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AF1q: a novel mediator of basal and 4-HPR-induced apoptosis in ovarian cancer cells.

Tiberio P, Cavadini E, Callari M, Daidone MG, Appierto V - PLoS ONE (2012)

Bottom Line: Through gene silencing, AF1q was found functionally involved in 4-HPR-induced apoptosis in A2780, an ovarian cancer cell line highly sensitive to retinoid growth inhibitory and apoptotic effects.Inhibition of the signaling intermediates of the 4-HPR apoptotic cascade showed that AF1q upregulation was depended on prior generation of ROS, induction of ER stress response, JNK activation, and PLAB upmodulation.The study expands the knowledge of the 4-HPR mechanism of action, which has not yet been completely elucidated, identifying AF1q as a novel mediator of retinoid anticancer activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. paola.tiberio@istitutotumori.mi.it

ABSTRACT

Background: Fenretinide (4-HPR) is a synthetic retinoid that exhibits potent antitumor and chemopreventive activities against different malignancies, including ovarian tumors. We previously showed that in ovarian cancer cells, 4-HPR induces apoptosis through a signaling cascade starting from reactive oxygen species (ROS) generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and induction of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Since recent studies have shown that the oncogene ALL1-fused from chromosome 1q (AF1q), a retinoic acid target gene, is implicated in apoptosis induction by several therapeutic agents, we investigated its possible involvement in the apoptosis induced by 4-HPR in ovarian cancer cells.

Methodology/principal findings: Protein expression analysis, performed in ovarian cancer cells and extended to other histotypes (breast, neuroblastoma, and cervical), revealed that 4-HPR enhanced AF1q expression in cancer cells sensitive to the retinoid but not in resistant cells. Through gene silencing, AF1q was found functionally involved in 4-HPR-induced apoptosis in A2780, an ovarian cancer cell line highly sensitive to retinoid growth inhibitory and apoptotic effects. Inhibition of the signaling intermediates of the 4-HPR apoptotic cascade showed that AF1q upregulation was depended on prior generation of ROS, induction of ER stress response, JNK activation, and PLAB upmodulation. Finally, we found that direct overexpression of AF1q, in the absence of external stimuli, increased apoptosis in ovarian cancer cell lines.

Conclusions/significance: The study expands the knowledge of the 4-HPR mechanism of action, which has not yet been completely elucidated, identifying AF1q as a novel mediator of retinoid anticancer activity. In addition, we demonstrate, for the first time, that AF1q plays a role in the onset of basal apoptosis in ovarian cancer cells, thus providing new information about the activity of this protein whose biologic functions are mostly unknown.

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Relationship between AF1q upregulation and 4-HPR-induced signaling cascade.Western blot analysis for AF1q expression in A2780 cells treated for 24 hours with 5 µM 4-HPR, with or without 100 µM vitamin C (A), 10 µM salubrinal (B), or 10 µM SP600125 (C). (D) Western blot analysis for AF1q expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blots were incubated with actin antibody.
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pone-0039968-g005: Relationship between AF1q upregulation and 4-HPR-induced signaling cascade.Western blot analysis for AF1q expression in A2780 cells treated for 24 hours with 5 µM 4-HPR, with or without 100 µM vitamin C (A), 10 µM salubrinal (B), or 10 µM SP600125 (C). (D) Western blot analysis for AF1q expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blots were incubated with actin antibody.

Mentions: We previously reported that 4-HPR-induced apoptosis occurs through a signaling cascade involving ROS generation, ER stress induction, JNK activation and PLAB upregulation (ROS → ER stress → JNK → PLAB) [11], [12]. We thus evaluated whether AF1q was involved in the aforementioned apoptotic pathway by testing the effects of the inhibition of these signaling intermediates on 4-HPR-induced AF1q upmodulation. To this aim, A2780 cells were co-treated with 4-HPR and, in turn, with the antioxidant vitamin C, the ER stress inhibitor salubrinal, and the pharmacological JNK inhibitor SP600125, which have been demonstrated to prevent ROS generation, eIF2α dephosphorylation, and JNK activation, respectively, and to strongly reduce 4-HPR-induced apoptosis in the same cell line [11]. The addition of 100 µM vitamin C to A2780 cells treated with 5 µM 4-HPR for 24 hours abrogated the 4-HPR-induced upregulation of AF1q (Figure 5A). Similarly, the addition of 10 µM salubrinal or 10 µM SP600125 strongly reduced 4-HPR-induced AF1q upregulation (Figure 5B and 5C). To investigate whether the upregulation of AF1q was a downstream event also of PLAB upregulation induced by 4-HPR, PLAB has been knocked down using a synthetic siRNA targeting PLAB mRNA. As shown in Figure 5D, the treatment of A2780 cells with the PLAB-specific siRNA was able to strongly reduce 4-HPR-induced PLAB as well as AF1q upmodulation compared with the cells transfected with a control siRNA. Conversely, AF1q silencing did not affect 4-HPR-induced PLAB upregulation (Figure 5E). The aforementioned data indicated that in A2780 cells AF1q upregulation was dependent on prior activation of the 4-HPR-induced-ROS-related signaling cascade and that its upmodulation occurred downstream of PLAB induction (Figure 6).


AF1q: a novel mediator of basal and 4-HPR-induced apoptosis in ovarian cancer cells.

