Inhibition of p38 MAPK-dependent excision repair cross-complementing 1 expression decreases the DNA repair capacity to sensitize lung cancer cells to etoposide

Etoposide (VP-16), a topoisomerase II inhibitor, is an effective anticancer drug currently used for the treatment of a wide range of cancers. Excision repair cross-complementary 1 (ERCC1) is a key protein involved in the process of nucleotide excision repair. High level of ERCC1 expression in cancers is associated with resistance to DNA damage-based chemotherapy. In this study, the effects of p38 mitogen-activated protein kinase (MAPK) signal on the ERCC1 expression induced by etoposide in non-small cell lung cancer (NSCLC) cell lines was investigated. Etoposide increased phosphorylated MAPK kinase 3/6 (MKK3/6)-p38 MAPK and ERCC1 protein and mRNA levels in A549 and H1975 cells. Moreover, SB202190, a p38 inhibitor, or knockdown of p38 expression by specific short interfering RNA (siRNA) significantly decreased the etoposide-induced ERCC1 protein levels and DNA repair capacity in etoposide-exposed NSCLC cells. Enhancement of p38 activation by constitutively active MKK6 (MKK6E) increased ERCC1 protein levels. Specific inhibition of ERCC1 by siRNA significantly enhanced the etoposide-induced cytotoxicity and hypoxanthine guanine phosphoribosyltransferase (hprt) gene mutation rate. Moreover, the Hsp90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) could decrease the etoposide-induced p38 MAPK-mediated ERCC1 expression and augment the cytotoxic effect and growth inhibition by etopsoside. 17-AAG and etoposide-induced synergistic cytotoxic effect and DNA repair capacity decrease could be abrogated in lung cancer cells with MKK6E or HA-p38 MAPK expression vector transfection. Our results suggest that in human NSCLC cells, ERCC1 is induced by etoposide through the p38 MAPK pathway, and this phenomenon is required for NSCLC survival and resistant DNA damage.     

Base excision repair proteins are required for integrin-mediated suppression of bleomycin-induced DNA breakage in murine lung endothelial cells

Engagement of integrin cell adhesion receptors suppresses bleomycin (BLM)-induced DNA strand breakage in endothelial cells. Previous investigation of cells from poly(ADP-ribose) polymerase (PARP)-1 knockout mice and with an inhibitor of the enzyme indicated that this facilitator of base excision repair (BER) is required for integrin-mediated suppression of DNA strand breakage. Here, small inhibitory RNA (siRNA) was used to assess the requirement for the BER proteins, DNA ligase III (Lig3) alpha, PARP-1, and X-ray repair complementing defective repair in Chinese hamster cells 1 (XRCC1), and for the long-patch BER ligase, DNA ligase I (Lig1), in integrin-mediated protection from BLM-induced DNA breakage. Murine lung endothelial cells (MLECs) were transfected with siRNA, treated with anti-beta1 integrin antibody, and then BLM. 3'-OH in DNA and accumulation of phosphorylated histone H2AX (gammaH2AX), which reflects double-strand breakage, were measured. Integrin antibody inhibited the increases in 3'-OH caused by BLM in MLECs transfected with either control or Lig1 siRNA. However, after knockdown of Lig3alpha, PARP-1, or XRCC1, suppression of DNA breakage by integrin antibody was limited. BLM increased gammaH2AX levels, and integrin treatment inhibited this by 57 to 73% in MLECs transfected with control siRNA. Integrin engagement also inhibited increases in gammaH2AX in BLM-treated cells transfected with Lig1 siRNA. In contrast, Lig3alpha, PARP-1, and XRCC1 siRNAs prevented integrin-mediated inhibition of BLM-induced gammaH2AX levels. The results suggest that the BER proteins, Lig3alpha, PARP-1, and XRCC1, are required for integrin-mediated suppression of BLM-induced DNA breakage.     

