PC14PE6 Cells Research Articles

Abstract 4599: PD-1/PD-L1 axis in cancer cells contributes to cellular EMT in lung adenocarcinoma

Abstract Background: Since intrinsic PD-1 receptor functions promote tumor growth was reported, we will investigate the role of PD-L1 in lung adenocarcinoma cells, and their impact on clinical outcome. Materials and methods: Lung adenocarcinoma CL1-5 cells, derived from CL1-0 cells have more invasive ability. We prepared PDL1-overexpression human lung adenocarcinoma cell line, derived from CL1-0 cells (CL1-0-PD1) and from PC14PE6 cells (PC14PE6-PD-L1). We observed the cellular morphology, and invasiveness; epithelial-mesenchymal transition (EMT) markers and regulators were also evaluated. To explore interaction between PD-1 and PD-L1, we treated the CL1-0, CL1-5, CL1-0-PDL1 cells, PC14PE6 cells, and PC14PE6-PD-L1 cells with anti-PD-1 antibody, and then evaluated cellular invasion. We also suppressed PD-1 to test whether PD-1/PDL-1 interaction contributed to the EMT change. Further, we evaluated cellular proliferation and chemosensitivity by MTT assay and colony formation assay. Finally, we correlated PD-L1 expression with clinical outcome in patients’ tumor specimen. Results: CL1-5 cells possessed higher PD-L1 expression than parental CL1-0 cells. CL1-0 cells with PD-L1 overexpression had more expression of EMT regulators and mesenchymal markers. Overexpression of PD-L1 in another lung adenocarcinoma PC14PE cells (PC14PE6-PD-L1 cells) become more aggressive. On the contrary, down-regulation of PD-L1 in CL1-5 cells and PC14-PE6-PD-L1 cells became indolent. Besides through the observation from confocal microscopy, PD-L1 expression was more co-localized with vimentin and slug, individually in CL1-5, CL1-0-PD-L1 and PC14PE6-PD-L1 cells. Therefore, PD-L1 promoted EMT in human lung adenocarcinoma cells. After adding anti-PD-1 antibody in CL1-5, CL1-0, and CL1-0-PDL1 cells, migration and invasion ability were decreased. Similar phenomenon was found when PD-1 antibody treatment in PC14PE6 cells, and PC14PE6-PD-L1 cells. Silencing PD-1 by siRNA also disrupt the cellular aggressivenss in CL1-0-PD-L1 and PC14PE6-PD-L1 cells. These results indicated PD-1/PD-L1 axis regulated cancer cells migration and invasiveness. PD-L1 expression also decreased cellular proliferation and had little influence on chemsensitivity. Finally, we found that higher PD-L1 expression was correlated with lymph node metastasis in clinical specimen. Conclusion: Lung adenocarcinoma cells with higher PD-L1 expression promote EMT via PD-1 receptor. PD-L1 expression lowers proliferation rate, but has little impact on chemosensitivity. Lung cancer patients with high PD-L1 expression in tumor have higher lymph node metastasis. Citation Format: Wen-Pin Su, Li-Chan Chang, Jing-Jou Yan, Wu-Chou Su. PD-1/PD-L1 axis in cancer cells contributes to cellular EMT in lung adenocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4599.

Ubiquitin-specific protease 4 controls metastatic potential through β-catenin stabilization in brain metastatic lung adenocarcinoma

Brain metastasis is the most common type of intracranial cancer and is the main cause of cancer-associated mortality. Brain metastasis mainly originates from lung cancer. Using a previously established in vitro brain metastatic model, we found that brain metastatic PC14PE6/LvBr4 cells exhibited higher expression of β-catenin and increased migratory activity than parental PC14PE6 cells. Knockdown of β-catenin dramatically suppressed the motility and invasiveness of PC14PE6/LvBr4 cells, indicating β-catenin is involved in controlling metastatic potential. Since β-catenin protein was increased without a significant change in its mRNA levels, the mechanism underlying increased β-catenin stability was investigated. We found that ubiquitin-specific protease 4 (USP4), recently identified as a β-catenin-specific deubiquitinylating enzyme, was highly expressed in PC14PE6/LvBr4 cells and involved in the increased stability of β-catenin protein. Similar to β-catenin knockdown, USP4-silenced PC14PE6/LvBr4 cells showed decreased migratory and invasive abilities. Moreover, knockdown of both USP4 and β-catenin inhibited clonogenicity and induced mesenchymal-epithelial transition by downregulating ZEB1 in PC14PE6/LvBr4 cells. Using bioluminescence imaging, we found that knockdown of USP4 suppressed brain metastasis in vivo and significantly increased overall survival and brain metastasis-free survival. Taken together, our results indicate that USP4 is a promising therapeutic target for brain metastasis in patients with lung adenocarcinoma.

