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Beneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer.

Lavergne M, Jourdan ML, Blechet C, Guyetant S, Pape AL, Heuze-Vourc'h N, Courty Y, Lerondel S, Sobilo J, Iochmann S, Reverdiau P - FEBS Open Bio (2013)

Bottom Line: In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer.Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2.We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3.

View Article: PubMed Central - PubMed

Affiliation: EA 6305, Université François Rabelais de Tours, Tours F-37032, France ; Centre d'Etude des Pathologies Respiratoires, UMR 1100/EA6305, Tours F-37032, France.

ABSTRACT
Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI-2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI-2 in tumour progression, TFPI-2 was overexpressed in NCI-H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI-2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2. We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3. Moreover, TFPI-2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP-1 was predominant in SCLC tissues and was inversely expressed with TFPI-2 in 35% of cases. These results suggest that downregulation of TFPI-2 expression could favour the development of SCLC.

No MeSH data available.


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Development of highly tumorigenic clones of NCI-H209 Luc cells from human SCLC expressing varying amounts of TFPI-2. (A) Immunostaining of the neuroendocrine differentiation markers, chromogranin, synaptophysin and CD56 (original magnification, ×40). (B) Relative quantification of TFPI-2 transcript levels in NCI-H209 Luc cells stably transfected with an empty vector (852) or a plasmid encoding TFPI-2 (T28 and T6) using qRT-PCR. Results expressed in 2/ΔΔCT/ (β-actin used as control gene) are presented as median ± 1st quartile/3rd quartile from six independent mRNA extractions and qRT-PCR performed in triplicate. (C) TFPI-2 protein expression in ECM extract using a rabbit polyclonal antibody against TFPI-2 by immunoblotting. (D) Immunohistochemistry staining of TFPI-2 (original magnification, ×40).
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fig0002: Development of highly tumorigenic clones of NCI-H209 Luc cells from human SCLC expressing varying amounts of TFPI-2. (A) Immunostaining of the neuroendocrine differentiation markers, chromogranin, synaptophysin and CD56 (original magnification, ×40). (B) Relative quantification of TFPI-2 transcript levels in NCI-H209 Luc cells stably transfected with an empty vector (852) or a plasmid encoding TFPI-2 (T28 and T6) using qRT-PCR. Results expressed in 2/ΔΔCT/ (β-actin used as control gene) are presented as median ± 1st quartile/3rd quartile from six independent mRNA extractions and qRT-PCR performed in triplicate. (C) TFPI-2 protein expression in ECM extract using a rabbit polyclonal antibody against TFPI-2 by immunoblotting. (D) Immunohistochemistry staining of TFPI-2 (original magnification, ×40).

Mentions: To study the potential involvement of TFPI-2 in the aggressiveness of these lung carcinomas, NCI-H209 cells from a SCLC specimen, positive for all three neuroendocrin markers (chromogranin, synaptophysin and CD56 (Fig. 2A)) were used. These cells were stably transfected to express firefly luciferase and a clone was then passaged twice subcutaneously in mice to enhance tumorogenicity. Cells were then transfected with an empty vector to obtain the 852 control clone or with a plasmid encoding TFPI-2, and the two clones expressing TFPI-2 (T6 and T28) were selected for further experiments. Maximum TFPI-2 level was achieved with the T6 clone as assessed by qPCR (Fig. 2B), the TFPI-2 triplet at 32, 30, and 27 kDa being observed from the extracellular matrix of cells by Western blotting (Fig. 2C) and by immunohistochemistry staining (Fig. 2D). TFPI-2 protein was less strongly expressed in the T28 clone than in the T6 clone although no TFPI-2 was found in 852 cells. These cells exhibited a very low level of TFPI-2 mRNA and a 2/ΔΔCT/ of 7.75 was obtained when the relative expression of TFPI-2 cDNA from 852 cells was compared to the reference cDNA. Moreover, TFPI-2 mRNA was increased 6,000-fold and 18,000-fold in the T28 and T6 clone respectively compared to 852 cells.


Beneficial role of overexpression of TFPI-2 on tumour progression in human small cell lung cancer.

