Ignite Creation Date:
2024-05-05 @ 4:59 PM
Last Modification Date:
2024-10-26 @ 9:26 AM
Study NCT ID:
NCT00364715
Status:
COMPLETED
Last Update Posted:
2006-08-17
First Post:
2006-08-15
Brief Title:
Study the Expression of Annexin A1 and Its Potential Usage as a Prognostic Marker in Oral Cancer
Sponsor:
National Taiwan University Hospital
Organization:
National Taiwan University Hospital
Study Overview
Official Title:
Study the Expression of Annexin A1 and Its Potential Usage as a Prognostic Marker in Oral Cancer
Status:
COMPLETED
Status Verified Date:
2006-08
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
False
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
None
Brief Summary:
Recent studies have shown that dysregulation of ANXA1 expression are associated with tumorigenesis Overexpression of ANXA1 protein is found in a wide variety of human tumors such as breast 10 liver 11 pancreatic cancer14 and glial tumors15 In contrast reduced levels of ANXA1 protein expression have been reported in ESCC4 5 gastric6 breast7 head and neck SCC8 and prostate cancer9 No previous study on ANXA1 protein expression has been reported in the cancer of oral cavity Furthermore although alterations in annexin expression in different types of tumors have been described no correlation has been established between ANXA1 and overall patient survival yet
ANXA1 is a major cellular substrate of the oncogenic tyrosine kinases such as EGF receptor and hepatocyte growth factor HGF receptor c-met Previously we have shown that expression of HGF and c-met is significantly associated with the progression of OSCC in Taiwan Kermorgant et al recently showed that PKC controls HGF-dependent c-met traffic signaling and cell migration Prior study indicate that the mitogen phorbol-12-myristate 13-acetate PMA induced ANXA1 nuclear translocation in a PKCdelta-dependent manner and ANXA1 nuclear translocation may participate in the regulation of cellular proliferation and the differentiation However it is not known whether HGF can induce ANXA1 nuclear translocation or not and how this relates to the pathogenesis of oral SCC
In this study we aimed to investigate whether HGF induced the translocation of ANXA1 protein to the nucleus in OSCC cells and the roles of ANXA1 nuclear localization in the carcinogenesis of OSCC using an immunohistochemical technique The data suggest a novel mechanism for HGF-induced ANXA1 protein nuclear translocation that may play an important role in the pathogenesis and prognosis in oral SCCs
ANXA1 is a major cellular substrate of the oncogenic tyrosine kinases such as EGF receptor and hepatocyte growth factor HGF receptor c-met Previously we have shown that expression of HGF and c-met is significantly associated with the progression of OSCC in Taiwan Kermorgant et al recently showed that PKC controls HGF-dependent c-met traffic signaling and cell migration Prior study indicate that the mitogen phorbol-12-myristate 13-acetate PMA induced ANXA1 nuclear translocation in a PKCdelta-dependent manner and ANXA1 nuclear translocation may participate in the regulation of cellular proliferation and the differentiation However it is not known whether HGF can induce ANXA1 nuclear translocation or not and how this relates to the pathogenesis of oral SCC
In this study we aimed to investigate whether HGF induced the translocation of ANXA1 protein to the nucleus in OSCC cells and the roles of ANXA1 nuclear localization in the carcinogenesis of OSCC using an immunohistochemical technique The data suggest a novel mechanism for HGF-induced ANXA1 protein nuclear translocation that may play an important role in the pathogenesis and prognosis in oral SCCs
Detailed Description:
MATERIAL AND METHODS
After approval by the Hospital Review Board we obtained formalin-fixed paraffin-embedded specimens from 115 patients with primary oral SCC and 66 patients with oral ED at the Department of Pathology National Taiwan University Hospital Taipei Taiwan Diagnosis of oral SCC and ED was based on histological examination of hematoxylin and eosin-stained tissue sections All patients received total surgical excision of the lesions at the Department of Oral and Maxillofacial Surgery or Department of Otolaryngology National Taiwan University Hospital during the period from 1997 to 2004 Specimens were obtained from either incisional biopsies or total surgical excision of the lesions If lymph node was diagnosed as positive for SCC neck dissection and postoperative radiation therapy were also included in the treatment protocol No patient had received any cancer therapy before initial biopsies Details of the patients oral habits including daily consumption of AQs cigarettes and alcohol as well as the duration of these habits were also recorded in the medical chart Twenty biopsy specimens of normal oral mucosa NOM were obtained from non-AQ-chewers and non-smokers during extraction of impacted permanent lower third molars after obtaining informed consent and used as the healthy controls
