Non-small-cell lung cancers with kinase domain mutations in the epidermal growth factor receptor are sensitive to ionizing radiation

Non-small cell lung cancers (NSCLCs) bearing mutations in the tyrosine kinase domain (TKD) of the epidermal growth factor receptor (EGFR) often exhibit dramatic sensitivity to the EGFR tyrosine kinase inhibitors gefitinib and erlotinib. Ionizing radiation (IR) is frequently used in the treatment of NSCLC, but little is known how lung tumor-acquired EGFR mutations affect responses to IR. Because this is of great clinical importance, we investigated and found that clonogenic survival of mutant EGFR NSCLCs in response to IR was reduced 500- to 1,000-fold compared with wild-type (WT) EGFR NSCLCs. Exogenous expression of either the L858R point mutant or the DeltaE746-E750 deletion mutant form of EGFR in immortalized human bronchial epithelial cells, p53 WT NSCLC (A549), or p53-null NSCLC (NCI-H1299) resulted in dramatically increased sensitivity to IR. We show that the majority of mutant EGFR NSCLCs, including those that contain the secondary gefitinib resistance T790M mutation, exhibit characteristics consistent with a radiosensitive phenotype, which include delayed DNA repair kinetics, defective IR-induced arrest in DNA synthesis or mitosis, and pronounced increases in apoptosis or micronuclei. Thus, understanding how activating mutations in the TKD domain of EGFR contribute to radiosensitivity should provide new insight into effective treatment of NSCLC with radiotherapy and perhaps avoid emergence of single agent drug resistance.     

Blockage of epidermal growth factor receptor-phosphatidylinositol 3-kinase-AKT signaling increases radiosensitivity of K-RAS mutated human tumor cells in vitro by affecting DNA repair.

PURPOSE: It is known that blockage of epidermal growth factor receptor (EGFR)/phosphatidylinositol 3-kinase (PI3K) activity enhances radiation sensitivity of human tumor cells presenting a K-RAS mutation. In the present study, we investigated whether impaired repair of DNA double-strand breaks (DSB) is responsible for the radiosensitizing effect of EGFR and PI3K inhibition in K-RAS mutated (K-RAS(mt)) cells. EXPERIMENTAL DESIGN: The effect of the EGFR tyrosine kinase inhibitor BIBX1382BS (BIBX) on cellular radiosensitivity was determined in K-RAS(mt) (A549) and K-RAS(wt) (FaDu) cell lines by clonogenic survival assay. Radiation-induced phosphorylation of H2AX (Ser139), ATM (Ser1981), and DNA-dependent protein kinase catalytic subunit (DNA-PKcs; Thr2609) was analyzed by immunoblotting. Twenty-four hours after irradiation, residual DSBs were quantified by identification of gammaH2AX foci and frequency of micronuclei. RESULTS: BIBX reduced clonogenic survival of K-RAS(mt)-A549 cells, but not of K-RAS(wt)-FaDu cells, after single-dose irradiation. Analysis of the radiation-induced H2AX phosphorylation revealed that BIBX, as well as the PI3K inhibitor LY294002, leads to a marked reduction of P-H2AX in K-RAS(mt)-A549 and MDA-MB-231 cells, but not in K-RAS(wt)-FaDu and HH4ded cells. Likewise, radiation-induced autophosphorylation of DNA-PKcs at Thr2609 was only blocked in A549 cells by these two inhibitors and AKT1 small interfering RNA transfection. However, neither in K-RAS(mt) nor in K-RAS(wt) cells the inhibitors did affect radiation-induced ATM phosphorylation. As a consequence of inhibitor treatment, a significant enhancement of both residual DSBs and frequency of micronuclei was apparent only in A549 but not in FaDu cells following radiation. CONCLUSION: Targeting of the EGFR-dependent PI3K-AKT pathway in K-RAS-mutated A549 cells significantly affects postradiation survival by affecting the activation of DNA-PKcs, resulting in a decreased DSB repair capacity.     

