SnoN/SkiL expression is modulated via arsenic trioxide-induced activation of the PI3K/AKT pathway in ovarian cancer cells

Abstract

SnoN/SkiL (TGFβ regulator) is dysregulated in ovarian cancer, a disease associated with acquired drug-resistance. Arsenic trioxide (As₂O₃, used in treating APL) induces SnoN to oppose the apoptotic response in ovarian cancer cells. We now report that As₂O₃ increases phosphorylation of EGFR/p66ShcA and EGFR degradation. As₂O₃ activates Src(Y416) whose activity (inhibited by PP2) modulates EGFR activation, its interaction with Shc/Grb2, and p-AKT. Inhibition of PI3K reduces SnoN and cell survival. Although EGFR or MAPK1 siRNA did not alter SnoN expression, As₂O₃-induced cleaved PARP was reduced together with increased XIAP. Collectively, As₂O₃ mediates an initial rise in pY-Src(416) to regulate the PI3K/AKT pathway which increases SnoN and cell survival; these early events may counter the cell death response associated with increased pY-EGFR/MAPK activation.

Fig. 1. As₂O₃ induces EGFR activation and interactions with ShcA/Grb2 adaptor proteins in HEY cells

A, HEY cells were treated with 100 ng/ml EGF from 1–30 h and then cell lysates were analyzed by western analyses. [n=4]. B, HEY cells were treated with 5, 10, or 25 µM As₂O₃ from 1–30 h and then cell lysates were analyzed by western analyses. [n=2]. C, HEY cells were treated with 5, 10, or 25 µM As₂O₃ for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (left panel). The percentage of viable and dead cells for data presented (right panel). [n=2]. D, HEY cells were treated with As₂O₃ in the presence/absence of cycloheximide (CHX, 0.05 ng/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, HEY cells were stimulated with EGF or As₂O₃ across time points indicated. Cell lysates were harvested (“inputs”) and western analyses performed. [n=4] F, Using lysates from E, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. IP CT refers to immunoprecipitation control using antibody and protein G beads. [n=4]. G, IP was performed using anti-ShcA rabbit polyclonal antibody followed by western analyses. H, Similar to experiments performed in E (“inputs”, left panel) and F (IP EGFR, right panel) except HEY cells were treated with As₂O₃ at 10µM at 9 and 18 h.

Fig. 1. As₂O₃ induces EGFR activation and interactions with ShcA/Grb2 adaptor proteins in HEY cells

A, HEY cells were treated with 100 ng/ml EGF from 1–30 h and then cell lysates were analyzed by western analyses. [n=4]. B, HEY cells were treated with 5, 10, or 25 µM As₂O₃ from 1–30 h and then cell lysates were analyzed by western analyses. [n=2]. C, HEY cells were treated with 5, 10, or 25 µM As₂O₃ for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (left panel). The percentage of viable and dead cells for data presented (right panel). [n=2]. D, HEY cells were treated with As₂O₃ in the presence/absence of cycloheximide (CHX, 0.05 ng/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, HEY cells were stimulated with EGF or As₂O₃ across time points indicated. Cell lysates were harvested (“inputs”) and western analyses performed. [n=4] F, Using lysates from E, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. IP CT refers to immunoprecipitation control using antibody and protein G beads. [n=4]. G, IP was performed using anti-ShcA rabbit polyclonal antibody followed by western analyses. H, Similar to experiments performed in E (“inputs”, left panel) and F (IP EGFR, right panel) except HEY cells were treated with As₂O₃ at 10µM at 9 and 18 h.