Cell culture Dulbeccos modifed Eagles medium DMEM and fetal bovine serum FBS were purchased from Life Technologies Inc Gaithersburg MDUSA PD98059 Ly294002 SB203580 and Hepatocyte growth factor HGF were products of Sigma St Louis MO USA Anti-ANX-1 monoclonal antibody was purchased from BD Biosciences Lexington KY USA Cell culture SAS cells were maintained in DMEM supplemented with 10 heat-inactivated FBS penicillin 100 UAmL1 and streptomycin 100 lgAmL1 at 37 o C under 5 CO2 atmosphere For Western blot analysis cells were seeded into 10 cm dishes at 2 x 106 cells per dish After 18-24 hr cells were further grown in the same medium supplemented without FBS for 24 h Serum-starved cells were treated with HGF in the indicated times For immunostaining 2 x 105 cells grown on cover slides 22 x 22 mm were starved for 24 h before stimulation with HGF
Pre-treatment with PD098059 Ly294002 and SB203580 The cells were plated overnight in complete medium and then washed The cells cultured in serum-free DMEM were pre-treated with 50M PD098059 inhibitor of ERK 30 M Ly294002 inhibitor of PI-3 kinase and 20 M SB203580inhibitor of p38 for 3 hrs respectively and then they were treated with 40ng HGF for 3hrs The cells were checked by immunohistochemistry and Western blot with ANXA1 antibody
ImmunocytochemistryIHC Formalin-fixed paraffin-embedded tumor samples were assessed for annexin A1 expressionThe IHC procedures was followed as the reference18 The monoclonal primary antibody anti-Annexin AI1600 dilution directed against the the N-terminus of annexin 1 were obtained from a signal source BD Biosciences Protein translocation as determined by two pathologists Jeng and Lin using immunohistochemistry was scored as negative score 0 positive score 1 and cytoplasmic protein expression was scored as negative score 0 weak score 1 moderate score 2 or strong score 3 using a system that has been previously validated
SAS cells grown on cover slides were fixed with 37 paraformaldehyde for 15 min and permeabilized with 02 Triton X-100 in PBS 058M Na2HPO4 017 M Na H2PO4 068 M NaCl pH74PBST for 5 min After washing the cells with PBST three times the cells were blocked for 30 min in PBS containing 1 bovine serum albumin Immunostaining was performed by incubation with anti-ANX-1 monoclonal antibody 1800 dilution for 2h After washing the cells with PBST three times the cells were incubated with TRITC-conjugated rabbit anti-mouse IgG Ig for 1 h Cover slides were washed with PBST mounted and examined using a Leica TCS SP2 Confocal microscope Leica Microsystems Wetzlar GmBH Germany
Cell fractionation SAS cells were seeded into 10cm dishes at 2x 106 cellsdish and cultured in DMEM for 18-24 h The cells were starved for 24 h in serum-free media After treatment with HGF for a given time the cells were harvested and washed with ice-cold PBS The cells were then resuspended in 100 lL of lysis buffer 10 mM Hepes 10 mM NaCl 01 mM EDTA 01 mM EGTA 1 NP-40 05 mM phenylmethylsulfonyl uoride 01 mM dithiothreitol 01 mM sodium orthovanadate and protease inhibitors and incubated on ice for 10 min The nuclei were collected by centrifugation at 2000 g for 5 min at 4 o C The supernatant was collected as a cytosolic fraction Protein concentration of each sample was determined
Evalution of annexin A1 Protein Expression The distributions of ANXA1 expression in four categories 0-25 26-50 51-75 and 76-100 with nuclear translocation were firstly showed by clinicopathological parameters of the oral ED and SCCs and the correlations between mean levels of expression and these clinicopathological parameters were further examined using ANOVA The Kaplan-Meier product-limit method was used to assess the prognostic significance of the ANXA1 nuclear staining in cancer cells as well as other clinicopathological parameters Comparison of cumulative survival between groups was performed using the log-rank test with the Statistica program StatSoft Inc USA Multivariable analyses were performed with Cox regression model to assess additional prognostic values of the different variables using SAS 80 SAS Institute Inc USA A P-value of less than 005 was considered as statistically significant
