厄洛替尼对非小细胞肺癌H1299细胞放射增敏研究

目的 探讨表皮生长因子受体酪氨酸激酶抑制剂对人非小细胞肺癌细胞放射敏感性的影响及其可能的机制。方法 体外培养人非小细胞肺癌细胞H1299。CCK-8检测厄洛替尼对H1299的毒性作用,并计算 IC50及 IC20,将 IC20作为后续试验的药物作用浓度。克隆形成实验检测射线联合厄洛替尼对H1299的作用,计算放射敏感性参数,绘制细胞存活曲线。流式细胞术检测细胞周期分布及凋亡。Western blot检测细胞EGFR/PI3K/AKT通路及凋亡相关蛋白表达。结果 厄洛替尼对H1299具有一定增殖抑制作用,IC50为27.3μmol/L、 IC20为3.3μmol/L。X射线联合 IC20浓度厄洛替尼能够降低H1299的克隆能力,使 G0/G1期、G2/M期比例增加,S期减少比例,细胞凋亡增加;抑制pEGFR及pAKT蛋白表达,增加凋亡相关蛋白Active Caspase 3、Cleaved PARP表达。结论 厄洛替尼对H1299具有放射增敏作用,其可能的机制是厄洛替尼联合射线抑制EGFR/PI3K/AKT通路,降低细胞损伤修复能力,改变细胞生长周期,诱导细胞凋亡。     

Enhanced radiosensitization of p53 mutant cells by oleamide

PURPOSE: Effect of oleamide, an endogenous fatty-acid primary amide, on tumor cells exposed to ionizing radiation (IR) has never before been explored. METHODS AND MATERIALS: NCI H460, human lung cancer cells, and human astrocytoma cell lines, U87 and U251, were used. The cytotoxicity of oleamide alone or in combination with IR was determined by clonogenic survival assay, and induction of apoptosis was estimated by FACS analysis. Protein expressions were confirmed by Western blotting, and immunofluorescence analysis of Bax by use of confocal microscopy was also performed. The combined effect of IR and oleamide to suppress tumor growth was studied by use of xenografts in the thighs of nude mice. RESULTS: Oleamide in combination with IR had a synergistic effect that decreased clonogenic survival of lung-carcinoma cell lines and also sensitized xenografts in nude mice. Enhanced induction of apoptosis of the cells by the combined treatment was mediated by loss of mitochondrial membrane potential, which resulted in the activation of caspase-8, caspase-9, and caspase-3 accompanied by cytochrome c release and Bid cleavage. The synergistic effects of the combined treatment were more enhanced in p53 mutant cells than in p53 wild-type cells. In p53 wild-type cells, both oleamide and radiation induced Bax translocation to mitochondria. On the other hand, in p53 mutant cells, radiation alone slightly induced Bax translocation to mitochondria, whereas oleamide induced a larger translocation. CONCLUSIONS: Oleamide may exhibit synergistic radiosensitization in p53 mutant cells through p53-independent Bax translocation to mitochondria.     

Studies on radiosensitization of Escherichia coli cells by cis-platinum complexes.

We recently reported that the antitumor drug cis-Pt(NH₃)₂CI₂ (cis-DDP) produces significant radiosensitization of anoxic E Coli C cells⁷. We have extended these studies to three other platinum drugs, all of which have been shown to be more effective antitumor drugs than cis-DDP. The drugs are: cis-dichloro bis(ethylene imine) Pt(II) (cis-DEP); cis-dichlorobicyclopentylamine Pt(II) (cis-PAD); and Pt-thymine blue (cis-PTB). Survival curve studies indicate that these drugs all produce greater anoxic radiosensitization of E coli C than cis-DDP at concentrations which are less toxic to the cells than similar concentrations of cis-DDP. If the cells are treated with any one of these drugs for two hours and then washed to remove the drug before irradiation, no detectable radiosensitization is found. We conclude that these drugs have the potential for being useful agents in combined modality therapy and that they warrant further study in mammalian systems.     

Radiosensitization of pulmonary metastases-I. In vivo testing of hycanthone, insulin and razoxane.

After the removal of a primary Lewis lung tumor from the flank of C57 Black mice, the metastases occurring in the lungs were used to investigate drugs as possible radiosensitizers. The left mouse lung was X-irradiated: the right lung acted as a reference. As selected and justified in our earlier paper (1978), the response measured was the ratio of the summed diameters of metastases per gram lung wt, left/right. This “metastases-only” model resembles the commonest clinical problem, the presence of potentially lethal metastases after excision of the primary. With this “model”, there was no chemotherapeutic action of single doses of the drugs or adjuvant solutions used. Doses of 12–24 Gray (1200–2400 rad) of X-irradiation were given alone or after a single injection of a drug.Statistically significant (p = < 0.05) radiosensitization of metastases was obtained using either 2mg/25g mouse of hycanthone methanesulfonate (an actinomycin D-like substance) or 56 milliunits (mU) of insulin. The latter reduced the mouse's blood sugar to about 30%. At these dose levels, “4-point” assays showed that the effect of 12 Gray of X-rays could be reproduced by 7 Gray plus hycanthone or 6.7 Gray plus insulin. Razoxane (ICRF 159) was also investigated, as single doses of 2.5–25 mg in 10% dimethylsulfoxide (DMSO) or 0.5% carboxymethylcellulose (CMC). In various experiments, it was given at 5 time intervals before X-irradiation. While it caused some decreases in the L/R ratio, these were never statistically significant nor was there any trend in the results. Hence, without ruling out the possibility of razoxane action under other circumstances, no radiosensitization was observed under our reported conditions.     

