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

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.


Introduction
Breast cancer is one of the most common causes of cancerrelated death in women, which accounts for one in four cancerrelated deaths in the United States [1]. In China, according to the most updated but limited cancer registries, breast cancer is the fifth leading cause of cancer-related death for females [2]. There is a decline in breast cancer mortality since 1995 [1][2][3], however, breast cancer is far from being cured because of delayed detection, the progressive growth, late detection of metastases and resistant to conventional therapies. Therefore, there is an urgent need to identify new biomarkers, which are warranted to provide more information on the tumor biology, chemotherapeutic effects, allowing a better prognostic and possibly predictive stratification of patients. Recent researches have reported that the growth of tumor cells may be inhibited via the mitochondrial apoptotic pathway in breast cancer [4,5].
Nicotinamide N-methyltransferase (NNMT, EC 2.1.1.1), a cytoplasmic enzyme belonging to Phase IIMetabolizing Enzymes, which catalyzes the methylation of nicotinamide and other pyridines to form pyridinium ions using S-adenosyl-L-methionine as methyl donor [6]. NNMT also plays a vital role in nicotinamide metabolism and the biotransformation of many drug and other xenobiotic compounds [7]. NNMT exhibits a high expression in the liver and follows a bimodal frequency distribution which might result in differences among individuals in the metabolism and therapeutic effect of drugs [8]. Proteomics analysis and DNA microarray analysis showed that NNMT was expressed at markedly higher levels in several kinds of cancers. It was identified as a novel serum tumor marker for colorectal cancer (CRC) in 2005 [9]. In addition to CRC, abnormal expression of NNMT was also reported in other tumors such as papillary thyroid cancer [10], glioblastoma [11], gastric cancer [12,13], renal carcinoma [14][15][16], oral squamous cell carcinoma [17,18], lung cancer [19], pancreatic cancer [20,21] and ovarian clear cell carcinoma [22]. Our previous studies have also shown that NNMT is overexpressed in a large proportion of renal cell cancers and that high expression of NNMT is significantly associated with unfavorable prognosis [23]. The most recent studies showed that knockdown of NNMT was able to inhibit the proliferation of KB cancer cells [24], renal carcinoma cells [25] and oral cancer cells [26], and NNMT expression was involved in maintaining cell proliferation by increasing the activity of Complex I (NADH:ubiquinone oxidoreductase) in SH-SY5Y neuroblastoma cells [27]. However, the mechanism of NNMT in cell proliferation is largely unknown and the functional role of NNMT in breast cancer has not been reported.
In the present study, we investigated the expression of NNMT in human breast cancer cell lines and found that shRNA targeted against NNMT significantly decreased cell growth, inhibited tumorigenicity in mice and induced apoptosis via the mitochondria-mediated pathway.

Ethics statement
All experiments in the present study were conducted in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The animal experiments were previously approved by the Animal Care and Use Committee at Sir RunRun Shaw hospital of Zhejiang University (Permit Number: 20120222-31). The number of animals used was minimized, and all necessary precautions were taken to mitigate pain or suffering.

Western blot analysis
Cell extracts were prepared with RIPA lysis buffer (Beyotime biotechnology, Shanghai, China). Protein concentrations were measured by a BCA Protein Assay Kit (Beyotime biotechnology, Shanghai, China) using bovine serum albumin (BSA) as the standard. A total of 50 mg of protein samples from each cell line was subjected to 10% sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to Immobilon P Transfer Membrane (Millipore, Bedford, MA, USA). After regular blocking and washing, the membranes were incubated with primary antibodies overnight at 4uC, followed by incubating with HRP-conjugated secondary antibodies for 1 h at room temperature. Signals were visualized using enhanced chemiluminescence detection reagents (Millipore, Billerica, MA, USA) and imaged using an Image Quant LAS-4000 (Fujifilm, Tokyo, Japan). All the experiments were independent and conducted at least three times. The protein quantification of the Western blot results were normalized to GAPDH and then compared to the control group, which was normalized as 1.