Somatic mutations in the tyrosine kinase domain of epidermal growth factor receptor (EGFR) abrogate EGFR-mediated radioprotection in non-small cell lung carcinoma

The epidermal growth factor receptor (EGFR) is an important determinant of radioresponse, whose elevated expression and activity frequently correlates with radioresistance in several cancers, including non-small cell lung carcinoma (NSCLC). We reported recently that NSCLC cell lines harboring somatic, activating mutations in the tyrosine kinase domain (TKD) of the EGFR exhibit significant delays in the repair of DNA double-strand breaks (DSB) and poor clonogenic survival in response to radiation. Here, we explore the mechanisms underlying mutant EGFR-associated radiosensitivity. In three representative NSCLC cell lines, we show that, unlike wild-type (WT) EGFR, receptors with common oncogenic TKD mutations, L858R or DeltaE746-E750, are defective in radiation-induced translocation to the nucleus and fail to bind the catalytic and regulatory subunits of the DNA-dependent protein kinase (DNA-PK), a key enzyme in the nonhomologous end-joining repair pathway. Moreover, despite the presence of WT EGFR, stable exogenous expression of either the L858R or the DeltaE746-E750 mutant forms of EGFR in human bronchial epithelial cells significantly delays repair of ionizing radiation (IR)-induced DSBs, blocks the resolution of frank or microhomologous DNA ends, and abrogates IR-induced nuclear EGFR translocation or binding to DNA-PK catalytic subunit. Our study has identified a subset of naturally occurring EGFR mutations that lack a critical radioprotective function of EGFR, providing valuable insights on how the EGFR mediates cell survival in response to radiation in NSCLC cell lines.     

人非小细胞肺癌细胞EGFR基因启动子甲基化与吉非替尼敏感性之间的相关性研究

目的 探讨EGFR基因启动子甲基化水平与人非小细胞肺癌（NSCLC）细胞株对吉非替尼敏感性之间的相关性。方法 用不同浓度吉非替尼分别作用于NSCLC细胞株HCC827、H1650、H1975、H358、H1299、A549后,CCK-8法检测细胞增殖抑制率,DNA直接测序法、实时荧光定量PCR法、免疫印迹法和甲基化特异性PCR法分别检测上述NSCLC细胞株EGFR基因突变、EGFR mRNA、EGFR蛋白表达和启动子区甲基化状态。结果 CCK-8法检测结果显示,19外显子缺失突变的HCC827细胞对吉非替尼最敏感,而同为19外显子缺失突变的H1650细胞对吉非替尼不敏感;野生型的H358细胞对吉非替尼中度敏感,其敏感性甚至超过19外显子突变的H1650细胞,而同为EGFR野生型的H1299、A549细胞对吉非替尼敏感性较差。吉非替尼处理72h后,HCC827细胞与H358细胞相比、HCC827细胞和H358细胞与其他4株细胞相比,IC50值均有显著性差异（P＜0.05）。HCC827细胞EGFR启动子为未甲基化状态,其EGFR蛋白和mRNA表达最高;H358细胞为部分甲基化,其EGFR蛋白和mRNA为中等表达;其他4个细胞株均为高甲基化状态,EGFR蛋白和mRNA呈低表达;HCC827细胞的EGFR表达水平较H358细胞高,HCC827和H358细胞的EGFR表达较其他4个细胞株高,差异均有统计学意义（P＜0.05）。结论 EGFR基因启动子区高甲基化可能下调EGFR基因的表达水平,从而降低NSCLC对吉非替尼的敏感性;对该基因的甲基化检测可能对预测吉非替尼治疗NSCLC疗效有一定的临床指导意义。     