Pronounced radiosensitization of cultured human cancer cells by COX inhibitor under acidic microenvironment

: To demonstrate the influence of pH on the cytotoxicity and radiosensitization by COX (cyclooxygenase) -1 and -2 inhibitors using established human cancer cells in culture.

: Nonselective COX inhibitor, ibuprofen (IB), and selective COX-2 inhibitor, SC-236, were used to determine the cytotoxicity and radiosensitization at varying pH of culture media. Human colon carcinoma cell line (HT-29) was exposed to the drug alone and in combination with radiation at different pH of the cell culture media. The end point was clonogenic ability of the single-plated cells after the treatment.

: Cytotoxicity and radiosensitization of IB increased with higher drug concentration and longer exposure time. The most significant radiosensitization was seen with IB (1.5 mM) for 2-h treatment at pH 6.7 before irradiation. The dose-modifying factor as defined by the ratio of radiation doses required to achieve the same effect on cell survival was 1.8 at 10% survival level. In contrast, SC-236 (50 μM for 2–8 h) showed no pH-dependent cytotoxicity. There was modest increase in the cell killing at lower doses of radiation.

: An acidic pH was an important factor affecting the increased cytotoxicity and radiosensitization by ibuprofen. Radiation response was enhanced at shoulder portion of the cell survival curve by selective COX-2 inhibitor.     

WT对辐射致DNA损伤修复的影响

目的 研究磷脂酰肌醇３－激酶的特异性抑制剂Ｗｏｒｔｍａｎｎｉｎ（ＷＴ）对小鼠胸腺细胞ＤＮＡ辐射损伤的修复的影响,以探索ＷＴ放增敏作用机制。方法 以５０μｍｏｌ／Ｌ ＷＴ作用小鼠胸腺细胞,以不同剂量γ－射线照射,照射后不同时间制取细胞ＤＮＡ糖浆胶电泳方法检测ＤＮＡ双链断裂（ｄｓｂ）。结果 ＷＴＯ单独对受照射细胞ｄｓｂ产额无明显影响,抑制照射后ｄｓｂ。结论 ＷＴ的放射增敏剂作用与其抑制照射后ｄｓｂ修复     

siRNA共转染沉默bcl-2和XIAP基因对UMSCC12细胞放射增敏作用的实验研究

目的 探讨同时沉默bcl-2和XIAP基因对头颈部鳞癌UMSCC12细胞放射敏感性的影响.方法 实验分为4组:对照siRNA转染组、siRNA-bcl-2转染组、siRNA-XIAP转染组和siRNA-bcl-2与siRNA-XIAP共转染组.采用siRNA bcl-2和siRNA-XIAP共转染头颈鳞癌细胞株UMSCC12,Western blot检测蛋白水平基因沉默的效果.以caspase-3和caspase-9活性检测自发和放疗诱导的凋亡,最后以克隆形成实验评价放射增敏效果.结果 共转染siRNA-bcl-2和siRNA-XIAP有效沉默了UMSCC12细胞bcl-2和XIAP的蛋白表达.caspase-3和caspase-9活性检测提示,单独沉默XIAP未增加细胞自发和放射诱导的凋亡.单独沉默bcl-2使放射诱导的凋亡增加,与对照siRNA转染组照射后相比,caspase-3和caspase-9的活性差异有统计学意义(t=5.32、6.27,P＜0.05),但未增加细胞自发的凋亡.共转染后的caspase-3和caspase-9活性在未照射时比对照siRNA转染组分别提高至1.36和1.34倍(t=11.47、6.22,P＜0.05),照射后分别提高至1.72和1.98倍(f=12.02、20.14,P＜0.05).克隆形成实验显示,与对照siRNA转染组比较,siRNA-XIAP的放射增敏比(SER)为1.06,siRNA-bcl-2的放射增敏比为1.15,siRNA-bcl-2与siRNA-XIAP共转染组的放射增敏比为1.41,比其他组显著升高.结论 与单独沉默bcl-2和XIAP相比,同时沉默bcl-2和XIAP增加了UMSCC12细胞的放射敏感性,其机制可能与增加了UNSCC12细胞自发和放射诱导的凋亡有关.