LPP inhibits collective cell migration during lung cancer dissemination.

Lipoma preferred partner (LPP) is a LIM domain protein, which has multiple functions as an actin-binding protein and a transcriptional coactivator, and it has been suggested that LPP has some roles in cell migration or invasion, however, its role in cancer cells remains to be elucidated. Here, we showed that LPP degraded N-cadherin in lung cancer, PC14PE6 cells via regulating the expression of matrix metalloproteinase 15 (MMP-15), and loss-of-LPP increases collective cell migration (CCM) and dissemination consequently. Knockdown of LPP and its functional partner, Etv5, markedly restores the full-length N-cadherin and increases cell-cell adhesion. We investigated the common target of LPP and Etv5, and found that MMP-15 is transcribed as their direct transcriptional target. Furthermore, MMP-15 could directly digest the N-cadherin extracellular domain. LPP knockdown in PC14PE6 cells increases N-cadherin-dependent CCM in the three-dimensional collagen gel invasion assays, and promoted the dissemination of cancer cells when they were orthotopically implanted in nude mice. Immunohistochemistry of lung adenocarcinoma specimens revealed the heterogeneity of LPP intensity and complementary expression of LPP and N-cadherin in the primary tumors. These findings suggest that loss-of-LPP, Etv5 or MMP-15 can be a prognostic marker of increasing malignancy.

Overexpression of microRNA-95-3p suppresses brain metastasis of lung adenocarcinoma through downregulation of cyclin D1.

Despite great efforts to improve survival rates, the prognosis of lung cancer patients is still very poor, mainly due to high invasiveness. We developed brain metastatic PC14PE6/LvBr4 cells through intracardiac injection of lung adenocarcinoma PC14PE6 cells. Western blot and RT-qPCR analyses revealed that PC14PE6/LvBr4 cells had mesenchymal characteristics and higher invasiveness than PC14PE6 cells. We found that cyclin D1 was upregulated, miR-95-3p was inversely downregulated, and pri-miR-95 and its host gene, ABLIM2, were consistently decreased in PC14PE6/LvBr4 cells. MiR-95-3p suppressed cyclin D1 expression through direct binding to the 3' UTR of cyclin D1 mRNA and suppressed invasiveness, proliferation, and clonogenicity of PC14PE6/LvBr4 cells. Ectopic cyclin D1 reversed miR-95-3p-mediated inhibition of invasiveness and clonogenicity, demonstrating cyclin D1 downregulation is involved in function of miR-95-3p. Using bioluminescence imaging, we found that miR-95-3p suppressed orthotopic tumorigenicity and brain metastasis in vivo and increased overall survival and brain metastasis-free survival. Consistent with in vitro metastatic cells, the levels of miR-95-3p, pri-miR-95, and ABLIM2 mRNA were decreased in brain metastatic tissues compared with lung cancer tissues and higher cyclin D1 expression was involved in poor prognosis. Taken together, our results demonstrate that miR-95-3p is a potential therapeutic target for brain metastasis of lung adenocarcinoma cells.

TopBP1 and Claspin contribute to the radioresistance of lung cancer brain metastases.

BackgroundRadiation therapy is one of the most effective therapeutic tools for brain metastasis. However, it is inevitable that some cancer cells become resistant to radiation. This study is focused on the identification of genes associated with radioresistance in metastatic brain tumor from lung cancer and the functional examination of the selected genes with regards to altered sensitivity of cancer cells to radiation.MethodsAfter establishing radioresistant cells from the xenograft model, we explored the significant transcriptional changes by performing DNA microarray profiling. Functional analyses in vitro and in vivo performed to validate the gene responsible for radioresistance.ResultsTranscriptional changes induced by radiation therapy are much more extensive in H460 cells than in PC14PE6 cells. The expression levels of TopBP1 and Claspin were increased in the cancer cells that survived radiation therapy. Depletion of TopBP1 or Claspin using shRNA showed an enhancement of sensitivity to radiation in radioresistant lung cancer cells (PC14PE6). Moreover, increased levels of TopBP1 or Claspin endowed cells a higher resistance to radiation. In xenograft models, the knock-down of TopBP1 or Claspin significantly prolonged the median survival time post radiation therapy.ConclusionsWe analyzed the gene expression profiles of the radiosensitive cells and the radioresistant cells to define a set of genes that may be involved in endowing lung cancer cells radioresistance post brain metastasis. Functional analyses indicated that the expression TopBP1 and Claspin positively affects the survival of cancer cells and thus negatively the xenograft metastasis model animals in response to radiation. These results show that TopBP1 and Claspin can be potential targets for the enhanced efficacy of radiotherapy.Electronic supplementary materialThe online version of this article (doi:10.1186/1476-4598-13-211) contains supplementary material, which is available to authorized users.

