- Email: [email protected]
P-294 Combined inhibition of epidermal growth factor receptor and COX-2 enhances growth inhibition in lung cancer cell lines
S166
Poster Session Z/Molecular
loci, we have identified 5 that show potential for distinguishing between MM, adenocarcinoma and/or non-cancer lung. Our observations support the strong promise of methylation markers as tools for accurate diagnosis of neoplasms in and around the lung.
ElP 292
Establishment of patient-like SCID mice model by orthotopically implanting lung cancer cell lines
Haruhiko Fuiino’, Kazuya Kondo’, Takanori Miyoshi’, Hisashi Ishikura’, Yuji Takahashi’, Naruhiko Sawada’, Yukiko Hirose’, Hiromitu Takizawa’, Syoji Sakiyama’ , Yasumasa Monden’ 1 Department of Oncological and Regenerative Surgery The University of Tokushima, School of Medicine, Tokushima, Japan; ‘Department of Oncological and Regenerative Surgery, The University of Tokushima, School of Medicine, Tokushima, japan
Background: Lymphogenous
and hematogenous metastasis occurs frequently with lung cancer. Suppression of these metastases provide an imin survival time in lung cancer patients. However, there is no patientmodel of these metastases of human lung cancer. We established a SCID mice model by orthotopic implantation. Material and methods: Suspensions of 2.0~10~ cancer cells with IOmg/ml Matrigel were injected into the left lung of SCID mice using a 30-gauge needle. We observed the progressive stages of development of lymphogenous and hematogenous metastasis in 6 lung cancer cell lines including 2 primary culture cells from resected lung cacner tissues from 2 week to 16 week. And we evaluate a differences of RNA expression of 57 genes (anticancer drug-metabolism, drug-resistant enzyme, and DNA repair gene protein, etc) between implanted lung cancer tissues and lung cancer cell lines using DNA arrey. Results: All cell lines made tumor in the implanted site. The tumor in the implanted site of the lung became larger according to the time. Three cell lines (Ma44-3, FT, and FM) had the mediastinal lymph node metastasis, Ma10 cell line had no metastasis. A549 cell line had both of mediastinal lymph node metastasis and multiple intrapulmonary metastases. Ma2 cell line had solitary distant metastasis (adrenal gland, ovary, or lung). The degree of RNA expression of 57 genes in each implanted tumor correlated to that in the each cancer cell line (Pearson’s correlation coefficient: r=0.814-0.917). Conclusion: The metastatic pattern of this model is very similar to that of clinical lung cancer, and the degree of expression of anticancer drug-related enzymes in the implanted tumor correlated to that of each cell line. We suppose that this model is useful for evaluating the inhibitory effect of anticancer drugs and the mechanism of lymphogenous and hematogenous metastasis. in patients provement like animal patient-like
ElP 293
Analysis of TNF-a, TNF-b, IL-6, IL-lo, and HSPJO polymorphisms in patients with lung cancer
Carola Seifart’ , Ulf Seifart2, Alexandra Plagens’, Christine Fischers, Claus Vogelmeier’ ’ Philipps-University of Marburg, internal Medicine, Div. Resp. Med., Marburg, Germany; 2 Philipps-University of Marburg, Internal Medicine, Div. of Heam./OncoL, Marburg, Germany; 3 University of Heidelberg, institute of Human Genetics, Heidelberg, Germany Lung cancer is a growing health problem. Apart from cigarette smoking, genetic factors seem to be of importance in the development of the disease. The present case-control study investigated the frequencies of five interleukin (IL) promoter and two heat shock protein (HSP) polymorphisms in 365 