Fig. 1. As₂O₃ induces EGFR activation and interactions with ShcA/Grb2 adaptor proteins in HEY cells

A, HEY cells were treated with 100 ng/ml EGF from 1–30 h and then cell lysates were analyzed by western analyses. [n=4]. B, HEY cells were treated with 5, 10, or 25 µM As₂O₃ from 1–30 h and then cell lysates were analyzed by western analyses. [n=2]. C, HEY cells were treated with 5, 10, or 25 µM As₂O₃ for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (left panel). The percentage of viable and dead cells for data presented (right panel). [n=2]. D, HEY cells were treated with As₂O₃ in the presence/absence of cycloheximide (CHX, 0.05 ng/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, HEY cells were stimulated with EGF or As₂O₃ across time points indicated. Cell lysates were harvested (“inputs”) and western analyses performed. [n=4] F, Using lysates from E, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. IP CT refers to immunoprecipitation control using antibody and protein G beads. [n=4]. G, IP was performed using anti-ShcA rabbit polyclonal antibody followed by western analyses. H, Similar to experiments performed in E (“inputs”, left panel) and F (IP EGFR, right panel) except HEY cells were treated with As₂O₃ at 10µM at 9 and 18 h.

Fig. 1. As₂O₃ induces EGFR activation and interactions with ShcA/Grb2 adaptor proteins in HEY cells

A, HEY cells were treated with 100 ng/ml EGF from 1–30 h and then cell lysates were analyzed by western analyses. [n=4]. B, HEY cells were treated with 5, 10, or 25 µM As₂O₃ from 1–30 h and then cell lysates were analyzed by western analyses. [n=2]. C, HEY cells were treated with 5, 10, or 25 µM As₂O₃ for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (left panel). The percentage of viable and dead cells for data presented (right panel). [n=2]. D, HEY cells were treated with As₂O₃ in the presence/absence of cycloheximide (CHX, 0.05 ng/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, HEY cells were stimulated with EGF or As₂O₃ across time points indicated. Cell lysates were harvested (“inputs”) and western analyses performed. [n=4] F, Using lysates from E, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. IP CT refers to immunoprecipitation control using antibody and protein G beads. [n=4]. G, IP was performed using anti-ShcA rabbit polyclonal antibody followed by western analyses. H, Similar to experiments performed in E (“inputs”, left panel) and F (IP EGFR, right panel) except HEY cells were treated with As₂O₃ at 10µM at 9 and 18 h.

Fig. 1. As₂O₃ induces EGFR activation and interactions with ShcA/Grb2 adaptor proteins in HEY cells

A, HEY cells were treated with 100 ng/ml EGF from 1–30 h and then cell lysates were analyzed by western analyses. [n=4]. B, HEY cells were treated with 5, 10, or 25 µM As₂O₃ from 1–30 h and then cell lysates were analyzed by western analyses. [n=2]. C, HEY cells were treated with 5, 10, or 25 µM As₂O₃ for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (left panel). The percentage of viable and dead cells for data presented (right panel). [n=2]. D, HEY cells were treated with As₂O₃ in the presence/absence of cycloheximide (CHX, 0.05 ng/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, HEY cells were stimulated with EGF or As₂O₃ across time points indicated. Cell lysates were harvested (“inputs”) and western analyses performed. [n=4] F, Using lysates from E, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. IP CT refers to immunoprecipitation control using antibody and protein G beads. [n=4]. G, IP was performed using anti-ShcA rabbit polyclonal antibody followed by western analyses. H, Similar to experiments performed in E (“inputs”, left panel) and F (IP EGFR, right panel) except HEY cells were treated with As₂O₃ at 10µM at 9 and 18 h.

Fig. 1. As₂O₃ induces EGFR activation and interactions with ShcA/Grb2 adaptor proteins in HEY cells

A, HEY cells were treated with 100 ng/ml EGF from 1–30 h and then cell lysates were analyzed by western analyses. [n=4]. B, HEY cells were treated with 5, 10, or 25 µM As₂O₃ from 1–30 h and then cell lysates were analyzed by western analyses. [n=2]. C, HEY cells were treated with 5, 10, or 25 µM As₂O₃ for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (left panel). The percentage of viable and dead cells for data presented (right panel). [n=2]. D, HEY cells were treated with As₂O₃ in the presence/absence of cycloheximide (CHX, 0.05 ng/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, HEY cells were stimulated with EGF or As₂O₃ across time points indicated. Cell lysates were harvested (“inputs”) and western analyses performed. [n=4] F, Using lysates from E, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. IP CT refers to immunoprecipitation control using antibody and protein G beads. [n=4]. G, IP was performed using anti-ShcA rabbit polyclonal antibody followed by western analyses. H, Similar to experiments performed in E (“inputs”, left panel) and F (IP EGFR, right panel) except HEY cells were treated with As₂O₃ at 10µM at 9 and 18 h.

