Overexpression of Cathepsin L is associated with gefitinib resistance in non-small cell lung cancer

Lung cancer, the most common malignancy, is still the leading cause of cancer-related death worldwide. Non-small-cell lung cancer (NSCLC) accounts for 80 % of all lung cancers. Recent studies showed Cathepsin L (CTSL) is overexpressed in various cancerous tissues; however, the association between CTSL expression and EGFR-TKI resistance remains unknown. In this study, we investigated the expression of CTSL in lung cancer specimens and matched normal tissues by quantitative real-time PCR and IHC. The functional role of CTSL in resistant PC-9/GR cell line was investigated by proliferation and apoptosis analysis compared with control PC-9 cells. Our results found that the level of CTSL expression was higher in NSCLC tissues compared with matched normal adjacent tissue samples, and CTSL was more highly expressed in PC-9/GR cells compared to PC-9 cells. Knocking-down of CTSL in PC-9/GR cells could decrease cell proliferation and potentiate apoptosis induced by gefitinib, suggesting CTSL may contribute to gefitinib resistance in NSCLC. CTSL might be explored as a candidate of therapeutic target for modulating EGFR-TKI sensitivity in NSCLC.     

Krüppel‐like factor 4 promotes c‐Met amplification‐mediated gefitinib resistance in non‐small‐cell lung cancer

Gefitinib has been widely used in the first‐line treatment of advanced EGFR‐mutated non‐small‐cell lung cancer (NSCLC). However, many NSCLC patients will acquire resistance to gefitinib after 9‐14 months of treatment. This study revealed that Krüppel‐like factor 4 (KLF4) contributes to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells. We observed that KLF4 was overexpressed in c‐Met‐overexpressing NSCLC cells and tissues. Knockdown of KLF4 increased tumorigenic properties in gefitinib‐resistant NSCLC cell lines without c‐Met overexpression, but it reduced tumorigenic properties and increased gefitinib sensitivity in gefitinib‐resistant NSCLC cells with c‐Met overexpression, whereas overexpression of KLF4 reduced gefitinib sensitivity in gefitinib‐sensitive NSCLC cells. Furthermore, Western blot analysis revealed that KLF4 contributed to the formation of gefitinib resistance in c‐Met‐overexpressing NSCLC cells by inhibiting the expression of apoptosis‐related proteins under gefitinib treatment and activating the c‐Met/Akt signaling pathway by decreasing the inhibition of β‐catenin on phosphorylation of c‐Met to prevent blockade by gefitinib. In summary, this study's results suggest that KLF4 is a promising candidate molecular target for both prevention and therapy of NSCLC with c‐Met overexpression.     

Establishment and Characterization of Non-small Cell Lung Cancer Cell Lines Resistant to Mitomycin C under Aerobic Conditions

To elucidate the mechanisms of acquired resistance to mitomycin C (MMC) in non-small cell lung cancer (NSCLC), we established two MMC-resistant NSCLC sublines by continuous exposure to MMC, using PC-9 as a parent cell line. The sublines, PC-9/MC2 and PC-9/MC4, were 6.4- and 10-fold more resistant to MMC than their parent cell line, respectively, at the IC₅₀ value as determined by MTT assay. They exhibited cross-resistance to EO9, but were not resistant to cisplatin, vindesine, etoposide, carboquone, or KW-2149, a novel MMC derivative. They were collaterally sensitive to adriamycin and menadione. Accumulation of the drug was decreased in the resistant sublines to about 60% of that in the parent cells. Cytosolic DT-diaphorase (DTD) activities were decreased to 13.5±3.2 in PC9/MC2 and 1.3±0.6 in PC-9/MC4 from 261.5±92.7 nmol/min/mg protein in the parent PC-9. NADH:cytochrome b ₅ reductase activities in both of the resistant cell lines were significantly decreased as compared to that in the parent cell line. Addition of dicumarol resulted in a two-fold increase in IC₅₀ value in PC-9, whereas the IC₅₀ value showed no change in PC-9/MC4. Moreover, dicumarol did not affect the sensitivities to KW-2149 but decreased the sensitivities to EO9 in both the parent and the resistant cell lines. Formation of an alkylating metabolite was significantly decreased in the resistant cells, in parallel to the degree of resistance. We concluded that deficient drug activation due to decreased DTD activity was important as a mechanism of resistance to MMC in PC-9, a relatively DTD-rich NSCLC cell line.     