Alpha-Smooth Muscle Actin (ACTA2) Is Required for Metastatic Potential of Human Lung Adenocarcinoma

Metastatic relapse of primary lung cancer leads to therapeutic resistance and unfavorable clinical prognosis; therefore, identification of key molecules associated with metastatic conversion has significant clinical implications. We previously identified a link between early brain metastasis of lung adenocarcinoma and amplification of the α-smooth muscle actin (ACTA2) gene. The aim of present study was to investigate the prognostic and functional significance of ACTA2 expression in cancer cells for the metastatic potential of lung adenocarcinomas. ACTA2 expression was analyzed in tumor cells from 263 patients with primary lung adenocarcinomas by immunohistochemistry, and was correlated with clinicopathologic parameters. The expression of ACTA2 in human lung adenocarcinoma cells was modulated with short hairpin RNAs (shRNA) and siRNAs specifically targeting ACTA2. The patients with lung adenocarcinomas with high ACTA2 expression in tumor cells showed significantly enhanced distant metastasis and unfavorable prognosis. ACTA2 downregulation remarkably impaired in vitro migration, invasion, clonogenicity, and transendothelial penetration of lung adenocarcinoma cells without affecting proliferation. Consistent with the in vitro results, depletion of ACTA2 in human lung adenocarcinoma PC14PE6 cells significantly reduced their metastatic potential without altering their tumorigenic potential. Expression of c-MET and FAK in lung adenocarcinoma cells was also reduced by ACTA2-targeting siRNAs and shRNAs, and was accompanied by a loss of mesenchymal characteristics. These findings indicate that ACTA2 regulates c-MET and FAK expression in lung adenocarcinoma cells, which positively and selectively influence metastatic potential. Therefore, ACTA2 could be a promising prognostic biomarker and/or therapeutic target for metastatic lung adenocarcinoma.

Effects of Src on proliferation and invasion of lung cancer cells

背景与目的Src蛋白在肿瘤的发生、发展和转移中具有重要作用。本研究旨在探讨Src蛋白在肺癌细胞中的表达活化情况及其对肺癌细胞增殖浸润的影响。方法采用Western blot和免疫沉淀法检测Src蛋白在肺癌细胞株中的表达和磷酸化情况；MT法检测抑制Src酪氨酸激酶活化对肺癌细胞体外增殖的影响；Boyden chamber法检测抑制Src酪氨酸激酶活化对肺癌细胞体外侵袭浸润的影响。结果本实验选用的肺癌细胞都存在pp60src的表达，非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞株Src蛋白的自主磷酸化水平（SrcpY418）明显增高，而人小细胞肺癌细胞株SBC5中几乎检测不到SrcpY418。抑制Src酪氨酸激酶活化对肺癌细胞体外增殖呈现出不同的反应，亚微摩尔Src蛋白酪氨酸激酶抑制剂对PC-9和A549细胞体外增殖表现出剂量依赖性抑制作用（P < 0.05）；而≤1 μM Src酪氨酸激酶抑制剂对H226、PC14PE6和RERFLCOK细胞增殖没有明显的抑制作用。Src酪氨酸激酶抑制剂对肺癌细胞体外侵袭浸润呈现明显的剂量依赖性抑制作用（P < 0.05）。结论Src蛋白的活化，而不是过度表达，在NSCLC细胞体外增殖和浸润中发挥着重要作用。

Inhibition of checkpoint kinase 1 sensitizes lung cancer brain metastases to radiotherapy

The most important therapeutic tool in brain metastasis is radiation therapy. However, resistance to radiation is a possible cause of recurrence or treatment failure. Recently, signal pathways about DNA damage checkpoints after irradiation have been noticed. We investigated the radiosensitivity can be enhanced with treatment of Chk1 inhibitor, AZD7762 in lung cancer cell lines and xenograft models of lung cancer brain metastasis. Clonogenic survival assays showed enhancement of radiosensitivity with AZD7762 after irradiation of various doses. AZD7762 increased ATR/ATM-mediated Chk1 phosphorylation and stabilized Cdc25A, suppressed cyclin A expression in lung cancer cell lines. In xenograft models of lung cancer (PC14PE6) brain metastasis, AZD7762 significantly prolonged the median survival time in response to radiation. Depletion of Chk1 using shRNA also showed an enhancement of sensitivity to radiation in PC14PE6 cells. The results of this study support that Chk1 can be a good target for enhancement of radiosensitivity.

