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Multi-Leu PACE4 Inhibitor Retention within Cells Is PACE4 Dependent and a Prerequisite for Antiproliferative Activity.

Couture F, Ly K, Levesque C, Kwiatkowska A, Ait-Mohand S, Desjardins R, Guérin B, Day R - Biomed Res Int (2015)

Bottom Line: The overexpression as well as the critical implication of the proprotein convertase PACE4 in prostate cancer progression has been previously reported and supported the development of peptide inhibitors.The multi-Leu peptide, a PACE4-specific inhibitor, was further generated and its capability to be uptaken by tumor xenograft was demonstrated with regard to its PACE4 expression status.PACE4-knockdown associated growth deficiencies were established on the knockdown HepG2, Huh7, and HT1080 cells as well as the antiproliferative effects of the multi-Leu peptide supporting the growth capabilities of PACE4 in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4 ; Department of Surgery/Urology Division, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4.

ABSTRACT
The overexpression as well as the critical implication of the proprotein convertase PACE4 in prostate cancer progression has been previously reported and supported the development of peptide inhibitors. The multi-Leu peptide, a PACE4-specific inhibitor, was further generated and its capability to be uptaken by tumor xenograft was demonstrated with regard to its PACE4 expression status. To investigate whether the uptake of this inhibitor was directly dependent of PACE4 levels, uptake and efflux from cancer cells were evaluated and correlations were established with PACE4 contents on both wild type and PACE4-knockdown cell lines. PACE4-knockdown associated growth deficiencies were established on the knockdown HepG2, Huh7, and HT1080 cells as well as the antiproliferative effects of the multi-Leu peptide supporting the growth capabilities of PACE4 in cancer cells.

No MeSH data available.


Related in: MedlinePlus

64Cu/NOTA-ML uptake and retention correlation with PACE4 levels in cells. (a) Percentage of uptake after 2-hour pulse and (b) percentage of retained radioactivity 2 h after the 2-hour pulse per 1 × 106 cells. (c) Plot of PACE4 expression levels as a function of the log2 of the mean percentage of retained radioactivity after 2-hour efflux. Log2 is applied only to scatter points to make it more visible. Name of the cell line is indicated above each corresponding point. Dashed line represents a linear regression for visual purposes.
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fig4: 64Cu/NOTA-ML uptake and retention correlation with PACE4 levels in cells. (a) Percentage of uptake after 2-hour pulse and (b) percentage of retained radioactivity 2 h after the 2-hour pulse per 1 × 106 cells. (c) Plot of PACE4 expression levels as a function of the log2 of the mean percentage of retained radioactivity after 2-hour efflux. Log2 is applied only to scatter points to make it more visible. Name of the cell line is indicated above each corresponding point. Dashed line represents a linear regression for visual purposes.

Mentions: To assess peptide uptake in these cells, 64Cu/NOTA-ML was prepared with high specific activity (1900–2000 Ci/mmol). Labeled peptide uptake on these cell lines was evaluated by the addition of 5μCi (around 2.5 pmol or 2 ng) to their culture medium. Cell uptake was further assessed during a 2-hour pulse by calculating both peptide at the surface and incorporation in the cells after medium removal and extensive cell washes with PBS. Following a 2-hour pulse, cells were also thoroughly washed from their peptide-containing medium and efflux of the unbound peptide fraction up to an additional 2 h was allowed in order to calculate compound retention to the cells (Figure 3). Interestingly, the 64Cu/NOTA-ML uptakes are significantly reduced in all PACE4-knockdown cells tested when compared to the uptakes in their respective control cells. In prostate cancer cells (Figure 3(a)), PACE4-knockdown diminished 64Cu/NOTA-ML uptake by 40% in both LNCaP (8 versus 13%/106 cells after 2 h) and DU145 cells (1.8 versus 3.6%/106 cells after 2 h). Interestingly, 64Cu/NOTA-ML uptakes in DU145 shPACE4 were comparable to the very low peptide uptake observed in the PACE4-deficient PC3 cells (1.8%/106 cells after 2 h). In HepG2 cell models, uptake reached 64Cu/NOTA-ML 9.6%/106 cells compared to 4.6% in the control and shPACE4 cells, respectively, yielding a disparity greater than 50% (Figure 3(b)). Interestingly, in the Huh7 cells this difference was even bigger (5.5 versus 1.6%/106 cells) representing more than 70% uptake inhibition. Surprisingly, the radiolabeled peptide had a very low uptake in HT1080 cells (1.46%/106 cells); however, it was still higher than the one of its PACE4-knockdown counterparts (1.22%/106 cells; Figure 3(c)). Since peptide radiotracer degradation is negligible during the 2-hour timeframe of this experiment [16] and due to the tight and stable 64Cu chelation in the NOTA moiety [20], the possibility of any artifacts associated with these parameters can be excluded. These differences can be clearly visualized when directly comparing the percentage of uptake after 2 h (Figure 4(a)) and even more clearly when comparing the percentage of 64Cu/NOTA-ML retained after a 2-hour efflux (Figure 4(b)) encompassing the fact that PACE4 levels are readily reflecting the ML peptide uptake. When percentage of a radiolabeled peptide retained in the cells 2 h postpulse was plotted together with PACE4 expression levels (Figure 4(c)), a positive and significant correlation was observed (Spearman r coefficient: 0.7091, P value: 0.0182), which was not the case with neither the percentages of uptake after 2 h (P value: 0.1457, data not shown) nor furin expression levels (P values: 0.1815 and 0.3578 for uptake and retention values resp.). These data indicate that a major part of the 64Cu/NOTA-ML retention directly correlates with PACE4 levels but not with cell entry or furin levels.