吉非替尼对肺癌细胞株HCC827和H358放射敏感性的影响及其机制研究

背景与目的表皮生长因子受体(epidermal growth factor receptor,EGFR)是决定放疗效应的一个重要因素,其过表达或是下游通路的激活与包括非小细胞肺癌在内的肿瘤的放疗抵抗相关,因而阻断EGFR的信号通路可能会增强放疗敏感性。本研究旨在探讨小分子EGFR酪氨酸激酶抑制剂吉非替尼能否增加肺癌细胞株HCC827和H358的放疗敏感性及其可能的机制。方法选取HCC827和H358这两个非小细胞肺癌细胞株,分为单纯X线组和X线+吉非替尼两组。单纯X线组采用单纯X线照射,X线+吉非替尼组经1μmol/L吉非替尼作用24h后再行X线照射。克隆形成实验比较两株细胞中不同分组细胞放射敏感性,免疫荧光激光共聚焦显微镜观察X线照射后各时间点细胞核中的磷酸化H2AX(γ-H2AX)及EGFR焦点在细胞中的定位情况,Western blot法检测放疗后胞质胞核蛋白中EGFR的表达。结果克隆形成实验中,H358细胞实验组与对照组在各放疗剂量点的SF2值分别为0.355和0.433;HCC827细胞实验组与对照组在各放疗剂量点的SF2值分别为0.223和0.242,差别不明显。激光共聚焦显微镜观察照射4Gy后各时间段实验组H358细胞核中g-H2AX斑点相比对照组要多,且持续时间更长。而对照组和实验组的HCC827细胞g-H2AX斑点在各时间段并无明显差异;激光共聚焦显微镜观察照射4Gy后对照组H358的EGFR蛋白在1h内入核,而经吉非替尼处理后EGFR蛋白几乎不入核;实验组及对照组HCC827细胞的EGFR表达位置均在细胞质中,胞核中很少或者没有,可以认为并无入核现象;Western blot结果显示,H358细胞在经4Gy放射处理后有入核现象,而预先经吉非替尼处理后,EGFR蛋白几乎不在核内表达而仍位于细胞浆内。对于HCC827细胞,实验组及对照组的EGFR蛋白均在细胞质中表达,胞核中很少或没有,且两组并无明显差异。结论吉非替尼可增加肺癌细胞株H358的放射敏感性,这可能与其阻止放疗后EGFR入核、影响放疗后双链断裂(double strand break,DSB)修复有关;而对HCC827细胞无影响,可能与其放疗后EGFR不入核相关。     

Radiosensitivity enhancement by combined treatment of celecoxib and gefitinib on human lung cancer cells.

PURPOSE: To characterize the radiation-enhancing effects and underlying mechanisms of combined treatment with celecoxib, a cyclooxygenase-2 selective inhibitor, and gefitinib, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, in human lung cancer cells. EXPERIMENTAL DESIGN: Clonogenic cytotoxicity assays and clonogenic radiation survival assays after treatments with celecoxib and gefitinib with or without radiation were done on three human lung cancer cell lines. Synergisms after combined treatment with celecoxib, gefitinib, and radiation were investigated using isobologram and statistical analyses according to an independent action model. Alterations in apoptosis and cell cycle were measured to identify the mechanisms underlying the cell killing or radiation-enhancing effects of celecoxib and gefitinib combination treatment. Western blots for phosphorylated EGFR, EGFR, cyclooxygenase-2, and G(2) checkpoint molecules were conducted after treatment with celecoxib and/or gefitinib with or without radiation. RESULTS: Combination celecoxib, gefitinib, and radiation treatments were shown to be synergistic in causing clonogenic cell deaths in all cell lines tested, but the nature of synergism was cell type specific. The combined drug treatments induced apoptosis in an additive manner in A549 cells and in a synergistic manner in NCI-H460 and VMRC-LCD cells. Celecoxib or gefitinib attenuated radiation-induced G(2)-M arrest, and combined drug treatment additively attenuated radiation-induced G(2)-M arrest in all cell lines. Radiation-induced checkpoint kinase (Chk) 1 and Chk2 phosphorylation were inhibited by celecoxib and gefitinib treatment, respectively. CONCLUSIONS: Combined celecoxib and gefitinib treatments were shown to synergistically enhance the effect of radiation on lung cancer cells. The mechanisms underlying these synergistic effects seem to involve the synergistic enhancement of apoptosis and cooperative attenuation of radiation-induced G(2)-M arrest, possibly via Chk1 and Chk2 inhibition, by the combined drug treatments.     

