- Email: [email protected]
P-318 Characterization of a human small-cell lung cancer cell line from in vivo multidrug resistant tumour
S172
I.
Results:
P 318
Freouencv of a oiven barameter (%I OverallSurvival ~3 years Overall survival > 5 years
Parameter
del lq Aneuploidy (cells number) Mutant p53 expression VEGF expression Bcl-2 expression Sax expression % of cells in S-phase
52 75 86 76 20 16 9,56
a,3 37,4 41.7 25 42 29 5,76
@<0,05) (P
(PCO,O5)
Conclusion: monofactorial analysis produced significant and independent prognostic factors for NSCLC: expression of mutant ~53, expression of VEGF, del Iq, aneuploidy of tumor cells and tumor cells in the S-phase of mitotic cycle. / P-316 1 Histone deacetylase inhibitor suppresses MMP-2 actwahon and cell invasion via RECK Wen-Chun Hunq’, Li-Teh Liu*, Hui-Chiu Chang3. ’ School of Technology for Medical Sciences, Kaohsiung, R.O.C.; *Institute of Medicine, Kaohsiung Medical University, Kaohsiung, R.O.C.; 3 Department of Physiology: Kaohsiung, R. 0. C. Histone deacetylase (HDAC) inhibitors are known to exert anti-metastatic and anti-angiogenic activity in vitro and in viva. RECK is a membrane-anchored glycoprotein that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor metastasis and angiogenesis. In this study, we test the possibility that HDAC inhibitor may increase RECK expression to inhibit MMP activation and cancer cell invasion. RT-PCR and western blot analysis demonstrated that trichostatin A (TSA) up-regulated RECK expression in CL-I human lung cancer cells. Promoter activity assay indicated that TSA enhanced RECK expression via transcriptional activation. Flow cytometric analysis showed that RECK protein on cell surface was increased after treatment of TSA. Moreover, upregulation of RECK expression by TSA attenuated MMP-2 activity as shown by gelatin zymography. To explore whether HDAC inhibitor-induced inhibition of MMP-2 activation is indeed mediated via RECK, we used small interference RNA (siRNA) to block RECK expression and our results demonstrated that inhibition of RECK by siRNA abolished the inhibitory effect of TSA on MMP-2 activation. In addition, TSA potently suppressed the invasive ability of CL-l cells. Taken together, this study reveals a novel mechanism by which HDAC inhibitors suppress tumor invasion and provides a new strategy for cancer therapy.
Ip-3171
Biology 2
Pus&r Session 2/Molecular
Luteolin induce apoptosis in human lung cell line (A549) through activation of caspase 3 and ~53
Te-Chun Hsia’, Jing-Gung Chungs, Hsueh-Fu Lu3, Liang-Wen Hang4, Wu-Huei Hsu’, Chuen-Ming Shih’. ’ Dept. of lntemal Medicine, China Medical College Hospital Taichung, Taiwan; ’ Dept. of Microbiology China Medical College, Taichung, Taiwan; 3 Dept. of Urology, China Medical College Hospital, Taichung, Taiwan; 4 Dept. of lnteranl Medicine, China Medical College Hospitab Taichung, Taiwan Luteolin (2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4H-l-benzopyran-4-one), is present in many plants (Combretaceae) in glycoside combination, e.g., as the arabinoside. It had been demonstrated that luteolin inhibit a series of human cancer cell lines (renal A-549, ovary SK-OV-3, melanoma SK-MEL-2, XF-498, HCT15, gastric HGC-27) but the mechanism of luteolin affect on human lung cancer cells was not addressed. Therefore, luetolin was used to study the biological activities on the human lung cancer cell line (A549). On MTT assay, luteolin showed obvious cytotoxic effects and also induced apoptosis on A549 cells. The cytotoxic effects of luteolin was accompanied by the dose- and timedependent appearance of characteristics of apoptosis including DNA fragmentation (gel electrophoresis) and sub-G1 ratio (flow cytometric assay). The A549 cells cotreated with luteolin caused a rapid transient induction of caspase 3 activity, but not caspase 1 activity. We also found out that cleavage of poly(ADPribose) polymerase (PARP) ans decrease of pro-caspase 3 protein were detected in luteolin -treated A549 cells. Increased the pro-apoptotic protein (bax) and decreased anti-apoptotic protein (MCI-I) were detected in A549 cells after cotreated with luteolin. We also used PCR and multiplex PCR methods to examine the caspase 3 gene expression and the results show that luteolin induced caspase 3 gene expression.