After approval by the Hospital Review Board we obtained formalin-fixed paraffin-embedded specimens from 115 patients with primary oral SCC and 66 patients with oral ED at the Department of Pathology National Taiwan University Hospital Taipei Taiwan Diagnosis of oral SCC and ED was based on histological examination of hematoxylin and eosin-stained tissue sections All patients received total surgical excision of the lesions at the Department of Oral and Maxillofacial Surgery or Department of Otolaryngology National Taiwan University Hospital during the period from 1997 to 2004 Specimens were obtained from either incisional biopsies or total surgical excision of the lesions If lymph node was diagnosed as positive for SCC neck dissection and postoperative radiation therapy were also included in the treatment protocol No patient had received any cancer therapy before initial biopsies Details of the patients oral habits including daily consumption of AQs cigarettes and alcohol as well as the duration of these habits were also recorded in the medical chart Twenty biopsy specimens of normal oral mucosa NOM were obtained from non-AQ-chewers and non-smokers during extraction of impacted permanent lower third molars after obtaining informed consent and used as the healthy controls
Cell culture Dulbeccos modifed Eagles medium DMEM and fetal bovine serum FBS were purchased from Life Technologies Inc Gaithersburg MDUSA PD98059 Ly294002 SB203580 and Hepatocyte growth factor HGF were products of Sigma St Louis MO USA Anti-ANX-1 monoclonal antibody was purchased from BD Biosciences Lexington KY USA Cell culture SAS cells were maintained in DMEM supplemented with 10 heat-inactivated FBS penicillin 100 UAmL1 and streptomycin 100 lgAmL1 at 37 o C under 5 CO2 atmosphere For Western blot analysis cells were seeded into 10 cm dishes at 2 x 106 cells per dish After 18-24 hr cells were further grown in the same medium supplemented without FBS for 24 h Serum-starved cells were treated with HGF in the indicated times For immunostaining 2 x 105 cells grown on cover slides 22 x 22 mm were starved for 24 h before stimulation with HGF
Pre-treatment with PD098059 Ly294002 and SB203580 The cells were plated overnight in complete medium and then washed The cells cultured in serum-free DMEM were pre-treated with 50M PD098059 inhibitor of ERK 30 M Ly294002 inhibitor of PI-3 kinase and 20 M SB203580inhibitor of p38 for 3 hrs respectively and then they were treated with 40ng HGF for 3hrs The cells were checked by immunohistochemistry and Western blot with ANXA1 antibody
ImmunocytochemistryIHC Formalin-fixed paraffin-embedded tumor samples were assessed for annexin A1 expressionThe IHC procedures was followed as the reference18 The monoclonal primary antibody anti-Annexin AI1600 dilution directed against the the N-terminus of annexin 1 were obtained from a signal source BD Biosciences Protein translocation as determined by two pathologists Jeng and Lin using immunohistochemistry was scored as negative score 0 positive score 1 and cytoplasmic protein expression was scored as negative score 0 weak score 1 moderate score 2 or strong score 3 using a system that has been previously validated
SAS cells grown on cover slides were fixed with 37 paraformaldehyde for 15 min and permeabilized with 02 Triton X-100 in PBS 058M Na2HPO4 017 M Na H2PO4 068 M NaCl pH74PBST for 5 min After washing the cells with PBST three times the cells were blocked for 30 min in PBS containing 1 bovine serum albumin Immunostaining was performed by incubation with anti-ANX-1 monoclonal antibody 1800 dilution for 2h After washing the cells with PBST three times the cells were incubated with TRITC-conjugated rabbit anti-mouse IgG Ig for 1 h Cover slides were washed with PBST mounted and examined using a Leica TCS SP2 Confocal microscope Leica Microsystems Wetzlar GmBH Germany
Cell fractionation SAS cells were seeded into 10cm dishes at 2x 106 cellsdish and cultured in DMEM for 18-24 h The cells were starved for 24 h in serum-free media After treatment with HGF for a given time the cells were harvested and washed with ice-cold PBS The cells were then resuspended in 100 lL of lysis buffer 10 mM Hepes 10 mM NaCl 01 mM EDTA 01 mM EGTA 1 NP-40 05 mM phenylmethylsulfonyl uoride 01 mM dithiothreitol 01 mM sodium orthovanadate and protease inhibitors and incubated on ice for 10 min The nuclei were collected by centrifugation at 2000 g for 5 min at 4 o C The supernatant was collected as a cytosolic fraction Protein concentration of each sample was determined
Evalution of annexin A1 Protein Expression The distributions of ANXA1 expression in four categories 0-25 26-50 51-75 and 76-100 with nuclear translocation were firstly showed by clinicopathological parameters of the oral ED and SCCs and the correlations between mean levels of expression and these clinicopathological parameters were further examined using ANOVA The Kaplan-Meier product-limit method was used to assess the prognostic significance of the ANXA1 nuclear staining in cancer cells as well as other clinicopathological parameters Comparison of cumulative survival between groups was performed using the log-rank test with the Statistica program StatSoft Inc USA Multivariable analyses were performed with Cox regression model to assess additional prognostic values of the different variables using SAS 80 SAS Institute Inc USA A P-value of less than 005 was considered as statistically significant
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None