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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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Down-regulation of NNMT expression decreased the plate efficiency and attenuated the capacity of colony formation in soft ager(A, B) To test plate colony formation of Bcap-37 and MDA-MB-231 cells infected with NNMT shRNAs, cells were placed in wells with media and incubated for 14 days before counting the number of colonies (foci>50 µm). The plate efficiency of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells were lower than that of Bcap-37/NC group. The similar result was found in MDA-MB-231 cell models (B). (C, D) Colony formation of Bcap-37 and MDA-MB-231 infected with NNMT shRNAs was carried out by placing cells in media containing soft ager for 14 days. The numbers of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA foci >100 µm were less than that of Bcap-37/NC group (**P<0.01). The similar result was found in MDA-MB-231 cell models (D). Values are expressed as means ± SD of four independent experiments.
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pone-0089202-g004: Down-regulation of NNMT expression decreased the plate efficiency and attenuated the capacity of colony formation in soft ager(A, B) To test plate colony formation of Bcap-37 and MDA-MB-231 cells infected with NNMT shRNAs, cells were placed in wells with media and incubated for 14 days before counting the number of colonies (foci>50 µm). The plate efficiency of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells were lower than that of Bcap-37/NC group. The similar result was found in MDA-MB-231 cell models (B). (C, D) Colony formation of Bcap-37 and MDA-MB-231 infected with NNMT shRNAs was carried out by placing cells in media containing soft ager for 14 days. The numbers of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA foci >100 µm were less than that of Bcap-37/NC group (**P<0.01). The similar result was found in MDA-MB-231 cell models (D). Values are expressed as means ± SD of four independent experiments.

Mentions: Plate colony formation and soft agar colony formation assay were conducted to validate the changes in cell proliferation observed using the MTT assay. The plate efficiency of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells were lower than that of Bcap-37/NC group (Figure 4A). The similar result was found in MDA-MB-231 cell models (Figure 4B). The results of soft agar colony formation assay indicated efficient down-regulation of NNMT attenuates the capacity of colony formation (Figure 4C and 4D) (p<0.01).


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)

Down-regulation of NNMT expression decreased the plate efficiency and attenuated the capacity of colony formation in soft ager(A, B) To test plate colony formation of Bcap-37 and MDA-MB-231 cells infected with NNMT shRNAs, cells were placed in wells with media and incubated for 14 days before counting the number of colonies (foci>50 µm). The plate efficiency of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells were lower than that of Bcap-37/NC group. The similar result was found in MDA-MB-231 cell models (B). (C, D) Colony formation of Bcap-37 and MDA-MB-231 infected with NNMT shRNAs was carried out by placing cells in media containing soft ager for 14 days. The numbers of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA foci >100 µm were less than that of Bcap-37/NC group (**P<0.01). The similar result was found in MDA-MB-231 cell models (D). Values are expressed as means ± SD of four independent experiments.
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pone-0089202-g004: Down-regulation of NNMT expression decreased the plate efficiency and attenuated the capacity of colony formation in soft ager(A, B) To test plate colony formation of Bcap-37 and MDA-MB-231 cells infected with NNMT shRNAs, cells were placed in wells with media and incubated for 14 days before counting the number of colonies (foci>50 µm). The plate efficiency of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells were lower than that of Bcap-37/NC group. The similar result was found in MDA-MB-231 cell models (B). (C, D) Colony formation of Bcap-37 and MDA-MB-231 infected with NNMT shRNAs was carried out by placing cells in media containing soft ager for 14 days. The numbers of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA foci >100 µm were less than that of Bcap-37/NC group (**P<0.01). The similar result was found in MDA-MB-231 cell models (D). Values are expressed as means ± SD of four independent experiments.
Mentions: Plate colony formation and soft agar colony formation assay were conducted to validate the changes in cell proliferation observed using the MTT assay. The plate efficiency of Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells were lower than that of Bcap-37/NC group (Figure 4A). The similar result was found in MDA-MB-231 cell models (Figure 4B). The results of soft agar colony formation assay indicated efficient down-regulation of NNMT attenuates the capacity of colony formation (Figure 4C and 4D) (p<0.01).

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