Effect of Src tyrosine kinase inhibition on secretion of MMP-2 and MMP-9 by non-small cell lung cancer cells

背景与目的Src酪氨酸激酶和基质金属蛋白酶在肺癌的浸润和转移中发挥重要作用。本研究旨在探讨抑制Src酪氨酸激酶对非小细胞肺癌（non-small cell lung cancer, NSCLC）细胞分泌基质金属蛋白酶-2（matrix metalloproteinase 2, MMP-2）和基质金属蛋白酶-9（matrix metalloproteinase 9, MMP-9）以及NSCLC细胞侵袭浸润的影响。方法采用ELISA法检测NSCLC细胞（PC14PE6、H226、PC-9、A549）培养上清中MMP-2和MMP-9含量以及抑制Src酪氨酸激酶对NSCLC细胞分泌MMP-2和MMP-9的影响；Boyden chamber法检测抑制Src酪氨酸激酶对NSCLC细胞体外侵袭浸润的影响。结果NSCLC细胞中PC14PE6和H226中MMP-2和MMP-9的水平较高，A549细胞中MMP-9的水平较低，而MMP-2和MMP-9在PC-9细胞中检测不到。Src酪氨酸激酶抑制剂对PC14PE6中的MMP-2水平以及PC14PE6、H226和A549细胞中的MMP-9水平呈剂量依赖性抑制关系。10 µM Src酪氨酸激酶抑制剂使PC14PE6细胞中的MMP-2水平、H226细胞和A549细胞中的MMP-9水平降低50%以上。10 µM Src酪氨酸激酶抑制剂对H226细胞中的MMP-2无明显抑制作用。Src酪氨酸激酶抑制剂对4种NSCLC细胞体外侵袭浸润的抑制程度略有差异，但均呈现明显的剂量依赖性抑制作用。3 µM Src酪氨酸激酶抑制剂对PC14PE6、H226、A549和PC-9细胞体外侵袭浸润的抑制率分别为79.1%、68.09%、90.96%和96.98%（P < 0.001）。结论通过抑制NSCLC细胞分泌MMP-2和MMP-9，抑制Src酪氨酸激酶可降低细胞的体外侵袭浸润能力。

Effect of Yeast-derived β-glucan in Conjunction With Bevacizumab for the Treatment of Human Lung Adenocarcinoma in Subcutaneous and Orthotopic Xenograft Models

Human lung cancer is the leading cause of cancer death worldwide. Bevacizumab, a monoclonal antibody against vascular endothelial growth factor (VEGF), in combination with chemotherapy showed significant therapeutic efficacy in human lung cancer patients. However, increased adverse effects limit its clinical utilization. Previous studies demonstrated that polysaccharide beta-glucan significantly augments antitumor monoclonal antibody-mediated efficacy via stimulation of the innate effector neutrophil complement receptor 3. Here, we explored combined beta-glucan with bevacizumab therapy for human lung cancer using murine xenograft models. To that end, human lung adenocarcinomas were screened for membrane-bound VEGF expression. Both subcutaneous and orthotopic lung cancer xenograft models were used to evaluate the combination therapy. We found that PC14PE6 adenocarcinoma cells express membrane-bound VEGF both in vitro and in vivo. Bevacizumab bound to surface VEGF on PC14PE6 cells and activated complement. In the subcutaneous PC14PE6 tumor model, beta-glucan plus bevacizumab showed augmented efficacy in terms of tumor progression and long-term survival compared with bevacizumab-treated alone. These effects were accompanied with massive complement deposition and neutrophil infiltration within tumors. However, this effect was not observed in surface-bound VEGF-negative human lung tumors. Therapeutic efficacy of beta-glucan with bevacizumab was further demonstrated in an orthotopic lung cancer model. Thus, our data suggest that beta-glucan enhances bevacizumab-mediated efficacy and may provide therapeutic benefits for lung cancers with membrane-bound VEGF expression.

