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Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells

View Article: PubMed Central - PubMed

ABSTRACT

AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin + Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells.

No MeSH data available.


Related in: MedlinePlus

Metformin + Akti-1/2 combo potently suppresses the propagation of adherent U87 cells and their tumor spheres.(A,E) Adherent parental U87 cells (A) or U87 tumor sphere cells (E) were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. The images were captured under an Olympus IX81 microscope at 100X (10X objective lens) or 200X (20X objective lens) magnification. (B,F) Adherent parental U87 cells (B) or U87 tumor sphere cells (F) were treated with the same manner as in (A,E) for up to 5 days. Samples were subjected to MTT assay at each time point. (C,D,G,H) Adherent parental U87 cells (C,D) or U87 tumor sphere cells (G,H) were treated with the same manner as in (A,E) for up to 5 days. Cells were collected at each time point and suspended with 100 μl PBS. Live and dead cells were counted by trypan blue exclusion using the Countess™ II FL Automated Cell Counter. Data were expressed as mean ± SD of triplicate measurements from one of three independent experiments which gave similar results. The colored asterisks indicate the difference between each treatment group and vehicle group by significance levels (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
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f4: Metformin + Akti-1/2 combo potently suppresses the propagation of adherent U87 cells and their tumor spheres.(A,E) Adherent parental U87 cells (A) or U87 tumor sphere cells (E) were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. The images were captured under an Olympus IX81 microscope at 100X (10X objective lens) or 200X (20X objective lens) magnification. (B,F) Adherent parental U87 cells (B) or U87 tumor sphere cells (F) were treated with the same manner as in (A,E) for up to 5 days. Samples were subjected to MTT assay at each time point. (C,D,G,H) Adherent parental U87 cells (C,D) or U87 tumor sphere cells (G,H) were treated with the same manner as in (A,E) for up to 5 days. Cells were collected at each time point and suspended with 100 μl PBS. Live and dead cells were counted by trypan blue exclusion using the Countess™ II FL Automated Cell Counter. Data were expressed as mean ± SD of triplicate measurements from one of three independent experiments which gave similar results. The colored asterisks indicate the difference between each treatment group and vehicle group by significance levels (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).

Mentions: Either Akti-1/2 or metformin led to certain morphological changes of adherent U87 cells (Fig. 4A) and decreases in their propagation as determined by MTT assay, with Akti-1/2 having more inhibitory effect on cell propagation (Fig. 4B). The combo treatment caused dramatic cell shrinkage, round up and detachment indicative of cell death (Fig. 4A), and almost completely blocked their propagation up to 5 days (Fig. 4B). To eliminate possible interference to the MTT assay caused by altered metabolic enzyme activities in those treated cells, we performed both manual cell counting under microscope (Fig. S7) and trypan blue exclusion-based live/dead cell counting using the Countess™ II FL Automated Cell Counter (Figs 4C, D and S8). Metformin alone blocked the increase in live cells (Fig. 4C) but did not induce cell death (Fig. 4D). Cell cycle analysis revealed that it induced a slight cell cycle arrest at the G1 phase (Fig. S9). In comparison, Akti-1/2 alone significantly reduced the live cell numbers (Fig. 4C) while increasing the proportions of dead cells (Fig. 4D) and cells arrested at the G2/M phase (Fig. S9). Remarkably, 5 days post treatment, the metformin + Akti-1/2 combo inhibited the expansion of live cells by greater than 99.5% (Fig. 4C) and greater than 95% of the cells in this group were dead (Fig. 4D). At an earlier point (2 days post treatment), the combo treatment dramatically increased the proportion of G2/M phase cells from 7% to 41% (Fig. S9). In a similar fashion, Akti-1/2 alone had much more prominent effects than metformin alone in reducing the sizes and the numbers of U87 tumor spheres enriched in CSCs (Fig. 4E), in inhibiting the propagation of tumor sphere cells (Fig. 4F), in decreasing the live sphere cell counts (Fig. 4G) and in raising the dead sphere cell proportions (Fig. 4H), and the combo had even greater effects (Fig. 4E–H).