Lentiviral shRNA-NNMT infection
Lentiviral vectors against NNMT were synthesized by Gene-Chem Co. Ltd (Shanghai, China). Table 2 showed the sequences of NNMT shRNA 1#, NNMT shRNA 2#, shRNA NC and the frame of lentiviral vectors. Bcap-37 and MDA-MB-231 cells were infected with lentivirus containing shRNA (NNMT shRNA 1#, NNMT shRNA 2#, shRNA NC; MOI = 100 for Bcap-37, MOI = 10 for MDA-MB-231) after seeded (2610 5 cells/well) in six-well plates for 24 h. Ten hours after co-culturing with lentivirus, the supernatant was replaced with fresh medium. 48 h after infection, the transduced cells were sorted for GFP-positive cell populations by BD FACS Aria II System (BD Biosciences, San Jose, CA, USA) and then subjected to functional assays. The efficiency of gene silencing was detected by real-time RT-PCR and Western blot analysis as described above. Cells infected with shRNA NC were used as negative control.

siRNA transfection
When detecting the ROS production, we chose specific siRNAs to silence NNMT expression in order to avoid the fluorescence of GFP in lentiviral vector interfering with the ROS measurement. 2610 5 cells (Bcap-37 and MDA-MB-231) were plated in 6-well plates in 2 ml antibiotic-free DMEM medium supplemented with FBS and 8 ml the NNMT specific siRNAs (10 mM) (sc-61213, Santa Cruz Biotechnology, CA, USA) were transfected into cultured cells at a final concentration of 80 nM using Lipofectamine 2000 transfection reagent (Invitrogen Life Technologies, Carlsbad, CA, USA) according to the manufacturer's instructions. Control siRNA contained a scrambled sequence that would not lead to the specific degradation of any known cellular mRNA.

MTT assay
Cell growth was assessed by the colorimetric MTT assay. All the cells were prepared at a concentration of 2610 4 cells/ml. Aliquots (200 mL) were dispensed into 96-well flat-bottom plates. The cells were allowed to attach for five hours at 37uC and 5% CO 2 , and cell growth was evaluated for up to 120 h. Subsequently, 20 ml of the 5 g/L MTT solution (Sigma, St. Louis, MO, USA) was added to each well. After incubation for 4 h at 37uC, the supernatant was removed carefully, and 150 ml of dimethyl sulfoxide (DMSO, Sigma, St. Louis, MO, USA) was added to each well. Ten minutes after incubation at 37uC, the absorbance value of each well was read at 490 nm using an ELISA plate reader instrument (Model 680, BIO RAD, Osaka, Japan). All experiments were repeated at least three times. The absorbance values at each time point were compared to that of control group at 0 h, which was normalized as 100%.

Plate colony formation assay
The ability of cells to form macroscopic colonies was determined by a plate colony formation assay. Cells in the

Soft agar colony formation assay
Soft agar clonogenic assays were performed at least three times to assess anchorage-independent growth. Cells (1610 4 /well in sixwell plates) were detached and plated in DMEM medium containing 0.3% low melting point (LMP) agar with a 0.5% LMP agar layer underlay. The cells were cultured at 37uC in 5%

Xenograft experiments
Male specific pathogen-free BALB/c Nude mice (6 weeks old, 18-20 g body weight) were handled under pathogen-free sterile conditions, maintained on a 12 hour light:dark cycle (lights on at 7:00am) with continuous access to sterile food and water. For tumorigenicity assays, 2610 6 cells each of the Bcap-37/NC, Bcap-37/NNMT shRNA 1# and Bcap-37/NNMT shRNA 2# cells were subcutaneously injected into the upper portion of the right hind limb of 6 BALB/c Nude mice for each group. Tumor size 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 6 SD of four independent experiments. *P,0.05 vs. control group. doi:10.1371/journal.pone.0089202.g007  respectively. All the experiment were repeated at least three times. The results of the cells treated with NNMT siRNA were compared to that of the ones treated with control siRNA, which were normalized as 1.