Modest Anti-Cancer Activity of a Bile Acid Acylated Heparin Derivative in a PC14PE6 Induced Orthotopic Lung Cancer Model

A novel chemically modified heparin derivative, heparin-deoxycholic acid nano-particles, has lower anticoagulant activity, and was recently reported to have significant anti-tumor effects on squamous head and neck cancer cells. Therefore, the aim of this study was to evaluate the anti-tumor effects of heparin-deoxycholic acid nano-particles in a human lung adenocarcinoma cell line. An orthotopic lung cancer model in 16 mice was developed using intra-thoracic injections of 0.5x10(6) PC14PE6 cells. Ten days after inoculation, the mice were divided into two groups. PBS and Heparin-DOCA particles were injected once a day every 3 days in the tail vein, for a total of 5 injections. The body weight and survival of each mouse were monitored and the tumor size in the lung was measured by SPECT-CT before and after heparin-DOCA nano-particle treatment. IThe HD particles had no significant cytotoxicity when the PC9 cells were treated in vitro. There was no statistical difference in tumor size, body weight and survival between the HD treated and control groups in vivo. Furthermore, there was no difference in the amount of CD31 between tumor tissues in the two study groups. HD synthesized with unfractionated heparin had no apparent inhibitory effects on tumor growth in a PC14PE6 cell induced orthotopic lung cancer mouse model. The HD particles did not significantly inhibit tumor-induced angiogenesis at the tumor sites.

Expression of epidermal growth factor (EGF)/transforming growth factor-α by human lung cancer cells determines their response to EGF receptor tyrosine kinase inhibition in the lungs of mice

Epidermal growth factor receptor (EGFR) has been extensively targeted in the treatment of non-small cell lung cancer, producing responses in a small number of patients. To study the role of ligand expression in mediating response to EGFR antagonism, we injected NCI-H441 [EGFR and EGF/transforming growth factor-alpha (TGF-alpha) positive] or PC14-PE6 (EGFR positive and EGF/TGF-alpha negative) human lung adenocarcinoma cells into the lungs of nude mice. We randomized the mice to receive treatment with the EGFR tyrosine kinase inhibitors gefitinib or AEE788 or vehicle. Treatment of mice bearing NCI-H441 but not PC14-PE6 lung tumors resulted in a significant reduction in primary tumor growth, pleural effusion, and lymph node metastasis. Immunohistochemical analyses revealed that NCI-H441 and PC14-PE6 cells expressed EGFR but that the expression of EGF/TGF-alpha was high in NCI-H441 cells and very low in PC14-PE6 cells. Consequently, EGFR was activated in both tumor and tumor-associated endothelial cells in the NCI-H441 tumors but not in the PC14-PE6 tumors. Antagonism of EGFR signaling by treatment of mice with AEE788 decreased proliferation and increased apoptosis of both tumor cells and tumor-associated endothelial cells in NCI-H441 tumors but not in PC14-PE6 tumors. However, after transfection of PC14-PE6 cells with TGF-alpha, lung tumors derived from the transfected cells expressed and activated EGFR in both tumor and tumor-associated endothelial cells and tumors responded to treatment with AEE788. Collectively, these results strongly suggest that the response of human lung cancers growing orthotopically in mice to the inhibition of EGFR signaling is determined by ligand (EGF/TGF-alpha) expression by tumor cells. Our findings provide an additional explanation for the susceptibility of lung cancers to treatment with EGFR tyrosine kinase inhibitors.

Hypoxia inducible factor‐1 influences sensitivity to paclitaxel of human lung cancer cell lines under normoxic conditions

Paclitaxel (PTX) is an anticancer drug that is effective against a wide range of solid tumors. The effect of PTX on two human lung cancer cell lines, PC14PE6 and NCI-H441 cells, was examined in an orthotopically transplanted animal model with an in vivo imaging devise. Although PTX effectively suppressed tumor growth and improved survival rate in NCI-H441, it did not influence these in PC14PE6. In vitro experiments confirmed that PC14PE6 cells are resistant to PTX under normoxic conditions and that both cell lines were resistant to PTX under hypoxic conditions. It was found that the expression level of endogenous hypoxia inducible factor (HIF)-1alpha in PC14PE6 is much higher than that in NCI-H441 cells under normoxic conditions. Furthermore, sensitivity to PTX in these cell lines was reversed when HIF-1alpha expression was decreased by siRNA specific to HIF-1alpha in PC14PE6 and increased by overexpression of the exogenous HIF-1alpha gene in NCI-H441. These results suggest that HIF-1 influences the PTX sensitivity of these cells. The authors further examined beta-tubulin, a target molecule of PTX, with western blotting and immunohistochemical analysis in these cells. The expression level of beta-tubulin was comparable in these cells under both normoxic and hypoxic conditions while the distribution of beta-tubulin and cell morphology were changed according to HIF-1alpha expression levels, suggesting that HIF-1 influences the conformation and dynamics of microtubules. These data support the potential development of HIF-1 targeted approaches in combination with PTX, where drug resistance tends to contribute to treatment failure.