Diagnostic value of HSP90α and related markers in lung cancer

PurposeTo investigate the expression of heat shock protein 90α (HSP90α) in patients with lung cancer (LC) and the clinical value of HSP90α and other related markers in the diagnosis of LC.MethodsOf 335 patients enrolled in the study cohort, 175 were screened for LC and 160 were healthy (HC). The plasma levels of HSP90α and related markers (CEA, NSE, CYFRA21‐1 and ProGRP) were detected in all individuals in the cohort by enzyme‐linked immunosorbent assay (ELISA). Groups were divided according to gender (male/female), age (≤60 years/>60 years), types of LC (small‐cell carcinoma, squamous carcinoma and adenocarcinoma), staging (I, II, III and IV) and metastasis (metastasis and non‐metastasis) separately. Wilcoxon Mann–Whitney test and Kruskal–Wallis test were used to compare statistical differences between two groups/among the multiple groups for each factor of HSP90α. The r‐value and Kappa were used to compare HSP90α with related markers, and the receiver operating curve (ROC) was used to evaluate the efficacy of plasma HSP90α in predicting LC.ResultsNo statistical difference was found in the plasma level of HSP90α among different age and gender groups (p > 0.05). In the group divided by LC type, staging and metastasis status, there were statistical differences among different groups in HSP90α level (p < 0.05). The levels of HSP90α, CEA, NSE, CYFRA21‐1 and ProGRP in LC groups were significantly higher than HC (p < 0.001). R values of HSP90α correlated with other related markers in the diagnosis of LC (p < 0.05). Although HSP90α and other related markers did not fit the satisfactory conformance, in terms of the positive rate of diagnosis, it was statistically differences in the diagnostic positive rate between HSP90α and each marker (p < 0.01). ROC analysis showed that a plasma HSP90α cut‐off point of 50.02 ng/ml had an optimal predictive value for LC.ConclusionsHSP90α has significant clinical value in early screening and diagnosis of LC. The combined application of HSP90α and related markers can improve the positive rate of early diagnosis of LC effectively.     

Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreatic, colon, and breast tumors

The receptor for epidermal growth factor (EGFR) is overexpressed in many cancers. One important signaling pathway regulated by EGFR is the phosphatidylinositol 3'-kinase (PI3K)-phosphoinositide-dependent kinase 1-Akt pathway. Activation of Akt leads to the stimulation of antiapoptotic pathways, promoting cell survival. Akt also regulates the mammalian target of rapamycin (mTOR)-S6K-S6 pathway to control cell growth in response to growth factors and nutrients. Recent reports have shown that the sensitivity of non-small-cell lung cancer cell lines to EGFR inhibitors such as erlotinib (Tarceva, OSI Pharmaceuticals) is dependent on inhibition of the phosphatidylinositol 3'-kinase-phosphoinositide-dependent kinase 1-Akt-mTOR pathway. There can be multiple inputs to this pathway as activity can be regulated by other receptors or upstream mutations. Therefore, inhibiting EGFR alone may not be sufficient for substantial inhibition of all tumor cells, highlighting the need for multipoint intervention. Herein, we sought to determine if rapamycin, an inhibitor of mTOR, could enhance erlotinib sensitivity for cell lines derived from a variety of tissue types (non-small-cell lung, pancreatic, colon, and breast). Erlotinib could inhibit extracellular signal-regulated kinase, Akt, and S6 only in cell lines that were the most sensitive. Rapamycin could fully inhibit S6 in all cell lines, but this was accompanied by activation of Akt phosphorylation. However, combination with erlotinib could down-modulate rapamycin-stimulated Akt activity. Therefore, in select cell lines, inhibition of both S6 and Akt was achieved only with the combination of erlotinib and rapamycin. This produced a synergistic effect on cell growth inhibition, observations that extended in vivo using xenograft models. These results suggest that combining rapamycin with erlotinib might be clinically useful to enhance response to erlotinib.     