Cytochrome c release measurement
The mitochondria fraction and cytosolic fraction were isolated using cytochrome c releasing apoptosis assay kit (BioVision, Inc., Mountain View, CA, USA) according to the manufacturer's instructions. The detection of Cyt c was performed by Western blot analysis as described above. All the experiments were independent and conducted at least three times. The protein quantification of the Western blot results were normalized to GAPDH and then compared to the NC group, which was normalized as 1.

Statistical analysis
All statistical analyses were carried out using the SPSS 19.0 statistical software package. Data were presented as mean 6 SD. The two-tailed independent-samples Student's t-test was performed to analyze the difference among groups. Statistical significance was define as *p,0.05 and **p,0.01.  increased apoptosis percentage were also found (refer to Figure  S1). However, MCF-7/ADR is not a natural cell line which grows poorly without doxorubicin. While some other studies indicated that MDA-MB-468 breast cancer cells could express NNMT [28], but we did not confirm this point in our study. Therefore, we did not use MCF-7/ADR and MDA-MB-468 cell lines to perform the further experiment. To study the effect of down-regulation or overexpression of NNMT on cell biological process, Bcap-37 and MDA-MB-231; MCF-7 and SK-BR-3 cell lines were selected for further study, respectively.

Down-regulation of NNMT expression inhibited the Cell Growth in vitro and in vitro
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.
To evaluate the effect of NNMT specific shRNAs on cell growth in vitro, colorimetric MTT assay was conducted. As shown in Figure 3A and 3B, efficient down-regulation of NNMT resulted in markedly reduced breast cancer cell growth both in Bcap-37 and MDA-MB-231 cell lines. A significant reduction of cell growth could be detected from 72 h when compared to the control cells (p,0.01). These results indicated that NNMT specific shRNAs attenuated the cell growth of breast cancer cells.
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 downregulation of NNMT attenuates the capacity of colony formation ( Figure 4C and 4D) (p,0.01).  Figure 5A). The similar result was found in tumor weight. The mean weight of tumors of Bcap-37/NNMT shRNA 1# (0.3660.07 g, n = 6) and Bcap-37/NNMT shRNA 2# (0.4260.05 g, n = 6) groups were significantly lower than Bcap-37/NC groups (0.6560.13 g, n = 6) (p,0.01, Figure 5B). These results indicated shRNA-mediated NNMT knockdown suppresses tumorigenicity in Bcap-37 cells. Taken together, down-regulation of NNMT expression inhibited the cell growth in vitro and in vivo.

Down-regulation of NNMT expression increased apoptosis and the ratio of Bax/Bcl-2
To determine the role of NNMT in cancer cell survival, MDA-MB-231 and Bcap-37 cells were treated with NNMT shRNAs and subsequently flow cytometry was used to quantify apoptosis. The extent of apoptosis was expressed as the sum of total percentages of annexin-positive populations, which represented the early apoptosis (Annexin V-PE positive/7-AAD negative) and late apoptosis (Annexin V-PE positive/7-AAD positive). As shown in Figure 6A Figure 6B). These results indicated down-regulation of NNMT increased apoptosis in both cell lines infected with NNMT shRNA 1# and shRNA 2# compared to negative control cells.
The Bcl-2 family of proteins, which shares homology in any of the four common Bcl-2 homology (BH) domains, was highly related with apoptosis [29]. Thus, we analyzed the changes of Bcl-2 family and found that the expression of Bax, Bcl-2, Bcl-xL and Puma significantly changed in NNMT down-regulated cells. As shown in Figure 7, the expression of pro-apoptotic proteins, Bax and Puma, was up-regulated significantly in both cell lines infected with NNMT shRNA 1# and NNMT shRNA 2# (p,0.01). On the contrary, the expression of Bcl-2 and Bcl-xL, which are identified as anti-apoptotic proteins, was significantly downregulated (p,0.01). As a result, the down-regulation of NNMT increased both mRNA and protein ratio of Bax/Bcl-2 compared to negative control (p,0.01). This result indicated that apoptosis might be induced by down-regulation of NNMT via regulating the Bax/Bcl-2 ratio in human breast cancer cells.
And as shown in Figure 9, overexpression of NNMT resulted in increased breast cancer cell growth both in MCF-7 and SK-BR-3 cell lines using MTT assay. A significant higher proliferation rate could be detected from 72 h when compared to the control cells (p,0.05). Consistent with the results of MTT assay, the colonies formed by NNMT-overexpressed cells were more numerous than those formed by control group (p,0.05, Figure 10).
To confirm the role of NNMT in cancer cell survival, the effect of NNMT overexpression on apoptosis was also quantified by flow cytometry. The data showed that overexpression of NNMT attenuates apoptosis in both NNMT overexpressed cell lines compared to negative control cells (p,0.05, Figure 11).
All these results showed that overexpression of NNMT could increase cell growth, tumorigenicity and inhibit cell apoptosis, which were consistent with the function of NNMT indicated by down-regulation of NNMT.

