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Down-regulation of nicotinamide N-methyltransferase induces apoptosis in human breast cancer cells via the mitochondria-mediated pathway.

Zhang J, Wang Y, Li G, Yu H, Xie X - PLoS ONE (2014)

Bottom Line: Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies.The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT.In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, Sir RunRun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT
Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies. However, the functional role of NNMT in breast cancer has not been elucidated. In the present study, we showed that NNMT was selectively expressed in some breast cancer cell lines, down-regulation of NNMT expression in Bcap-37 and MDA-MB-231 cell lines by NNMT shRNA significantly inhibited cell growth in vitro, decreased tumorigenicity in mice and induced apoptosis. The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT. In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2. These data suggest that down-regulation of NNMT induces apoptosis via the mitochondria-mediated pathway in breast cancer cells.

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Lentiviral shRNA-NNMT efficaciously down-regulated expression of NNMT.Real-Time RT-PCR analysis (A, D) and Western blot (B, C, E, F) were used to analyze NNMT expression in Bcap-37, Bcap-37/NC, Bcap-37/NNMT shRNA 1#, Bcap-37/NNMT shRNA 2#, MDA-MB-231, MDA-MB-231/NC, MDA-MB-231/NNMT shRNA 1# and MDA-MB-231/NNMT shRNA 2# cells after infected for 24 h and 48 h. GAPDH was used as an internal control. (A, B, C) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in Bcap-37 cells compared to the Bcap-37/NC. (D, E, F) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in MDA-MB-231 cells compared to the MDA-MB-231/NC. (C) and (F) shows the protein quantification of the Western blot results shown in (B) and (E), respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (A, C, D, F) are expressed as means ± SD of six independent experiments. There was no statistical significance between cells infected with shRNA NC and wild type cells both in Bcap-37 cells and MDA-MB-231 cells. No significant difference of effectiveness was found between shRNA#1 and shRNA#2 in MDA-MB-231 cells. **P<0.01 vs. NC.
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pone-0089202-g002: Lentiviral shRNA-NNMT efficaciously down-regulated expression of NNMT.Real-Time RT-PCR analysis (A, D) and Western blot (B, C, E, F) were used to analyze NNMT expression in Bcap-37, Bcap-37/NC, Bcap-37/NNMT shRNA 1#, Bcap-37/NNMT shRNA 2#, MDA-MB-231, MDA-MB-231/NC, MDA-MB-231/NNMT shRNA 1# and MDA-MB-231/NNMT shRNA 2# cells after infected for 24 h and 48 h. GAPDH was used as an internal control. (A, B, C) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in Bcap-37 cells compared to the Bcap-37/NC. (D, E, F) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in MDA-MB-231 cells compared to the MDA-MB-231/NC. (C) and (F) shows the protein quantification of the Western blot results shown in (B) and (E), respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (A, C, D, F) are expressed as means ± SD of six independent experiments. There was no statistical significance between cells infected with shRNA NC and wild type cells both in Bcap-37 cells and MDA-MB-231 cells. No significant difference of effectiveness was found between shRNA#1 and shRNA#2 in MDA-MB-231 cells. **P<0.01 vs. NC.

Mentions: The efficacy in down-regulating expression of NNMT gene was detected by real-time quantitative RT-PCR and Western blot (Figure 2). Compared with shRNA NC, mRNA and protein levels of NNMT were reduced significantly after NNMT shRNA 1# and NNMT shRNA 2# lentivirus infected into Bcap-37 and MDA-MB-231 cell lines (p<0.01). There was no statistical significance between cells infected with shRNA NC and wild type cells both in Bcap-37 cells and MDA-MB-231 cells.


Down-regulation of nicotinamide N-methyltransferase induces apoptosis in human breast cancer cells via the mitochondria-mediated pathway.

