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ApoG2 induces cell cycle arrest of nasopharyngeal carcinoma cells by suppressing the c-Myc signaling pathway.

Hu ZY, Sun J, Zhu XF, Yang D, Zeng YX - J Transl Med (2009)

Bottom Line: The results of cell cycle analysis showed that the downregulation of c-Myc signaling pathway by siRNA interference could cause a significant arrest of NPC cell at S phase of the cell cycle.In CNE-2 xenografts, ApoG2 significantly downregulated the expression of c-Myc and suppressed tumor growth in vivo.This data suggested that the inhibitory effect of ApoG2 on NPC cell cycle proliferation might contribute to its use in anticancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Oncology in South China and Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, PR China. huzheyu24@gmail.com

ABSTRACT

Background: apogossypolone (ApoG2) is a novel derivate of gossypol. We previously have reported that ApoG2 is a promising compound that kills nasopharyngeal carcinoma (NPC) cells by inhibiting the antiapoptotic function of Bcl-2 proteins. However, some researchers demonstrate that the antiproliferative effect of gossypol on breast cancer cells is mediated by induction of cell cycle arrest. So this study was aimed to investigate the effect of ApoG2 on cell cycle proliferation in NPC cells.

Results: We found that ApoG2 significantly suppressed the expression of c-Myc in NPC cells and induced arrest at the DNA synthesis (S) phase in a large percentage of NPC cells. Immunoblot analysis showed that expression of c-Myc protein was significantly downregulated by ApoG2 and that the expression of c-Myc's downstream molecules cyclin D1 and cyclin E were inhibited whereas p21 was induced. To further identify the cause-effect relationship between the suppression of c-Myc signaling pathway and induction of cell cycle arrest, the expression of c-Myc was interfered by siRNA. The results of cell cycle analysis showed that the downregulation of c-Myc signaling pathway by siRNA interference could cause a significant arrest of NPC cell at S phase of the cell cycle. In CNE-2 xenografts, ApoG2 significantly downregulated the expression of c-Myc and suppressed tumor growth in vivo.

Conclusion: Our findings indicated that ApoG2 could potently disturb the proliferation of NPC cells by suppressing c-Myc signaling pathway. This data suggested that the inhibitory effect of ApoG2 on NPC cell cycle proliferation might contribute to its use in anticancer therapy.

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The effect c-Myc siRNA transfection on c-Myc downstream molecules and cell cycle distribution. (A) Comparison of the effect of c-Myc siRNA and scrambled (nontargeting) siRNA on the expression of c-Myc downstream molecules. Transfection of CNE-2 cells with c-Myc or scrambled (nontargeting) siRNA for 48 h. Cells were then subjected to Western blotting using anti-c-Myc, -cyclin D1, -p21, and -p53 antibodies as described in Materials and Methods. Comparison of the effect of scrambled (nontargeting) siRNA (B) and c-Myc siRNA (C) on cell cycle distribution of CNE-2 cells using PI staining and flow cytometry. Each histogram is representative of three experiments. (D) Analysis of cell cycle distributions showed that, compared to srambled siRNA, c-Myc siRNA induced a conspicuous increasing of cells in S phase in CNE-2 cells at 48 h. Bar heights, average of three independent experiments; bars, SD.
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Figure 4: The effect c-Myc siRNA transfection on c-Myc downstream molecules and cell cycle distribution. (A) Comparison of the effect of c-Myc siRNA and scrambled (nontargeting) siRNA on the expression of c-Myc downstream molecules. Transfection of CNE-2 cells with c-Myc or scrambled (nontargeting) siRNA for 48 h. Cells were then subjected to Western blotting using anti-c-Myc, -cyclin D1, -p21, and -p53 antibodies as described in Materials and Methods. Comparison of the effect of scrambled (nontargeting) siRNA (B) and c-Myc siRNA (C) on cell cycle distribution of CNE-2 cells using PI staining and flow cytometry. Each histogram is representative of three experiments. (D) Analysis of cell cycle distributions showed that, compared to srambled siRNA, c-Myc siRNA induced a conspicuous increasing of cells in S phase in CNE-2 cells at 48 h. Bar heights, average of three independent experiments; bars, SD.

Mentions: Authors have reported that the oncoprotein c-Myc regulates the expression of p21 and cyclins, increases cyclin D-CDK4 activity, and facilitates cell cycle progression [15]. Also, Fan et al. found that upregulated expression of c-Myc protein in NPC cells contributed to unrestricted cell proliferation, metastasis, and tumor progression [16]. In our study, the immunoblots data indicated that suppression of the c-Myc pathway might be responsible for ApoG2-induced cell cycle arrest in NPC cells. To test this hypothesis, we used three siRNA oligonucleotides (Ribobio, Guangzhou, China) to knock down c-Myc protein in CNE-2 cells. As shown in fig. 4A, all these three oligonucleotides significantly suppressed the expression of c-Myc protein; the reduction in c-Myc expression led to upregulation of p21 expression and downregulation of cyclin D expression. Cell cycle analysis showed that incubation with scrambled siRNA resulted in a significantly lower CNE-2 cell population arrested at S phase than did incubation with c-Myc siRNA (Fig. 4B and 4C). Compared to srambled siRNA, c-Myc siRNAs induced conspicuous increasing of cells in S phase in CNE-2 cells at 48 h (Fig. 4D). Based on these results, we suggested that suppression of the c-Myc pathway by ApoG2 leads directly to cell cycle arrest in NPC cells.


ApoG2 induces cell cycle arrest of nasopharyngeal carcinoma cells by suppressing the c-Myc signaling pathway.