SU11274逆转肝细胞生长因子诱导不同EGFR基因型非小细胞肺癌细胞株对吉非替尼耐药

背景与目的：肝细胞生长因子(hepatocyte growth factor,HGF)诱导敏感非小细胞肺癌(nonsmall cell lung cancer,NSCLC)细胞对表皮生长因子受体酪氨酸激酶抑制剂(epidermal growth factor receptor-tyrosine kinase inhibitor,EGFR-TKI)耐药,其机制与c-Met激活有关。本研究探讨c-Met抑制剂SU11274逆转HGF诱导的不同EGFR基因型NSCLC细胞株对吉非替尼耐药及逆转耐药机制。方法：选择人NSCLC细胞株PC9(EGFR突变型)、H292(EGFR野生型)和A549(EGFR野生型),应用吉非替尼和SU11274单独或联合作用于HGF诱导的细胞株。实验分为6组：C组(不加药对照组)、H组(HGF处理组)、G组(吉非替尼处理组)、S(SU11274处理组)、HG组(HGF+吉非替尼处理组)和HGS组(HGF+吉非替尼+SU11274处理组)。MTT法检测对细胞增殖的影响,流式细胞术检测细胞凋亡的影响;应用蛋白质印迹法(Western blot)检测细胞中c-Met及其下游通道Stat3、Akt和Erk1/2蛋白表达水平。结果：吉非替尼对3种细胞的生长抑制作用均呈浓度依赖性,HGF处理能够缓解吉非替尼的增殖抑制作用(P<0.05);不同浓度吉非替尼联合SU11274作用于HGF诱导细胞时,3种细胞株存活率比吉非替尼单独作用于HGF诱导细胞时明显降低(P<0.05);HGS组的细胞凋亡比HG组明显增加(P<0.05);HGS组的c-Met、Stat3、Akt和Erk1/2活化蛋白量比HG组明显减少。结论：c-Met抑制剂SU11274可逆转HGF诱导的不同EGFR基因型NSCLC细胞株对吉非替尼耐药,其机制可能与抑制HGF活化的c-Met及其下游通道蛋白表达有关。     

Additive interaction of gefitinib ('Iressa', ZD1839) and ionising radiation in human tumour cells in vitro

Cultures of human carcinoma A-431, A-549 and HeLa cells were challenged with gamma-rays without or with concomitant exposure to gefitinib, a potent inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR). The outcome of treatment was determined from cell and colony count, cell cycle progression and DNA double-strand break formation and rejoining. Apoptosis was measured in parallel from hypodiploid DNA and using an annexin V assay. Gefitinib developed a cytostatic effect in all cell lines, with drug sensitivity correlating the level of EGFR expression. A weak cytotoxicity of gefitinib was observed in HeLa cells only, although the drug was unable to induce significant cell cycle redistribution in this cell line. In contrast, substantial G1 block and S-phase depletion was observed in A-431 and A-549 cells exposed to gefitinib. The drug brought about additive to subadditive interaction with radiation with regard to growth inhibition, clonogenic death and induction of apoptosis. Consistently, gefitinib did not hinder the rejoining of radiation-induced DNA double-strand breaks in any cell line. The results demonstrate that gefitinib may elicit cytotoxicity at high concentration, but does not act as a radiosensitiser in vitro in concomitant association with radiation.     

Enhancement of Radiosensitivity by Eurycomalactone in Human NSCLC Cells Through G₂ /M Cell Cycle Arrest and Delayed DNA Double-Strand Break Repair

Radiotherapy (RT) is an important treatment for non-small cell lung cancer (NSCLC). However, the major obstacles to successful RT include the low radiosensitivity of cancer cells and the restricted radiation dose, which is given without damaging normal tissues. Therefore, the sensitizer that increases RT efficacy without dose escalation will be beneficial for NSCLC treatment. Eurycomalactone (ECL), an active quassinoid isolated from Eurycoma longifolia Jack, has been demonstrated to possess anticancer activity. In this study, we aimed to investigate the effect of ECL on sensitizing NSCLC cells to X-radiation (X-ray) as well as the underlying mechanisms. The results showed that ECL exhibited selective cytotoxicity against the NSCLC cells A549 and COR-L23 compared to the normal lung fibroblast. Clonogenic survival results indicated that ECL treatment prior to irradiation synergistically decreased the A549 and COR-L23 colony number. ECL treatment reduced the expression of cyclin B1 and CDK1/2 leading to induce cell cycle arrest at the radiosensitive G₂ /M phase. Moreover, ECL markedly delayed the repair of radiation-induced DNA double-strand breaks (DSBs). In A549 cells, pretreatment with ECL not only delayed the resolving of radiation-induced -H2AX foci but also blocked the formation of 53BP1 foci at the DSB sites. In addition, ECL pretreatment attenuated the expression of DNA repair proteins Ku-80 and KDM4D in both NSCLC cells. Consequently, these effects led to an increase in apoptosis in irradiated cells. Thus, ECL radiosensitized the NSCLC cells to X-ray via G2 /M arrest induction and delayed the repair of X-ray-induced DSBs. This study offers a great potential for ECL as an alternative safer radiosensitizer for increasing the RT efficiency against NSCLC.     