ZD6474, an Inhibitor of Vascular Endothelial Growth Factor Receptor Tyrosine Kinase, Inhibits Growth of Experimental Lung Metastasis and Production of Malignant Pleural Effusions in a Non-Small Cell Lung Cancer Model

ZD6474 is a novel, orally active inhibitor of vascular endothelial growth factor receptor-2 (VEGFR-2) tyrosine kinase, with some additional activity against epidermal growth factor receptor (EGFR) tyrosine kinase. The purpose of this study was to determine the potential of ZD6474 in the control of established experimental lung metastasis and pleural effusions produced by human non-small cell lung cancer (NSCLC) cells. PC14PE6 (adenocarcinoma) and H226 (squamous cell carcinoma) cells express high levels of EGFR and only PC14PE6 cells overexpress VEGF. Neither ZD6474 nor the EGFR tyrosine kinase inhibitor gefitinib inhibit proliferation of PC14PE6 or H226 cells in vitro. Both PC14PE6 and H226 cells inoculated intravenously into nude mice induced multiple lung nodules after 5-7 weeks. In addition, PC14PE6 cells produced bloody pleural effusions. Daily oral treatment with ZD6474 did not reduce the number of lung nodules produced by PC14PE6 or H226 cells, but did reduce the lung weight and the size of lung nodules. ZD6474 also inhibited the production of pleural effusions by PC14PE6 cells. Histological analyses of lung lesions revealed that ZD6474 treatment inhibited activation of VEGFR-2 and reduced tumor vascularization and tumor cell proliferation. Therapeutic effects of ZD6474 were considered likely to be due to inhibition of VEGFR-2 tyrosine kinase because gefitinib was inactive in this model. These results indicate that ZD6474, an inhibitor of VEGFR-2, may be useful in controlling the growth of established lung metastasis and pleural effusions by NSCLC.

Mouse Orthotopic Lung Cancer Model Induced by PC14PE6

This study was undertaken to investigate in detail the xenograft mouse orthotopic lung cancer model induced by PC14PE6 adenocarcinoma cells. Three cell doses (0.5x10(6); 1x10(6); 2x10(6)) of PC14PE6 cells were injected into the lungs of male BALB/c nude mice by the intrathoracic injection method. The lung and other organs, including brain, liver, spleen, kidney, muscle, adrenal gland, and lymph node on knee, were removed and stained with H/E to detect the presence of tumor cells. The reliable tumorigenicity time in the PC14PE6 adenocarcinoma cell-inoculated BALB/c nude mouse was 10 days after intrathoracic injection. The average life span of the three groups after inoculation was 14 days in the 2x10(6) cells inoculum group; 25 days in the 1x10(6) cells inoculum group; and 32 days in the 0.5x10(6) cells inoculum group. The PC14PE6 adenocarcinoma cells induced orthotopic lung cancer limited within the thorax. This orthotopic lung cancer model is an efficient cancer model with easy inoculation methods, rapid and high tumorigenicity, and simple monitoring methods for metastasis.

Src Tyrosine Kinase Inhibitor, M475271, Suppresses Subcutaneous Growth and Production of Lung Metastasis Via Inhibition of Proliferation, Invasion, and Vascularization of Human Lung Adenocarcinoma Cells

