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Fenretinide (4-HPR) Targets Caspase-9, ERK 1/2 and the Wnt3a/β-Catenin Pathway in Medulloblastoma Cells and Medulloblastoma Cell Spheroids.

Bassani B, Bartolini D, Pagani A, Principi E, Zollo M, Noonan DM, Albini A, Bruno A - PLoS ONE (2016)

Bottom Line: Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed.Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB.Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities.

View Article: PubMed Central - PubMed

Affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.

ABSTRACT
Medulloblastoma (MB), a neuroectodermal tumor arising in the cerebellum, represents the most frequent childhood brain malignancy. Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl) retinamide (4-HPR, fenretinide), a synthetic analogue of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopreventive and chemotherapeutic agent for various neoplasms, from breast cancer to neuroblastoma. Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB. Flow cytometry analysis was performed to evaluate 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects. Functional analysis to determine 4-HPR ability to interfere with MB cell migration and invasion were performed. Western Blot analysis were used to investigate the crucial molecules involved in selected signaling pathways associated with apoptosis (caspase-9 and PARP-1), cell survival (ERK 1/2) and tumor progression (Wnt3a and β-catenin). We show that 4-HPR induces caspase 9-dependent cell death in DAOY and ONS-76 cells, associated with increased ROS generation, suggesting that free radical intermediates might be directly involved. We observed 4-HPR induction of cell cycle arrest in G1/S phase, inactivated β-catenin, and inhibition of MB cell migration and invasion. We also evaluated the ability of 4-HPR to target MB cancer-stem/cancer-initiating cells, using an MB spheroids model, followed by flow cytometry and quantitative real-time PCR. 4-HPR treatment reduced DAOY and ONS-76 spheroid formation, in term of number and size. Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities. Finally, we analyzed 4-HPR ability to inhibit MB tumor cell growth in vivo in nude mice. Taken together, our data suggest that 4-HPR targets both parental and MB tumor stem/initiating cell-like populations. Since 4-HPR exerts low toxicity, it could represent a valid compound in the treatment of human MB.

No MeSH data available.


Related in: MedlinePlus

Effects of fenretinide (4-HPR) on MB cells migration and invasion.Fenretinide (4-HPR) reduces DAOY and ONS-76 cell migration (A, B) and invasion (C, D). Results are representative of 3 independent experiments and showed as Mean ± SEM *p <0.05; **p<0.001; ***p<0.001(one-way ANOVA).
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pone.0154111.g004: Effects of fenretinide (4-HPR) on MB cells migration and invasion.Fenretinide (4-HPR) reduces DAOY and ONS-76 cell migration (A, B) and invasion (C, D). Results are representative of 3 independent experiments and showed as Mean ± SEM *p <0.05; **p<0.001; ***p<0.001(one-way ANOVA).

Mentions: We also investigated the effect of 4-HPR on MB cell lines invasion and migration in vitro, using the chemoinvasion and chemotaxis assays, as previously described [55, 56]. 4-HPR (2.5–10 μM) inhibited both DAOY and ONS-76 migration and invasion in a dose-dependent manner (Fig 4A, 4B, 4C and 4D). Since Wnt3a/β-Catenin pathway is one of the major signaling pathways involved cancer cell migration and invasion (ref), we studied whether 4-HPR was able to interfere with the Wnt3a pathway in MB cells. 4-HPR induced a down-regulation of Wnt3a levels in both DAOY and ONS-76 cells (Fig 5A and 5B) as western blot analyses. We also investigated the effects of 4-HPR on the modulation of Axin-1, GSκ3β and β-Catenin, key downstream factors in Wnt pathway. 5 and 10 μM 4-HPR decreased the expression levels of Axin-1 (Fig 5C and 5D) and phospho-GSκ3β (Fig 5E and 5F) after 24 hours of treatment of both MB cell lines. Decreased levels of β-Catenin (Fig 5G and 5H) were also observed following 4-HPR treatment in both cells This could be correlated with a reduced invasion and migration of MB cells and to increased levels of apoptotic cell rates after 4-HPR treatment observed in Fig 4.


Fenretinide (4-HPR) Targets Caspase-9, ERK 1/2 and the Wnt3a/β-Catenin Pathway in Medulloblastoma Cells and Medulloblastoma Cell Spheroids.