活性氧在导致吉非替尼耐药细胞凋亡中的作用

研究针对K-ras突变小分子NSC-741909是否可特异性杀伤吉非替尼原发耐药细胞,并对活性氧（reactive oxygen species,ROS）在其中的作用进行探讨。方法:选取H322（K-ras野生）及A549（K-ras突变）人源非小细胞肺癌细胞系,观察吉非替尼对不同细胞系生长的影响;NSC-741909作用于吉非替尼耐药细胞,观察细胞增殖与凋亡的变化;DCFH-DA荧光探针标记细胞内ROS,FCM检测细胞内ROS水平的变化,Western blot检测细胞内JNK通路蛋白变化。结果:K-ras突变型细胞对吉非替尼原发耐药,但NSC-741909可使这种原发耐药细胞出现凋亡;细胞内产生ROS,p-JNK表达增加而总JNK无改变,MKP1表达受抑制。结论:针对K-ras突变的小分子NSC-741909可使吉非替尼耐药细胞产生凋亡,NSC-741909通过使细胞内产生ROS持续激活JNK通路,是其导致吉非替尼耐药细胞凋亡的可能机制之一。      

Combined inhibition of IGFR enhances the effects of gefitinib in H1650: a lung cancer cell line with EGFR mutation and primary resistance to EGFR-TK inhibitors

Purpose

H1650 non-small cell lung cancer (NSCLC) cells display primary resistance to epidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) although they have a deletion mutation on exon 19 of the EGFR gene. We investigated the effect of inhibition of both insulin-like growth factor receptor (IGFR) and EGFR signaling considering that IGFR signaling pathway has been implicated in the development and progression with therapeutic resistance of various cancers including lung cancer.

Methods

Three human NSCLC cell lines with an EGFR mutation of PC-9, HCC827 and H1650 were used for experiment. Cell viability and proliferative activity were assessed by MTT and three-dimensional culture assay. Combination index was obtained by CalcuSyn software. The change of EGFR- and IGFR-related signals was evaluated by western blots.

Results

H1650 cells were 1,000 times more resistant to gefitinib and erlotinib than HCC827 and PC-9 cells possessing the same EGFR mutation. Phosphatase and tensin homolog loss and sustained phosphorylation of Akt in spite of treatment with gefitinib were evident only in H1650 cells. Interestingly, IGFR phosphorylation was decreased by gefitinib in HCC827 and PC-9 cells while being maintained in H1650 cells. Combined treatment with the IGFR inhibitors α-IR3 and AG1024 enhanced gefitinib-induced growth inhibition and apoptosis, and down-regulated phosphorylation of Akt, EGFR and IGFR.

Conclusion

Combined inhibition of IGFR signaling enhances the growth inhibitory and apoptosis-inducing effects of gefitinib, suggesting that this approach could be useful to overcome the primary resistance to EGFR-TKIs in lung cancer.     

Gefitinib induces premature senescence in non-small cell lung cancer cells with or without EGFR gene mutation

Despite its tremendous antitumor effect in a subset of patients with non-small cell lung cancer (NSCLC), the exact mechanism of gefitinib-induced cell death has not been fully determined. In this study, forms of cell death in various NSCLC cell lines after gefitinib exposure was analyzed to elucidate the cell death mechanism of gefitinib. Though higher concentration of gefitinib (10 microM) induced extensive apoptosis in two cell lines (EGFR-mutated PC-9 cells and EGFR wild- type EBC-2/R cells), clinically relevant concentrations of gefitinib (1 microM) induced prominent premature senescence instead of apoptosis in these cells. This induction of senescence was preceded by immediate increase of p16INK4A, p21WAF1/Cip1 and p27Kip1 levels and subsequent G1 cell cycle arrest. These phenomena were not observed in gefitinib-resistant (RERF-LC-MS) cells. Additionally, ex vivo exposure to gefitinib induced senescence in short-term cultured tumor cells that were obtained from malignant pleural effusion of a patient with NSCLC, whose tumor was later revealed to be clinically sensitive to gefitinib. Our results indicate that senescence might be a major anti-tumor mechanism of gefitinib in these NSCLC cells regardless of the EGFR gene mutation status.     