Characterization of A Human Small-Cell Lung Cancer Cell Lme From In Vivo Multidrug Resistant Tumour
M.J. Valladares-Averbes’, Antolin S. Novoas, P lglesias Diaz3, Haz Mar“, Brandon Inmaculadas, Goyanes Vicentec, Reboredo Margarita’, Alonso Guillermo”, Calvo Lourdes*, L.M Anton Aparicio7. ’ Complejo Hospitalario Universitario Juan Canalejo. Oncology and Research Units, LA Corufia, SPAIN; 2 Oncology LA Corufia, SPAIN; 3 Pathology, LA Corufia, SPA/N; 4 Research Unit, LA CoruAa, SPAIN; 5 Research Unit, La Coruna, SPAIN; 6 Genetic Unit, LA Corufia, SPAIN; 7 Dept. of Medicine. CH Universitario Juan Canalejo. Universidade da Coruiia, LA Corutia, SPAIN
Background: Cell-lines derived from human tumors play a pivotal role to study genetic and phenotypic changes associated with progression and drugresistance in cancer. We describe the development and characterization of a new human small-cell lung carcinoma (SCLC) cell-line (CL) from in vivo multidrug-resistant tumor. Material and Methods: A 59 years-old male with extensive-disease SCLC received chemotherapy including cisplatin, etoposide, ifosfamide and the topoisomerase I inhibitor topotecan. Disease progressed and he developed metastatic pleural effusion (PE). Mononuclear cells were isolated from PE and suspended in culture flasks with serum supplemented medium. Cells were maintained in continuous culture as suspensions and survived cryopreservation. After successive passages the cell-line (named JCA-OJCS) was considered as established. Phenotype was analyzed by immunocytochemistry (ICQ) in comparison with SCLC-CL HI46 and DMS92. Gene-expression for metastasis related markers were examined using reverse transcriptase (RT)-PCR. Karyotype was studied by G-banding. Results: SCLC cell-line JCA-OJC3 grew as floating aggregates even in serum and high calcium-containing mediums; it may be the results of the expression of neural cell adhesion molecule (N-CAM). JCA-OJC3 cells stained also positively for epithelial (Ep)-CAM, N-cadherin, cytokeratins and thyroid transcription factor-i (TTF-1). Syntaxin 1, a synaptic protein essential for exocytosis of biogenic amines and neurotransmitter release, was highly expressed by ICQ in JCA-OJC3 and the other SCLC-CL. RT-PCR showed specific amplicon for pituitary-tumor transforming gene (PTTGI) in JCA-OJC3, H146 and DMS92. Beta I,6 N acetyl glucosaminyl transferase V (GNTV) mRNA was also amplified in JCA-OJC3 and DMS92. EGF-receptor was not found in JCA-OJC3 in ICQ and RT-PCR analysis. G-banded karyotype of JCA-OJC3 cells demonstrated clonal abnormalities with modal number 49-50 and a marker chromosome (chr) derived from chr 5. One copy of chrs 3 and 5 were loosed in 40% of cells. Additional copies of groups C, E and G were found in 85%. Adhesive behavior and tissue-specific homing properties were investigated in vitro on cryostat-cut sections of rat organs. Conclusions: JCA-OJC3 cell-line displays in vitro properties that are representative of SCLC. It may provide a useful model for the study of molecular and phenotypic changes associated with SCLC progression. I P 319
Presence of the epidermal growth factor receptor family and epidermal growth factor receptor binding ligands in small cell lung cancer cell lines
