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Insulin-like-growth-factor-binding-protein-3 (IGFBP-3) contrasts melanoma progression in vitro and in vivo.

Naspi A, Panasiti V, Abbate F, Roberti V, Devirgiliis V, Curzio M, Borghi M, Lozupone F, Carotti S, Morini S, Gaudio E, Calvieri S, Londei P - PLoS ONE (2014)

Bottom Line: Insulin-like-factor-binding-protein 3 (IGFBP-3) is known to modulate the activity of insulin-like growth factors (IGFs) besides having a number of IGF-independent effects on cell growth and survival.In the present work, we have evaluated the levels of IGFBP-3 in the blood serum and tissues of patients affected by cutaneous melanoma, showing that loss of IGFBP-3 from both is strongly correlated with disease progression and reduced survival.In summary, IGFBP-3 appears to exert a specific inhibitory effect on melanoma growth and dissemination, suggesting that it may qualify as a useful therapeutic agent in melanomas and perhaps other cancers, at the least as a valid adjuvant therapy during treatment with conventional anti-tumoral drugs.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci-Bolognetti, Dpt. Biotecnologie Cellulari ed Ematologia, University of Rome Sapienza, Rome, Italy.

ABSTRACT
Insulin-like-factor-binding-protein 3 (IGFBP-3) is known to modulate the activity of insulin-like growth factors (IGFs) besides having a number of IGF-independent effects on cell growth and survival. IGFBP-3 has been reported to decrease significantly in the blood serum of patients affected by certain cancers. In the present work, we have evaluated the levels of IGFBP-3 in the blood serum and tissues of patients affected by cutaneous melanoma, showing that loss of IGFBP-3 from both is strongly correlated with disease progression and reduced survival. In vitro treatment with IGFBP-3 of human and murine metastatic melanoma cell lines specifically inhibited the cells' migratory and invasive behaviour, inducing up-regulation of melanocytic differentiation markers such as tyrosinase activity and melanin content. A molecular analysis of the cellular pathways transducing the effect of IGFBP-3 implicated the Akt-GSK3β axis. Moreover, administration of IGFBP-3 in vivo to SCID mice inoculated with human metastatic melanoma cells strongly reduced or completely inhibited tumor growth. In summary, IGFBP-3 appears to exert a specific inhibitory effect on melanoma growth and dissemination, suggesting that it may qualify as a useful therapeutic agent in melanomas and perhaps other cancers, at the least as a valid adjuvant therapy during treatment with conventional anti-tumoral drugs.

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IGFBP-3 up-regulates tyrosinase activity and increases melanin content in Me501 cells.(A) Tyrosinase activity measured by L-DOPA oxidation (left panel) and melanin content determined by measuring the absorbance at 405 nm (right panel) of lysates of Me501 cells grown in 2% serum, untreated or treated for 96 h with IGFBP-3 (2 µg/mL). (B) Tyrosinase activity of lysates of Me501 cell grown in the absence of serum for 24 and 48 h, treated and untreated with IGFBP-3 (C) Western blot analysis of tyrosinase content in Me501 cells after 0, 24 and 48 h of treatment with IGFBP-3 (2 µg/mL). (D) Phase-contrast microscopy of untreated and IGFBP-3-treated (2 µg/mL) Me501 cells, grown in the presence and in the absence of serum. Original magnification X100. (E, F) IGFBP-3 immuno-histochemical positivity in primary melanoma correlates positively with melanin content. IGFBP-3 is stained red, while melanin stains as dark-brown spots (E) X200 magnification (F) X400 magnification. The asterisks mark the IGFBP-3 immuno-positive melanocyte nests, the white and black arrows indicate the clusters of melanin and IGFBP–3 positive melanocytes, respectively. (G) Box plots showing the correlation between IGFBP-3 tumor score (calculated as described in the Methods) and sample pigmentation. P value was calculated by Mann-Whitney U test.
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pone-0098641-g008: IGFBP-3 up-regulates tyrosinase activity and increases melanin content in Me501 cells.(A) Tyrosinase activity measured by L-DOPA oxidation (left panel) and melanin content determined by measuring the absorbance at 405 nm (right panel) of lysates of Me501 cells grown in 2% serum, untreated or treated for 96 h with IGFBP-3 (2 µg/mL). (B) Tyrosinase activity of lysates of Me501 cell grown in the absence of serum for 24 and 48 h, treated and untreated with IGFBP-3 (C) Western blot analysis of tyrosinase content in Me501 cells after 0, 24 and 48 h of treatment with IGFBP-3 (2 µg/mL). (D) Phase-contrast microscopy of untreated and IGFBP-3-treated (2 µg/mL) Me501 cells, grown in the presence and in the absence of serum. Original magnification X100. (E, F) IGFBP-3 immuno-histochemical positivity in primary melanoma correlates positively with melanin content. IGFBP-3 is stained red, while melanin stains as dark-brown spots (E) X200 magnification (F) X400 magnification. The asterisks mark the IGFBP-3 immuno-positive melanocyte nests, the white and black arrows indicate the clusters of melanin and IGFBP–3 positive melanocytes, respectively. (G) Box plots showing the correlation between IGFBP-3 tumor score (calculated as described in the Methods) and sample pigmentation. P value was calculated by Mann-Whitney U test.