Adenoviral-mediated PTEN expression radiosensitizes non-small cell lung cancer cells by suppressing DNA repair capacity

Expression of the PTEN tumor suppressor gene is abnormal in many human cancers. Loss of PTEN expression leads to the activation of downstream signaling pathways that have been associated with resistance to radiation. In non-small cell lung carcinoma (NSCLC), suppressed expression of PTEN is frequently due to methylation of its promoter region. In this study, we tested whether gene transfer of wild-type PTEN into an NSCLC cell line with a known methylated PTEN promoter, H1299, would increase its sensitivity to ionizing radiation. Pretreating H1299 cells with an adenoviral-mediated PTEN (Ad-PTEN)-expressing vector sensitized H1299 cells to radiation. To determine the mechanism responsible for radiosensitization, we first examined radiation-induced apoptosis, which was enhanced but did not correlate with radiosensitizing effect of Ad-PTEN. Therefore, we next examined the ability of Ad-PTEN to modulate the repair of radiation-induced DNA double-strand breaks (DSBs) using the detection of repair foci positive for gamma-H2AX, a protein that becomes evident at the sites of each DSB and that can be visualized by immunofluorescent staining. Compared with controls, the repair of radiation-induced DSBs was retarded in H1299 cells pretreated with Ad-PTEN, consistent with the radiosensitizing effect of the vector. We conclude that signal transduction pathways residing primarily in the cytoplasm may intersect with DNA damage and repair pathways in the nucleus to modulate cellular responses to radiation. Elucidating the mechanisms responsible for this intersection may lead to novel strategies for improving therapy for cancers with defective PTEN.     

Enhancement of the antitumor activity of ionising radiation by nimotuzumab, a humanised monoclonal antibody to the epidermal growth factor receptor, in non-small cell lung cancer cell lines of differing epidermal growth factor receptor status

The expression and activity of the epidermal growth factor receptor (EGFR) are determinants of radiosensitivity in several tumour types, including non-small cell lung cancer (NSCLC). However, little is known of whether genetic alterations of EGFR in NSCLC cells affect the therapeutic response to monoclonal antibodies (mAbs) to EGFR in combination with radiation. We examined the effects of nimotuzumab, a humanised mAb to EGFR, in combination with ionising radiation on human NSCLC cell lines of differing EGFR status. Flow cytometry revealed that H292 and Ma-1 cells expressed high and moderate levels of EGFR on the cell surface, respectively, whereas H460, H1299, and H1975 cells showed a low level of surface EGFR expression. Immunoblot analysis revealed that EGFR phosphorylation was inhibited by nimotuzumab in H292 and Ma-1 cells but not in H460, H1299, or H1975 cells. Nimotuzumab augmented the cytotoxic effect of radiation in H292 and Ma-1 cells in a clonogenic assay in vitro, with a dose enhancement factor of 1.5 and 1.3, respectively. It also enhanced the antitumor effect of radiation on H292 and Ma-1 cell xenografts in nude mice, with an enhancement factor of 1.3 and 4.0, respectively. Nimotuzumab did not affect the radioresponse of H460 cells in vitro or in vivo. Nimotuzumab enhanced the antitumor efficacy of radiation in certain human NSCLC cell lines in vitro and in vivo. This effect may be related to the level of EGFR expression on the cell surface rather than to EGFR mutation.     

CD133+ cells contribute to radioresistance via altered regulation of DNA repair genes in human lung cancer cells

Background

Radioresistance in human tumors has been linked in part to a subset of cells termed cancer stem cells (CSCs). The prominin 1 (CD133) cell surface protein is proposed to be a marker enriching for CSCs. We explore the importance of DNA repair in contributing to radioresistance in CD133+ lung cancer cells.

