Limits...
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 apoptosis of MB cell lines.Flow cytometry analysis showed that treatment with 4-HPR (1–20 μM, 6–24 hours) resulted in DAOY (A, B) and ONS-76 (C, D) increased rates in apoptotic (AnnexinV+7-AAD-/+) and necrotic (7-AAD+) cells. Western blotting analysis (WB) for caspase-9 and PARP on cell lysate obtained from DAOY (E, G) and ONS-76 (F, H) cells treated for 24 hours with 4-HPR (2.5–10 μM) show an increase of full length/cleaved Caspase-9 and cleaved PARP-1, confirming data obtained by flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4930187&req=5

pone.0154111.g001: Effects of fenretinide (4-HPR) on apoptosis of MB cell lines.Flow cytometry analysis showed that treatment with 4-HPR (1–20 μM, 6–24 hours) resulted in DAOY (A, B) and ONS-76 (C, D) increased rates in apoptotic (AnnexinV+7-AAD-/+) and necrotic (7-AAD+) cells. Western blotting analysis (WB) for caspase-9 and PARP on cell lysate obtained from DAOY (E, G) and ONS-76 (F, H) cells treated for 24 hours with 4-HPR (2.5–10 μM) show an increase of full length/cleaved Caspase-9 and cleaved PARP-1, confirming data obtained by flow cytometry.

Mentions: To evaluate whether 4-HPR triggers apoptosis in MB cells, we performed flow cytometry analyses by staining control and treated cells with Annexin V and 7-AAD. The data showed that increased concentrations of 4-HPR treatment enhanced cell death of both cell lines (Fig 1A–1D). Following 6h treatment, ONS-76 cell line seemed to be more sensitive to 4-HPR than DAOY cells. Even thougth, after 24 h treatment, a lower percentage of DAOY viable cells was observed compared with ONS-76, suggesting that longer exposition might be needed to induce apoptosis in DAOY cells (Fig 1B). Apoptosis appeared to be a key mechanism induced by 4-HPR, as confirmed by Western Blot analysis for selected mitochondrial pathway associated with caspase-9 and poly ADP-ribose polymerase (PARP) levels, on both DAOY and ONS-76 cell lines treated with 2.5, 5 and 10 μM of 4-HPR for 24h (Fig 1E–1H). 4-HPR increased the expression of full length and active caspase-9 as well as cleaved PARP expression in a dose-dependent manner after 24hs of treatment in both DAOY and ONS-76 cells (Fig 1E–1H). Enhanced levels of Caspase-9 were particularly evident in ONS-76 cells (Fig 1F). Together, these results suggested that 4-HPR triggers apoptosis in MB cells by activating a mitochondrial pathway.


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 apoptosis of MB cell lines.Flow cytometry analysis showed that treatment with 4-HPR (1–20 μM, 6–24 hours) resulted in DAOY (A, B) and ONS-76 (C, D) increased rates in apoptotic (AnnexinV+7-AAD-/+) and necrotic (7-AAD+) cells. Western blotting analysis (WB) for caspase-9 and PARP on cell lysate obtained from DAOY (E, G) and ONS-76 (F, H) cells treated for 24 hours with 4-HPR (2.5–10 μM) show an increase of full length/cleaved Caspase-9 and cleaved PARP-1, confirming data obtained by flow cytometry.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0154111.g001: Effects of fenretinide (4-HPR) on apoptosis of MB cell lines.Flow cytometry analysis showed that treatment with 4-HPR (1–20 μM, 6–24 hours) resulted in DAOY (A, B) and ONS-76 (C, D) increased rates in apoptotic (AnnexinV+7-AAD-/+) and necrotic (7-AAD+) cells. Western blotting analysis (WB) for caspase-9 and PARP on cell lysate obtained from DAOY (E, G) and ONS-76 (F, H) cells treated for 24 hours with 4-HPR (2.5–10 μM) show an increase of full length/cleaved Caspase-9 and cleaved PARP-1, confirming data obtained by flow cytometry.
Mentions: To evaluate whether 4-HPR triggers apoptosis in MB cells, we performed flow cytometry analyses by staining control and treated cells with Annexin V and 7-AAD. The data showed that increased concentrations of 4-HPR treatment enhanced cell death of both cell lines (Fig 1A–1D). Following 6h treatment, ONS-76 cell line seemed to be more sensitive to 4-HPR than DAOY cells. Even thougth, after 24 h treatment, a lower percentage of DAOY viable cells was observed compared with ONS-76, suggesting that longer exposition might be needed to induce apoptosis in DAOY cells (Fig 1B). Apoptosis appeared to be a key mechanism induced by 4-HPR, as confirmed by Western Blot analysis for selected mitochondrial pathway associated with caspase-9 and poly ADP-ribose polymerase (PARP) levels, on both DAOY and ONS-76 cell lines treated with 2.5, 5 and 10 μM of 4-HPR for 24h (Fig 1E–1H). 4-HPR increased the expression of full length and active caspase-9 as well as cleaved PARP expression in a dose-dependent manner after 24hs of treatment in both DAOY and ONS-76 cells (Fig 1E–1H). Enhanced levels of Caspase-9 were particularly evident in ONS-76 cells (Fig 1F). Together, these results suggested that 4-HPR triggers apoptosis in MB cells by activating a mitochondrial pathway.

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