Lars Damstrup’ , Mikkel W. Pedersens, Hans Skovgaard Poulsens. ’ University Hospital Copenhagen, Department of Oncology and Radiation Biology, Copenhagen, Denmark; 2 Department of Radiation Biology, Copenhagen, Denmark The family of ligands binding to the epidermal growth factor receptor (EGFR) is epidermal growth factor (EGF), transforming growth factor c1 (TGFol), amphiregullin (AR), betacelullin (BTC) and heparin binding EGF like growth factor (HB-EGF). In this study we have examined our panel of 21 human small cell lung cancer (SCLC) cell lines for the presence of the EGF receptor family and EGFR binding ligands. Initial screening using RT-PCR demonstrated that all 21 SCLC cell lines expressed one or more EGFR binding ligands. Furthermore, all 21 cell lines expressed at least 2 EGFR family members (EGFR, erbB2, erbB3 and erbB4). All the SCLC cell lines expressed more than one of the receptors. None of 15 examined SCLC cell lines expressed the EGFR mutation type Ill. As a consequence of the endogenous production of the EGFR binding ligands, the addition of EGF/TGFcl did not significantly alter DNA synthesis in examined EGFR positive SCLC cell lines. However, addition of the EGFR neutralizing antibody (moAb528) significantly attenuated baseline DNA synthesis. Furthermore, we have examined the effect of the tyrosine kinase inhibitor, erbstatin. Erbstatin was used for different time periods at different concentrations. Proliferation was evaluated using the MTT assay. In the examined EGFR positive SCLC cell lines, we found a dose-dependent reduction in proliferation after administration of erbstatin. The significant attenuation in proliferation was associated with changes in morphology. Our results indicate that the majority of SCLC cell lines express EGFR binding ligands, which acts as autocrine growth factors in EGFR positive SCLC cell lines.
I.
Results:
P 318
Freouencv of a oiven barameter (%I OverallSurvival ~3 years Overall survival > 5 years
Parameter
del lq Aneuploidy (cells number) Mutant p53 expression VEGF expression Bcl-2 expression Sax expression % of cells in S-phase
52 75 86 76 20 16 9,56
a,3 37,4 41.7 25 42 29 5,76
@<0,05) (P
(PCO,O5)
Conclusion: monofactorial analysis produced significant and independent prognostic factors for NSCLC: expression of mutant ~53, expression of VEGF, del Iq, aneuploidy of tumor cells and tumor cells in the S-phase of mitotic cycle. / P-316 1 Histone deacetylase inhibitor suppresses MMP-2 actwahon and cell invasion via RECK Wen-Chun Hunq’, Li-Teh Liu*, Hui-Chiu Chang3. ’ School of Technology for Medical Sciences, Kaohsiung, R.O.C.; *Institute of Medicine, Kaohsiung Medical University, Kaohsiung, R.O.C.; 3 Department of Physiology: Kaohsiung, R. 0. C. Histone deacetylase (HDAC) inhibitors are known to exert anti-metastatic and anti-angiogenic activity in vitro and in viva. RECK is a membrane-anchored glycoprotein that negatively regulates matrix metalloproteinases (MMPs) and inhibits tumor metastasis and angiogenesis. In this study, we test the possibility that HDAC inhibitor may increase RECK expression to inhibit MMP activation and cancer cell invasion. RT-PCR and western blot analysis demonstrated that trichostatin A (TSA) up-regulated RECK expression in CL-I human lung cancer cells. Promoter activity assay indicated that TSA enhanced RECK expression via transcriptional activation. Flow cytometric analysis showed that RECK protein on cell surface was increased after treatment of TSA. Moreover, upregulation of RECK expression by TSA attenuated MMP-2 activity as shown by gelatin zymography. To explore whether HDAC inhibitor-induced inhibition of MMP-2 activation is indeed mediated via RECK, we used small interference RNA (siRNA) to block RECK expression and our results demonstrated that inhibition of RECK by siRNA abolished the inhibitory effect of TSA on MMP-2 activation. In addition, TSA potently suppressed the invasive ability of CL-l cells. Taken together, this study reveals a novel mechanism by which HDAC inhibitors suppress tumor invasion and provides a new strategy for cancer therapy.