individuals: 117 patients with lung cancer, matched controls and 131 healthy individuals. We analysed TNF-a-308, TNF-b-lntronl-252, IL-6-174, IL-10-819, IL-10-1082, HSP-70-1 and HSP-70-horn polymorphisms using RFLP based converted PCR. The study population consisted of 77 patients with NSCLC (including 40 patients with squamous cell carcinoma and 26 with adenocarcinoma), 40 patients with SCLC, matched controls without pulmonary disease (control group) and 131 healthy individuals (population control). Genotype analyses revealed, next to smoking and gender, significant difference in allele distribution of IL10-1082-G between SCLC and population control (p=O.O04, OR=3.74 [95%CI: 1.52-9.17]), while other differences were only seen by NSCLC-subgroup analysis. These showed the same allele (IL-10-1082-G) to be increased in the adenocarcinoma group (p=O.O41, OR=2.85 [95%CI: 1.04-7.801) and TNF-11-308-A (p=O.O28, OR=2.33 [95%CI: 1.09-4.981) and IL-10-819-T (p=O.O22, ORz2.37 [95%CI: 1.14-4.891) with significantly higher frequency compared to the control group in patients with squamous cell carcinoma. No association were seen for TNF-B-lntronl-252, IL-6-174, HSP-70-I and HSP--/O-horn polymorphisms with lung cancer or lung cancer subgroups. Because both, the IL-IO (-1082) and TNF-ol (-308) promoter polymorphisms are known to be functional, it could be speculated that these polymorphisms are related to altered inflammatory response thereby influencing susceptibility to lung cancer subgroups. Funded by BMBF 1 GC690115
Biology
IP 294
I
Combined Inhibition of Epidermal Growth Factor Receptor and COX-2 Enhances Growth Inhibition in Lung Cancer Cell Lines
Jae Cheol Lee’,‘, Michael J. Kelley’. ’ Department of Medicine, Thoracic Oncology Program, Duke University Medical Center; and Durham VA Hospital, Durham, NC, USA; 2 Laboratory of Experimental Therapeutics, Korea Cancer Center Hospital, Seoul, Korea Both epidermal growth factor receptor (EGFR) and the inducible cyclooxygenase 2 (COX-2) are important for cell proliferation and survival. PGE2, a byproduct of COX-2 expression, can transactivate EGFR in colon cancer cells; and TGF-c(, an EGFR ligand, increases COX-2 expression indicating that these pathways are closely related. We investigated the combined effect of selective EGFR inhibitors (AG1478 or ZD1839) and COX-2 inhibitors (celecoxib or NS398) on the growth of the A549 lung cancer cell line. ZD1839 reduced TGF-a-induced COX-2 expression and PGE2 production by 50% and 95%, respectively. TGF-a-induced phosphorylation of EGFR was reduced by celecoxib 40 uM, supporting the close relationship between these pathways. Using the MTS assay, the IC50 of AG1478 and ZD1839 alone was 15 uM and 10 uM, respectively, while that of celecoxib and NS398 alone was 40 uM and 120 uM, respectively. The addition of 2-10 uM of NS398 or IO-20 uM celecoxib to IO uM of AG1478 produced additive or, for many concentrations, more than additive growth inhibition. More than additive growth inhibition was observed at concentrations of NS398 that had no growth inhibitory effect when used alone. The apoptotic fraction at 48hr increased from 9.75% with IO uM of ZD1839 and 7.62% with 20 uM of celecoxib to 32.4% with combined treatment. Similar results were found with AG1478 and ZD1839. In summary, there is a close relationship between EGFR signaling and COX-2 signaling in lung cancer cells. The combined use of selective epidermal growth factor receptor and COX-2 inhibitors enhances the growth inhibition of either agent used alone in lung cancer cells and may be a useful therapeutic or preventive strategy for lung cancer.
