吉非替尼联合顺铂对肺癌细胞增殖的协同抑制作用

目的：观察表皮生长因子受体酪氨酸激酶抑制剂吉非替尼（Gefitinib,G）与顺铂（Cisplatin,DDP）联合对肺癌细胞株A549增殖抑制的协同效应。方法：采用四甲基偶氮唑蓝（MTT）法检测不同浓度吉非替尼单药、顺铂单药以及两药联用对肺癌细胞株A549的抑制程度,并应用两药相互作用指数评价药物的联合效应。结果：在药物作用时间足够时,两药联用相对单药组能显著地提高肿瘤细胞的抑制率,并且抑制效率随药物浓度的增加而提高。当作用时间≥48h时,联用组A值相对于单药组明显降低（P〈0．05）。联用对肿瘤细胞增长的抑制作用在48h时较24h明显增高（P〈0．05）,并随着时间的延长呈现递增趋势,至72h后逐渐进入平台期（P〉0．05）。两药协同效应在作用24h时表现不明显,在48h后才开始出现（CDI〈1）。结论：在药物作用时间足够时,吉非替尼与顺铂联用对肺癌细胞的增殖具有协同抑制效应,可为临床应用提供参考。     

阻断表皮生长因子受体和环氧合酶-2信号途径对肺腺癌细胞的抗增殖作用

目的:探讨表皮生长因子受体抑制剂吉非替尼(gefitinib)联合新型环氧合酶2（cyclooxygenase2, COX2）抑制剂塞来昔布(celecoxib)对肺腺癌A549细胞株的抑制作用及其可能机制。方法:肺腺癌A549细胞培养于RPMI 1640 培养液中,实验分为正常对照组、吉非替尼5 μmol/L组、塞来昔布25 μmol/L组、吉非替尼5 μmol/L加塞来昔布25 μmol/L组。药物干预细胞48 h后,倒置相差显微镜观察细胞形态学变化,锥虫蓝拒染法检测药物对细胞生长的影响,Annexin V/PI法和Hoechst33258染色法检测细胞凋亡,流式细胞术检测药物作用周期,免疫荧光和Realtime PCR法检测EGFR、COX2蛋白的表达及EGFR mRNA的表达情况。结果:吉非替尼联合塞来昔布组相比单药组,A549 细胞明显出现大量颗粒和空泡,细胞变圆并开始脱落。吉非替尼与塞来昔布单药对A549细胞抑制作用具有时间和剂量依赖性,加药48 h时吉非替尼（5 μmol/L）联合塞来昔布（25 μmol/L）组抑制率为(58.2±4.6)%,明显高于单药组（P<0.01）。联合用药组A549细胞凋亡率明显高于单独用药组（33.9% vs 6.0%,8.8%）,其S期细胞明显减少、G0/G1期明显增加（P<0.01）;EGFR、COX2蛋白的表达明显减弱（P<005）,EGFR mRNA相对表达量（028±0.05）明显高于单独用药组（P<0.05）。结论:吉非替尼和塞来昔布联合对肺腺癌A549 细胞增殖的抑制具有明显协同作用,其可能机制是诱导凋亡、增强G0/G1期阻滞和进步下调活化的EGFR与COX2的表达。     

Epidermal growth factor receptor dimerization status determines skin toxicity to HER-kinase targeted therapies

Skin toxicity, a common drug-related adverse event observed in cancer patients treated with epidermal growth factor receptor (EGFR)-directed therapies is rarely seen with therapies targeting HER2. This study reports the significance of the EGFR and HER2 dimerization status in skin with regard to these dermatologic side effects. We demonstrate the differential effect of HER-directed therapies on the ligand driven activation status of EGFR, HER2 and MAPK in normal human epidermal keratinocytes. EGFR-directed therapies, such as gefitinib and cetuximab, inhibited ligand-induced activation of EGFR and MAPK in human keratinocytes. Pertuzumab, an antibody interfering with functional HER2 heterodimerization, failed to block ligand-induced HER signaling in primary keratinocytes. Using a novel proximity-based dimerization assay (eTagtrade mark) we show that EGFR homodimers are the predominant HER dimer pair in normal primary kertinocytes and in normal skin tissue from 16 patients with solid malignancies. The presence of [p]EGFR and [p]MAPK, but the absence of [p]HER2, demonstrates productive signaling via EGFR but not HER2 in human skin. These data illustrate the importance of the EGFR dimerization partner in human skin and suggests that inhibition of EGFR homodimer signaling rather than EGFR/HER2 heterodimer signaling maybe the key molecular event determining dermatologic toxicity discrepancies observed between EGFR-targeted versus HER2-targeted therapies.     