Ip-3171
Biology 2
Pus&r Session 2/Molecular
Luteolin induce apoptosis in human lung cell line (A549) through activation of caspase 3 and ~53
Te-Chun Hsia’, Jing-Gung Chungs, Hsueh-Fu Lu3, Liang-Wen Hang4, Wu-Huei Hsu’, Chuen-Ming Shih’. ’ Dept. of lntemal Medicine, China Medical College Hospital Taichung, Taiwan; ’ Dept. of Microbiology China Medical College, Taichung, Taiwan; 3 Dept. of Urology, China Medical College Hospital, Taichung, Taiwan; 4 Dept. of lnteranl Medicine, China Medical College Hospitab Taichung, Taiwan Luteolin (2-(3,4-Dihydroxyphenyl)-5,7-dihydroxy-4H-l-benzopyran-4-one), is present in many plants (Combretaceae) in glycoside combination, e.g., as the arabinoside. It had been demonstrated that luteolin inhibit a series of human cancer cell lines (renal A-549, ovary SK-OV-3, melanoma SK-MEL-2, XF-498, HCT15, gastric HGC-27) but the mechanism of luteolin affect on human lung cancer cells was not addressed. Therefore, luetolin was used to study the biological activities on the human lung cancer cell line (A549). On MTT assay, luteolin showed obvious cytotoxic effects and also induced apoptosis on A549 cells. The cytotoxic effects of luteolin was accompanied by the dose- and timedependent appearance of characteristics of apoptosis including DNA fragmentation (gel electrophoresis) and sub-G1 ratio (flow cytometric assay). The A549 cells cotreated with luteolin caused a rapid transient induction of caspase 3 activity, but not caspase 1 activity. We also found out that cleavage of poly(ADPribose) polymerase (PARP) ans decrease of pro-caspase 3 protein were detected in luteolin -treated A549 cells. Increased the pro-apoptotic protein (bax) and decreased anti-apoptotic protein (MCI-I) were detected in A549 cells after cotreated with luteolin. We also used PCR and multiplex PCR methods to examine the caspase 3 gene expression and the results show that luteolin induced caspase 3 gene expression.
Characterization of A Human Small-Cell Lung Cancer Cell Lme From In Vivo Multidrug Resistant Tumour
M.J. Valladares-Averbes’, Antolin S. Novoas, P lglesias Diaz3, Haz Mar“, Brandon Inmaculadas, Goyanes Vicentec, Reboredo Margarita’, Alonso Guillermo”, Calvo Lourdes*, L.M Anton Aparicio7. ’ Complejo Hospitalario Universitario Juan Canalejo. Oncology and Research Units, LA Corufia, SPAIN; 2 Oncology LA Corufia, SPAIN; 3 Pathology, LA Corufia, SPA/N; 4 Research Unit, LA CoruAa, SPAIN; 5 Research Unit, La Coruna, SPAIN; 6 Genetic Unit, LA Corufia, SPAIN; 7 Dept. of Medicine. CH Universitario Juan Canalejo. Universidade da Coruiia, LA Corutia, SPAIN
Background: Cell-lines derived from human tumors play a pivotal role to study genetic and phenotypic changes associated with progression and drugresistance in cancer. We describe the development and characterization of a new human small-cell lung carcinoma (SCLC) cell-line (CL) from in vivo multidrug-resistant tumor. Material and Methods: A 59 years-old male with extensive-disease SCLC received chemotherapy including cisplatin, etoposide, ifosfamide and the topoisomerase I inhibitor topotecan. Disease progressed and he developed metastatic pleural effusion (PE). Mononuclear cells were isolated from PE and suspended in culture flasks with serum supplemented medium. Cells were maintained in continuous culture as suspensions and survived cryopreservation. After successive passages the cell-line (named JCA-OJCS) was considered as established. Phenotype was analyzed by immunocytochemistry (ICQ) in comparison with SCLC-CL HI46 and