Tiberio P, Cavadini E, Callari M, Daidone MG, Appierto V - PLoS ONE (2012)

Relationship between AF1q upregulation and 4-HPR-induced signaling cascade.Western blot analysis for AF1q expression in A2780 cells treated for 24 hours with 5 µM 4-HPR, with or without 100 µM vitamin C (A), 10 µM salubrinal (B), or 10 µM SP600125 (C). (D) Western blot analysis for AF1q expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blots were incubated with actin antibody.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3383705&req=5

pone-0039968-g005: Relationship between AF1q upregulation and 4-HPR-induced signaling cascade.Western blot analysis for AF1q expression in A2780 cells treated for 24 hours with 5 µM 4-HPR, with or without 100 µM vitamin C (A), 10 µM salubrinal (B), or 10 µM SP600125 (C). (D) Western blot analysis for AF1q expression in A2780 cells stably transfected with a plasmid containing a PLAB siRNA or a scrambled nonsilencing siRNA following addition of 5 µM 4-HPR for 24 hours. As a control for loading, the blots were incubated with actin antibody.
Mentions: We previously reported that 4-HPR-induced apoptosis occurs through a signaling cascade involving ROS generation, ER stress induction, JNK activation and PLAB upregulation (ROS → ER stress → JNK → PLAB) [11], [12]. We thus evaluated whether AF1q was involved in the aforementioned apoptotic pathway by testing the effects of the inhibition of these signaling intermediates on 4-HPR-induced AF1q upmodulation. To this aim, A2780 cells were co-treated with 4-HPR and, in turn, with the antioxidant vitamin C, the ER stress inhibitor salubrinal, and the pharmacological JNK inhibitor SP600125, which have been demonstrated to prevent ROS generation, eIF2α dephosphorylation, and JNK activation, respectively, and to strongly reduce 4-HPR-induced apoptosis in the same cell line [11]. The addition of 100 µM vitamin C to A2780 cells treated with 5 µM 4-HPR for 24 hours abrogated the 4-HPR-induced upregulation of AF1q (Figure 5A). Similarly, the addition of 10 µM salubrinal or 10 µM SP600125 strongly reduced 4-HPR-induced AF1q upregulation (Figure 5B and 5C). To investigate whether the upregulation of AF1q was a downstream event also of PLAB upregulation induced by 4-HPR, PLAB has been knocked down using a synthetic siRNA targeting PLAB mRNA. As shown in Figure 5D, the treatment of A2780 cells with the PLAB-specific siRNA was able to strongly reduce 4-HPR-induced PLAB as well as AF1q upmodulation compared with the cells transfected with a control siRNA. Conversely, AF1q silencing did not affect 4-HPR-induced PLAB upregulation (Figure 5E). The aforementioned data indicated that in A2780 cells AF1q upregulation was dependent on prior activation of the 4-HPR-induced-ROS-related signaling cascade and that its upmodulation occurred downstream of PLAB induction (Figure 6).

Bottom Line: Through gene silencing, AF1q was found functionally involved in 4-HPR-induced apoptosis in A2780, an ovarian cancer cell line highly sensitive to retinoid growth inhibitory and apoptotic effects.Inhibition of the signaling intermediates of the 4-HPR apoptotic cascade showed that AF1q upregulation was depended on prior generation of ROS, induction of ER stress response, JNK activation, and PLAB upmodulation.The study expands the knowledge of the 4-HPR mechanism of action, which has not yet been completely elucidated, identifying AF1q as a novel mediator of retinoid anticancer activity.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Oncology and Molecular Medicine, Fondazione IRCCS Istituto Nazionale dei Tumori, Milan, Italy. paola.tiberio@istitutotumori.mi.it

ABSTRACT

Background: Fenretinide (4-HPR) is a synthetic retinoid that exhibits potent antitumor and chemopreventive activities against different malignancies, including ovarian tumors. We previously showed that in ovarian cancer cells, 4-HPR induces apoptosis through a signaling cascade starting from reactive oxygen species (ROS) generation and involving endoplasmic reticulum (ER) stress response, Jun N-terminal Kinase (JNK) activation, and induction of the proapoptotic PLAcental Bone morphogenetic protein (PLAB). Since recent studies have shown that the oncogene ALL1-fused from chromosome 1q (AF1q), a retinoic acid target gene, is implicated in apoptosis induction by several therapeutic agents, we investigated its possible involvement in the apoptosis induced by 4-HPR in ovarian cancer cells.

Methodology/principal findings: Protein expression analysis, performed in ovarian cancer cells and extended to other histotypes (breast, neuroblastoma, and cervical), revealed that 4-HPR enhanced AF1q expression in cancer cells sensitive to the retinoid but not in resistant cells. Through gene silencing, AF1q was found functionally involved in 4-HPR-induced apoptosis in A2780, an ovarian cancer cell line highly sensitive to retinoid growth inhibitory and apoptotic effects. Inhibition of the signaling intermediates of the 4-HPR apoptotic cascade showed that AF1q upregulation was depended on prior generation of ROS, induction of ER stress response, JNK activation, and PLAB upmodulation. Finally, we found that direct overexpression of AF1q, in the absence of external stimuli, increased apoptosis in ovarian cancer cell lines.

Conclusions/significance: The study expands the knowledge of the 4-HPR mechanism of action, which has not yet been completely elucidated, identifying AF1q as a novel mediator of retinoid anticancer activity. In addition, we demonstrate, for the first time, that AF1q plays a role in the onset of basal apoptosis in ovarian cancer cells, thus providing new information about the activity of this protein whose biologic functions are mostly unknown.

Show MeSH
Related in: MedlinePlus