Preclinical study of a "tailor-made" combination of NK4-expressing gene therapy and gefitinib (ZD1839, Iressa) for disseminated peritoneal scirrhous gastric cancer

We evaluated the effect of a "tailor-made" chemo-gene therapy in scirrhous gastric cancer (SGC)-bearing nude mice. For this tailor-made approach, we first selected gefitinib (epidermal growth factor receptor-tyrosine kinase inhibitor)-sensitive SGC cell lines, and 5/8 cell lines demonstrated various degrees of gefitinib-sensitivity. In the highly gefitinib-sensitive NUGC-4, the biological response to NK4 (HGF antagonist/angiogenesis inhibitor) was examined. Subsequently, the composition of an NK4-expressing ternary complex (cationic lipid/nucleic acid/HMG-1, 2 protein) was optimized for maximum transfection activity in NUGC-4. Finally, mice were peritoneally coinoculated with NUGC-4 and scirrhous-associated gastric fibroblasts, NF22, on day 0. Animal models were orally administrated gefitinib (50 mg/kg/day, on days 7-28), and peritoneally NK4-expressing ternary complex (on days 14, 21 and 28). NK4-expression suppressed the gefitinib-resistance induced by the interaction between fibroblasts and SGC, and eventually, this tailor-made combination synergistically decelerated the disease progression by inhibiting proliferative, angiogenic and antiapoptotic effects in tumor tissues. On day 28, both the hemoglobin concentration (g/dl) (control (n = 8), 11.9; treated (n = 8), 17.3; p = 0.0014) and the numbers of mice in good condition (control, 2; treated, 8; p = 0.0012) were significantly greater, and the abdominal girth (mm) (control, 81.1; treated, 70.3; p = 0.0036) was significantly reduced. The median points of bloody ascite-free survival time (days) (control, 22; treated, 44; p < 0.0001) and time to euthanasia (days) (control, 36.5; treated, 56; p < 0.0001) were also significantly prolonged. This combination is a potentially useful approach to the treatment of peritoneal gefitinib-sensitive SGC dissemination.     

A combination of gefitinib and FOLFOX-4 as first-line treatment in advanced colorectal cancer patients. A GISCAD multicentre phase II study including a biological analysis of EGFR overexpression, amplification and NF-kB activation

Interesting activity has been reported by combining chemotherapy with cetuximab. An alternative approach for blocking EGFR function has been the development of small-molecule inhibitors of tyrosine kinase domain such as gefitinib. We designed a multicentre phase II study in advanced colorectal cancer combining gefitinib+FOLFOX in order to determine the activity and to relate EGFR expression and gene amplification and NF-kB activation to therapeutic results. Patients received FOLFOX-4 regimen plus gefitinib as first-line treatment. Tumour samples were analysed for EGFR protein expression by immunohistochemical analysis and for EGFR gene amplification by fluorescence in situ hybridisation (FISH), chromogenic in situ hybridisation (CISH) and NF-kB activation. Forty-three patients were enrolled into this study; 15 patients experienced a partial response (response rate=34.9%), whereas other 12 (27.9%) had a stable disease. Median progression-free survival (PFS) was 7.8 months and median overall survival (OS) was 13.9 months. We did not find any relationship with EGFR overexpression, gene amplification, while NF-kB activation was associated with a resistance to therapy. Gefitinib does not seem to increase the activity of FOLFOX in advanced colorectal cancer even in patients overexpressing EGFR or with EGFR amplification. Furthermore, while NF-kB activation seems to predict resistance to chemotherapy as demonstrated 'in vitro' models, gefitinib does not overcome this mechanism of resistance, as reported for cetuximab.     

Multicenter phase II trial of gefitinib first-line therapy followed by chemotherapy in advanced non-small-cell lung cancer (NSCLC): SAKK protocol 19/03.

BACKGROUND: Gefitinib is active in patients with pretreated non-small-cell lung cancer (NSCLC). We evaluated the activity and toxicity of gefitinib first-line treatment in advanced NSCLC followed by chemotherapy at disease progression. PATIENTS AND METHODS: In all, 63 patients with chemotherapy-naive stage IIIB/IV NSCLC received gefitinib 250 mg/day. At disease progression, gefitinib was replaced by cisplatin 80 mg/m(2) on day 1 and gemcitabine 1250 mg/m(2) on days 1, 8 for up to six 3-week cycles. Primary end point was the disease stabilization rate (DSR) after 12 weeks of gefitinib. RESULTS: After 12 weeks of gefitinib, the DSR was 24% and the response rate (RR) was 8%. Median time to progression (TtP) was 2.5 months and median overall survival (OS) 11.5 months. Never smokers (n = 9) had a DSR of 56% and a median OS of 20.2 months; patients with epidermal growth factor receptor (EGFR) mutation (n = 4) had a DSR of 75% and the median OS was not reached after the follow-up of 21.6 months. In all, 41 patients received chemotherapy with an overall RR of 34%, DSR of 71% and median TtP of 6.7 months. CONCLUSIONS: First-line gefitinib monotherapy led to a DSR of 24% at 12 weeks in an unselected patients population. Never smokers and patients with EGFR mutations tend to have a better outcome; hence, further trials in selected patients are warranted.     