DMS92. Gene-expression for metastasis related markers were examined using reverse transcriptase (RT)-PCR. Karyotype was studied by G-banding. Results: SCLC cell-line JCA-OJC3 grew as floating aggregates even in serum and high calcium-containing mediums; it may be the results of the expression of neural cell adhesion molecule (N-CAM). JCA-OJC3 cells stained also positively for epithelial (Ep)-CAM, N-cadherin, cytokeratins and thyroid transcription factor-i (TTF-1). Syntaxin 1, a synaptic protein essential for exocytosis of biogenic amines and neurotransmitter release, was highly expressed by ICQ in JCA-OJC3 and the other SCLC-CL. RT-PCR showed specific amplicon for pituitary-tumor transforming gene (PTTGI) in JCA-OJC3, H146 and DMS92. Beta I,6 N acetyl glucosaminyl transferase V (GNTV) mRNA was also amplified in JCA-OJC3 and DMS92. EGF-receptor was not found in JCA-OJC3 in ICQ and RT-PCR analysis. G-banded karyotype of JCA-OJC3 cells demonstrated clonal abnormalities with modal number 49-50 and a marker chromosome (chr) derived from chr 5. One copy of chrs 3 and 5 were loosed in 40% of cells. Additional copies of groups C, E and G were found in 85%. Adhesive behavior and tissue-specific homing properties were investigated in vitro on cryostat-cut sections of rat organs. Conclusions: JCA-OJC3 cell-line displays in vitro properties that are representative of SCLC. It may provide a useful model for the study of molecular and phenotypic changes associated with SCLC progression. I P 319
Presence of the epidermal growth factor receptor family and epidermal growth factor receptor binding ligands in small cell lung cancer cell lines
Lars Damstrup’ , Mikkel W. Pedersens, Hans Skovgaard Poulsens. ’ University Hospital Copenhagen, Department of Oncology and Radiation Biology, Copenhagen, Denmark; 2 Department of Radiation Biology, Copenhagen, Denmark The family of ligands binding to the epidermal growth factor receptor (EGFR) is epidermal growth factor (EGF), transforming growth factor c1 (TGFol), amphiregullin (AR), betacelullin (BTC) and heparin binding EGF like growth factor (HB-EGF). In this study we have examined our panel of 21 human small cell lung cancer (SCLC) cell lines for the presence of the EGF receptor family and EGFR binding ligands. Initial screening using RT-PCR demonstrated that all 21 SCLC cell lines expressed one or more EGFR binding ligands. Furthermore, all 21 cell lines expressed at least 2 EGFR family members (EGFR, erbB2, erbB3 and erbB4). All the SCLC cell lines expressed more than one of the receptors. None of 15 examined SCLC cell lines expressed the EGFR mutation type Ill. As a consequence of the endogenous production of the EGFR binding ligands, the addition of EGF/TGFcl did not significantly alter DNA synthesis in examined EGFR positive SCLC cell lines. However, addition of the EGFR neutralizing antibody (moAb528) significantly attenuated baseline DNA synthesis. Furthermore, we have examined the effect of the tyrosine kinase inhibitor, erbstatin. Erbstatin was used for different time periods at different concentrations. Proliferation was evaluated using the MTT assay. In the examined EGFR positive SCLC cell lines, we found a dose-dependent reduction in proliferation after administration of erbstatin. The significant attenuation in proliferation was associated with changes in morphology. Our results indicate that the majority of SCLC cell lines express EGFR binding ligands, which acts as autocrine growth factors in EGFR positive SCLC cell lines.