Role of dt-diaphorase as a determinant of sensitivity to mitomycin analogs in nonsmall cell lung-cancer cell-lines

DT-diaphorase (DT-D) is regarded as a two-electron reductase that plays an important role in the biotransformation of mitomycin C (MMC) to antitumor metabolites, which is enhanced under hypoxic conditions. To evaluate the role of DT-D as a bioactivator of MMC and its analogue, KW-2149, in non-small cell lung cancer (NSCLC) cell lines under an aerobic or hypoxic condition, we examined the inhibitory effect of dicumarol which was regarded as an inhibitor of DT-D on the sensitivity to these anticancer agents in vitro. In this study, we used an MMC-resistant NSCLC cell line (PC-9/MC4) which was established in our laboratory from a PC-9 cell line as a parent cell line by continuous exposure to MMC. The subline PC-9/MC4 was 6.7-fold more resistant to MMC than PC-9, the parent cell line, under aerobic conditions, and 5.2-fold more resistant even under hypoxic conditions. PC-9/MC4 cell lines did not show collateral resistance to KW-2149, a newly developed MMC analogue. The IC50 value of PC-9 against MMC significantly decreased by co-incubation with dicumarol under aerobic, but not under hypoxic conditions. KW-2149 was cytotoxic to PC-9/MC4 as well as PC-9 cells, and the sensitivity to KW-2149 was not altered by coincubation with dicumarol or exposure to hypoxia. There were no significant differences in intracellular uptake of MMC and the activities of cytosolic detoxification enzymes, GST and GSH, between PC-9 and PC-9/MC4 cell lines under aerobic conditions. These findings suggest a partial role of DT-D in bioactivation of MMC, but not of KW-2149, under aerobic conditions. However, the detailed mechanisms of drug resistance to MMC under hypoxic conditions are still not clear.     

MicroRNA-34a overcomes HGF-mediated gefitinib resistance in EGFR mutant lung cancer cells partly by targeting MET

In non-small-cell lung cancer (NSCLC) that harbours an activating epidermal growth factor receptor (EGFR) mutation, over-expression of hepatocyte growth factor (HGF) is an important mechanism involved in the acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) by restoring activity of the PI3K/Akt pathway via phosphorylation of MET. In our study, we found that the forced expression of miR-34a inhibited cell growth and induced apoptosis partly by targeting MET in HGF-induced gefitinib-resistant HCC827 and PC-9 cells. Furthermore, dramatic tumour regression was observed in the miR-34a plus gefitinib group in HGF-induced gefitinib resistant mouse xenograft models. This study demonstrates for the first time that miR-34a rescues HGF-induced gefitinib resistance in EGFR mutant NSCLC cells.     

MiR-130a Overcomes Gefitinib Resistance by Targeting Met in Non-Small Cell Lung Cancer Cell Lines

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer and the most common cause oflung cancer death. Currently, the epidermal growth factor receptor inhibitor gefitinib is used for its treatment;however, drug resistance is a major obstacle. Expression of Met has been associated with both primary andacquired resistance to gefitinib, but the mechanisms regulating its expression are not fully understood. Recently,miRNAs such as miR-130a have been shown to play a role in gefitinib resistance, but importance in NSCLC andrelationships with Met have not been fully explored. Here we show that miR-130a is over-expressed in gefitinibsensitiveNSCLC cell lines, but is low in gefitinib-resistant NSCLC cell lines. Moreover, miR-130a expressionwas negatively correlated with that of Met. Further analysis revealed that over-expression of miR-130a increasedcell apoptosis and inhibited proliferation of NSCLC cells treated with gefitinib, whereas lowering the expressionof miR-130a decreased cell apoptosis and promoted cell proliferation after treatment with gefitinib in bothgefitinib-sensitive and -resistant NSCLC cell lines, suggesting that miR-130a overcomes gefitinib resistance.We also demonstrated that miR-130a binds to the 3’-UTR of Met and significantly suppresses its expression.Finally, our results showed that over-expressing Met could “rescue” the functions of miR-130a regarding cellapoptosis and proliferation after cells are treated with gefitinib. These findings indicate that the miR-130a/Metaxis plays an important role in gefitinib resistance in NSCLC. Thus, the miR-130a/Met axis may be an effectivetherapeutic target in gefitinib-resistant lung cancer patients.     