Down-regulation of NNMT expression increased ROS production
ROS production was associated with apoptosis [30] and increasing intracellular ROS levels was highly related to apoptosis induction [31]. The mitochondria-mediated apoptotic pathway of cell death is especially susceptible to ROS. To assess the ROS production in NNMT knockdown breast cancer cells, the NNMT specific siRNAs, instead of shRNAs, was used to avoid interference of fluorescence of GFP in lentiviral vector. The efficacy in downregulated expression of NNMT gene by siRNA was confirmed by real-time quantitative RT-PCR and Western blot (p,0.01, Figure 12A and 12B). And as shown in Figure 12C and 12D, down-regulation of NNMT significantly increased ROS production in both of Bcap-37 and MDA-MB-231 cell lines transfected with NNMT siRNAs (p,0.01).

Down-regulation of NNMT expression activated the mitochondria-mediated apoptotic pathway
Down-regulation of NNMT increased mRNA and protein ratio of Bax/Bcl-2 and the production of intracellular ROS, which suggested that down-regulation of NNMT may be involved in mitochondria-mediated pathway. To determine whether downregulation of NNMT induces apoptosis via the mitochondriamediated pathway, we detected the release of Cyt c and the activation of related caspases, such as caspase-9 and caspase-3, which were key events in the mitochondria-mediated apoptotic pathway. As shown in Figure 13, Cyt c was observed to accumulate in cytosolic compartment in NNMT down-regulated Bcap-37 and MDA-MB-231 cells, while the amount of mitochondrial Cyt c was obviously decreased. In addition, the caspase-9, caspase-3 and PARP were decreased in NNMT knockdown Bcap-37 and MDA-MB-231 cells, while the cleaved ones were found significantly increased. Taken together, these results indicated that the down-regulation of NNMT in breast cancer cells resulted in activation of the mitochondria-mediated apoptotic pathway. , 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 6 SD of six independent experiments. **P,0.01 vs. NC. doi:10.1371/journal.pone.0089202.g013 Down-Regulation of NNMT Induces Apoptosis Down-regulation of NNMT expression inactivated Akt and ERK1/2 PI3K/Akt and MAPK pathways are the well known signaling cascade which participated in the regulation of cell progression and survival via protein phosphorylation [32,33]. We tested key signaling components in PI3K/Akt and MAPK pathways and found that the phosphorylation of Akt and ERK 1/2 was decreased in NNMT shRNA treated cells. As shown in Figure 14A, 14B, 14C and 14D, down-regulation of NNMT decreased the expression levels of p-Akt and p-ERK1/2 in Bcap-37 and MDA-MB-231 cells and also decreased the ratio of p-AKT/AKT and p-ERK1/2/ERK1/2. Furthermore, it was confirmed by IGF-1 (Sigma, St. Louis, MO, USA), a potent activator of PI3K/Akt. 100 ng/ml IGF-1 partially decreased the apoptosis in NNMT shRNA treated cells ( Figure 14E and 14F), suggesting the Akt pathway involved in apoptosis induced by down-regulation of NNMT. Discussion NNMT is predominantly expressed in liver, catalyzes the Nmethylation of nicotinamide, pyridines, and other structural analogues that are involved in the biotransformation and detoxification of many drugs and xenobiotic compounds. It plays a pivotal role in cellular events by regulating nicotinamide balance such as energy production, longevity, and cellular resistance to stress or injury [6,[34][35][36]. To the best of our knowledge, there is no direct study on the biological process of NNMT in breast cancer up to now. The only public correlation of NNMT and breast cancer was that NNMT had been found over-expressed in adriamycin-resistant breast cancer cell line MCF-7/ADR compared with its parent cell line MCF-7 [37]. We confirmed high expression of NNMT in some of the breast cancer cell lines, however, the role of NNMT in breast cancer is largely unknown.
