-SIRT1及si-NC转染到SU-DHL-4细胞中并检测转染后SIRT1的表达;MTT法和克隆形成实验检测经放射处理后细胞放射敏感性的变化。成组t检验差异或单因素方差分析。结果 SIRT1在DLBCL组织中的阳性率为72.6%(103/140),明显高于SIRT1在RLH中的阳性率(26.5%,8/25)(P=0.001);SIRT1在DLBCL细胞系中的表达水平显著高于人正常B细胞永生化细胞系HMy2.CIR细胞(P=0.020);SIRT1基因沉默后,SIRT1在SU-DHL-4细胞中的表达量明显降低(P=0.008)。放射处理后,SIRT1基因沉默组的细胞增殖率和克隆形成率显著低于对照组(P=0.030)。结论 SIRT1基因沉默明显提高了DLBCL细胞的放射敏感性,为DLBCL细胞的治疗提供了一个新的靶标。     

microRNA-7 increases radiosensitivity of human cancer cells with activated EGFR-associated signaling

Many microRNAs (miRNAs) play crucial roles in regulating expression of oncogenes or tumor suppressor genes. The epidermal growth factor receptor (EGFR) is frequently overexpressed in a wide range of solid tumors and is an important therapeutic target; however, the therapeutic outcome of currently available anti-EGFR agents is often limited due to diverse molecular resistance mechanisms. In this study, we evaluated the potential of targeting miRNA-7 for overcoming radio-resistance of cancer cells with activated EGFR-associated signaling. A panel of human cancer cell lines with increased EGFR-PI3K-Akt signaling was transfected with pre-miR-7 or control miRNA. Ectopic overexpression of miR-7 attenuated EGFR and Akt expression and radiosensitized SQ20B squamous cell carcinoma of the larynx, MDA-MB-468 breast cancer cells, A549 lung carcinoma cells, and U251 and U87 malignant glioma cells. In contrast, antisense-mediated inhibition of mature miR-7 expression led up-regulation of EGFR and its downstream effectors, and increased radio-resistance of U251 glioma cells. Overexpression of miR-7 prolonged radiation-induced γH2AX foci formation and downregulation of DNA-dependent protein kinases (DNA-PKcs). miR-7 may be a useful therapeutic target for overcoming the radio-resistance of human cancers with activated EGFR-PI3K-AKT signaling.     

STAT3基因沉默对胰腺癌细胞放射敏感性的影响及其机制

目的:探讨信号转导和转录激活因子3（STAT3）基因沉默对人胰腺癌细胞AsPC-1和PANC-1放射敏感性的影响及其机制。方法:体外培养人胰腺癌细胞AsPC-1和PANC-1，通过脂质体转染STAT3 shRNA作为实验组（shSTAT3组），设置阴性对照（shNC组）和空白对照组（NC组）。采用qRT-PCR检测转染后各组AsPC-1和PANC-1细胞中STAT3 mRNA表达水平，Western blot检测细胞中STAT3蛋白表达水平。MTT实验检测各组细胞增殖能力，流式细胞仪检测各组细胞电子射线照射后的凋亡情况，qRT-PCR和Western blot分别检测各组细胞电子射线照射后的Bax、Bcl-2和Caspase-3 mRNA和蛋白的表达情况。结果:shSTAT3组AsPC-1和PANC-1细胞中STAT3 mRNA和蛋白表达水平比NC组和shNC组明显降低（P<0.05）。接受电子射线照射后，shSTAT3组AsPC-1和PANC-1细胞增殖率显著低于shNC组（P<0.05），其细胞凋亡率较shNC组明显升高（P<0.05）。接受电子射线照射后，转染STAT3 shRNA后能够明显上调细胞中Bax和Caspase-3的表达和下调Bcl-2的表达（P<0.05）。结论:沉默STAT3基因能够通过上调Bax和Caspase-3的表达和下调Bcl-2的表达促进细胞凋亡，增强胰腺癌细胞的放射敏感性。     