MEK inhibitors reverse resistance in epidermal growth factor receptor mutation lung cancer cells with acquired resistance to gefitinib

Lung adenocarcinoma cells harboring epidermal growth factor receptor (EGFR) mutations are sensitive to EGFR tyrosine kinase inhibitors (TKIs), including gefitinib. Acquired resistance to EGFR-TKIs develops after prolonged treatments. The study was prompt to explore effective strategies against resistance to EGFR-TKIs. We established gefitinib resistant PC-9 cells which harbor EGFR exon 19 deletion. Known mechanisms for intrinsic or acquired EGFR-TKI resistance, including KRAS mutation, HER2 mutation, EGFR T790M mutation and MET gene amplification, were studied, and we did not observe any known mechanisms for intrinsic or acquired resistance to EGFR-TKIs in the resistant cells. In the parental PC-9 cells, labeled as PC-9/wt, gefitinib completely inhibited EGF-induced phosphorylation of EGFR, AKT and ERK. Gefitinib inhibited EGFR phosphorylation, but was unable to block EGF-induced phosphorylation of ERK in resistant cells, labeled as PC-9/gef cells, including PC-9/gefB4, PC-9/gefE3, and PC-9/gefE7 subclones. We detected NRAS Q61K mutation in the PC-9/gef cells but not the PC-9/wt cells. MEK inhibitors, either AZD6244 or CI1040, inhibited ERK phosphorylation and sensitized gefitinib-induced cytotoxicity in PC-9/gef cells. Whereas MEK inhibitors or gefitinib alone did not activate caspases in PC-9/gef cells, combination of gefitinib and AZD6244 or CI1040 induced apoptosis. Our in vivo studies showed that gefitinib inhibited growth of PC-9/wt xenografts but not PC-9/gef xenografts. Furthermore, combination of a MEK inhibitor and gefitinib inhibited growth of both PC-9/wt xenografts and PC-9/gefB4 xenografts. To conclude, persistent activation of ERK pathway contributes to the acquired gefitinib-resistance. Combined treatment of gefitinib and MEK inhibitors may be therapeutically useful for acquired gefitinib-resistance lung adenocarcinoma cells harboring EGFR mutations.     

Establishment of a human non-small cell lung cancer cell line resistant to gefitinib

The epidermal growth factor receptor (EGFR) tyrosine-kinase inhibitor gefitinib (Iressa, ZD1839) has shown promising activity preclinically and clinically. Because comparative investigations of drug-resistant sublines with their parental cells are useful approaches to identifying the mechanism of gefitinib resistance and select factors that determine sensitivity to gefitinib, we established a human non-small cell lung carcinoma subline (PC-9/ZD) that is resistant to gefitinib. PC-9/ZD cells are approximately 180-fold more resistant to gefitinib than their parental PC-9 cells and PC-9/ZD cells do not exhibit cross-resistance to conventional anticancer agents or other tyrosine kinase inhibitors, except AG-1478, a specific inhibitor of EGFR. PC-9/ZD cells also display significant resistance to gefitinib in a tumor-bearing animal model. To elucidate the mechanism of resistance, we characterized PC-9/ZD cells. The basal level of EGFR in PC-9 and PC-9/ZD cells was comparable. A deletion mutation was identified within the kinase domain of EGFR in both PC-9 and PC-9/ZD, but no difference in the sequence of EGFR cDNA was detected in either cell line. Increased EGFR/HER2 (and EGFR/HER3) heterodimer formations were demonstrated in PC-9/ZD cells by chemical cross-linking and immunoprecipitation analysis in cells unexposed to gefitinib. Exposure to gefitinib increased heterodimer formation in PC-9 cells, but not in PC-9/ZD cells. Gefitinib inhibits EGFR autophosphorylation in a dose-dependent manner in PC-9 cells but not in PC-9/ZD cells. A marked difference in inhibition of site-specific phosphorylation of EGFR was observed at Tyr1068 compared to other tyrosine residues (Tyr845, 992 and 1045). To elucidate the downstream signaling in the PC9/ZD cellular machinery, complex formation between EGFR and its adaptor proteins GRB2, SOS, and Shc was examined. A marked reduction in the GRB2-EGFR complex and absence of SOS-EGFR were observed in PC-9/ZD cells, even though the protein levels of GRB2 and SOS in PC-9 and PC-9/ZD cells were comparable. Expression of phosphorylated AKT was increased in PC-9 cells and inhibited by 0.02 microM gefitinib. But the inhibition was not significant in PC-9/ZD cells. These results suggest that alterations of adaptor-protein-mediated signal transduction from EGFR to AKT is a possible mechanism of the resistance to gefitinib in PC-9/ZD cells. These phenotypes including EGFR-SOS complex and heterodimer formation of HER family members are potential biomarkers for predicting resistance to gefitinib.     