Multi-Leu PACE4 Inhibitor Retention within Cells Is PACE4 Dependent and a Prerequisite for Antiproliferative Activity.

Couture F, Ly K, Levesque C, Kwiatkowska A, Ait-Mohand S, Desjardins R, Guérin B, Day R - Biomed Res Int (2015)

64Cu/NOTA-ML uptake and retention correlation with PACE4 levels in cells. (a) Percentage of uptake after 2-hour pulse and (b) percentage of retained radioactivity 2 h after the 2-hour pulse per 1 × 106 cells. (c) Plot of PACE4 expression levels as a function of the log2 of the mean percentage of retained radioactivity after 2-hour efflux. Log2 is applied only to scatter points to make it more visible. Name of the cell line is indicated above each corresponding point. Dashed line represents a linear regression for visual purposes.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig4: 64Cu/NOTA-ML uptake and retention correlation with PACE4 levels in cells. (a) Percentage of uptake after 2-hour pulse and (b) percentage of retained radioactivity 2 h after the 2-hour pulse per 1 × 106 cells. (c) Plot of PACE4 expression levels as a function of the log2 of the mean percentage of retained radioactivity after 2-hour efflux. Log2 is applied only to scatter points to make it more visible. Name of the cell line is indicated above each corresponding point. Dashed line represents a linear regression for visual purposes.
Mentions: To assess peptide uptake in these cells, 64Cu/NOTA-ML was prepared with high specific activity (1900–2000 Ci/mmol). Labeled peptide uptake on these cell lines was evaluated by the addition of 5μCi (around 2.5 pmol or 2 ng) to their culture medium. Cell uptake was further assessed during a 2-hour pulse by calculating both peptide at the surface and incorporation in the cells after medium removal and extensive cell washes with PBS. Following a 2-hour pulse, cells were also thoroughly washed from their peptide-containing medium and efflux of the unbound peptide fraction up to an additional 2 h was allowed in order to calculate compound retention to the cells (Figure 3). Interestingly, the 64Cu/NOTA-ML uptakes are significantly reduced in all PACE4-knockdown cells tested when compared to the uptakes in their respective control cells. In prostate cancer cells (Figure 3(a)), PACE4-knockdown diminished 64Cu/NOTA-ML uptake by 40% in both LNCaP (8 versus 13%/106 cells after 2 h) and DU145 cells (1.8 versus 3.6%/106 cells after 2 h). Interestingly, 64Cu/NOTA-ML uptakes in DU145 shPACE4 were comparable to the very low peptide uptake observed in the PACE4-deficient PC3 cells (1.8%/106 cells after 2 h). In HepG2 cell models, uptake reached 64Cu/NOTA-ML 9.6%/106 cells compared to 4.6% in the control and shPACE4 cells, respectively, yielding a disparity greater than 50% (Figure 3(b)). Interestingly, in the Huh7 cells this difference was even bigger (5.5 versus 1.6%/106 cells) representing more than 70% uptake inhibition. Surprisingly, the radiolabeled peptide had a very low uptake in HT1080 cells (1.46%/106 cells); however, it was still higher than the one of its PACE4-knockdown counterparts (1.22%/106 cells; Figure 3(c)). Since peptide radiotracer degradation is negligible during the 2-hour timeframe of this experiment [16] and due to the tight and stable 64Cu chelation in the NOTA moiety [20], the possibility of any artifacts associated with these parameters can be excluded. These differences can be clearly visualized when directly comparing the percentage of uptake after 2 h (Figure 4(a)) and even more clearly when comparing the percentage of 64Cu/NOTA-ML retained after a 2-hour efflux (Figure 4(b)) encompassing the fact that PACE4 levels are readily reflecting the ML peptide uptake. When percentage of a radiolabeled peptide retained in the cells 2 h postpulse was plotted together with PACE4 expression levels (Figure 4(c)), a positive and significant correlation was observed (Spearman r coefficient: 0.7091, P value: 0.0182), which was not the case with neither the percentages of uptake after 2 h (P value: 0.1457, data not shown) nor furin expression levels (P values: 0.1815 and 0.3578 for uptake and retention values resp.). These data indicate that a major part of the 64Cu/NOTA-ML retention directly correlates with PACE4 levels but not with cell entry or furin levels.

Bottom Line: The overexpression as well as the critical implication of the proprotein convertase PACE4 in prostate cancer progression has been previously reported and supported the development of peptide inhibitors.The multi-Leu peptide, a PACE4-specific inhibitor, was further generated and its capability to be uptaken by tumor xenograft was demonstrated with regard to its PACE4 expression status.PACE4-knockdown associated growth deficiencies were established on the knockdown HepG2, Huh7, and HT1080 cells as well as the antiproliferative effects of the multi-Leu peptide supporting the growth capabilities of PACE4 in cancer cells.

View Article: PubMed Central - PubMed

Affiliation: Institut de Pharmacologie de Sherbrooke, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4 ; Department of Surgery/Urology Division, Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4.

ABSTRACT
The overexpression as well as the critical implication of the proprotein convertase PACE4 in prostate cancer progression has been previously reported and supported the development of peptide inhibitors. The multi-Leu peptide, a PACE4-specific inhibitor, was further generated and its capability to be uptaken by tumor xenograft was demonstrated with regard to its PACE4 expression status. To investigate whether the uptake of this inhibitor was directly dependent of PACE4 levels, uptake and efflux from cancer cells were evaluated and correlations were established with PACE4 contents on both wild type and PACE4-knockdown cell lines. PACE4-knockdown associated growth deficiencies were established on the knockdown HepG2, Huh7, and HT1080 cells as well as the antiproliferative effects of the multi-Leu peptide supporting the growth capabilities of PACE4 in cancer cells.

No MeSH data available.


Related in: MedlinePlus