Mentions: Besides losing the ability of migrate and invade, the IGFBP-3 treated cells kept in culture for a few days acquired a more dendritic appearance and looked visibly blacker, suggesting that they were steered towards melanocytic differentiation (Fig. 8D). This behaviour is in agreement with the fact that one the downstream targets of Akt most affected by IGFBP-3 treatment was GSK3β, a factor whose activation by dephosphorylation triggers melanin synthesis and differentiation in melanocytes [25]. Accordingly, we tested whether IGFBP-3 treatment resulted in activation of the melanin synthesis pathway, by measuring the tyrosinase activity and the amount of melanin in treated and untreated Me501 cells. As shown in Fig. 8 (A,B,C), IGFBP-3 treated Me501 cells had a significantly higher tyrosinase activity and melanin content compared to untreated cells. These in vitro results were corroborated by immuno-staining of tumor samples taken from patients. As shown in Fig. 8 (E,F,G), only tumor tissues positive for IGFBP-3 contained significant amounts of melanin.


Insulin-like-growth-factor-binding-protein-3 (IGFBP-3) contrasts melanoma progression in vitro and in vivo.

Naspi A, Panasiti V, Abbate F, Roberti V, Devirgiliis V, Curzio M, Borghi M, Lozupone F, Carotti S, Morini S, Gaudio E, Calvieri S, Londei P - PLoS ONE (2014)

IGFBP-3 up-regulates tyrosinase activity and increases melanin content in Me501 cells.(A) Tyrosinase activity measured by L-DOPA oxidation (left panel) and melanin content determined by measuring the absorbance at 405 nm (right panel) of lysates of Me501 cells grown in 2% serum, untreated or treated for 96 h with IGFBP-3 (2 µg/mL). (B) Tyrosinase activity of lysates of Me501 cell grown in the absence of serum for 24 and 48 h, treated and untreated with IGFBP-3 (C) Western blot analysis of tyrosinase content in Me501 cells after 0, 24 and 48 h of treatment with IGFBP-3 (2 µg/mL). (D) Phase-contrast microscopy of untreated and IGFBP-3-treated (2 µg/mL) Me501 cells, grown in the presence and in the absence of serum. Original magnification X100. (E, F) IGFBP-3 immuno-histochemical positivity in primary melanoma correlates positively with melanin content. IGFBP-3 is stained red, while melanin stains as dark-brown spots (E) X200 magnification (F) X400 magnification. The asterisks mark the IGFBP-3 immuno-positive melanocyte nests, the white and black arrows indicate the clusters of melanin and IGFBP–3 positive melanocytes, respectively. (G) Box plots showing the correlation between IGFBP-3 tumor score (calculated as described in the Methods) and sample pigmentation. P value was calculated by Mann-Whitney U test.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048209&req=5