Bassani B, Bartolini D, Pagani A, Principi E, Zollo M, Noonan DM, Albini A, Bruno A - PLoS ONE (2016)

Effects of fenretinide (4-HPR) on MB cells migration and invasion.Fenretinide (4-HPR) reduces DAOY and ONS-76 cell migration (A, B) and invasion (C, D). Results are representative of 3 independent experiments and showed as Mean ± SEM *p <0.05; **p<0.001; ***p<0.001(one-way ANOVA).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4930187&req=5

pone.0154111.g004: Effects of fenretinide (4-HPR) on MB cells migration and invasion.Fenretinide (4-HPR) reduces DAOY and ONS-76 cell migration (A, B) and invasion (C, D). Results are representative of 3 independent experiments and showed as Mean ± SEM *p <0.05; **p<0.001; ***p<0.001(one-way ANOVA).
Mentions: We also investigated the effect of 4-HPR on MB cell lines invasion and migration in vitro, using the chemoinvasion and chemotaxis assays, as previously described [55, 56]. 4-HPR (2.5–10 μM) inhibited both DAOY and ONS-76 migration and invasion in a dose-dependent manner (Fig 4A, 4B, 4C and 4D). Since Wnt3a/β-Catenin pathway is one of the major signaling pathways involved cancer cell migration and invasion (ref), we studied whether 4-HPR was able to interfere with the Wnt3a pathway in MB cells. 4-HPR induced a down-regulation of Wnt3a levels in both DAOY and ONS-76 cells (Fig 5A and 5B) as western blot analyses. We also investigated the effects of 4-HPR on the modulation of Axin-1, GSκ3β and β-Catenin, key downstream factors in Wnt pathway. 5 and 10 μM 4-HPR decreased the expression levels of Axin-1 (Fig 5C and 5D) and phospho-GSκ3β (Fig 5E and 5F) after 24 hours of treatment of both MB cell lines. Decreased levels of β-Catenin (Fig 5G and 5H) were also observed following 4-HPR treatment in both cells This could be correlated with a reduced invasion and migration of MB cells and to increased levels of apoptotic cell rates after 4-HPR treatment observed in Fig 4.

Bottom Line: Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed.Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB.Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities.

View Article: PubMed Central - PubMed

Affiliation: Scientific and Technological Pole, IRCCS MultiMedica, Milano, Italy.

ABSTRACT
Medulloblastoma (MB), a neuroectodermal tumor arising in the cerebellum, represents the most frequent childhood brain malignancy. Current treatments for MB combine radiation and chemotherapy and are often associated with relevant side effects; novel therapeutic strategies are urgently needed. N-(4-Hydroxyphenyl) retinamide (4-HPR, fenretinide), a synthetic analogue of all-trans retinoic acid, has emerged as a promising and well-tolerated cancer chemopreventive and chemotherapeutic agent for various neoplasms, from breast cancer to neuroblastoma. Here we investigated the effects of 4-HPR on MB cell lines and identified the mechanism of action for a potential use in therapy of MB. Flow cytometry analysis was performed to evaluate 4-HPR induction of apoptosis and oxygen reactive species (ROS) production, as well as cell cycle effects. Functional analysis to determine 4-HPR ability to interfere with MB cell migration and invasion were performed. Western Blot analysis were used to investigate the crucial molecules involved in selected signaling pathways associated with apoptosis (caspase-9 and PARP-1), cell survival (ERK 1/2) and tumor progression (Wnt3a and β-catenin). We show that 4-HPR induces caspase 9-dependent cell death in DAOY and ONS-76 cells, associated with increased ROS generation, suggesting that free radical intermediates might be directly involved. We observed 4-HPR induction of cell cycle arrest in G1/S phase, inactivated β-catenin, and inhibition of MB cell migration and invasion. We also evaluated the ability of 4-HPR to target MB cancer-stem/cancer-initiating cells, using an MB spheroids model, followed by flow cytometry and quantitative real-time PCR. 4-HPR treatment reduced DAOY and ONS-76 spheroid formation, in term of number and size. Decreased expression of the surface markers CD133+ and ABCG2+ as well as Oct-4 and Sox-2 gene expression were observed on BTICs treated with 4-HPR further reducing BITIC invasive activities. Finally, we analyzed 4-HPR ability to inhibit MB tumor cell growth in vivo in nude mice. Taken together, our data suggest that 4-HPR targets both parental and MB tumor stem/initiating cell-like populations. Since 4-HPR exerts low toxicity, it could represent a valid compound in the treatment of human MB.

No MeSH data available.


Related in: MedlinePlus