Lavergne M, Jourdan ML, Blechet C, Guyetant S, Pape AL, Heuze-Vourc'h N, Courty Y, Lerondel S, Sobilo J, Iochmann S, Reverdiau P - FEBS Open Bio (2013)

Development of highly tumorigenic clones of NCI-H209 Luc cells from human SCLC expressing varying amounts of TFPI-2. (A) Immunostaining of the neuroendocrine differentiation markers, chromogranin, synaptophysin and CD56 (original magnification, ×40). (B) Relative quantification of TFPI-2 transcript levels in NCI-H209 Luc cells stably transfected with an empty vector (852) or a plasmid encoding TFPI-2 (T28 and T6) using qRT-PCR. Results expressed in 2/ΔΔCT/ (β-actin used as control gene) are presented as median ± 1st quartile/3rd quartile from six independent mRNA extractions and qRT-PCR performed in triplicate. (C) TFPI-2 protein expression in ECM extract using a rabbit polyclonal antibody against TFPI-2 by immunoblotting. (D) Immunohistochemistry staining of TFPI-2 (original magnification, ×40).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3722576&req=5

fig0002: Development of highly tumorigenic clones of NCI-H209 Luc cells from human SCLC expressing varying amounts of TFPI-2. (A) Immunostaining of the neuroendocrine differentiation markers, chromogranin, synaptophysin and CD56 (original magnification, ×40). (B) Relative quantification of TFPI-2 transcript levels in NCI-H209 Luc cells stably transfected with an empty vector (852) or a plasmid encoding TFPI-2 (T28 and T6) using qRT-PCR. Results expressed in 2/ΔΔCT/ (β-actin used as control gene) are presented as median ± 1st quartile/3rd quartile from six independent mRNA extractions and qRT-PCR performed in triplicate. (C) TFPI-2 protein expression in ECM extract using a rabbit polyclonal antibody against TFPI-2 by immunoblotting. (D) Immunohistochemistry staining of TFPI-2 (original magnification, ×40).
Mentions: To study the potential involvement of TFPI-2 in the aggressiveness of these lung carcinomas, NCI-H209 cells from a SCLC specimen, positive for all three neuroendocrin markers (chromogranin, synaptophysin and CD56 (Fig. 2A)) were used. These cells were stably transfected to express firefly luciferase and a clone was then passaged twice subcutaneously in mice to enhance tumorogenicity. Cells were then transfected with an empty vector to obtain the 852 control clone or with a plasmid encoding TFPI-2, and the two clones expressing TFPI-2 (T6 and T28) were selected for further experiments. Maximum TFPI-2 level was achieved with the T6 clone as assessed by qPCR (Fig. 2B), the TFPI-2 triplet at 32, 30, and 27 kDa being observed from the extracellular matrix of cells by Western blotting (Fig. 2C) and by immunohistochemistry staining (Fig. 2D). TFPI-2 protein was less strongly expressed in the T28 clone than in the T6 clone although no TFPI-2 was found in 852 cells. These cells exhibited a very low level of TFPI-2 mRNA and a 2/ΔΔCT/ of 7.75 was obtained when the relative expression of TFPI-2 cDNA from 852 cells was compared to the reference cDNA. Moreover, TFPI-2 mRNA was increased 6,000-fold and 18,000-fold in the T28 and T6 clone respectively compared to 852 cells.

Bottom Line: In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer.Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2.We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3.

View Article: PubMed Central - PubMed

Affiliation: EA 6305, Université François Rabelais de Tours, Tours F-37032, France ; Centre d'Etude des Pathologies Respiratoires, UMR 1100/EA6305, Tours F-37032, France.

ABSTRACT
Tissue factor pathway inhibitor-2 (TFPI-2) is a potent inhibitor of plasmin, a protease which is involved in tumour progression by activating (MMPs). This therefore makes TFPI-2 a potential inhibitor of invasiveness and the development of metastases. In this study, low levels of TFPI-2 expression were found in 65% of patients with small cell lung cancer (SCLC), the most aggressive type of lung cancer. To study the impact of TFPI-2 in tumour progression, TFPI-2 was overexpressed in NCI-H209 SCLC cells which were orthotopically implanted in nude mice. Investigations showed that TFPI-2 inhibited lung tumour growth. Such inhibition could be explained in vitro by a decrease in tumour cell viability, blockade of G1/S phase cell cycle transition and an increase in apoptosis shown in NCI-H209 cells expressing TFPI-2. We also demonstrated that TFPI-2 upregulation in NCI-H209 cells decreased MMP expression, particularly by downregulating MMP-1 and MMP-3. Moreover, TFPI-2 inhibited phosphorylation of the MAPK signalling pathway proteins involved in the induction of MMP transcripts, among which MMP-1 was predominant in SCLC tissues and was inversely expressed with TFPI-2 in 35% of cases. These results suggest that downregulation of TFPI-2 expression could favour the development of SCLC.

No MeSH data available.


Related in: MedlinePlus