Fig. 2. Src tyrosine kinase inhibitor (PP2) alters As₂O₃-mediated EGFR activation, interaction of EGFR with ShcA/Grb2 adaptor proteins, and SnoN expression

A, HEY cells were pretreated with PP2 (10 µM), U0126 (10 µM), or PD153035 (100 nM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=4] B, HEY cells were treated with PP2 (10 µM) only or for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. C, Densitometric analyses of SnoN (6 h) is shown for data presented in B. D, HEY cells were treated with 25 µM As₂O₃ in the presence/absence of actinomycin D (ActD, 0.5 µg/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, Following seeding and adherence of HEY, cells were pretreated with PP2 (10 µM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. RNA was isolated and used for qPCR. Relative RNA-fold changes are presented for SnoN. [n=3]. F, HEY cells were pretreated with SU6656 (5 or 10 µM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. G, HEY cells were pretreated with PP2 (10 µM) for 2 h followed by treatment with EGF for either 5 min or 1 h or As₂O₃ for either 1 h or 6 h. Cell lysates were collected (“inputs”) and western analyses performed. [n=2]. H, Using lysates presented from G, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses.

Fig. 2. Src tyrosine kinase inhibitor (PP2) alters As₂O₃-mediated EGFR activation, interaction of EGFR with ShcA/Grb2 adaptor proteins, and SnoN expression

A, HEY cells were pretreated with PP2 (10 µM), U0126 (10 µM), or PD153035 (100 nM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=4] B, HEY cells were treated with PP2 (10 µM) only or for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. C, Densitometric analyses of SnoN (6 h) is shown for data presented in B. D, HEY cells were treated with 25 µM As₂O₃ in the presence/absence of actinomycin D (ActD, 0.5 µg/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, Following seeding and adherence of HEY, cells were pretreated with PP2 (10 µM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. RNA was isolated and used for qPCR. Relative RNA-fold changes are presented for SnoN. [n=3]. F, HEY cells were pretreated with SU6656 (5 or 10 µM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. G, HEY cells were pretreated with PP2 (10 µM) for 2 h followed by treatment with EGF for either 5 min or 1 h or As₂O₃ for either 1 h or 6 h. Cell lysates were collected (“inputs”) and western analyses performed. [n=2]. H, Using lysates presented from G, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses.

Fig. 2. Src tyrosine kinase inhibitor (PP2) alters As₂O₃-mediated EGFR activation, interaction of EGFR with ShcA/Grb2 adaptor proteins, and SnoN expression

A, HEY cells were pretreated with PP2 (10 µM), U0126 (10 µM), or PD153035 (100 nM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=4] B, HEY cells were treated with PP2 (10 µM) only or for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. C, Densitometric analyses of SnoN (6 h) is shown for data presented in B. D, HEY cells were treated with 25 µM As₂O₃ in the presence/absence of actinomycin D (ActD, 0.5 µg/ml) across time points indicated. Cell lysates were harvested and western analyses performed. [n=2]. E, Following seeding and adherence of HEY, cells were pretreated with PP2 (10 µM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. RNA was isolated and used for qPCR. Relative RNA-fold changes are presented for SnoN. [n=3]. F, HEY cells were pretreated with SU6656 (5 or 10 µM) for 2 h prior to stimulation with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. G, HEY cells were pretreated with PP2 (10 µM) for 2 h followed by treatment with EGF for either 5 min or 1 h or As₂O₃ for either 1 h or 6 h. Cell lysates were collected (“inputs”) and western analyses performed. [n=2]. H, Using lysates presented from G, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses.

