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Knockdown of Akt sensitizes osteosarcoma cells to apoptosis induced by cisplatin treatment.

Zhang G, Li M, Zhu X, Bai Y, Yang C - Int J Mol Sci (2011)

Bottom Line: Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin.Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA).It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; E-Mails: Zhangguoyou7@gmail.com (G.Z.); zhgych@yahoo.com.cn (X.Z.); baiyushu@21cn.com (Y.B.); changwei_y@yahoo.com.cn (W.Y.).

ABSTRACT
Akt plays an important role in the inhibition of apoptosis induced by chemotherapy and other stimuli. We therefore investigated if knockdown of Akt2 promoted drug-induced apoptosis in cultured osteosarcoma cells in vitro. SAOS-2 cells were transfected with Akt2 siRNA. The sensitivity of the transformed cell line to the chemotherapeutic drug cisplatin was assessed. Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin. Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA). It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation. The results of this study suggest that knockdown of Akt2 expression may have therapeutic applications in enhancing the efficacy of chemotherapy in patients with osteosarcoma.

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DAPI staining and ELISA for detection of apoptotic cells. (A) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells were treated with cisplatin (1 μM) for 48 h and then fixed and stained with DAPI. Morphological changes were visualized by fluorescence microscopy. Nuclear fragmentation and apoptotic bodies were clearly apparent in Akt2-siRNA-transfected cells, while no nuclear fragmentation or apoptotic bodies were seen in Akt-3m-siRNA-transfected or untransfected SAOS-2 cells treated with cisplatin (1 μM); (B) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells treated with cisplatin (1 μM) for 48 h. At the end of incubation cells were harvested and apoptosis assays were performed using a cell death detection ELISAPLUS kit. Data in each set represents the mean ± S.D. of three independent experiments.
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f4-ijms-12-02994: DAPI staining and ELISA for detection of apoptotic cells. (A) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells were treated with cisplatin (1 μM) for 48 h and then fixed and stained with DAPI. Morphological changes were visualized by fluorescence microscopy. Nuclear fragmentation and apoptotic bodies were clearly apparent in Akt2-siRNA-transfected cells, while no nuclear fragmentation or apoptotic bodies were seen in Akt-3m-siRNA-transfected or untransfected SAOS-2 cells treated with cisplatin (1 μM); (B) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells treated with cisplatin (1 μM) for 48 h. At the end of incubation cells were harvested and apoptosis assays were performed using a cell death detection ELISAPLUS kit. Data in each set represents the mean ± S.D. of three independent experiments.

Mentions: Treatment of Akt2-siRNA-transfected SAOS-2 cells with low concentrations of cisplatin (1 μM) resulted in morphological changes typical of apoptosis, such as cell shrinkage, rounding and detachment of the cells from the plate, as observed by phase contrast microscopy (not shown). DAPI staining was used to visualize nucleosomal DNA damage. Nuclear fragmentation and apoptotic bodies were clearly apparent in Akt2-siRNA-transfected SAOS-2 treated with cisplatin (1 μM), while no nuclear fragmentation or apoptotic bodies were seen in Akt-3m-siRNA-transfected or untransfected SAOS-2 cells treated with cisplatin (1 μM) (Figure 4A). ELISA analysis determined that the apoptosis rate in Akt2-siRNA-transfected SAOS-2 cells treated with cisplatin (1 μM) was higher than in similarly-treated Akt-3m-siRNA-transfected or untransfected SAOS-2 cells (P < 0.05) (Figure 4B).


Knockdown of Akt sensitizes osteosarcoma cells to apoptosis induced by cisplatin treatment.

Zhang G, Li M, Zhu X, Bai Y, Yang C - Int J Mol Sci (2011)

DAPI staining and ELISA for detection of apoptotic cells. (A) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells were treated with cisplatin (1 μM) for 48 h and then fixed and stained with DAPI. Morphological changes were visualized by fluorescence microscopy. Nuclear fragmentation and apoptotic bodies were clearly apparent in Akt2-siRNA-transfected cells, while no nuclear fragmentation or apoptotic bodies were seen in Akt-3m-siRNA-transfected or untransfected SAOS-2 cells treated with cisplatin (1 μM); (B) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells treated with cisplatin (1 μM) for 48 h. At the end of incubation cells were harvested and apoptosis assays were performed using a cell death detection ELISAPLUS kit. Data in each set represents the mean ± S.D. of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3116170&req=5

f4-ijms-12-02994: DAPI staining and ELISA for detection of apoptotic cells. (A) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells were treated with cisplatin (1 μM) for 48 h and then fixed and stained with DAPI. Morphological changes were visualized by fluorescence microscopy. Nuclear fragmentation and apoptotic bodies were clearly apparent in Akt2-siRNA-transfected cells, while no nuclear fragmentation or apoptotic bodies were seen in Akt-3m-siRNA-transfected or untransfected SAOS-2 cells treated with cisplatin (1 μM); (B) Untransfected, Akt2-siRNA-transfected, and Akt-3m-siRNA-transfected SAOS-2 cells treated with cisplatin (1 μM) for 48 h. At the end of incubation cells were harvested and apoptosis assays were performed using a cell death detection ELISAPLUS kit. Data in each set represents the mean ± S.D. of three independent experiments.
Mentions: Treatment of Akt2-siRNA-transfected SAOS-2 cells with low concentrations of cisplatin (1 μM) resulted in morphological changes typical of apoptosis, such as cell shrinkage, rounding and detachment of the cells from the plate, as observed by phase contrast microscopy (not shown). DAPI staining was used to visualize nucleosomal DNA damage. Nuclear fragmentation and apoptotic bodies were clearly apparent in Akt2-siRNA-transfected SAOS-2 treated with cisplatin (1 μM), while no nuclear fragmentation or apoptotic bodies were seen in Akt-3m-siRNA-transfected or untransfected SAOS-2 cells treated with cisplatin (1 μM) (Figure 4A). ELISA analysis determined that the apoptosis rate in Akt2-siRNA-transfected SAOS-2 cells treated with cisplatin (1 μM) was higher than in similarly-treated Akt-3m-siRNA-transfected or untransfected SAOS-2 cells (P < 0.05) (Figure 4B).

Bottom Line: Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin.Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA).It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedics, Changhai Hospital, Second Military Medical University, Shanghai 200433, China; E-Mails: Zhangguoyou7@gmail.com (G.Z.); zhgych@yahoo.com.cn (X.Z.); baiyushu@21cn.com (Y.B.); changwei_y@yahoo.com.cn (W.Y.).

ABSTRACT
Akt plays an important role in the inhibition of apoptosis induced by chemotherapy and other stimuli. We therefore investigated if knockdown of Akt2 promoted drug-induced apoptosis in cultured osteosarcoma cells in vitro. SAOS-2 cells were transfected with Akt2 siRNA. The sensitivity of the transformed cell line to the chemotherapeutic drug cisplatin was assessed. Reduced expression of Akt2 did not directly inhibit the growth rate of the transfected cells; however, it significantly increased their sensitivity to cisplatin. Knockdown of Akt2, together with cisplatin treatment, promoted the expression of p53 up-regulated modulator of apoptosis (PUMA). It is possible that the augmentation of cisplatin cytotoxicity may be mediated by PUMA activation. The results of this study suggest that knockdown of Akt2 expression may have therapeutic applications in enhancing the efficacy of chemotherapy in patients with osteosarcoma.

Show MeSH
Related in: MedlinePlus