Dual inhibiting OCT4 and AKT potently suppresses the propagation of human cancer cells
Metformin + Akti-1/2 combo potently suppresses the propagation of adherent U87 cells and their tumor spheres.(A,E) Adherent parental U87 cells (A) or U87 tumor sphere cells (E) were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. The images were captured under an Olympus IX81 microscope at 100X (10X objective lens) or 200X (20X objective lens) magnification. (B,F) Adherent parental U87 cells (B) or U87 tumor sphere cells (F) were treated with the same manner as in (A,E) for up to 5 days. Samples were subjected to MTT assay at each time point. (C,D,G,H) Adherent parental U87 cells (C,D) or U87 tumor sphere cells (G,H) were treated with the same manner as in (A,E) for up to 5 days. Cells were collected at each time point and suspended with 100 μl PBS. Live and dead cells were counted by trypan blue exclusion using the Countess™ II FL Automated Cell Counter. Data were expressed as mean ± SD of triplicate measurements from one of three independent experiments which gave similar results. The colored asterisks indicate the difference between each treatment group and vehicle group by significance levels (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
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f4: Metformin + Akti-1/2 combo potently suppresses the propagation of adherent U87 cells and their tumor spheres.(A,E) Adherent parental U87 cells (A) or U87 tumor sphere cells (E) were treated with DMSO (Vehicle), 10 mM metformin (Metformin), 5 μM Akti-1/2 (Akti-1/2), or 10 mM metformin + 5 μM Akti-1/2 (Metformin + Akti-1/2), for 5 days. The images were captured under an Olympus IX81 microscope at 100X (10X objective lens) or 200X (20X objective lens) magnification. (B,F) Adherent parental U87 cells (B) or U87 tumor sphere cells (F) were treated with the same manner as in (A,E) for up to 5 days. Samples were subjected to MTT assay at each time point. (C,D,G,H) Adherent parental U87 cells (C,D) or U87 tumor sphere cells (G,H) were treated with the same manner as in (A,E) for up to 5 days. Cells were collected at each time point and suspended with 100 μl PBS. Live and dead cells were counted by trypan blue exclusion using the Countess™ II FL Automated Cell Counter. Data were expressed as mean ± SD of triplicate measurements from one of three independent experiments which gave similar results. The colored asterisks indicate the difference between each treatment group and vehicle group by significance levels (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001).
Mentions: Either Akti-1/2 or metformin led to certain morphological changes of adherent U87 cells (Fig. 4A) and decreases in their propagation as determined by MTT assay, with Akti-1/2 having more inhibitory effect on cell propagation (Fig. 4B). The combo treatment caused dramatic cell shrinkage, round up and detachment indicative of cell death (Fig. 4A), and almost completely blocked their propagation up to 5 days (Fig. 4B). To eliminate possible interference to the MTT assay caused by altered metabolic enzyme activities in those treated cells, we performed both manual cell counting under microscope (Fig. S7) and trypan blue exclusion-based live/dead cell counting using the Countess™ II FL Automated Cell Counter (Figs 4C, D and S8). Metformin alone blocked the increase in live cells (Fig. 4C) but did not induce cell death (Fig. 4D). Cell cycle analysis revealed that it induced a slight cell cycle arrest at the G1 phase (Fig. S9). In comparison, Akti-1/2 alone significantly reduced the live cell numbers (Fig. 4C) while increasing the proportions of dead cells (Fig. 4D) and cells arrested at the G2/M phase (Fig. S9). Remarkably, 5 days post treatment, the metformin + Akti-1/2 combo inhibited the expansion of live cells by greater than 99.5% (Fig. 4C) and greater than 95% of the cells in this group were dead (Fig. 4D). At an earlier point (2 days post treatment), the combo treatment dramatically increased the proportion of G2/M phase cells from 7% to 41% (Fig. S9). In a similar fashion, Akti-1/2 alone had much more prominent effects than metformin alone in reducing the sizes and the numbers of U87 tumor spheres enriched in CSCs (Fig. 4E), in inhibiting the propagation of tumor sphere cells (Fig. 4F), in decreasing the live sphere cell counts (Fig. 4G) and in raising the dead sphere cell proportions (Fig. 4H), and the combo had even greater effects (Fig. 4E–H).

View Article: PubMed Central - PubMed

ABSTRACT

AKT serves as an epigenetic modulator that links epigenetic regulation to cell survival and proliferation while the epigenetic mediator OCT4 critically controls stem cell pluripotency and self-renewal. Emerging evidence indicated their complicated interplays in cancer cells and cancer stem cells (CSCs), and inhibiting either one may activate the other. Thus, in this study, we propose a strategy to targeting both factors simultaneously. Firstly, a combination of an OCT4-specific shRNA and the specific AKT inhibitor Akti-1/2 potently suppressed the propagation of human embryonal carcinoma cells, adherent cancer cells and stem-like cancer cells, establishing the proof-of-concept that dual inhibiting OCT4 and AKT can effectively target various cancer cells. Next, we combined Akti-1/2 with metformin, a widely-prescribed drug for treating type 2 diabetes, which was reported to down-regulate OCT4 expression. The metformin&thinsp;+&thinsp;Akti-1/2 combo significantly altered multiple signaling and epigenetic pathways, induced growth arrest and cell death of adherent and stem-like glioblastoma U87 cells, and attenuated their tumorigenicity in vivo. Taken together, we demonstrate here that simultaneously targeting an epigenetic mediator and an epigenetic modulator, by dual inhibiting OCT4 and AKT, can have significantly improved efficacies over single treatment in suppressing the propagation of CSCs as well as the entire bulk of differentiated cancer cells.

No MeSH data available.


Related in: MedlinePlus