Polymorphisms in base excision DNA repair genes and association with melanoma risk in a pilot study on Central-South Italian population

Base excision repair plays a key role in the removing of DNA damage from exposure to endogenous and exogenous carcinogens. The BER pathway removes alterations of a single oxidized, reduced or methylated base. Recently some studies have explored the association between risk for cutaneous melanoma and non-synonymous single-nucleotide polymorphisms (nsSNPs) in DNA-repair genes, although with contradictory results. We hypothesized that common nsSNPs of BER genes, specifically ADPRT rs1136410, XRCC1 rs25487, rs25489, rs1799782, APEX1 rs1130409, OGG1 rs1052133, LIG3 rs3136025 and MUTYH rs3219466, may contribute to risk of melanoma. The aim of this study is to investigate whether or not a correlation between these nsSNPs and melanoma risk and/or aggressiveness is present. 167 melanoma patients and 186 healthy control subjects were analysed. By multivariate statistical analysis no association was found between nsSNP and melanoma aggressiveness, while only the two XRCC1 (rs25487 and rs25489) nsSNPs showed a strong correlation (p<0.001) with melanoma risk. To our knowledge this is the first study reporting an association between BER nsSNPs and melanoma risk in Central-South Italian individuals. Our findings, if confirmed in larger population studies, will allow the inclusion of these XRCC1 nsSNPs in a screening panel for those individuals at higher risk for melanoma.     

Ku protein targeting by Ku70 small interfering RNA enhances human cancer cell response to topoisomerase II inhibitor and gamma radiation

Ku protein is a heterodimer (Ku70 and Ku86) known to play an important role in V(D)J recombination, apoptosis, telomere fusion, and double-strand break repair. Its role in double-strand breaks is relevant to cancer therapy because lack of Ku86 causes one of the most radiation-responsive phenotypes (hamster cells, XRS5). Although it is known that the heterodimer is necessary for the various functions of this protein, the impact of targeting Ku in human cancer cells has not been shown due to lack of appropriate approaches. It is also not known whether complete knock-out of Ku protein is required to enhance the sensitivity of human cells to gamma radiation as Ku protein is much more abundant in human cells than in hamster cells. In the current article, we have investigated the direct effect of Ku70 depletion in human cervical epithelioid (HeLa) and colon carcinoma (HCT116) cells. We specifically targeted Ku70 mRNA by use of small interfering RNA (siRNA). Of the five Ku70 siRNA synthesized, three inhibited the expression of Ku70 by up to 70% in HeLa cells. We have tested the effect of chemically synthesized siRNAs for target sequence 5 (CS #5) on the response of HeLa cells 72 hours after transfection to gamma radiation and etoposide, as this showed the maximum inhibition of Ku70 expression. Ku70 siRNA induced a decrease in the surviving fraction of irradiated HeLa cells by severalfold. Similar sensitizing effects were observed for etoposide, a topoisomerase II inhibitor. Studies with HCT116 cells using the same Ku70 siRNA (CS #5) showed a direct correlation between expression of Ku70 and sensitization to radiation and etoposide treatments.     

Hsp90 inhibitor-mediated disruption of chaperone association of ATR with hsp90 sensitizes cancer cells to DNA damage