I P 295
Dramatically Different Roles for Caveolin-l in the Development of Non-Small Cell Lung Cancer (NSCLC) vs. Small Cell Lung Cancer (SCLC)
Noriaki Sunaqa’, Kuniharu Miyajima ‘, Makoto Suzuki’, Mitsuo Sate’ , Michael A. Whites, Adi F. Gazdar’ , John D. Minna’ ’ Hamon Center for Therapeutic Oncology Research, Univ of Texas Southwestern Medical Center at Da//as, Da//as, USA; 8 Department of Cell Biology, Univ of Texas Southwestern Medical Center at Da//as, Da//as, USA Caveolin- is an essential structural constituent of caveolae that plays an important role in cellular processes including transport, signaling, and tumorigenesis. Previous studies have implicated caveolin-l in the development of several human cancers including lung cancer; however, it is still unclear how caveolin1 plays a role in the development of lung cancer. In the present study, we first examined endogenous expression of caveolin-l protein in 46 lung cancer cell lines including 25 NSCLCs and 21 SCLCs. Caveolin-l protein expression was dramatically reduced or absent in 95% (20/21) of the SCLCs, whereas it was retained in 76% (19/25) of NSCLCs compared with normal human lung epithelial cultures where it was abundantly expressed. Treatment with 5-aza2’deoxycytidine restored caveolin-l expression in 2 SCLC cell lines (NCI-Hi46 and H2171) lacking caveolin-l expression, showing that the promoter region of the caveolin-l gene is functionally methylated in these SCLC cell lines. In addition, bisulfite treatment of genomic DNA followed by sequencing and methylation-specific PCR analysis revealed caveolin-l promoter hypermethylation in 93% of SCLCs and 9% of NSCLCs. We then assessed the role of caveolinin the NSCLCs that retain caveolin-l protein expression using the RNA interference (RNAi) technology. Two days after introduction of small interfering RNA (siRNA) of caveolin-l into the NCI-H358, H1299, H2009 and HCCIS NSCLC cell lines, we could demonstrate dramatic down regulation of caveolin 1. These cells were plated for colony formation assays and we found significantly inhibited colony formation, demonstrating that caveolinexpression is indispensable for the growth of NSCLCs. These results suggest dramatically different roles for caveolin-l in SCLC where it has the properties of a tumor suppressor while in NSCLC it appears required for growth.
Poster Session Z/Molecular
loci, we have identified 5 that show potential for distinguishing between MM, adenocarcinoma and/or non-cancer lung. Our observations support the strong promise of methylation markers as tools for accurate diagnosis of neoplasms in and around the lung.
ElP 292
Establishment of patient-like SCID mice model by orthotopically implanting lung cancer cell lines
Haruhiko Fuiino’, Kazuya Kondo’, Takanori Miyoshi’, Hisashi Ishikura’, Yuji Takahashi’, Naruhiko Sawada’, Yukiko Hirose’, Hiromitu Takizawa’, Syoji Sakiyama’ , Yasumasa Monden’ 1 Department of Oncological and Regenerative Surgery The University of Tokushima, School of Medicine, Tokushima, Japan; ‘Department of Oncological and Regenerative Surgery, The University of Tokushima, School of Medicine, Tokushima, japan
Background: Lymphogenous
and hematogenous metastasis occurs frequently with lung cancer. Suppression of these metastases provide an imin survival time in lung cancer patients. However, there is no patientmodel of these metastases of human lung cancer. We established a SCID mice model by orthotopic implantation. Material and methods: Suspensions of 2.0~10~ cancer cells with IOmg/ml Matrigel were injected into the left lung of SCID mice using a 30-gauge needle. We observed the progressive stages of development of lymphogenous and hematogenous metastasis in 6 lung cancer cell lines including 2 primary culture cells from resected lung cacner tissues from 2 week to 16 week. And we evaluate a differences of RNA expression of 57 genes (anticancer drug-metabolism, drug-resistant