Fig. 3. PI3K inhibitors (LY294002 and GDC0941) and PIK3CA siRNA alter SnoN expression and the apoptotic response to As₂O₃

A, Following seeding and adherence of HEY, cells were co-treated with LY294002 (1–25 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3] B, Following seeding and adherence of HEY cells, cells were co-treated with GDC0941 (100 nM-10 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=2] C, Following seeding and adherence of HEY, cells were co-treated with LY294002 and As₂O₃ for 18 h. RNA was harvested and qPCR performed. [n=3] D, Caspase activity assay was performed on HEY cells treated with As₂O₃, As₂O₃ with LY294002, or As₂O₃ with GDC0941 for 18 h. [n=3]. E, Following seeding and adherence of HEY, cells were co-treated with LY294002 (25 µM) or GDC0941 (10 µM) with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. F, Following seeding and adherence of HEY, cells were treated with PP2 10 µM), SU6656 (10 µM), LY294002 (25 µM), or GDC0941 (10 µM) with As₂O₃ (10 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3]. G, HEY cells were treated with non-targeting (control) siRNA or PIK3CA siRNA. At 72 h after transfection, cells were treated with As₂O₃ for 6 h. Cell lysates were then collected and western analyses performed. H, Densitometric analyses of the SnoN from data presented in E is shown. I, HEY cells were treated with As₂O₃ alone or in combination with LY294002 for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. J, HEY cells were treated with LY294002 alone for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. K, The data presented in I is also represented in tabular format showing % of late apoptotic/necrotic, alive, early apoptotic and dead cells. [n=2]

Fig. 3. PI3K inhibitors (LY294002 and GDC0941) and PIK3CA siRNA alter SnoN expression and the apoptotic response to As₂O₃

A, Following seeding and adherence of HEY, cells were co-treated with LY294002 (1–25 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3] B, Following seeding and adherence of HEY cells, cells were co-treated with GDC0941 (100 nM-10 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=2] C, Following seeding and adherence of HEY, cells were co-treated with LY294002 and As₂O₃ for 18 h. RNA was harvested and qPCR performed. [n=3] D, Caspase activity assay was performed on HEY cells treated with As₂O₃, As₂O₃ with LY294002, or As₂O₃ with GDC0941 for 18 h. [n=3]. E, Following seeding and adherence of HEY, cells were co-treated with LY294002 (25 µM) or GDC0941 (10 µM) with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. F, Following seeding and adherence of HEY, cells were treated with PP2 10 µM), SU6656 (10 µM), LY294002 (25 µM), or GDC0941 (10 µM) with As₂O₃ (10 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3]. G, HEY cells were treated with non-targeting (control) siRNA or PIK3CA siRNA. At 72 h after transfection, cells were treated with As₂O₃ for 6 h. Cell lysates were then collected and western analyses performed. H, Densitometric analyses of the SnoN from data presented in E is shown. I, HEY cells were treated with As₂O₃ alone or in combination with LY294002 for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. J, HEY cells were treated with LY294002 alone for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. K, The data presented in I is also represented in tabular format showing % of late apoptotic/necrotic, alive, early apoptotic and dead cells. [n=2]

Fig. 3. PI3K inhibitors (LY294002 and GDC0941) and PIK3CA siRNA alter SnoN expression and the apoptotic response to As₂O₃

A, Following seeding and adherence of HEY, cells were co-treated with LY294002 (1–25 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3] B, Following seeding and adherence of HEY cells, cells were co-treated with GDC0941 (100 nM-10 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=2] C, Following seeding and adherence of HEY, cells were co-treated with LY294002 and As₂O₃ for 18 h. RNA was harvested and qPCR performed. [n=3] D, Caspase activity assay was performed on HEY cells treated with As₂O₃, As₂O₃ with LY294002, or As₂O₃ with GDC0941 for 18 h. [n=3]. E, Following seeding and adherence of HEY, cells were co-treated with LY294002 (25 µM) or GDC0941 (10 µM) with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. F, Following seeding and adherence of HEY, cells were treated with PP2 10 µM), SU6656 (10 µM), LY294002 (25 µM), or GDC0941 (10 µM) with As₂O₃ (10 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3]. G, HEY cells were treated with non-targeting (control) siRNA or PIK3CA siRNA. At 72 h after transfection, cells were treated with As₂O₃ for 6 h. Cell lysates were then collected and western analyses performed. H, Densitometric analyses of the SnoN from data presented in E is shown. I, HEY cells were treated with As₂O₃ alone or in combination with LY294002 for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. J, HEY cells were treated with LY294002 alone for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. K, The data presented in I is also represented in tabular format showing % of late apoptotic/necrotic, alive, early apoptotic and dead cells. [n=2]