Materials and methods

A549 and H1299 lung cancer cell lines were used. Sorted CD133+ cells were exposed to either single 4 Gy or 8 Gy doses and clonogenic survival measured. ?-H2AX immunofluorescence and quantitative real time PCR was performed on sorted CD133+ cells both in the absence of IR and after two single 4 Gy doses. Lentiviral shRNA was used to silence repair genes.

Results

A549 but not H1299 cells expand their CD133+ population after single 4 Gy exposure, and isolated A549 CD133+ cells demonstrate IR resistance. This resistance corresponded with enhanced repair of DNA double strand breaks (DSBs) and upregulated expression of DSB repair genes in A549 cells. Prior IR exposure of two single 4 Gy doses resulted in acquired DNA repair upregulation and improved repair proficiency in both A549 and H1299. Finally Exo1 and Rad51 silencing in A549 cells abrogated the CD133+ IR expansion phenotype and induced IR sensitivity in sorted CD133+ cells.

Conclusions

CD133 identifies a population of cells within specific tumor types containing altered expression of DNA repair genes that are inducible upon exposure to chemotherapy. This altered gene expression contributes to enhanced DSB resolution and the radioresistance phenotype of these cells. We also identify DNA repair genes which may serve as promising therapeutic targets to confer radiosensitivity to CSCs.     

Nontoxic concentration of DNA‐PK inhibitor NU7441 radio‐sensitizes lung tumor cells with little effect on double strand break repair

High‐linear energy transfer (LET) heavy ions have been increasingly employed as a useful alternative to conventional photon radiotherapy. As recent studies suggested that high LET radiation mainly affects the nonhomologous end‐joining (NHEJ) pathway of DNA double strand break (DSB) repair, we further investigated this concept by evaluating the combined effect of an NHEJ inhibitor (NU7441) at a non‐toxic concentration and carbon ions. NU7441‐treated non‐small cell lung cancer (NSCLC) A549 and H1299 cells were irradiated with X‐rays and carbon ions (290 MeV/n, 50 keV/μm). Cell survival was measured by clonogenic assay. DNA DSB repair, cell cycle distribution, DNA fragmentation and cellular senescence induction were studied using a flow cytometer. Senescence‐associated protein p21 was detected by western blotting. In the present study, 0.3 μM of NU7441, nontoxic to both normal and tumor cells, caused a significant radio‐sensitization in tumor cells exposed to X‐rays and carbon ions. This concentration did not seem to cause inhibition of DNA DSB repair but induced a significant G2/M arrest, which was particularly emphasized in p53‐null H1299 cells treated with NU7441 and carbon ions. In addition, the combined treatment induced more DNA fragmentation and a higher degree of senescence in H1299 cells than in A549 cells, indicating that DNA‐PK inhibitor contributes to various modes of cell death in a p53‐dependent manner. In summary, NSCLC cells irradiated with carbon ions were radio‐sensitized by a low concentration of DNA‐PK inhibitor NU7441 through a strong G2/M cell cycle arrest. Our findings may contribute to further effective radiotherapy using heavy ions.     

Adenovirus-mediated p16INK4a gene expression radiosensitizes non-small cell lung cancer cells in a p53-dependent manner

We examined the influence of adenovirus-mediated wild-type p16INK4a (Ad/p16) expression on the radiation sensitivity of NSCLC cell lines, all of which lacked constitutive p16INK4a but each of which varied in p53 status: A549 (-p16INK4a/ +pRb/wt-p53), H322 (-p16INK4a/ +pRb/mt-p53), and H1299 (-p16INK4a/ +pRb/deleted-p53). The in vitro clonogenic survival results indicate that Ad/p16 enhanced the radiosensitivity of A549 but not H322 or H1299. Further analysis indicated that the apoptosis induced by combination therapy using Ad/p16 plus irradiation was dependent on the endogenous p53 status of the cancer cells. We performed Western blotting to analyse the p53 protein expression of A549 cells treated with either Ad/p16 or Ad/Luc. Endogenous p53 protein levels were higher in A549 cells transfected with Ad/p16 than in those transfected with Ad/Luc. Furthermore, when wt-p53 protein expression was restored in H1299 using Ad/ p53, Ad/p16 stabilized p53 protein expression and radiosensitized the cells. These results suggest that Ad/ p16-induced stabilization of p53 protein may play an important role in Ad/p16 mediated radiosensitization by enhancing or restoring apoptosis properties. Thus, Ad/ p16 plus radiation in combination may be a useful gene therapy strategy for tumors that have wt-p53 but nonfunctional p16INK4a.     

