Down-regulation of NNMT expression inhibited the cell growth <i>in vitro</i>.

<p>(A, B) Cell growth was analyzed using the MTT assay. As shown in (A), remarkably low proliferation rates were observed in Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells compared to Bcap-37/NC cells after 72 h after seeding the cells in plates; the similar results were found in MDA-MB-231 cell models (B). The absorbance values at each time point were compared to that of control group at 0 h, which was normalized as 100%. Values are expressed as means ± SD of six independent experiments. **<i>P</i><0.01 vs. NC.</p

Down-Regulation of Nicotinamide N-methyltransferase Induces Apoptosis in Human Breast Cancer Cells via the Mitochondria-Mediated Pathway

<div><p>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 <i>in vitro</i>, 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.</p></div

Effect of down-regulated NNMT on cytochrome c releasing and caspase processing.

<p>Cytochrome c in cytosolic and mitochondrial extracts and the processing of caspases-9, 3 and PARP in Bcap-37 cell (A) and MDA-MB-231 cell (C) were analyzed by Western blot. GAPDH was used as an internal control to ensure that equal amounts of proteins were loaded in each lane. (B) and (D) shows the protein quantification of the western blot results shown in (A) and (C), respectively. Cyt c in cytosolic compartment was significantly increased, while the amount of mitochondrial Cyt c was significantly decreased in both cell lines infected with NNMT shRNA 1# and shRNA 2# compared to negative control. In addition, the caspase-9, caspase-3 and PARP were significantly decreased, while the cleaved ones were found significantly increased compared to negative control. The protein levels were normalized to GAPDH level and all values were shown compared to the NC, which was normalized as 1. Values in (B, D) are expressed as means ± SD of six independent experiments. **<i>P</i><0.01 vs. NC.</p

Down-regulation of NNMT expression decreased the plate efficiency and attenuated the capacity of colony formation in soft ager

<p>(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 (**<i>P</i><0.01). The similar result was found in MDA-MB-231 cell models (D). Values are expressed as means ± SD of four independent experiments.</p

Down-regulation of NNMT expression inactivated Akt and ERK1/2.

<p>(A, B, C, D) The expression of Akt, p-Akt, ERK1/2 and p-ERK1/2 in Bcap-37 and MDA-MB-231 cells were analyzed by Western blot. GAPDH was used as an internal control. (C) and (D) show the ratio of p-AKT/AKT and p-ERK1/2/ERK1/2 after protein quantification of the western blot results shown in (A) and (B), respectively. The protein levels were normalized to GAPDH level and all values were shown compared to the NC, which was normalized as 1. Values in (C, D) are expressed as means ± SD of six independent experiments. The phosphorylation of Akt and ERK 1/2 was decreased significantly in both cell lines infected with NNMT shRNA 1# and shRNA 2# compared to negative control (*<i>P</i><0.05; **<i>P</i><0.01). (E, F) Effects of IGF-1 on apoptosis induced by down-regulation of NNMT were detected by Annexin V-PE and 7-AAD staining method. Each group of cells were seeded into 6-well plates with or without IGF-1 at a final concentration of 100 ng/ml. Apoptotic cells population was determined after incubation for 48 h. 100 ng/ml IGF-1 decreased the apoptosis significantly in NNMT knockdown Bcap-37 and MDA-MB-231 cells compared to negative control. Values are expressed as means ± SD of six independent experiments. (**<i>P</i><0.01).</p

Overexpression of NNMT increased the plate efficiency and enhanced the capacity of colony formation in soft ager.

<p>(A, B) To test plate colony formation of MCF-7 and SK-BR-3 cells transfected with pcDNA3.1/NNMT, 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 MCF-7/NNMT-1 and MCF-7/NNMT-2 cells was higher than that of MCF-7/Vector group. The similar result was found in SK-BR-3 cell models (B). (C, D) The colony formation numbers of MCF-7/NNMT-1 and MCF-7/NNMT-2 cells foci >100 µm after 14 days were more numerous than that of MCF-7/Vector group (*<i>P</i><0.05). The similar result was found in SK-BR-3 cell models (D). Values are expressed as means ± SD of four independent experiments.</p

Primer series of NNMT, Bcl-2, Bax, Bcl-xL, Puma and GAPDH gene.

<p>Primer series of NNMT, Bcl-2, Bax, Bcl-xL, Puma and GAPDH gene.</p

Down-regulation of NNMT expression inhibited the tumor growth <i>in vivo</i>

<p>(A, B) Xenograft experiment was used to assess the effect of down-regulation of NNMT expression on the cell growth of Bcap-37 <i>in vivo</i> (n = 6 for each group). Mice in all groups developed tumors. (A) The xenograft tumor volume was measured using calipers every three days. The average xenograft tumor volume was significantly smaller in Bcap-37 cells infected with NNMT shRNAs (NNMT shRNA 1# and NNMT shRNA 2#). (B) The average tumor weight was significantly lower in Bcap-37 cells infected with NNMT shRNAs at day 30. Values are expressed as means ± SD. There was no statistical significance between cells infected with NNMT shRNA 1# and shRNA 2# (*<i>P</i><0.05; **<i>P</i><0.01).</p

Construction of MCF-7 and SK-BR-3 cell strains expressing NNMT stably.

<p>Real-Time RT-PCR analysis (A, C) and Western blot (B, D) were used to analyze NNMT expression in MCF-7, MCF-7/Vector, MCF-7/NNMT-1, MCF-7/NNMT-2, SK-BR-3, SK-BR-3/Vector, SK-BR-3/NNMT-1 and SK-BR-3/NNMT-2. GAPDH was used as an internal control. (A, B) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in MCF-7 cells compared to the MCF-7/Vector. (C, D) NNMT mRNA and protein levels were increased significantly after transfected with pcDNA3.1/NNMT in SK-BR-3 cells compared to the SK-BR-3/Vector. The differences between cells transfected with pcDNA3.1 and wild type cells were not significant both in MCF-7 cells and SK-BR-3 cells. (B) and (D) shows the protein quantification of the western blot results, respectively. The mRNA and protein levels were normalized to GAPDH level and are shown relative to the control groups (normalized as 1). Values in (B, D) are expressed as means ± SD of four independent experiments. *<i>P</i><0.05 vs. control group.</p

Overexpression of NNMT promoted the cell growth <i>in vitro</i>.

<p>(A, B) Cell growth was analyzed using the MTT assay. As shown in (A), higher proliferation rates were observed in MCF-7/NNMT-1 and MCF-7/NNMT-2 cells compared to MCF-7/Vector cells after 72 h after seeding the cells in plates; the similar results were found in SK-BR-3 cell models (B). The absorbance values at each time point were compared to that of control group at 0 h, which was normalized as 100%. Values are expressed as means ± SD of four independent experiments. **<i>P</i><0.01 vs. control group; *<i>P</i><0.05 vs. control group.</p