Hu ZY, Sun J, Zhu XF, Yang D, Zeng YX - J Transl Med (2009)

The effect c-Myc siRNA transfection on c-Myc downstream molecules and cell cycle distribution. (A) Comparison of the effect of c-Myc siRNA and scrambled (nontargeting) siRNA on the expression of c-Myc downstream molecules. Transfection of CNE-2 cells with c-Myc or scrambled (nontargeting) siRNA for 48 h. Cells were then subjected to Western blotting using anti-c-Myc, -cyclin D1, -p21, and -p53 antibodies as described in Materials and Methods. Comparison of the effect of scrambled (nontargeting) siRNA (B) and c-Myc siRNA (C) on cell cycle distribution of CNE-2 cells using PI staining and flow cytometry. Each histogram is representative of three experiments. (D) Analysis of cell cycle distributions showed that, compared to srambled siRNA, c-Myc siRNA induced a conspicuous increasing of cells in S phase in CNE-2 cells at 48 h. Bar heights, average of three independent experiments; bars, SD.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2742515&req=5

Figure 4: The effect c-Myc siRNA transfection on c-Myc downstream molecules and cell cycle distribution. (A) Comparison of the effect of c-Myc siRNA and scrambled (nontargeting) siRNA on the expression of c-Myc downstream molecules. Transfection of CNE-2 cells with c-Myc or scrambled (nontargeting) siRNA for 48 h. Cells were then subjected to Western blotting using anti-c-Myc, -cyclin D1, -p21, and -p53 antibodies as described in Materials and Methods. Comparison of the effect of scrambled (nontargeting) siRNA (B) and c-Myc siRNA (C) on cell cycle distribution of CNE-2 cells using PI staining and flow cytometry. Each histogram is representative of three experiments. (D) Analysis of cell cycle distributions showed that, compared to srambled siRNA, c-Myc siRNA induced a conspicuous increasing of cells in S phase in CNE-2 cells at 48 h. Bar heights, average of three independent experiments; bars, SD.
Mentions: Authors have reported that the oncoprotein c-Myc regulates the expression of p21 and cyclins, increases cyclin D-CDK4 activity, and facilitates cell cycle progression [15]. Also, Fan et al. found that upregulated expression of c-Myc protein in NPC cells contributed to unrestricted cell proliferation, metastasis, and tumor progression [16]. In our study, the immunoblots data indicated that suppression of the c-Myc pathway might be responsible for ApoG2-induced cell cycle arrest in NPC cells. To test this hypothesis, we used three siRNA oligonucleotides (Ribobio, Guangzhou, China) to knock down c-Myc protein in CNE-2 cells. As shown in fig. 4A, all these three oligonucleotides significantly suppressed the expression of c-Myc protein; the reduction in c-Myc expression led to upregulation of p21 expression and downregulation of cyclin D expression. Cell cycle analysis showed that incubation with scrambled siRNA resulted in a significantly lower CNE-2 cell population arrested at S phase than did incubation with c-Myc siRNA (Fig. 4B and 4C). Compared to srambled siRNA, c-Myc siRNAs induced conspicuous increasing of cells in S phase in CNE-2 cells at 48 h (Fig. 4D). Based on these results, we suggested that suppression of the c-Myc pathway by ApoG2 leads directly to cell cycle arrest in NPC cells.

Bottom Line: The results of cell cycle analysis showed that the downregulation of c-Myc signaling pathway by siRNA interference could cause a significant arrest of NPC cell at S phase of the cell cycle.In CNE-2 xenografts, ApoG2 significantly downregulated the expression of c-Myc and suppressed tumor growth in vivo.This data suggested that the inhibitory effect of ApoG2 on NPC cell cycle proliferation might contribute to its use in anticancer therapy.

View Article: PubMed Central - HTML - PubMed

Affiliation: State Key Laboratory of Oncology in South China and Department of Experimental Research, Sun Yat-sen University Cancer Center, Guangzhou, PR China. huzheyu24@gmail.com

ABSTRACT

Background: apogossypolone (ApoG2) is a novel derivate of gossypol. We previously have reported that ApoG2 is a promising compound that kills nasopharyngeal carcinoma (NPC) cells by inhibiting the antiapoptotic function of Bcl-2 proteins. However, some researchers demonstrate that the antiproliferative effect of gossypol on breast cancer cells is mediated by induction of cell cycle arrest. So this study was aimed to investigate the effect of ApoG2 on cell cycle proliferation in NPC cells.

Results: We found that ApoG2 significantly suppressed the expression of c-Myc in NPC cells and induced arrest at the DNA synthesis (S) phase in a large percentage of NPC cells. Immunoblot analysis showed that expression of c-Myc protein was significantly downregulated by ApoG2 and that the expression of c-Myc's downstream molecules cyclin D1 and cyclin E were inhibited whereas p21 was induced. To further identify the cause-effect relationship between the suppression of c-Myc signaling pathway and induction of cell cycle arrest, the expression of c-Myc was interfered by siRNA. The results of cell cycle analysis showed that the downregulation of c-Myc signaling pathway by siRNA interference could cause a significant arrest of NPC cell at S phase of the cell cycle. In CNE-2 xenografts, ApoG2 significantly downregulated the expression of c-Myc and suppressed tumor growth in vivo.

Conclusion: Our findings indicated that ApoG2 could potently disturb the proliferation of NPC cells by suppressing c-Myc signaling pathway. This data suggested that the inhibitory effect of ApoG2 on NPC cell cycle proliferation might contribute to its use in anticancer therapy.

Show MeSH
Related in: MedlinePlus