吉非替尼对胃癌细胞株放射增敏的作用

背景与目的:表皮生长因子受体(epidermal growth factor receptor,EGFR)在绝大部分人类上皮肿瘤中都有表达,其表达高低与放疗抗拒有关.我们检测EGFR酪氨酸激酶抑制剂吉非替尼(gefitinib)对高表达胃癌细胞株放射增敏的作用,并初步探讨其机制.方法:Western blot法测定7株人胃癌细胞株(MKN45、SGC7901、SNU-1、N87、AGS、SNU-16、KATO-Ⅲ)中EGFR蛋白的表达,选取2株EGFR相对高表达的胃癌细胞用于后续实验.采用MTT法测定吉非替尼的半数抑制浓度(50%inhibition concentration,IC50),克隆形成实验计算细胞存活率,拟合生存曲线并计算放射生物学参数,流式细胞仪检测吉非替尼联合放疗的凋亡率及细胞周期分布.结果:选取7株胃癌细胞中EGFR表达最高的MKN45和SGC7901细胞,发现其存活率均随吉非替尼浓度或放射剂量的增加而明显下降(P＜0.05).MTT法检测吉非替尼对MKN45及SGC7901细胞的IC50分别为0.4mmol/L和0.8 mmol/L.MKN45细胞在0.1×IC50及0.2×IC50剂量下的增敏比(SER)分别为1.102和1.154,SGC7901则为1.092和1.176.吉非替尼或照射均可增加凋亡率,减少S期细胞比例及增加G2/M期细胞比例(P＜0.01).结论:吉非替尼序贯照射应用可提高EGFR高表达胃癌细胞的放射敏感性并阻碍细胞增殖、促进凋亡和干扰细胞周期分布.吉非替尼有望成为EGFR高表达胃癌的放射增敏剂.     

p53 enhances gefitinib-induced growth inhibition and apoptosis by regulation of Fas in non-small cell lung cancer

Treatment with gefitinib, a specific inhibitor of epidermal growth factor receptor tyrosine kinase (EGFR-TK), has resulted in dramatic responses in some patients with non-small cell lung cancer (NSCLC). Most patients who respond to gefitinib have EGFR-TK mutations; however, >10% of patients with EGFR-TK mutations do not respond. Similarly, some patients without EGFR-TK mutations respond to this drug, suggesting that other factors determine sensitivity to gefitinib. Aberrations of the tumor suppressor gene p53 are frequently associated with drug resistance. In this study, we investigated the role of p53 in growth-inhibitory and apoptotic effects of gefitinib in the human NSCLC cell lines NCI-H1299 and A549, which have no EGFR-TK mutations. NCI-H1299 cells, which had a p53-null genotype, were more resistant to gefitinib compared with A549 cells, which were wild-type p53 (IC(50), 40 micromol/L in NCI-H1299 and 5 micromol/L in A549). Treatment of A549 with gefitinib resulted in the translocation of p53 from cytosol to nucleus and the up-regulation of Fas, which was localized to the plasma membrane. In the stable H1299 cell line with tetracycline-inducible p53 expression, induced p53 enhanced growth inhibition and apoptosis by gefitinib through the up-regulation of Fas and restoration of caspase activation. A caspase inhibitor, Z-VAD-fmk, reduced these effects. Conversely, inhibition of p53 using antisense oligonucleotide in A549 caused a significant decrease in apoptosis by gefitinib and down-regulation of Fas under the same conditions. In conclusion, p53 may play a role in determining gefitinib sensitivity by regulating Fas expression in NSCLC.     