HMGB1 siRNA干扰对食管鳞癌细胞放射敏感性的影响

目的： 采用siRNA技术干扰人食管鳞癌细胞中高迁移率族蛋白B1（HMGB1）基因表达，观察经X射线照射后细胞增殖活性、存活能力及细胞凋亡率的变化。方法： 针对HMGB1 mRNA序列，设计合成有效的干扰序列HMGB1 siRNA，并转染食管鳞癌KYSE30细胞，同时设置转染阴性对照序列的阴性对照组以及未进行转染的空白对照组。采用实时荧光定量PCR（qPCR）和Western blot法检测各组细胞HMGB1 mRNA和蛋白的表达；分别用MTS比色、克隆形成实验、流式细胞术和Western blot法检测HMGB1 siRNA干扰对食管鳞癌细胞KYSE30细胞的增殖活力、存活能力、细胞凋亡率及凋亡相关蛋白表达的影响。结果： HMGB1 siRNA干扰后，KYSE30细胞HMGB1 mRNA和蛋白的表达水平明显降低（P均 < 0.01）；MTS数据显示HMGB1 siRNA干扰后经X射线照射显著抑制了食管鳞癌细胞的增殖水平（与空白对照组和阴性对照组比较，P < 0.05）；克隆形成实验结果显示空白对照组、阴性对照组、HMGB1 siRNA组的D₀值分别为2.57、2.54、1.55 Gy；D_q值分别为1.69、1.65、1.30 Gy；SF₂值分别为0.27、0.27、0.13；外推数N值分别为1.93、1.91、2.31；X射线照射后HMGB1 siRNA组肿瘤细胞的凋亡率明显增加，同时bcl-2蛋白表达显著降低，而bax、caspase-3蛋白的表达明显增加（与空白对照组和阴性对照组比较，P < 0.01）。结论： siRNA干扰技术可用来抑制食管鳞癌细胞中HMGB1基因的表达，联合X射线照射降低了肿瘤细胞的增殖水平和存活能力，诱导了细胞凋亡，增加了放射敏感性，该作用可能与调控bcl-2、bax和caspase-3基因表达有关。     

Nanoparticle-mediated down-regulation of TWIST increases radiosensitivity of nasopharyngeal carcinoma cells via ERK pathway

Evidence suggests that over-expression of TWIST, an epithelial-mesenchymal transition inducer, might have a correlation with cancer progression and chemoresistance. However, its roles in radioresistance of cancer have rarely been reported. High TWIST expression was detected in nasopharyngeal carcinoma (NPC) and associated with poor prognosis. Thus, in the present study, we aimed to determine whether knockdown of TWIST can increase radiosensitivity of NPC cells. Chitosan-encapsulated TWIST-siRNA nanoparticles were constructed and used to silence TWIST expression in CNE2 cells. The cell viability and apoptosis as well as possible MAPKs pathways were assessed after irradiation treatment. The results showed that the nanoparticles successfully suppressed TWIST expression in CNE2 cells, and TWIST depletion significantly sensitized CNE2 cells to irradiation by inducing activation of ERK pathway but not JNK or p-38 pathways. The data suggested that TWIST depletion might be a promising approach sensitizing NPC cells to irradiation. Further investigations are needed to confirm the results.     

尼妥珠单抗对肺癌A549细胞系的体外放射增敏效应

目的 研究尼妥珠单抗对肺腺癌A549细胞系的放射增敏作用及潜在的分子机制。方法 体外培养人肺腺癌A549细胞系,克隆形成实验测定尼妥珠单抗和辐射联合作用于A549细胞系的细胞存活率,并根据数学模型拟合细胞存活曲线,求出放射增敏比(SER)。其次,采用流式细胞术检测尼妥珠单抗联合2 Gy、8 GyX线照射后的细胞周期变化。最后,Western blot检测尼妥珠单抗与放射联合作用后细胞EGFR磷酸化水平的差异,再检测A549细胞接受辐射后p27、AKT及其磷酸化形式在不同时间点的表达情况,与单纯放射组比较表达的差异。结果 克隆形成实验显示尼妥珠单抗显著增加了A549细胞的放射敏感性,放射增敏比(SER)为1.36。尼妥珠单抗使细胞周期阻滞于G₁/S期,而辐射后细胞周期阻滞于G₂/M期,二者联合作用后S期比例较单纯放射组细胞显著减少。尼妥珠单抗与放射联合作用后,辐射诱导的EGFR磷酸化被抑制,AKT在放射后各时间点Thr308位点磷酸化也受到显著抑制,p27蛋白在放射后各时间点表达水平均显著下降,而尼妥珠单抗作用后p27表达明显增加,再行照射后仍有较高表达。结论 尼妥珠单抗对体外培养的肺腺癌细胞系具有放射增敏作用,其机制在于通过对EGFR功能的抑制,抑制了其下游信号通路中AKT的激活,而p27的表达增加,可能与细胞周期分布发生改变相关。     

沉默 ATM基因对人胃癌细胞 BGC823放射敏感性的影响

目的：探讨毛细血管扩张突变基因（ATM）对人胃癌细胞放射敏感性的影响。方法：将 ATM基因敲减后获取得到的稳定转染克隆细胞和空载细胞同时通过不同剂量的照射后,观察敲减 ATM基因前后 BGC823细胞平板克隆形成率,并使用流式细胞术,观察 ATM被敲减后对细胞周期和凋亡的影响。结果：BGC823细胞中的 ATM被敲减后,经不同剂量的照射（2、4、6和8Gy）克隆形成率分别为（15．4±0．9）％、（9．2±0．3）％、（2．2±0．1）％和（1．0±0．1）％,远低于空载细胞的克隆形成率分别为（28．1±0．6）％、（15．6±0．8）％、（5．3±0．1）％和（1．8±0．1）％,P 均小于0．05,有显著性差异。采用相同剂量（4Gy）照射后,流式细胞术检测发现,BGC823细胞中的 ATM被敲减后,细胞周期被阻滞,细胞停止生长,并在12h 后出现凋亡的现象。结论：ATM基因的表达可影响胃癌细胞的放射敏感性,ATM的表达被敲减后对 BGC823细胞的放射起到增敏作用。ATM基因在胃癌放射敏感性方面可能有非常重要的作用。     