enzyme, and DNA repair gene protein, etc) between implanted lung cancer tissues and lung cancer cell lines using DNA arrey. Results: All cell lines made tumor in the implanted site. The tumor in the implanted site of the lung became larger according to the time. Three cell lines (Ma44-3, FT, and FM) had the mediastinal lymph node metastasis, Ma10 cell line had no metastasis. A549 cell line had both of mediastinal lymph node metastasis and multiple intrapulmonary metastases. Ma2 cell line had solitary distant metastasis (adrenal gland, ovary, or lung). The degree of RNA expression of 57 genes in each implanted tumor correlated to that in the each cancer cell line (Pearson’s correlation coefficient: r=0.814-0.917). Conclusion: The metastatic pattern of this model is very similar to that of clinical lung cancer, and the degree of expression of anticancer drug-related enzymes in the implanted tumor correlated to that of each cell line. We suppose that this model is useful for evaluating the inhibitory effect of anticancer drugs and the mechanism of lymphogenous and hematogenous metastasis. in patients provement like animal patient-like
ElP 293
Analysis of TNF-a, TNF-b, IL-6, IL-lo, and HSPJO polymorphisms in patients with lung cancer
Carola Seifart’ , Ulf Seifart2, Alexandra Plagens’, Christine Fischers, Claus Vogelmeier’ ’ Philipps-University of Marburg, internal Medicine, Div. Resp. Med., Marburg, Germany; 2 Philipps-University of Marburg, Internal Medicine, Div. of Heam./OncoL, Marburg, Germany; 3 University of Heidelberg, institute of Human Genetics, Heidelberg, Germany Lung cancer is a growing health problem. Apart from cigarette smoking, genetic factors seem to be of importance in the development of the disease. The present case-control study investigated the frequencies of five interleukin (IL) promoter and two heat shock protein (HSP) polymorphisms in 365 individuals: 117 patients with lung cancer, matched controls and 131 healthy individuals. We analysed TNF-a-308, TNF-b-lntronl-252, IL-6-174, IL-10-819, IL-10-1082, HSP-70-1 and HSP-70-horn polymorphisms using RFLP based converted PCR. The study population consisted of 77 patients with NSCLC (including 40 patients with squamous cell carcinoma and 26 with adenocarcinoma), 40 patients with SCLC, matched controls without pulmonary disease (control group) and 131 healthy individuals (population control). Genotype analyses revealed, next to smoking and gender, significant difference in allele distribution of IL10-1082-G between SCLC and population control (p=O.O04, OR=3.74 [95%CI: 1.52-9.17]), while other differences were only seen by NSCLC-subgroup analysis. These showed the same allele (IL-10-1082-G) to be increased in the adenocarcinoma group (p=O.O41, OR=2.85 [95%CI: 1.04-7.801) and TNF-11-308-A (p=O.O28, OR=2.33 [95%CI: 1.09-4.981) and IL-10-819-T (p=O.O22, ORz2.37 [95%CI: 1.14-4.891) with significantly higher frequency compared to the control group in patients with squamous cell carcinoma. No association were seen for TNF-B-lntronl-252, IL-6-174, HSP-70-I and HSP--/O-horn polymorphisms with lung cancer or lung cancer subgroups. Because both, the IL-IO (-1082) and TNF-ol (-308) promoter polymorphisms are known to be functional, it could be speculated that these polymorphisms are related to altered inflammatory response thereby influencing susceptibility to lung cancer subgroups. Funded by BMBF 1 GC690115
Biology
IP 294
I
Combined Inhibition of Epidermal Growth Factor Receptor and COX-2 Enhances Growth Inhibition in Lung Cancer Cell Lines
