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Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole-imidazole polyamide, which targets an E-box motif.

Taniguchi M, Fujiwara K, Nakai Y, Ozaki T, Koshikawa N, Toshio K, Kataba M, Oguni A, Matsuda H, Yoshida Y, Tokuhashi Y, Fukuda N, Ueno T, Soma M, Nagase H - FEBS Open Bio (2014)

Bottom Line: Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors.In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo.Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

View Article: PubMed Central - PubMed

Affiliation: Division of Orthopedic Surgery, Nihon University School of Medicine, 30-1 Oyaguchi Kami-Cho, Itabashi, Tokyo 173-8610, Japan.

ABSTRACT
Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors. In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc-6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

No MeSH data available.


Related in: MedlinePlus

Myc-6-dependent induction of apoptosis. (A) Tali image-based cytometric analysis. MG63 cells were treated with the indicated concentrations of Myc-6 or left untreated. Following 72 h of Myc-6 exposure, the adherent and attached cells were collected and processed for Tali image-based cytometric analysis. Differences versus cells treated with or without Myc-6 were considered significant at p < 0.05. ∗p < 0.0001. The columns represent means ± SD. (B) Apopxin violet staining. Cells were treated as in (A). Forty-eight hours after the treatment, cells were incubated with apopxin violet 500 solution for 30 min, and then number of apopxin-positive cells was counted. ∗p < 0.01. (C) Cleavage of pro-apoptotic caspase-3. Cells were treated as in (A). Forty-eight hours after the treatment, cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies.
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f0010: Myc-6-dependent induction of apoptosis. (A) Tali image-based cytometric analysis. MG63 cells were treated with the indicated concentrations of Myc-6 or left untreated. Following 72 h of Myc-6 exposure, the adherent and attached cells were collected and processed for Tali image-based cytometric analysis. Differences versus cells treated with or without Myc-6 were considered significant at p < 0.05. ∗p < 0.0001. The columns represent means ± SD. (B) Apopxin violet staining. Cells were treated as in (A). Forty-eight hours after the treatment, cells were incubated with apopxin violet 500 solution for 30 min, and then number of apopxin-positive cells was counted. ∗p < 0.01. (C) Cleavage of pro-apoptotic caspase-3. Cells were treated as in (A). Forty-eight hours after the treatment, cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies.

Mentions: Since these results indicated that Myc-6 has an ability to prohibit MG63 cell growth, we asked whether Myc-6 could affect the cell cycle distribution. After 72 h of incubation with or without Myc-6, both floating and adherent cells were collected and their cell cycle distribution was examined by Tali image-based cytometer. As seen in Fig. 2A, a dose-dependent massive increase in number of cells with sub-G1 or G1 DNA content was detectable, and also number of cells bearing S phase DNA content was remarkably reduced in a dose-dependent fashion. These observations indicate that MG63 cells undergo apoptosis and/or G1 cell cycle arrest following Myc-6 exposure.


Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole-imidazole polyamide, which targets an E-box motif.

Taniguchi M, Fujiwara K, Nakai Y, Ozaki T, Koshikawa N, Toshio K, Kataba M, Oguni A, Matsuda H, Yoshida Y, Tokuhashi Y, Fukuda N, Ueno T, Soma M, Nagase H - FEBS Open Bio (2014)

Myc-6-dependent induction of apoptosis. (A) Tali image-based cytometric analysis. MG63 cells were treated with the indicated concentrations of Myc-6 or left untreated. Following 72 h of Myc-6 exposure, the adherent and attached cells were collected and processed for Tali image-based cytometric analysis. Differences versus cells treated with or without Myc-6 were considered significant at p < 0.05. ∗p < 0.0001. The columns represent means ± SD. (B) Apopxin violet staining. Cells were treated as in (A). Forty-eight hours after the treatment, cells were incubated with apopxin violet 500 solution for 30 min, and then number of apopxin-positive cells was counted. ∗p < 0.01. (C) Cleavage of pro-apoptotic caspase-3. Cells were treated as in (A). Forty-eight hours after the treatment, cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies.
© Copyright Policy - CC BY-NC-ND
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4048845&req=5

f0010: Myc-6-dependent induction of apoptosis. (A) Tali image-based cytometric analysis. MG63 cells were treated with the indicated concentrations of Myc-6 or left untreated. Following 72 h of Myc-6 exposure, the adherent and attached cells were collected and processed for Tali image-based cytometric analysis. Differences versus cells treated with or without Myc-6 were considered significant at p < 0.05. ∗p < 0.0001. The columns represent means ± SD. (B) Apopxin violet staining. Cells were treated as in (A). Forty-eight hours after the treatment, cells were incubated with apopxin violet 500 solution for 30 min, and then number of apopxin-positive cells was counted. ∗p < 0.01. (C) Cleavage of pro-apoptotic caspase-3. Cells were treated as in (A). Forty-eight hours after the treatment, cell lysates were prepared and analyzed by immunoblotting with the indicated antibodies.
Mentions: Since these results indicated that Myc-6 has an ability to prohibit MG63 cell growth, we asked whether Myc-6 could affect the cell cycle distribution. After 72 h of incubation with or without Myc-6, both floating and adherent cells were collected and their cell cycle distribution was examined by Tali image-based cytometer. As seen in Fig. 2A, a dose-dependent massive increase in number of cells with sub-G1 or G1 DNA content was detectable, and also number of cells bearing S phase DNA content was remarkably reduced in a dose-dependent fashion. These observations indicate that MG63 cells undergo apoptosis and/or G1 cell cycle arrest following Myc-6 exposure.

Bottom Line: Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors.In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo.Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

View Article: PubMed Central - PubMed

Affiliation: Division of Orthopedic Surgery, Nihon University School of Medicine, 30-1 Oyaguchi Kami-Cho, Itabashi, Tokyo 173-8610, Japan.

ABSTRACT
Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors. In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc-6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

No MeSH data available.


Related in: MedlinePlus