Gefitinib-induced disseminated intravascular coagulation in a patient with non-small cell lung cancer

In February 2005, Gefitinib （Iressa）, a small-molecular .epidermal growth factor receptor and tyrosine kinaseinhibitor, was approved in China as an anticancer agent for patients with advanced （local or metastatic） non-small cell lung cancer （NSCLC）, who failed prior chemotherapy. The common adverse events of the drug include acne-like skin rash, paronychia, pruritus, diarrhea, nausea/vomiting, anorexia, hepatitis, and hyperbilirubinemia.1 However, these adverse events are generally mild in severity and reversible on cessation of the treatment. Therefore, gefitinib has been regarded as a relatively safe agent,     

Second‐line treatments after first‐line gefitinib therapy in advanced nonsmall cell lung cancer

Gefitinib is effective as first‐line therapy for advanced nonsmall cell lung cancer (NSCLC). However, after failure of gefitinib, it is unknown whether any second‐line regimens could lead to better outcomes. To study the influence of different second‐line antitumor regimens on the outcomes of patients with NSCLC after failure of first‐line gefitinib, we carried out a retrospective study in a tertiary referral medical center to investigate the prognosis of patients with NSCLC receiving second‐line antitumor treatment after gefitinib therapy. Clinical data and epidermal growth factor receptor (EGFR) mutational status of tumors were collected. A total of 195 patients with Stage IIIb or IV NSCLC receiving first‐line gefitinib and at least 1 subsequent line therapy were identified. A second‐line therapy with a platinum‐based combination or taxane‐containing regimen were associated with a higher therapy response, whereas a platinum‐based combination was linked to better overall survival. Ninety‐five patients had tumors with known EGFR mutation status; 61 had EGFR mutations and 34 had wild‐type EGFR. A second‐line therapy with a gemcitabine/platinum combination regimen resulted in better overall survival than erlotinib in patients with EGFR mutations (p = 0.035) but not in patients with wild‐type EGFR (p = 0.785). The study suggested that, after failure of first‐line gefitinib therapy, second‐line platinum‐based combination regimens were associated with a better overall survival than other regimens, including erlotinib. The survival benefit of platinum‐based combination regimens existed in patients with mutant EGFR but not wild‐type EGFR.     

The relationship between tyrosine kinase inhibitor therapy and overall survival in patients with non-small cell lung cancer carrying EGFR mutations

For patients with epidermal growth factor receptor (EGFR) mutation-positive lung cancer, the relationship between the dose or duration of treatment with tyrosine kinase inhibitor (TKI) and overall survival remains unclear. Here, we analyzed clinical data of 39 patients who were diagnosed with EGFR mutation-positive non-small cell lung cancer and treated with TKI, but subsequently died. Several parameters were measured in this study: overall survival; first, second, and overall TKI therapy durations; first TKI intensity (actual dose/normal dose); and TKI rate (overall TKI therapy duration/overall survival). The response rate to TKI therapy was 50% , and the median survival was 553 days. After TKI therapy failed, 38.5% patients were re-challenged with TKI. We observed a moderate relationship [r = 0.534, 95% confidential interval (CI) = 0.263 to 0.727, P 0.001] between overall TKI therapy duration and overall survival. However, we found no relationship between overall survival and first TKI intensity (r = 0.073, 95% CI = -0.380 to 0.247, P = 0.657) or TKI rate (r = 0.0345, 95% CI = -0.284 to 0.346, P = 0.835). Nonsmall cell lung cancer patients with mutation-positive tumors remained on TKI therapy for, on average, 33% of the overall survival time. These findings suggest that patients with EGFR mutation-positive tumors should not stick to using TKIs.     

Downregulation of HOXC6 by miR-27a ameliorates gefitinib resistance in non-small cell lung cancer

Non-small cell lung cancer (NSCLC) is a major type of lung cancer. Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), represented by gefitinib (Gef), are targeted drugs used for the treatment of NSCLC. However, NSCLC patients often develop resistance to tyrosine kinase inhibitors, which limits their efficacy. Homeobox gene HOXC6 is dysregulated in many cancers and contributes to chemoresistance in cancer cells. However, the role and mechanism of HOXC6 in the development of Gef resistance in NSCLC remains unclear. In the present study, we found that HOXC6 was highly expressed in Gef-resistant NSCLC cells. Further experiments showed that silencing of HOXC6 ameliorated Gef resistance in PC9/G cells whereas overexpression of HOXC6 promoted Gef resistance in PC9 cells. HOXC6 influenced Gef sensitivity in NSCLC cells by regulating cell proliferation, colony formation, cell apoptosis, cell cycle, cell mobility and other related signaling molecules or pathways. HOXC6 was also found to be a direct target of miR-27a. As expected, overexpression of miR-27a ameliorated Gef resistance by inhibiting HOXC6 expression in vitro and in vivo. Clinical analysis revealed that high HOXC6 levels and low miR-27a levels were significantly correlated with more malignant clinical features and poorer survival of NSCLC patients. In summary, the present study demonstrates that HOXC6 may be a potential therapeutic target for overcoming Gef resistance in NSCLC patients. A combination of Gef and miR-27a agomirs may be an effective intervention for Gef-resistant NSCLC.