Following DNA damage that results in stalled replication fork, activation of ATR-CHK1 signaling induces the DNA damage response (DDR) in transformed cells. In the present studies on human cervical and breast cancer cells, we determined the effects of hsp90 inhibition on the levels and accumulation of DNA damage/repair-associated proteins following exposure to γ-ionizing radiation (IR; 4 Gy). We show that hsp90 inhibition with 17-allylamino-demehoxygeldanamycin or the novel, nongeldanamycin analogue AUY922 (resorcinylic isoxazole amide; Novartis Pharma) dose-dependently reduced the levels of ATR and CHK1 without affecting ATM levels. AUY922-mediated depletion of ATR and CHK1 was associated with an increase in their polyubiquitylation and decreased binding to hsp90. Cotreatment with bortezomib partially restored AUY922-mediated depletion of ATR and CHK1 levels. Additionally, treatment with AUY922 reduced the accumulation of ATR, p53BP1, and CHK1 but not γ-H2AX to the sites of DNA damage. Following exposure to IR, AUY922 treatment abrogated IR-induced phospho (p)-ATR and p-CHK1 levels, but significantly enhanced γ-H2AX levels. AUY922 treatment also increased IR-induced accumulation of the cells in G(2)-M phase of the cell cycle, inhibited the repair of IR-induced DNA damage, and augmented IR-mediated loss of clonogenic survival. Short hairpin RNA-mediated depletion of ATR also inhibited IR-induced p-ATR and p-CHK1, but increased γ-H2AX levels, sensitizing cancer cells to IR-induced apoptosis and loss of clonogenic survival. These findings indicate that ATR is a bona fide hsp90 client protein and post-IR administration of AUY922, by inhibiting ATR-CHK1-mediated DDR, sensitizes cancer cells to IR.     

Targeting of AKT1 enhances radiation toxicity of human tumor cells by inhibiting DNA-PKcs-dependent DNA double-strand break repair

We have already reported that epidermal growth factor receptor/phosphatidylinositol 3-kinase/AKT signaling is an important pathway in regulating radiation sensitivity and DNA double-strand break (DNA-dsb) repair of human tumor cells. In the present study, we investigated the effect of AKT1 on DNA-dependent protein kinase catalytic subunit (DNA-PKcs) activity and DNA-dsb repair in irradiated non-small cell lung cancer cell lines A549 and H460. Treatment of cells with the specific AKT pathway inhibitor API-59 CJ-OH (API; 1-5 micromol/L) reduced clonogenic survival between 40% and 85% and enhanced radiation sensitivity of both cell lines significantly. As indicated by fluorescence-activated cell sorting analysis (sub-G(1) cells) and poly(ADP-ribose) polymerase cleavage, API treatment or transfection with AKT1-small interfering RNA (siRNA) induced apoptosis of H460 but not of A549 cells. However, in either apoptosis-resistant A549 or apoptosis-sensitive H460 cells, API and/or AKT1-siRNA did not enhance poly(ADP-ribose) polymerase cleavage and apoptosis following irradiation. Pretreatment of cells with API or transfection with AKT1-siRNA strongly inhibited radiation-induced phosphorylation of DNA-PKcs at T2609 and S2056 as well as repair of DNA-dsb as measured by the gamma-H2AX foci assay. Coimmunoprecipitation experiments showed a complex formation of activated AKT and DNA-PKcs, supporting the assumption that AKT plays an important regulatory role in the activation of DNA-PKcs in irradiated cells. Thus, targeting of AKT enhances radiation sensitivity of lung cancer cell lines A549 and H460 most likely through specific inhibition of DNA-PKcs-dependent DNA-dsb repair but not through enhancement of radiation-induced apoptosis.     

Radiosensitization of lung cancer by nutlin, an inhibitor of murine double minute 2

p53 plays a critical role in cell cycle arrest and induction of apoptosis. Certain malignancies carry wild-type p53, which is frequently down-regulated by murine double minute 2 (MDM2) overexpression. Availability of a small-molecule inhibitor against MDM2, nutlin, has made it feasible to evaluate the anti-MDM2-based therapeutic strategies. The rationale for the current study is that functional p53 has been linked with improved responses to radiation treatment. Hence, this study evaluates the use of nutlin, a small-molecule inhibitor that blocks the interaction of p53 and MDM2, in sensitizing cancer cells to radiation. Expression of MDM2, p53, and p21 in both p53 wild-type and p53-defective lung cancer cell lines was examined. Clonogenic and 7-amino-actinomycin D studies were used to determine possible mechanisms of cell death. The combined effect of MDM2 inhibition and radiation on cell cycle was also studied. We found that radiosensitization by nutlin occurs in lung cancer cells with wild-type p53. There were increased apoptosis and cell cycle arrest following administration of nutlin and radiation. Furthermore, the combination of nutlin and radiation decreased the ability of endothelial cells to form vasculature, as shown by Matrigel assays. Our data suggest that nutlin is an effective radiosensitizer of p53 wild-type cells. The radiosensitizing effect seems to be at least partially due to induction of apoptosis and cell cycle arrest. In addition, nutlin may be an effective radiosensitizer of tumor vasculature.     