In the present study, we investigated the biological function of NNMT in breast cancer cell lines (Bcap-37 and MDA-MB-231). ShRNA lentiviral vector against NNMT was designed to inhibit endogenous NNMT expression in both cell lines. Accompany with the down-regulation of NNMT expression, a significant inhibition of cell growth of Bcap-37 and MDA-MB-231 cells was found. The results of nude mice tumorigenesis experiments on Bcap-37 also showed that down-regulation of NNMT expression inhibited cancer cells tumorigenicity in vivo. 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. Our data are consistent with the results derived from bladder cancer cells [38], KB cancer cells [24], oral carcinoma cells [26] and renal cancer cells [25], which strongly suggested that NNMT plays an important role in cancer cell growth in vitro and in vivo.
Defective apoptotic machinery often confers survival advantage of cancer cells [29], and apoptosis attenuation is important in progressing to states of high-grade malignancy and resistance to therapy in tumors [39,40]. Thus,we analyzed the effect of downregulation of NNMT on apoptosis. There was a higher percentage of apoptosis population in Bcap-37 and MDA-MB-231 cells infected with NNMT shRNA. The cleaved-caspase-3 and cleaved PARP, which are reliable markers of apoptosis, were also showed increased by down-regulation of NNMT. On the contrary, overexpression of NNMT in the MCF-7 and SK-BR-3 breast cancer cell lines showed attenuated apoptosis when compared to negative control cells. Those results together demonstrated that down-regulation of NNMT induces apoptosis in Bcap-37 and MDA-MB-231, which also suppose that NNMT may play a vital role in breast cancer development via apoptosis. The underlying molecular mechanisms of the apoptosis promoted by downregulation of NNMT in breast cancer cells would further clear the role of NNMT in cancer cells.
The Bcl-2 family of proteins, main apoptosis regulators, was designed to explain the mechanism of apoptosis induced by downregulation of NNMT. In the present study, we observed that the expression of Bax and Puma was up-regulated, while the expression of Bcl-2 and Bcl-xL was significantly down-regulated in NNMT shRNA infected breast cancer cells, which resulted in the increase of the ratio of Bax/Bcl-2. Among the Bcl-2 family members, anti-apoptotic Bcl-2 and Bcl-xL have been reported to protect the cells by interacting with mitochondrial proteins such as the adenine nucleotide translocase (ANT) or the voltage dependent anion channel (VDAC), thus preventing them from forming mitochondrial pores, protecting membrane integrity, and inhibiting the release of apoptogenic factors such as Cyt c [41]. On the contrary, Bax can homodimerize or heterodimerize with other pro-apoptotic members such as Bak or truncated Bid, disrupting the integrity of the outer mitochondrial membrane (OMM) by forming mitochondrial pores and increasing its permeability, which can then lead to the release of apoptogenic factors such as Cyt c [42]. Puma, a Bcl-2 family member acting as neutralizing anti-apoptotic proteins, can heterodimerize with Bcl-2 and Bcl-xL and sequester them, thereby blocking their anti-apoptotic action at the mitochondria [29]. Interestingly, down-regulation of NNMT increased ROS production in human breast cancer cell lines was found. It has been reported that increasing ROS production can damage mitochondrial membranes, leading to the opening of mitochondrial permeability transition pore (MPTP) and releasing Cyt c [43,44]. Taken those results together, we infered that downregulation of NNMT in human breast cancer may cause the mitochondria dysfunction and release of Cyt c from mitochondria. The ratio of Bax/Bcl-2 partially showed the response to proximal death and survival signals of cells as reported by Oltvai ZN, et al [45].