Jae Cheol Lee’,‘, Michael J. Kelley’. ’ Department of Medicine, Thoracic Oncology Program, Duke University Medical Center; and Durham VA Hospital, Durham, NC, USA; 2 Laboratory of Experimental Therapeutics, Korea Cancer Center Hospital, Seoul, Korea Both epidermal growth factor receptor (EGFR) and the inducible cyclooxygenase 2 (COX-2) are important for cell proliferation and survival. PGE2, a byproduct of COX-2 expression, can transactivate EGFR in colon cancer cells; and TGF-c(, an EGFR ligand, increases COX-2 expression indicating that these pathways are closely related. We investigated the combined effect of selective EGFR inhibitors (AG1478 or ZD1839) and COX-2 inhibitors (celecoxib or NS398) on the growth of the A549 lung cancer cell line. ZD1839 reduced TGF-a-induced COX-2 expression and PGE2 production by 50% and 95%, respectively. TGF-a-induced phosphorylation of EGFR was reduced by celecoxib 40 uM, supporting the close relationship between these pathways. Using the MTS assay, the IC50 of AG1478 and ZD1839 alone was 15 uM and 10 uM, respectively, while that of celecoxib and NS398 alone was 40 uM and 120 uM, respectively. The addition of 2-10 uM of NS398 or IO-20 uM celecoxib to IO uM of AG1478 produced additive or, for many concentrations, more than additive growth inhibition. More than additive growth inhibition was observed at concentrations of NS398 that had no growth inhibitory effect when used alone. The apoptotic fraction at 48hr increased from 9.75% with IO uM of ZD1839 and 7.62% with 20 uM of celecoxib to 32.4% with combined treatment. Similar results were found with AG1478 and ZD1839. In summary, there is a close relationship between EGFR signaling and COX-2 signaling in lung cancer cells. The combined use of selective epidermal growth factor receptor and COX-2 inhibitors enhances the growth inhibition of either agent used alone in lung cancer cells and may be a useful therapeutic or preventive strategy for lung cancer.
I P 295
Dramatically Different Roles for Caveolin-l in the Development of Non-Small Cell Lung Cancer (NSCLC) vs. Small Cell Lung Cancer (SCLC)
Noriaki Sunaqa’, Kuniharu Miyajima ‘, Makoto Suzuki’, Mitsuo Sate’ , Michael A. Whites, Adi F. Gazdar’ , John D. Minna’ ’ Hamon Center for Therapeutic Oncology Research, Univ of Texas Southwestern Medical Center at Da//as, Da//as, USA; 8 Department of Cell Biology, Univ of Texas Southwestern Medical Center at Da//as, Da//as, USA Caveolin- is an essential structural constituent of caveolae that plays an important role in cellular processes including transport, signaling, and tumorigenesis. Previous studies have implicated caveolin-l in the development of several human cancers including lung cancer; however, it is still unclear how caveolin1 plays a role in the development of lung cancer. In the present study, we first examined endogenous expression of caveolin-l protein in 46 lung cancer cell lines including 25 NSCLCs and 21 SCLCs. Caveolin-l protein expression was dramatically reduced or absent in 95% (20/21) of the SCLCs, whereas it was retained in 76% (19/25) of NSCLCs compared with normal human lung epithelial cultures where it was abundantly expressed. Treatment with 5-aza2’deoxycytidine restored caveolin-l expression in 2 SCLC cell lines (NCI-Hi46 and H2171) lacking caveolin-l expression, showing that the promoter region of the caveolin-l gene is functionally methylated in these SCLC cell lines. In addition, bisulfite treatment of genomic DNA followed by sequencing and methylation-specific PCR analysis revealed caveolin-l promoter hypermethylation in 93% of SCLCs and 9% of NSCLCs. We then assessed the role of caveolinin the NSCLCs that retain caveolin-l protein expression using the RNA interference (RNAi) technology. Two days after introduction of small interfering RNA (siRNA) of caveolin-l into the NCI-H358, H1299, H2009 and HCCIS NSCLC cell lines, we could demonstrate dramatic down regulation of caveolin 1. These cells were plated for colony formation assays and we found significantly inhibited colony formation, demonstrating that caveolinexpression is indispensable for the growth of NSCLCs. These results suggest dramatically different roles for caveolin-l in SCLC where it has the properties of a tumor suppressor while in NSCLC it appears required for growth.