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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.


Related in: MedlinePlus

Impact of TFPI-2 on MMP expression in 852, T28 and T6 cells (A) Relative expression of MMP-9 transcripts normalised to β-actin transcripts using qRT-PCR. Gelatinase activity of MMP-2 and MMP-9 in 80-fold concentrated conditioned media from cells cultured for 4 days and analysed on gelatin zymography. (B) qRT-PCR for MMP-1 transcripts and representative Western blotting analysis of cell lysate proteins extracted from each clone using β-actin as the loading control. (C) qRT-PCR for MMP-3 transcripts and Western blotting analysis of cell lysate proteins. Data for relative quantifications of mRNA represent median and quartiles (Q1 and Q3) from at least 4 independent experiments. Mann Whitney statistical analysis was used to compare results from 852 cells and cells expressing TFPI-2 (T28 and T6 cells) with *p < 0.05, **p < 0.01, ***p < 0.001. Western blotting and gelatin zymography are representative of at least 4 independent experiments.
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fig0006: Impact of TFPI-2 on MMP expression in 852, T28 and T6 cells (A) Relative expression of MMP-9 transcripts normalised to β-actin transcripts using qRT-PCR. Gelatinase activity of MMP-2 and MMP-9 in 80-fold concentrated conditioned media from cells cultured for 4 days and analysed on gelatin zymography. (B) qRT-PCR for MMP-1 transcripts and representative Western blotting analysis of cell lysate proteins extracted from each clone using β-actin as the loading control. (C) qRT-PCR for MMP-3 transcripts and Western blotting analysis of cell lysate proteins. Data for relative quantifications of mRNA represent median and quartiles (Q1 and Q3) from at least 4 independent experiments. Mann Whitney statistical analysis was used to compare results from 852 cells and cells expressing TFPI-2 (T28 and T6 cells) with *p < 0.05, **p < 0.01, ***p < 0.001. Western blotting and gelatin zymography are representative of at least 4 independent experiments.

Mentions: The proteases known to be affected by TFPI-2 expression are matrix metalloproteinases which are involved in ECM degradation and the spread of cancer cells. Levels of MMP-1, -2, -3 and -9 transcripts were therefore quantified in 852, T28 and T6 cells using RT and qPCR (Fig. 6). Varying amounts of MMP transcripts were quantified in all cell clones, except MMP-2 mRNA which was not detected in any of the cell clones studied. A low level of MMP-9 mRNA was expressed in 852 cells and the transcripts decreased in T28 (p < 0.01) and T6 cells (Fig. 6A). However, gelatinolytic activity of proMMP-9 and proMMP-2 was found in the conditioned medium of all clones, with a higher signal for proMMP-2. Increased activity was obtained with T28 and T6 clones expressing TFPI-2. In contrast, a decrease in MMP-1 mRNA was found for the two clones expressing TFPI-2, but the difference was significant only for the T6 clone (p < 0.001) compared to the 852 control clone (Fig. 6B). Similar results were obtained with MMP-3, as mRNA levels were significantly decreased (p < 0.01) in T28 and T6 cells (13-fold and 5-fold, respectively) (Fig. 6C). Western blotting also clearly showed that this decrease in MMP-1 and MMP-3 transcripts was associated with lower protein expression (Fig. 6B and C). The proform of MMP-1 at 52 kDa was observed, while MMP-3 was visualised as both pro and active forms in the 3 clones (54 and 44 kDa, respectively).


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)

Impact of TFPI-2 on MMP expression in 852, T28 and T6 cells (A) Relative expression of MMP-9 transcripts normalised to β-actin transcripts using qRT-PCR. Gelatinase activity of MMP-2 and MMP-9 in 80-fold concentrated conditioned media from cells cultured for 4 days and analysed on gelatin zymography. (B) qRT-PCR for MMP-1 transcripts and representative Western blotting analysis of cell lysate proteins extracted from each clone using β-actin as the loading control. (C) qRT-PCR for MMP-3 transcripts and Western blotting analysis of cell lysate proteins. Data for relative quantifications of mRNA represent median and quartiles (Q1 and Q3) from at least 4 independent experiments. Mann Whitney statistical analysis was used to compare results from 852 cells and cells expressing TFPI-2 (T28 and T6 cells) with *p < 0.05, **p < 0.01, ***p < 0.001. Western blotting and gelatin zymography are representative of at least 4 independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig0006: Impact of TFPI-2 on MMP expression in 852, T28 and T6 cells (A) Relative expression of MMP-9 transcripts normalised to β-actin transcripts using qRT-PCR. Gelatinase activity of MMP-2 and MMP-9 in 80-fold concentrated conditioned media from cells cultured for 4 days and analysed on gelatin zymography. (B) qRT-PCR for MMP-1 transcripts and representative Western blotting analysis of cell lysate proteins extracted from each clone using β-actin as the loading control. (C) qRT-PCR for MMP-3 transcripts and Western blotting analysis of cell lysate proteins. Data for relative quantifications of mRNA represent median and quartiles (Q1 and Q3) from at least 4 independent experiments. Mann Whitney statistical analysis was used to compare results from 852 cells and cells expressing TFPI-2 (T28 and T6 cells) with *p < 0.05, **p < 0.01, ***p < 0.001. Western blotting and gelatin zymography are representative of at least 4 independent experiments.
Mentions: The proteases known to be affected by TFPI-2 expression are matrix metalloproteinases which are involved in ECM degradation and the spread of cancer cells. Levels of MMP-1, -2, -3 and -9 transcripts were therefore quantified in 852, T28 and T6 cells using RT and qPCR (Fig. 6). Varying amounts of MMP transcripts were quantified in all cell clones, except MMP-2 mRNA which was not detected in any of the cell clones studied. A low level of MMP-9 mRNA was expressed in 852 cells and the transcripts decreased in T28 (p < 0.01) and T6 cells (Fig. 6A). However, gelatinolytic activity of proMMP-9 and proMMP-2 was found in the conditioned medium of all clones, with a higher signal for proMMP-2. Increased activity was obtained with T28 and T6 clones expressing TFPI-2. In contrast, a decrease in MMP-1 mRNA was found for the two clones expressing TFPI-2, but the difference was significant only for the T6 clone (p < 0.001) compared to the 852 control clone (Fig. 6B). Similar results were obtained with MMP-3, as mRNA levels were significantly decreased (p < 0.01) in T28 and T6 cells (13-fold and 5-fold, respectively) (Fig. 6C). Western blotting also clearly showed that this decrease in MMP-1 and MMP-3 transcripts was associated with lower protein expression (Fig. 6B and C). The proform of MMP-1 at 52 kDa was observed, while MMP-3 was visualised as both pro and active forms in the 3 clones (54 and 44 kDa, respectively).

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