Radiosensitizing effect of YM155, a novel small-molecule survivin suppressant, in non-small cell lung cancer cell lines

PURPOSE: Survivin, a member of the inhibitor of apoptosis protein family, is an attractive target for cancer therapy. We have now investigated the effect of YM155, a small-molecule inhibitor of survivin expression, on the sensitivity of human non-small cell lung cancer (NSCLC) cell lines to gamma-radiation. EXPERIMENTAL DESIGN: The radiosensitizing effect of YM155 was evaluated on the basis of cell death, clonogenic survival, and progression of tumor xenografts. Radiation-induced DNA damage was evaluated on the basis of histone H2AX phosphorylation and foci formation. RESULTS: YM155 induced down-regulation of survivin expression in NSCLC cells in a concentration- and time-dependent manner. A clonogenic survival assay revealed that YM155 increased the sensitivity of NSCLC cells to gamma-radiation in vitro. The combination of YM155 and gamma-radiation induced synergistic increases both in the number of apoptotic cells and in the activity of caspase-3. Immunofluorescence analysis of histone gamma-H2AX also showed that YM155 delayed the repair of radiation-induced double-strand breaks in nuclear DNA. Finally, combination therapy with YM155 and gamma-radiation delayed the growth of NSCLC tumor xenografts in nude mice to a greater extent than did either treatment modality alone. CONCLUSIONS: These results suggest that YM155 sensitizes NSCLC cells to radiation both in vitro and in vivo, and that this effect of YM155 is likely attributable, at least in part, to the inhibition of DNA repair and enhancement of apoptosis that result from the down-regulation of survivin expression. Combined treatment with YM155 and radiation warrants investigation in clinical trials as a potential anticancer strategy.     

Niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase inhibitor,radiosensitizes human lung and breast cancer cells

The aim of this study was to assess niraparib (MK-4827), a novel poly(ADP-Ribose) polymerase (PARP) inhibitor, for its ability to radiosensitize human tumor cells. Human tumor cells derived from lung, breast and prostate cancers were tested for radiosensitization by niraparib using clonogenic survival assays. Both p53 wild-type and p53-defective lines were included. The ability of niraparib to alter the repair of radiation-induced DNA double strand breaks (DSBs) was determined using detection of γ-H₂AX foci and RAD51 foci. Clonogenic survival analyses indicated that micromolar concentrations of niraparib radiosensitized tumor cell lines derived from lung, breast, and prostate cancers independently of their p53 status but not cell lines derived from normal tissues. Niraparib also sensitized tumor cells to H₂O₂ and converted H₂O₂-induced single strand breaks (SSBs) into DSBs during DNA replication. These results indicate that human tumor cells are significantly radiosensitized by the potent and selective PARP-1 inhibitor, niraparib, in the in vitro setting. The mechanism of this effect appears to involve a conversion of sublethal SSBs into lethal DSBs during DNA replication due to the inhibition of base excision repair by the drug. Taken together, our findings strongly support the clinical evaluation of niraparib in combination with radiation.     

Gefitinib induces apoptosis in the EGFRL858R non-small-cell lung cancer cell line H3255

Somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) have recently been described in patients with non-small-cell lung cancer (NSCLC) who achieve radiographic regressions to the EGFR inhibitor gefitinib. One of these mutations, L858R (Leu-->Arg), is also found in NSCLC cell line H3255, which is very sensitive to gefitinib treatment. We characterized nine NSCLC cell lines (three isolated from patients with bronchioloalveolar carcinoma and six isolated from patients with adenocarcinoma) for their in vitro sensitivity to gefitinib. Of these, only H3255 (EGFR(L858R)) and H1666 (EGFR(WT)) are sensitive to gefitinib with IC(50) values of 40 nmol/L and 2 micromol/L, respectively. We examined the effects of gefitinib on H3255 and cell lines containing wild-type EGFR that are either sensitive (H1666) or resistant (A549 and H441) to gefitinib exposure in vitro. Gefitinib treatment (1 micromol/L) leads to significant apoptosis accompanied by increased poly(ADP-ribose) polymerase cleavage only in the H3255 cell line, leads to G(1)-S arrest in H1666, and has no effects in the A549 and H441 cell lines. Although EGFR and AKT are constitutively phosphorylated in H3255, H1666, and H441 cell lines, AKT is completely inhibited by gefitinib treatment only in the H3255 cell line. These findings further characterize a mechanism by which gefitinib treatment of NSCLC harboring EGFR(L858R) leads to a dramatic response to gefitinib.