Prevalence and screening of dyspnea interfering with daily life activities in ambulatory patients with advanced lung cancer

This study aimed to identify 1) the prevalence of “clinical dyspnea,” defined here as dyspnea interfering with any daily life activities, 2) the impact of dyspnea on daily life activities, and 3) the screening ability of the Cancer Dyspnea Scale (CDS) and the Dyspnea Numeric Scale (DNS). A total of 157 outpatients with advanced lung cancer completed the two scales (CDS and DNS) along with a questionnaire about interference with daily life activities (normal work, walking, sleep, mood, relation with other people, enjoyment of life, and general activities). Over half of this population (55%) experienced “clinical dyspnea.” Dyspnea interfered with not only physical domain (52%), such as walking and work, but also with psychological domain (23%), such as mood and enjoyment. Both scales were feasible for screening of clinical dyspnea. Applying a screening protocol may contribute to avoiding underestimation of clinical dyspnea and lead to appropriate interventions for it.     

应用症状自评量表评估肺癌与乳腺癌患者心理健康水平的差异

目的：了解肺癌与乳腺癌患者心理健康状况的差异，并将肺癌患者与全国常模的测试结果进行比较。方法：选择2002—01／10在解放军总医院中医科确诊的肺癌患者50例，乳腺癌患者34例、应用症状由评越表（以躯体化、强迫、人际关系、抑郁、焦虑、敌对、恐怖、偏执、精神病性9个因子作为观察指标，按照0-4的5级评分，0=无，1=轻度，2=中度，3=相当重，4=严重），根据各自的实际情况做出独立的测试。将肺癌患者分别与全国常模和乳腺癌患者的心理测试结果进行比较。结果：纳入病例总数84例，进入结果分析84例，无脱落者。①肺癌患者与国内常模症状自评量表评分比较：肺癌患者总分、总均分、阳性项目数、阳性项目均分、躯体化、抑郁、焦虑、恐怖等因子分值均高于国内常模（145．12&;#177;40．36、129．96&;#177;38．76；1．61&;#177;0.41，1．44&;#177;0.43；31．86&;#177;18.32，2492&;#177;18．41：2．82&;#177;0.45，2．60&;#177;0.59；1．55&;#177;0.49，1．37&;#177;0.48；1．72&;#177;0.65，1．50&;#177;0.59：1．73&;#177;0.40，1．39&;#177;0.43；1.38&;#177;0.45，1．23&;#177;0.41）。②肺癌患者与乳腺癌患者症状自评量表评分比较：肺癌患者焦虑、恐怖因子高于乳腺癌患者（1．73&;#177;0.40．1．54&;#177;0.46；1.38&;#177;0.45．1．19&;#177;0.39，P〈0.05）。结论：肺癌患者存在不同程度的心理问题，肺癌患者的总体精神状况和躯体化。抑郁、焦虑，恐怖等方面与正常人比较均有显著差异，心身健康状态较差。肺癌患者焦虑、恐怖状况高于乳腺癌患者，可能与肺癌患者普遍病情较重，身体状况差，化疗反应较大，及预后差有关。而乳腺癌病情相对糖锌．瘫状自评甘嘉测试与伞围常樟比较善异较小。     