The melanoma specific 9.2.27PE immunotoxin efficiently kills melanoma cells in vitro

Malignant melanomas are generally drug resistant and have a very poor prognosis. We have studied the effects of a chemical conjugate of pseudomonas exotoxin A (PE) and the antibody 9.2.27, which recognizes the high molecular weight melanoma associated antigen (HMW‐MAA) expressed in most malignant melanomas and melanoma cell lines. We demonstrate that the 9.2.27PE immunotoxin (IT) induces cell death in malignant melanoma cells through protein synthesis inhibition followed by some morphological and biochemical features of apoptosis, like rounding up of cells, chromatin condensation and inactivation of PARP. Unlike previous results with the 425.3PE IT in breast cancer cells, we detected no depolarization of the mitochondrial membrane after 9.2.27PE IT treatment. This is likely due to the lack of strong activation of caspase‐8 and caspase‐3. The lack of depolarization suggests that cytochrome c, a molecule that triggers activation of caspase‐3, was retained within the mitochondria. In addition, the protein level of the antiapoptotic Bcl‐2 did not decrease in contrast to other antiapoptotic molecules belonging to the inhibitor of apoptosis and the Bcl‐2 family. This suggests that Bcl‐2 may play a role in maintaining the mitochondrial membrane integrity in the 9.2.27PE‐treated cells. Nevertheless, 9.2.27PE IT efficiently killed malignant melanoma cells that can be ascribed to inhibition of protein synthesis followed by some morphological and biochemical features of apoptosis. © 2009 UICC     

RNAi-mediated silencing of CD147 inhibits tumor cell proliferation,invasion and increases chemosensitivity to cisplatin in SGC7901 cells in vitro

Background

CD147 is a widely distributed cell surface glycoprotein that belongs to the Ig superfamily. CD147 has been implicated in numerous physiological and pathological activities. Enriched on the surface of many tumor cells, CD147 promotes tumor growth, invasion, metastasis and angiogenesis and confers resistance to some chemotherapeutic drugs. In this study, we investigated the possible role of CD147 in the progression of gastric cancer.

Methods

Short hairpin RNA (shRNA) expressing vectors targeting CD147 were constructed and transfected into human gastric cancer cells SGC7901 and CD147 expression was monitored by quantitative realtime RT-PCR and Western blot. Cell proliferation, the activities of MMP-2 and MMP-9, the invasive potential and chemosensitivity to cisplatin of SGC7901 cells were determined by MTT, gelatin zymography, Transwell invasion assay and MTT, respectively.

Results

Down-regulation of CD147 by RNAi approach led to decreased cell proliferation, MMP-2 and MMP-9 activities and invasive potential of SGC7901 cells as well as increased chemosensitivity to cisplatin.

Conclusion

CD147 involves in proliferation, invasion and chemosensitivity of human gastric cancer cell line SGC7901, indicating that CD147 may be a promising therapeutic target for gastric cancer.     

siRNA沉默Annexin A2基因表达对鼻咽癌细胞放射敏感性的影响

目的 探讨siRNA沉默Annexin A2基因表达对鼻咽癌CNE-2(R743)细胞放射敏感性的影响。方法 化学合成Annexin A2基因的siRNA经HiPerFect转染入R743细胞。RT-PCR和蛋白印迹法检测转染前后Annexin A2的mRNA和蛋白表达,克隆形成实验分析转染前后对细胞放射敏感性的影响,流式细胞仪和TUNEL实验分别检测转染前后经X线照射后细胞周期、细胞凋亡情况。结果 RT-PCR和蛋白印迹法结果显示转染组细胞Annexin A2基因及蛋白表达下调。克隆形成实验分析结果表明转染组D₀、D_q、SF₂值均低于单纯照射组和转染对照组,其相应放射增敏比(D₀值比)分别为1.30和1.27。X线照射后转染组细胞G₂+M期比例增加并高于单纯照射组和转染对照组(32.46%、9.42%、9.17%,P=0.000、0.000),转染组凋亡率也高于单纯照射组和转染对照组(35.20%、10.87%、11.33%,P=0.000、0.000)。结论 siRNA沉默Annexin A2基因表达可增强R743细胞放射敏感性,可能与DNA损伤修复、细胞周期时相分布变化有关。