Fig. 3. PI3K inhibitors (LY294002 and GDC0941) and PIK3CA siRNA alter SnoN expression and the apoptotic response to As₂O₃

A, Following seeding and adherence of HEY, cells were co-treated with LY294002 (1–25 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3] B, Following seeding and adherence of HEY cells, cells were co-treated with GDC0941 (100 nM-10 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=2] C, Following seeding and adherence of HEY, cells were co-treated with LY294002 and As₂O₃ for 18 h. RNA was harvested and qPCR performed. [n=3] D, Caspase activity assay was performed on HEY cells treated with As₂O₃, As₂O₃ with LY294002, or As₂O₃ with GDC0941 for 18 h. [n=3]. E, Following seeding and adherence of HEY, cells were co-treated with LY294002 (25 µM) or GDC0941 (10 µM) with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. F, Following seeding and adherence of HEY, cells were treated with PP2 10 µM), SU6656 (10 µM), LY294002 (25 µM), or GDC0941 (10 µM) with As₂O₃ (10 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3]. G, HEY cells were treated with non-targeting (control) siRNA or PIK3CA siRNA. At 72 h after transfection, cells were treated with As₂O₃ for 6 h. Cell lysates were then collected and western analyses performed. H, Densitometric analyses of the SnoN from data presented in E is shown. I, HEY cells were treated with As₂O₃ alone or in combination with LY294002 for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. J, HEY cells were treated with LY294002 alone for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. K, The data presented in I is also represented in tabular format showing % of late apoptotic/necrotic, alive, early apoptotic and dead cells. [n=2]

Fig. 3. PI3K inhibitors (LY294002 and GDC0941) and PIK3CA siRNA alter SnoN expression and the apoptotic response to As₂O₃

A, Following seeding and adherence of HEY, cells were co-treated with LY294002 (1–25 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3] B, Following seeding and adherence of HEY cells, cells were co-treated with GDC0941 (100 nM-10 µM) and As₂O₃ (25 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=2] C, Following seeding and adherence of HEY, cells were co-treated with LY294002 and As₂O₃ for 18 h. RNA was harvested and qPCR performed. [n=3] D, Caspase activity assay was performed on HEY cells treated with As₂O₃, As₂O₃ with LY294002, or As₂O₃ with GDC0941 for 18 h. [n=3]. E, Following seeding and adherence of HEY, cells were co-treated with LY294002 (25 µM) or GDC0941 (10 µM) with As₂O₃ (25 µM) for 6 h. Cells lysates were harvested and western analyses performed. [n=2]. F, Following seeding and adherence of HEY, cells were treated with PP2 10 µM), SU6656 (10 µM), LY294002 (25 µM), or GDC0941 (10 µM) with As₂O₃ (10 µM) for 18 h. Cells lysates were harvested and western analyses performed. [n=3]. G, HEY cells were treated with non-targeting (control) siRNA or PIK3CA siRNA. At 72 h after transfection, cells were treated with As₂O₃ for 6 h. Cell lysates were then collected and western analyses performed. H, Densitometric analyses of the SnoN from data presented in E is shown. I, HEY cells were treated with As₂O₃ alone or in combination with LY294002 for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. J, HEY cells were treated with LY294002 alone for 18 h at which time both the floating and adherent cells were collected. Cells were stained with annexin V-FITC and propidium iodide (PI) followed by flow cytometric analysis. Raw data plots are shown (top panels). The percentage of viable and dead cells are shown in lower panel. K, The data presented in I is also represented in tabular format showing % of late apoptotic/necrotic, alive, early apoptotic and dead cells. [n=2]

Fig. 4. Knockdown of EGFR/MAPK1/pp60 c-Src modulate cell survival in response to As₂O₃