Zhang J, Wang Y, Li G, Yu H, Xie X - PLoS ONE (2014)

Lentiviral shRNA-NNMT efficaciously down-regulated expression of NNMT.Real-Time RT-PCR analysis (A, D) and Western blot (B, C, E, F) were used to analyze NNMT expression in Bcap-37, Bcap-37/NC, Bcap-37/NNMT shRNA 1#, Bcap-37/NNMT shRNA 2#, MDA-MB-231, MDA-MB-231/NC, MDA-MB-231/NNMT shRNA 1# and MDA-MB-231/NNMT shRNA 2# cells after infected for 24 h and 48 h. GAPDH was used as an internal control. (A, B, C) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in Bcap-37 cells compared to the Bcap-37/NC. (D, E, F) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in MDA-MB-231 cells compared to the MDA-MB-231/NC. (C) and (F) shows the protein quantification of the Western blot results shown in (B) and (E), respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (A, C, D, F) are expressed as means ± SD of six independent experiments. There was no statistical significance between cells infected with shRNA NC and wild type cells both in Bcap-37 cells and MDA-MB-231 cells. No significant difference of effectiveness was found between shRNA#1 and shRNA#2 in MDA-MB-231 cells. **P<0.01 vs. NC.
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pone-0089202-g002: Lentiviral shRNA-NNMT efficaciously down-regulated expression of NNMT.Real-Time RT-PCR analysis (A, D) and Western blot (B, C, E, F) were used to analyze NNMT expression in Bcap-37, Bcap-37/NC, Bcap-37/NNMT shRNA 1#, Bcap-37/NNMT shRNA 2#, MDA-MB-231, MDA-MB-231/NC, MDA-MB-231/NNMT shRNA 1# and MDA-MB-231/NNMT shRNA 2# cells after infected for 24 h and 48 h. GAPDH was used as an internal control. (A, B, C) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in Bcap-37 cells compared to the Bcap-37/NC. (D, E, F) NNMT mRNA and protein levels were reduced significantly after infected with NNMT shRNA 1# and NNMT shRNA 2# in MDA-MB-231 cells compared to the MDA-MB-231/NC. (C) and (F) shows the protein quantification of the Western blot results shown in (B) and (E), respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (A, C, D, F) are expressed as means ± SD of six independent experiments. There was no statistical significance between cells infected with shRNA NC and wild type cells both in Bcap-37 cells and MDA-MB-231 cells. No significant difference of effectiveness was found between shRNA#1 and shRNA#2 in MDA-MB-231 cells. **P<0.01 vs. NC.
Mentions: The efficacy in down-regulating expression of NNMT gene was detected by real-time quantitative RT-PCR and Western blot (Figure 2). Compared with shRNA NC, mRNA and protein levels of NNMT were reduced significantly after NNMT shRNA 1# and NNMT shRNA 2# lentivirus infected into Bcap-37 and MDA-MB-231 cell lines (p<0.01). There was no statistical significance between cells infected with shRNA NC and wild type cells both in Bcap-37 cells and MDA-MB-231 cells.

Bottom Line: Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies.The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT.In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2.

View Article: PubMed Central - PubMed

Affiliation: Clinical Laboratory, Sir RunRun Shaw Hospital, School of Medicine, Zhejiang University, Hangzhou, China.

ABSTRACT
Nicotinamide N-methyltransferase (NNMT) has been found involved in cell proliferation of several malignancies. However, the functional role of NNMT in breast cancer has not been elucidated. In the present study, we showed that NNMT was selectively expressed in some breast cancer cell lines, down-regulation of NNMT expression in Bcap-37 and MDA-MB-231 cell lines by NNMT shRNA significantly inhibited cell growth in vitro, decreased tumorigenicity in mice and induced apoptosis. The silencing reciprocal effect of NNMT was confirmed by over-expressing NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines which lack constitutive expression of NNMT. In addition, down-regulation of NNMT expression resulted in reducing expression of Bcl-2 and Bcl-xL, up-regulation of Bax, Puma, cleaved caspase-9, cleaved caspase-3 and cleaved PARP, increasing reactive oxygen species production and release of cytochrome c from mitochondria, and decreasing the phosphorylation of Akt and ERK1/2. These data suggest that down-regulation of NNMT induces apoptosis via the mitochondria-mediated pathway in breast cancer cells.

Show MeSH
Related in: MedlinePlus