应用症状自评量表评估肺癌与乳腺癌患者心理健康水平的差异

目的:了解肺癌与乳腺癌患者心理健康状况的差异,并将肺癌患者与全国常模的测试结果进行比较。方法:选择2002-01/10在解放军总医院中医科确诊的肺癌患者50例,乳腺癌患者34例,应用症状自评量表(以躯体化、强迫、人际关系、抑郁、焦虑、敌对、恐怖、偏执、精神病性9个因子作为观察指标,按照0～4的5级评分,0=无,l=轻度,2=中度,3=相当重,4=严重),根据各自的实际情况做出独立的测试。将肺癌患者分别与全国常模和乳腺癌患者的心理测试结果进行比较。结果:纳入病例总数84例,进入结果分析84例,无脱落者。①肺癌患者与国内常模症状自评量表评分比较:肺癌患者总分、总均分、阳性项目数、阳性项目均分、躯体化、抑郁、焦虑、恐怖等因子分值均高于国内常模(145.12±40.36,129.96±38.76;1.61±0.41,1.44±0.43;31.86±18.32,24.92±18.41;2.82±0.45,2.60±0.59;1.55±0.49,1.37±0.48;1.72±0.65,1.50±0.59;1.73±0.40,1.39±0.43;1.38±0.45,1.23±0.41)。②肺癌患者与乳腺癌患者症状自评量表评分比较:肺癌患者焦虑、恐怖因子高于乳腺癌患者(1.73±0.40,1.54±0.46;1.38±0.45,1.19±0.39,P<0.05)。结论:肺癌患者存在不同程度的心理问题,肺癌患者的总体精神状况和躯体化、抑郁、焦虑、恐怖等方面与正常人比较均有显著差异,心身健康状态较差。肺癌患者焦虑、恐怖状况高于乳腺癌患者,可能与肺癌患者普遍病情较重,身体状况差,化疗反应较大,及预后差有关。而乳腺癌病情相对较轻,症状自评量表测试与全国常模比较差异较小。     

17-DMAG体外对结肠癌HT-29细胞的影响及细胞内活性氧的研究

目的 观察17-二甲胺乙胺基-17-去甲氧基格尔德霉素（17-DMAG）对人结肠癌HT-29细胞增殖、细胞凋亡的影响及其细胞内活性氧（ROS）的变化.方法 用CCK-8检测17-DMAG对结肠癌HT-29细胞增殖影响;AnnexinV-FITC/PI双染法流式细胞检测细胞凋亡率;酶标仪检测细胞内ROS的变化.结果 17-DMAG呈时间-剂量依赖性抑制HT-29细胞增殖.0.25 μmol/L、0.5 μmol/L、1.0 μmol/L、2.5μmol/L作用于HT-29细胞24h后,细胞增殖抑制率分别为（22.17±1.15）％、（28.45±1.16）％、（35.04±1.58）％和（46.85±2.44）％,作用48 h后,细胞增殖抑制率分别为（27.55±0.65）％、（33.33±1.23）％、（46.20±4.76）％和（55.45±4.47）％,作用72h,细胞增殖抑制率分别为（39.19±1.74）％、（44.29±2.00）％、（50.66±2.17）％和（58.84±3.18）％.正常对照组HT-29细胞24h的自然总凋亡率（早期＋晚期）为（2.72±0.57）％,0.25 μmol/L、0.5μmol/L、1.0 μmol/L和2.5μmol/L 17-DMAG干预24h后细胞总凋亡率分别为（5.38±0.46）％、（6.88±0.52）％、（10.44±0.32）％与（17.87 ±4.66）％,17-DMAG作用HT-29细胞24h的凋亡率与对照组细胞凋亡率相比差异有统计学意义（P＜0.05）.0.25 μmoL/L、0.5μmol/L、1.0 μmol/L和2.5 μmol/L 17-DMAG作用HT-29细胞12 h与24h,其活性氧水平均较对照组有不同程度的上升,差异有统计学意义（P＜0.05）.结论 17-DMAG体外呈时间-剂量依赖性抑制HT-29细胞增殖,诱导其凋亡,可能部分与细胞内的ROS升高有关.     