A, HEY cells were treated with siRNA presented followed by treatment with As₂O₃ for 6 or 18 h. Cell lysates were collected and western analyses performed. [n=2] B, The data from the apoptosis assay for cells as described in A is represented in tabular format. [n=2] C, HEY cells were treated with EGF or with As₂O₃. Cell lysates were collected (“inputs”) and western analyses performed. [n=2] D, Using lysates presented from C, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. [n=2]. E, Proposed model of the action of As₂O₃ on regulation of SnoN levels. In response to As₂O₃, the Src and AKT pathways are activated. Inhibition via LY294002 (and PP2 but not SU6656) leads to a reduction in As₂O₃-induced SnoN mRNA and protein levels. As₂O₃ also leads to tyrosine phosphorylation of the EGF receptor and phosphorylation of p66ShcA together with increased binding of Grb2. Inhibition of the MAPK pathway via siRNA targeting MAPK1 leads to increased cellular survival in response to As₂O₃.

Fig. 4. Knockdown of EGFR/MAPK1/pp60 c-Src modulate cell survival in response to As₂O₃

A, HEY cells were treated with siRNA presented followed by treatment with As₂O₃ for 6 or 18 h. Cell lysates were collected and western analyses performed. [n=2] B, The data from the apoptosis assay for cells as described in A is represented in tabular format. [n=2] C, HEY cells were treated with EGF or with As₂O₃. Cell lysates were collected (“inputs”) and western analyses performed. [n=2] D, Using lysates presented from C, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. [n=2]. E, Proposed model of the action of As₂O₃ on regulation of SnoN levels. In response to As₂O₃, the Src and AKT pathways are activated. Inhibition via LY294002 (and PP2 but not SU6656) leads to a reduction in As₂O₃-induced SnoN mRNA and protein levels. As₂O₃ also leads to tyrosine phosphorylation of the EGF receptor and phosphorylation of p66ShcA together with increased binding of Grb2. Inhibition of the MAPK pathway via siRNA targeting MAPK1 leads to increased cellular survival in response to As₂O₃.

Fig. 4. Knockdown of EGFR/MAPK1/pp60 c-Src modulate cell survival in response to As₂O₃

A, HEY cells were treated with siRNA presented followed by treatment with As₂O₃ for 6 or 18 h. Cell lysates were collected and western analyses performed. [n=2] B, The data from the apoptosis assay for cells as described in A is represented in tabular format. [n=2] C, HEY cells were treated with EGF or with As₂O₃. Cell lysates were collected (“inputs”) and western analyses performed. [n=2] D, Using lysates presented from C, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. [n=2]. E, Proposed model of the action of As₂O₃ on regulation of SnoN levels. In response to As₂O₃, the Src and AKT pathways are activated. Inhibition via LY294002 (and PP2 but not SU6656) leads to a reduction in As₂O₃-induced SnoN mRNA and protein levels. As₂O₃ also leads to tyrosine phosphorylation of the EGF receptor and phosphorylation of p66ShcA together with increased binding of Grb2. Inhibition of the MAPK pathway via siRNA targeting MAPK1 leads to increased cellular survival in response to As₂O₃.

Fig. 4. Knockdown of EGFR/MAPK1/pp60 c-Src modulate cell survival in response to As₂O₃

A, HEY cells were treated with siRNA presented followed by treatment with As₂O₃ for 6 or 18 h. Cell lysates were collected and western analyses performed. [n=2] B, The data from the apoptosis assay for cells as described in A is represented in tabular format. [n=2] C, HEY cells were treated with EGF or with As₂O₃. Cell lysates were collected (“inputs”) and western analyses performed. [n=2] D, Using lysates presented from C, immunoprecipitation (IP) was performed using anti-EGFR rabbit polyclonal antibody followed by western analyses. [n=2]. E, Proposed model of the action of As₂O₃ on regulation of SnoN levels. In response to As₂O₃, the Src and AKT pathways are activated. Inhibition via LY294002 (and PP2 but not SU6656) leads to a reduction in As₂O₃-induced SnoN mRNA and protein levels. As₂O₃ also leads to tyrosine phosphorylation of the EGF receptor and phosphorylation of p66ShcA together with increased binding of Grb2. Inhibition of the MAPK pathway via siRNA targeting MAPK1 leads to increased cellular survival in response to As₂O₃.