pone-0098641-g008: IGFBP-3 up-regulates tyrosinase activity and increases melanin content in Me501 cells.(A) Tyrosinase activity measured by L-DOPA oxidation (left panel) and melanin content determined by measuring the absorbance at 405 nm (right panel) of lysates of Me501 cells grown in 2% serum, untreated or treated for 96 h with IGFBP-3 (2 µg/mL). (B) Tyrosinase activity of lysates of Me501 cell grown in the absence of serum for 24 and 48 h, treated and untreated with IGFBP-3 (C) Western blot analysis of tyrosinase content in Me501 cells after 0, 24 and 48 h of treatment with IGFBP-3 (2 µg/mL). (D) Phase-contrast microscopy of untreated and IGFBP-3-treated (2 µg/mL) Me501 cells, grown in the presence and in the absence of serum. Original magnification X100. (E, F) IGFBP-3 immuno-histochemical positivity in primary melanoma correlates positively with melanin content. IGFBP-3 is stained red, while melanin stains as dark-brown spots (E) X200 magnification (F) X400 magnification. The asterisks mark the IGFBP-3 immuno-positive melanocyte nests, the white and black arrows indicate the clusters of melanin and IGFBP–3 positive melanocytes, respectively. (G) Box plots showing the correlation between IGFBP-3 tumor score (calculated as described in the Methods) and sample pigmentation. P value was calculated by Mann-Whitney U test.
Mentions: Besides losing the ability of migrate and invade, the IGFBP-3 treated cells kept in culture for a few days acquired a more dendritic appearance and looked visibly blacker, suggesting that they were steered towards melanocytic differentiation (Fig. 8D). This behaviour is in agreement with the fact that one the downstream targets of Akt most affected by IGFBP-3 treatment was GSK3β, a factor whose activation by dephosphorylation triggers melanin synthesis and differentiation in melanocytes [25]. Accordingly, we tested whether IGFBP-3 treatment resulted in activation of the melanin synthesis pathway, by measuring the tyrosinase activity and the amount of melanin in treated and untreated Me501 cells. As shown in Fig. 8 (A,B,C), IGFBP-3 treated Me501 cells had a significantly higher tyrosinase activity and melanin content compared to untreated cells. These in vitro results were corroborated by immuno-staining of tumor samples taken from patients. As shown in Fig. 8 (E,F,G), only tumor tissues positive for IGFBP-3 contained significant amounts of melanin.

Bottom Line: Insulin-like-factor-binding-protein 3 (IGFBP-3) is known to modulate the activity of insulin-like growth factors (IGFs) besides having a number of IGF-independent effects on cell growth and survival.In the present work, we have evaluated the levels of IGFBP-3 in the blood serum and tissues of patients affected by cutaneous melanoma, showing that loss of IGFBP-3 from both is strongly correlated with disease progression and reduced survival.In summary, IGFBP-3 appears to exert a specific inhibitory effect on melanoma growth and dissemination, suggesting that it may qualify as a useful therapeutic agent in melanomas and perhaps other cancers, at the least as a valid adjuvant therapy during treatment with conventional anti-tumoral drugs.

View Article: PubMed Central - PubMed

Affiliation: Istituto Pasteur-Fondazione Cenci-Bolognetti, Dpt. Biotecnologie Cellulari ed Ematologia, University of Rome Sapienza, Rome, Italy.

ABSTRACT
Insulin-like-factor-binding-protein 3 (IGFBP-3) is known to modulate the activity of insulin-like growth factors (IGFs) besides having a number of IGF-independent effects on cell growth and survival. IGFBP-3 has been reported to decrease significantly in the blood serum of patients affected by certain cancers. In the present work, we have evaluated the levels of IGFBP-3 in the blood serum and tissues of patients affected by cutaneous melanoma, showing that loss of IGFBP-3 from both is strongly correlated with disease progression and reduced survival. In vitro treatment with IGFBP-3 of human and murine metastatic melanoma cell lines specifically inhibited the cells' migratory and invasive behaviour, inducing up-regulation of melanocytic differentiation markers such as tyrosinase activity and melanin content. A molecular analysis of the cellular pathways transducing the effect of IGFBP-3 implicated the Akt-GSK3β axis. Moreover, administration of IGFBP-3 in vivo to SCID mice inoculated with human metastatic melanoma cells strongly reduced or completely inhibited tumor growth. In summary, IGFBP-3 appears to exert a specific inhibitory effect on melanoma growth and dissemination, suggesting that it may qualify as a useful therapeutic agent in melanomas and perhaps other cancers, at the least as a valid adjuvant therapy during treatment with conventional anti-tumoral drugs.

Show MeSH
Related in: MedlinePlus