Src, a proto-oncogene, has been strongly implicated in the growth, progression and metastasis of a number of human cancers. Its role in lung cancer is, however, still unknown. In the present study, we assessed the expression of Src in three different human lung adenocarcinoma cell lines (PC-9, PC14PE6, A549), and explored the effect of a novel Src kinase inhibitor, M475271, on the behavior of the cell lines. The three cell lines expressed various levels of auto-phosphorylated Src. While M475271 reduced Src-phosphorylation and invasiveness of all three cell lines, it inhibited the proliferation of PC-9 and A549 cells with highly phosphorylated Src, but not PC14PE6 cells. We further examined the effect of M475271 on subcutaneous tumors and lung metastasis caused by PC-9 and/or A549 cells in NK-cell depleted SCID mice. Daily oral treatment with M475271 inhibited the growth of subcutaneous tumors with PC-9 and A549 cells via inhibition of tumor cells proliferation, VEGF production and/or vascularization in the mice in a dose-dependent manner. In the metastasis model with A549 cells, the lung weight in the M475271 (50 mg/kg)-treated group was less than that of the control group, despite no difference in the number of metastatic nodules. Our results suggest that inhibition of tyrosine kinase Src by M475271 could reduce the growth, invasion and VEGF-mediated neovascularization of lung adenocarcinoma cells, resulting in inhibition of growth of subcutaneous tumors and lung metastasis. Therefore, a novel Src tyrosine kinase inhibitor, M475271, might be helpful for controlling the progression of human lung adenocarcinoma.

Sensitization of Tumor-Associated Endothelial Cell Apoptosis by the Novel Vascular-Targeting Agent ZD6126 in Combination with Cisplatin

ZD6126 is a novel vascular-targeting agent that selectively disrupts the tubulin cytoskeleton of endothelial cells. In the immature vessels characteristic of tumor vasculature, this leads to endothelial cell contraction, blood vessel congestion, and, consequently, tumor cell death. ZD6126 has been shown to delay tumor growth in a range of xenograft models. The antitumor effect of ZD6126 can be increased in combination with cisplatin or radiation therapy, although the precise mechanism of this enhancement has not been demonstrated. ZD6126 treatment has also been shown to inhibit lung metastasis, and the present study has explored the potential to increase the antimetastatic effect of ZD6126 by combining with cisplatin, and the underlining mechanism has been investigated. Human lung adenocarcinoma PC14PE6 cells were injected into the tail vein of nude mice. Five weeks after injection animals were treated with ZD6126 (200 mg/kg i.p.), cisplatin (6 mg/kg i.v.), or a combination of the two agents. The animals were sacrificed 24 hours later, and the extent of lung metastases and the presence of apoptotic cells were assessed. Histologic analysis revealed that the ZD6126/cisplatin combination resulted in a 2 to 4-fold increase in the total number of tumor-associated apoptotic cells compared with either treatment alone. ZD6126 alone induced apoptosis of tumor-associated endothelial cells in tumors, and the extent of apoptosis was increased 2-fold in combination with cisplatin. The lung weight was significantly reduced, and the number of metastatic nodules significantly was lower in the combined treatment group than in the control group. These data suggest that the antimetastatic effect of the vascular-targeting agent ZD6126 can be increased by use in combination with cisplatin, which increases the incidence of endothelial cell apoptosis.