吉非替尼联合鸦胆子油乳对肺腺癌细胞的作用及其机制

目的 探讨吉非替尼（Gefitinib, G）联合鸦胆子油乳（Bruceolic oil emulsion, B）对肺腺癌PC-9、A549细胞增殖与凋亡的作用及其机制。方法 取对数生长期的PC-9、A549细胞进行实验,两细胞株在实验中均分为对照组和实验组。采用MTT比色法、TUNEL法及流式细胞技术检测各实验组对肺腺癌细胞的增殖抑制、诱导细胞凋亡及细胞周期阻滞等作用。结果 吉非替尼、鸦胆子油乳单药均能抑制PC-9、A549细胞的生长、增殖并诱导其凋亡,呈浓度和时间依赖效应。与鸦胆子油乳联合后,吉非替尼对PC-9和A549细胞的IC50较单用时分别下降了50.72%和66.56%。两药联合后对两细胞的增殖抑制及凋亡诱导作用进一步增强。结论 吉非替尼联合鸦胆子油乳能显著抑制肺腺癌PC-9、A549细胞增殖、诱导细胞凋亡,可能与药物对细胞周期的阻滞作用有关。两者合用时鸦胆子油乳可对吉非替尼起到增效增敏的作用。     

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.     

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。     

吉非替尼获得性耐药细胞株对不同化疗药物的敏感性分析

目的 探讨吉非替尼获得性耐药细胞株对不同化疗药物的敏感性,为分子靶向治疗失败的患者选择化疗方案提供临床前的依据.方法 体外培养人肺腺癌细胞株PC9和吉非替尼获得性耐药株PC9/G,采用二苯基溴化四氮唑蓝(MTT)法测定PC9和PCg/G细胞对不同药物的敏感性以及细胞的增殖抑制率;采用流式细胞仪检测药物对PC9和PCg/G细胞凋亡的影响及P-170蛋白的表达;采用基因芯片技术分析PC9和PC9/G细胞的表达基因谱差异;采用Western blot法检测PC9和PC9/G细胞中总Akt、磷酸化Akt和整合素β1的表达.结果 MTT和凋亡检测的结果 表明,与PC9细胞相比,吉非替尼获得性耐药细胞株PC9/G对顺铂的耐药指数为5.4,细胞凋亡减少,联合LY294002后对顺铂的敏感性显著增加(P＜0.05).PC9/G细胞对多西紫杉醇较PC9细胞更为敏感,培美曲塞对两株细胞的IC50及凋亡影响的差异无统计学意义(P＞0.05).PC9/G细胞中P-170蛋白的表达水平为5.32,与PC9细胞(7.18)比较,差异无统计学意义(P＞0.05).基因芯片分析显示,PC9/G细胞中,整合素β1及DNA修复基因的表达上调,有丝分裂期基因表达下调.PC9/G细胞中总Akt、磷酸化Akt及整合素β1的蛋白表达水平分别为1.32、1.82和1.59,PC9细胞巾总Akt、磷酸化Akt及整合素β1的蛋白表达水平分别为0.83、0.87和0.57.结论 在吉非替尼获得件耐药细胞株中存在P13K的表达上调及激活、整合素β1及DNA修复基因的表达上调,其表达土调与顺铂耐药相关;表皮生长因子受体-酪氨酸激酶抑制剂(EGFR-TKI)治疗失败的患者在选择化疗方案时应避免使用铂类药物,可选择给予多西紫杉醇或培美曲塞治疗.     

Enhanced cytotoxicity induced by gefitinib and specific inhibitors of the Ras or phosphatidyl inositol-3 kinase pathways in non-small cell lung cancer cells