Protection of mammalian cells against chemotherapeutic agents thiotepa, 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea, and mafosfamide using the DNA base excision repair genes Fpg and alpha-hOgg1: implications for protective gene therapy applications

Chemotherapeutic agents used in the treatment of cancer often lead to dose-limiting bone marrow suppression and may initiate secondary leukemia. N,N',N"-triethylenethiophosphoramide (thiotepa), a polyfunctional alkylating agent, is used in the treatment of breast, ovarian, and bladder carcinomas and is also being tested for efficacy in the treatment of central nervous system tumors. Thiotepa produces ring-opened bases such as formamidopyrimidine and 7-methyl-formamidopyrimidine, which can be recognized and repaired by the formamidopyrimidine glycosylase/AP lyase (Fpg) enzyme of Escherichia coli. Using this background information, we have created constructs using the E. coli fpg gene along with the functional equivalent human ortholog alpha-hOgg1. Although protection with the Fpg protein has been previously observed in Chinese hamster ovary cells, we demonstrate significant (100-fold) protection against thiotepa using the E. coli Fpg or the human alpha-hOgg1 cDNA in NIH3T3 cells. We have also observed a 10-fold protection by both the Fpg and alpha-hOgg1 transgenes against 1,3-N,N'-bis(2-chloroethyl)-N-nitrosourea (BCNU) and, to a lesser extent, mafosfamide (2-fold), an active form of the clinical agent cyclophosphamide. These latter two findings are novel and are particularly significant since the added protection was in an O(6)-methylguanine-DNA methyltransferase-positive background. These results support our general approach of using DNA base excision repair genes in gene therapy for cellular protection of normal cells during chemotherapy, particularly against the severe myelosuppressive effect of agents such as thiotepa, BCNU, and cyclophosphamide.     

Potentiation of paclitaxel activity by the HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin in human ovarian carcinoma cell lines with high levels of activated AKT

Activation of the phosphatidylinositol-3-kinase (PI3K)/AKT survival pathway is a mechanism of cytotoxic drug resistance in ovarian cancer, and inhibitors of this pathway can sensitize to cytotoxic drugs. The HSP90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17-AAG) depletes some proteins involved in PI3K/AKT signaling, e.g., ERBB2, epidermal growth factor receptor (EGFR), and phosphorylated AKT (p-AKT). 17-AAG and paclitaxel were combined (at a fixed 1:1 ratio of their IC(50)) in four ovarian cancer cell lines that differ in expression of p-AKT, EGFR, and ERBB2. The EGFR-overexpressing A431 and KB epidermoid cell lines were also included. Combination indices (CI) were calculated using the median-effect equation and interpreted in the context of 17-AAG-mediated inhibition of PI3K signaling. Synergy was observed in IGROV-1- and ERBB2-overexpressing SKOV-3 ovarian cancer cells that express a high level of constitutively activated p-AKT [CI at fraction unaffected (fu)(0.5) = 0.50 and 0.53, respectively]. Slight synergy was observed in A431 cells (moderate p-AKT/overexpressed EGFR; CI at fu(0.5) = 0.76) and antagonism in CH1 (moderate p-AKT), HX62 cells (low p-AKT), and KB cells (low p-AKT/overexpressed EGFR; CI at fu(50) = 3.0, 3.5, and 2.0, respectively). The observed effects correlated with changes in the rate of apoptosis induction. 17-AAG induced a decrease in HSP90 client proteins (e.g., C-RAF, ERBB2, and p-AKT) or in downstream markers of their activity (e.g., phosphorylated extracellular signal-regulated kinase or p-AKT) in SKOV-3, IGROV-1, and CH1 cells at IC(50) concentrations. A non-growth-inhibitory concentration (6 nmol/L) reduced the phosphorylation of AKT (but not extracellular signal-regulated kinase) and sensitized SKOV-3 cells to paclitaxel. In conclusion, 17-AAG may sensitize a subset of ovarian cancer to paclitaxel, particularly those tumors in which resistance is driven by ERBB2 and/or p-AKT.     