Cyt c plays a crucial role for the execution of the mitochondrialmediated intrinsic pathway apoptosis because it can form apoptosome with apoptosis-activating factor 1(Apaf-1) and caspase-9 after releasing into the cytoplasm and activate the executioner caspases-3 and 7, which finally causes cell apoptosis through nuclear fragmentation of cells [46][47][48][49]. To confirm whether down-regulation of NNMT induces apoptosis via the mitochondria-mediated pathway, we analyzed the release of Cyt c and the activation of related caspases, such as caspase-9 and caspase-3, which were key events in the mitochondria-mediated apoptotic pathway. As expected, we have shown that Cyt c was released from mitochondrial fraction into cytosolic fraction and the cleaved caspase-9, caspase-3 and PARP were found significantly increased in NNMT shRNA infected cells. These results indicated that down-regulation of NNMT in breast cancer cells induces apoptosis via the mitochondria-mediated pathway by increasing the ratio of Bax/Bcl-2 and ROS production, resulting in releasing Cyt c from mitochondrial fraction into cytosolic to activate the executioner caspases-3 and 7.
In our study, we also found that the phosphorylation of Akt and ERK 1/2 was decreased in NNMT shRNA treated cells. Akt can inhibit apoptosis through multiple mechanisms and preventing AKT activation can induce apoptosis [50,51]. The result of IGF-1 decreased the apoptosis in NNMT shRNA treated cells indicated that the apoptosis induced by down-regulation of NNMT can be attributed, at least partially to Akt inactivation. This also suggests that the Akt pathway involved in the effect of NNMT on cancer cells. Our results of down-regulation of NNMT on Akt pathway are in line with the most recent reports [52,53]. Win KT, et al reported that NNMT overexpression was significantly positively associated with phosphorylation of Akt and indicated worse prognosis in patients with nasopharyngeal carcinoma recently [53]. Another report shown that NNMT expression regulates neurone morphology in vitro via the sequential activation of ephrin-B2 (EFNB2) and Akt cellular signaling pathways [52]. Diverse cellular functions, ranging from cell survival to cell death, are regulated by activation of ERK pathway [54]. We don't know the exact mechanisms of How NNMT phosphorylates ERK and AKT in breast cancer so far. Ulanovskaya et al (Nat Chem Biol) recently reported that the methylation events regulated by NNMT can alter histone-dependent gene expression, but also extend beyond histones to include tumor suppressor proteins like PP2A. We suppose that the production of ROS and condition of PP2A methylation and demethylation regulated by NNMT may contribute to the phosphorylates ERK and AKT in breast cancer, however, this needs more detailed experiments to confirm.
In summary, we found that down-regulation of NNMT expression significantly inhibited cell growth, decreased tumorigenicity in mice and induced apoptosis via the mitochondriamediated pathway. Although the definite mechanism of its role needs to be further studied, NNMT may become a promising candidate for breast cancer therapy. Figure S1 Down-regulation of NNMT expression inhibited cell growth and induced apoptosis in MCF-7/ADR cells. (A) Western blot were used to analyze NNMT expression in MCF-7/ADR, MCF-7/ADR/NC, MCF-7/ADR/NNMT shRNA 1# and MCF-7/ADR/NNMT shRNA 2# after infected for 48 h. GAPDH was used as an internal control. (B) Cell growth was analyzed using the MTT assay. As shown, remarkably low proliferation rates were observed in MCF-7/ADR/NNMT shRNA 1# and MCF-7/ADR/NNMT shRNA 2# cells compared to MCF-7/ADR/NC cells after 72 h after seeding the cells in plates. 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 6 SD of four independent experiments. (C) Apoptosis was detected by flow cytometric analysis using the Annexin V-PE/7-AAD Apoptosis Detection Kit after seeded for 48 h. The extent of apoptosis is expressed as the sum total percentages of annexin-positive populations. The percentage of apoptosis populations was increased in both cell lines infected with NNMT shRNA 1# and shRNA 2# compared to negative control cells. Values are expressed as means 6 SD of four independent experiments. **P, 0.01 vs. NC. (TIF)