In this study, we have characterized a panel of NSCLC cell lines with differential sensitivity to gefitinib for activating mutations in egfr, pik3ca, and k-ras, and basal protein expression levels of PTEN. The egfr mutant NSCLC cell line H1650 as well as the egfr wild type cell lines H292 and A431 were highly sensitive to gefitinib treatment, indicating that other factors determine gefitinib-sensitivity in egfr wild type cells. Activating k-ras mutations were specifically detected in gefitinib-resistant cells, suggesting that the occurrence of k-ras mutations is correlated with resistance to EGFR antagonists. No pik3ca mutations were detected within the panel of cell lines, and PTEN protein expression levels did not correlate with gefitinib sensitivity. Gefitinib effectively blocked Akt and Erk phosphorylation in two gefitinib-sensitive NSCLC cell lines, further supporting our previous findings that persistent activity of the PI3K/Akt and/or Ras/Erk pathways is associated with gefitinib-resistance of NSCLC cell lines. Gefitinib-resistant NSCLC cell lines, showing EGFR-independent activity of the PI3K/Akt or Ras/Erk pathways, were treated with gefitinib in combination with specific inhibitors of mTOR, P13K, Ras, and MEK. Additive cytotoxicity was observed in A549 cells co-treated with gefitinib and the MEK inhibitor U0126 or the farnesyl transferase inhibitor SCH66336 and in H460 cells treated with gefitinib and the PI3K inhibitor LY294002, but not in H460 cells treated with gefitinib and rapamycin. These data suggest that combination treatment of NSCLC cells with gefitinib and specific inhibitors of the PI3K/Akt and Ras/Erk pathways may provide a successful strategy.     

Cytotoxicity of a novel indoloquinone eo9 in hypoxic non-small-cell lung-cancer cell-lines

3-Hydroxymethyl-5-aziridinyl-1-methyl-[1H-indole-4,7-dione]-prop-beta-en -alpha-ol (EO9) is a bioreductive anticancer agent active for non-small cell lung cancer (NSCLC) and structurally related to mitomycin C (MMC). DT-diaphorase (DTD) is regarded as a two electron reductase that plays an important role in the biotransformation of MMC to antitumor metabolites. To evaluate the role of DTD as a bioactivator of EO9 in NSCLC cell lines under oxic and hypoxic conditions, we examined the inhibitory effect of dicumarol which was regarded as a selective inhibitor of DTD on the sensitivity to EO9 in vitro. In this study, we used an MMC-resistant NSCLC cell line (PC-9/MC4) which was established from a PC-9 cell line as a parent cell line by continuous exposure to MMC in our laboratory. We reported previously that the subline PC-9/MC4 was 6.7-fold more resistant to MMC than PC-9 with decreased DTD activity. The IC50 value of PC-9 against EO9 was significantly increased by co-incubation with dicumarol under oxic conditions. EO9 was more cytotoxic against PC-9/MC4 than against PC-9 cells and the enhancement was impaired by tempol under hypoxic conditions. These findings suggest a suppressive role of DTD against one-electron reduction pathway in the bioactivation of EO9 under hypoxic conditions and EO9 may be more active against oxygen-deficient solid tumors especially in MMC-resistant NSCLC cells with low levels of DTD activity.     

A small molecule pan-Bcl-2 family inhibitor, GX15-070, induces apoptosis and enhances cisplatin-induced apoptosis in non-small cell lung cancer cells

Purpose Overexpression of Bcl-2 family members as well as deregulated apoptosis pathways are known hallmarks of lung cancer. Non-small cell lung cancer (NSCLC) cells are typically resistant to cytotoxic chemotherapy and approaches that alter the balance between pro-survival and pro-death Bcl-2 family members have shown promise in preclinical models of NSCLC. Methods Here we evaluated the effects of a novel pan-Bcl-2 inhibitor GX15-070 on NSCLC survival and when combined with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors as well as traditional cytotoxic agents. GX15-070 is a small molecule agent that binds anti-apoptotic Bcl-2 proteins and interferes with their ability to interact with pro-apoptotic proteins. We evaluated the effect of GX15-070 and correlated the effect on EGFR status as well as Bcl-2 family protein expression. Results We show that GX15-070 can disrupt Mcl-1:Bak interactions in lung cancer cells. We identified differential sensitivity of a panel of lung cancer cells to GX15-070 and no clear relationship existed between EGFR status or Bcl-2 family protein expression and sensitivity to GX15-070. GX15-070 could induce apoptosis in a subset of lung cancer cell lines and this correlated with the effects on cell viability. GX15-070 combined with gefitinib was synergistic in a cell line dependent on EGFR for survival but GX15-070 could not reverse resistance to gefitinib in cell lines not dependent on EGFR for survival. Finally, we observed synergy between GX15-070 and cisplatin in NSCLC cells. Conclusions Based on these results, GX15-070 can trigger apoptosis in NSCLC cells and can enhance chemotherapy-induced death. These data suggest that clinical trials with GX15-070 in combination with cytotoxic chemotherapy are indicated.