晚期非小细胞肺癌患者外周血细胞核苷酸切除修复互补基因1表达水平与铂类化疗敏感性相关研究

目的 建立检测晚期非小细胞肺癌(NSCLC)患者外周血核苷酸切除修复互补基因1(ERCC1 mRNA)表达水平的实时荧光定量逆转录聚合酶链反应(RT-PCR)方法,并探讨该基因表达水平与铂类化疗敏感性的关系.方法 符合入选条件的晚期NSCLC患者60例,化疗前利用实时荧光定量RT-PCR技术对其外周血标本进行ERCC1 mRNA相对定量分析.患者分别接受4～6个周期的含铂类第三代方案的化疗(具体方案为吉西他滨+顺铂或多西他赛+顺铂),并采用实体瘤疗效评价标准(RECIST标准)对患者化疗的有效率(response rate,RR)进行评估,疗效分为完全缓解(CR)、部分缓解(PR)、稳定(SD)和进展(PD).结果 60例患者外周血均检测到ERCC1mRNA的表达.化疗总有效率(PR/PR+SD+PD)30.0％(18/60),总疾病控制率(PR+SD/PR+SD+PD)86.7％(52/60).有效(PR)患者的ERCC1mRNA表达水平(中位值＝1.790)与无效(SD+PD)患者的ERCC1 mRNA表达水平(中位值=2.779)差异有统计学意义(Z=2.376,P＜0.05).疗效为PR,SD,PD患者的ERCC1 mRNA表达水平呈逐渐升高的趋势(中位值分别为1.790,2.883,3.545),差异有统计学意义(H=6.319,P＜0.05).ERCC1 mRNA表达水平低于中位值患者(“低”表达)的有效率(53.3％)与高于中位值患者(“高”表达)的有效率(6.6％)差异有统计学意义(P＜0.05).ERCC1 mRNA表达水平与年龄、性别、ECOG PS评分、临床分期、组织细胞病理学分型等因素之间无显著相关性(P＞0.05).结论 ①外周血细胞ERCC1 mRNA表达水平与铂类化疗敏感性具有显著负相关.②所建实时荧光定量RT-PCR方法能成功检测晚期非小细胞肺癌患者外周血细胞ERCC1 mRNA表达水平,有较高的敏感性与特异性,可应用于临床检测工作,指导临床化疗.     

MEK/ERK inhibitor U0126 increases the radiosensitivity of rhabdomyosarcoma cells in vitro and in vivo by downregulating growth and DNA repair signals

Multimodal treatment has improved the outcome of many solid tumors, and in some cases the use of radiosensitizers has significantly contributed to this gain. Activation of the extracellular signaling kinase pathway (MEK/ERK) generally results in stimulation of cell growth and confers a survival advantage playing the major role in human cancer. The potential involvement of this pathway in cellular radiosensitivity remains unclear. We previously reported that the disruption of c-Myc through MEK/ERK inhibition blocks the expression of the transformed phenotype; affects in vitro and in vivo growth and angiogenic signaling; and induces myogenic differentiation in the embryonal rhabdomyosarcoma (ERMS) cell lines (RD). This study was designed to examine whether the ERK pathway affects intrinsic radiosensitivity of rhabdomyosarcoma cancer cells. Exponentially growing human ERMS, RD, xenograft-derived RD-M1, and TE671 cell lines were used. The specific MEK/ERK inhibitor, U0126, reduced the clonogenic potential of the three cell lines, and was affected by radiation. U0126 inhibited phospho-active ERK1/2 and reduced DNA protein kinase catalytic subunit (DNA-PKcs) suggesting that ERKs and DNA-PKcs cooperate in radioprotection of rhabdomyosarcoma cells. The TE671 cell line xenotransplanted in mice showed a reduction in tumor mass and increase in the time of tumor progression with U0126 treatment associated with reduced DNA-PKcs, an effect enhanced by radiotherapy. Thus, our results show that MEK/ERK inhibition enhances radiosensitivity of rhabdomyosarcoma cells suggesting a rational approach in combination with radiotherapy.