In this issue

The matrix metalloproteases, or MMPs, are a family of zinc-dependent proteolytic enzymes that degrade the extracellular matrix. As such, they are important during cell migration, angiogenesis and wound healing. However, they are also necessary for the invasion and migration of tumor cells. Therefore, MMP expression is often upregulated in invasive metastatic cancers. MMP-19 lacks several structural features present in other MMP subclasses, notably the Asp, Tyr and Gly residues close to the zinc-binding site, the fibronectin-like domain, the transmembrane domain and the furin-activation sequence. On pages 709–716 of this issue, Impola et al. demonstrate yet another unique feature of MMP-19: unlike some of its family members, it is present in normal, proliferating, but not migrating keratinocytes; it disappears from these cells over the course of neoplastic dedifferentiation. Because MMP-19 is induced by TNF-α, because it degrades many basal membrane components, including type IV collage, laminin-1 and nidogen, and because it is present in normal cells, they hypothesize that MMP-19 plays a role in normal tissue remodeling such as that which occurs during wound repair. Because the intrauterine environment may play a role in the development of adult diseases, Leong et al. endeavor to characterize the relationship between birth weight (as a measure of said intrauterine environment) and adult obesity (as a known risk factor for cancer). They collected information on birth weight, adult height and adult weight from randomly selected women aged 50–79 living in the states of Massachusetts, New Hampshire, and Wisconsin via telephone interviews. In their paper on pages 789–791 of this issue, they report their results that both low and high birth weights are associated with higher adult body mass index, and they conclude that fetal experience may influence adult obesity and therefore contribute to cancer risk. Glutathione-S-transferases (GSTs) catalyze the glutathione-dependent detoxification of several chemotherapeutic agents or their metabolites. Therefore, Sweeney et al. hypothesized that polymorphisms resulting in a reduction or elimination of enzymatic activity may modify the effectiveness of chemotherapy and thus predict differences in treatment outcomes. GSTA1 is highly active in the glutathione conjugation of metabolites of cyclophosphamide (CP), a nitrogen mustard chemotherapeutic drug often used in combination chemotherapy for breast cancer. A newly recognized polymorphism, the GSTA1*B allele, reduces hepatic expression of this enzyme. On pages 810–814 of this issue, they report that GSTA1*B/*B homozygotes receiving combination chemotherapy for breast cancer had a longer overall survival during the first 5 years following their diagnosis, probably because their reduced ability to detoxify CP means that they received a higher effective dose. However, after 5 years there was no evidence of a continued survival difference based on genotype. Similar results were reported for patients with the GST1105Val polymorphism who received combination chemotherapy for advanced colorectal cancer. Overall survival among breast cancer patients treated by chemotherapy, by genotype at the GSTA1 proximal promoter polymorphism. Micrometastases are one of the worst problems facing cancer patients who undergo successful resection of their primary tumors, for even if no metastases are detected at the time of surgery, micrometastases may have sprouted and can become manifest years later. The lungs are one of the most common sites for these micrometastases. MMPs play a pivotal role in metastasis by promoting tumor invasion and angiogenesis; thus, a number of inhibitors (MMPIs) have been developed to counteract them. ONO-4817 is a selective spectrum MMPI that inhibits the gelatinases MMP-2 and MMP-9, but not MMP-1, by binding reversibly to the zinc-binding region of these MMPs. On pages 822–828, Yamamoto et al. test its efficacy in combination with a cytotoxic anticancer drug, docetaxel (DOC), against established lung micrometastases. They injected nude mice with either PC14PE6 cells, human non-small-cell lung adenocarcinoma cells, or H226 cells, human squamous cell carcinoma cells, both of which express MMP-1, MMP-2 and/or MMP-9. The mice were then treated with DOC and ONO-4817 after micrometastases had been established. They found that the combination therapy significantly suppressed the tumor burden and prolonged the survival of the mice. Effect of treatment with ONO-4817 and DOC on the survival of nude mice inoculated with PC14PE6 cells. Expression of the RARβ subtype of the retinoic acid receptor (RARβ) is selectively lost in most cultured cancer cell lines and premalignant and malignant lesions, implying that its loss is associated with human cancer development. Its loss also renders the cells resistant to the growth inhibitory effects of retinoic acid treatment. In their report on pages 833–839 of this issue, Teraishi et al. report that recombinant adenovirus-mediated gene transfer of the G1 phase cell cycle inhibitor p21 into H1299 human non-small-cell lung cancer cells and DLD-1 human colorectal cancer cells transcriptionally upregulates RARβ expression, thereby rendering these cells more sensitive to the apoptosis-promoting effects of treatment with all-trans-retinoic acid. Transcriptional activation of the RARβ promoter by Ad5CMV-p21 infection.

Production of Experimental Malignant Pleural Effusions Is Dependent on Invasion of the Pleura and Expression of Vascular Endothelial Growth Factor/Vascular Permeability Factor by Human Lung Cancer Cells

We determined the molecular mechanisms that regulate the pathogenesis of malignant pleural effusion (PE) associated with advanced stage of human, non-small-cell lung cancer. Intravenous injection of human PC14 and PC14PE6 (adenocarcinoma) or H226 (squamous cell carcinoma) cells into nude mice yielded numerous lung lesions. PC14 and PC14PE6 lung lesions invaded the pleura and produced PE containing a high level of vascular endothelial growth factor (VEGF)-localized vascular hyperpermeability. Lung lesions produced by H226 cells were confined to the lung parenchyma with no PE. The level of expression of VEGF mRNA and protein by the cell lines directly correlated with extent of PE formation. Transfection of PC14PE6 cells with antisense VEGF165 gene did not inhibit invasion into the pleural space but reduced PE formation. H226 cells transfected with either sense VEGF 165 or sense VEGF 121 genes induced localized vascular hyperpermeability and produced PE only after direct implantation into the thoracic cavity. The production of PE was thus associated with the ability of tumor cells to invade the pleura, a property associated with expression of high levels of urokinase-type plasminogen activator and low levels of TIMP-2. Collectively, the data demonstrate that the production of malignant PE requires tumor cells to invade the pleura and express high levels of VEGF/VPF.