NGX6 expression improves the sensitivity of tamoxifen-resistant MCF-7 cells through modulation of the Smad signaling pathway

  • Authors:
    • Wen-Jing Zhao
    • Ke Wang
  • View Affiliations

  • Published online on: April 8, 2013     https://doi.org/10.3892/ijo.2013.1886
  • Pages: 2060-2068
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Abstract

The effects of nasopharyngeal carcinoma-associated gene 6 (NGX6) in breast cancer was studied. We demonstrated that the levels of the NGX6 protein and mRNA were lower in patients with tamoxifen-resistant tumors compared to patients with tamoxifen-sensitive tumors. Tamoxifen was able to decrease proliferation, increase apoptosis and induce G1 arrest in NGX6-expressing TRM-7 cells in vitro. In order to detect the mechanism(s) of tamoxifen action, we first obtain the three-dimensional structure of the NGX6 protein by using Protein Homology/analogY Recognition Engine (PHYRE). Prediction of the docking between the NGX6 protein and tamoxifen was performed using SYBYL-X 1.3. Furthermore, we found that tamoxifen activated Smad2/3, and increased the expression of Smad4 in NGX6-expressing TRM-7 cells as evaluated by western blot analysis. Smad2/3-targeted siRNA was used to confirm the mechanism(s) of tamoxifen action in NGX6-expressing cells. These results indicated that NGX6 may increase the sensitivity of breast cancer cells to tamoxifen.

Introduction

Various cancers associated with the short arm of chromosome 9 for the region contains tumor suppressors (15). Nasopharyngeal carcinoma-associated gene 6 (NGX6) localized on chromosome 9p21–22 was originally cloned from nasopharyngeal carcinoma cells (6). The full-length of the NGX6 cDNA is 2134 bp in length and encodes a putative protein of 338 amino acids (7,8). NGX6 expressed at higher levels in normal nasopharyngeal epithelial tissues than cancer tissues (7). Another study showed similar results in colorectal carcinoma (8). Moreover, NGX6 caused the regression of tumorigenesis and metastasis of colon and nasopharyngeal cancer in vivo(8,9). Accumulating evidence indicates that NGX6 plays a role as suppressor in cancer, however, the effects of NGX6 on breast cancer remain unclear. To better understand the role and mechanism(s) of the NGX6 gene, we upregulated NGX6 expression in breast cancer cells using pCDNA3.1-NGX6 transfection. Then the combined effects of tamoxifen and NGX6 were evaluated in tamoxifen-resistant MCF-7 cells.

Breast cancer is the most frequently diagnosed cancer and the leading cause of cancer death in females worldwide, accounting for 23% (1.38 million) of the total new cancer cases and 14% (458,400) of the total cancer deaths in 2008 (10). In China, breast cancer is the most frequently diagnosed cancer in women and is the second leading cause of cancer-related female deaths (1113). Estrogen action through estrogen receptor (ER)-α is a critical regulator of breast cancer cell proliferation and survival (14). ER-positive breast cancers have low sensitivity to chemotherapy (15). In the past 30 years, tamoxifen has been used for ER-positive breast cancer patients as the primary therapeutic choice. Unfortunately, tamoxifen has no significant efficacy for >50% of ER-positive breast cancer patients (16). An adjuvant therapy is required for ER-positive breast cancer treatment.

In the present study, we demonstrate that overexpression of NGX6 promotes the antitumor effects of tamoxifen on tamoxifen-resistant MCF-7 cells, which may be due at least partially to activation of Smad signaling pathway.

Materials and methods

Breast cancer patient specimens

Specimens were derived from 24 patients with no chemotherapy or radiotherapy before the resection at the Department of Breast Surgery, The First Affiliated Hospital of China Medical University between July 2008 and July 2011. Breast cancer tissue specimens were derived from 12 patients showing tamoxifen resistance (samples 1–12). The control specimens were obtained from other 12 patients who showed no tamoxifen resistance (samples 13–24). Basic patient information is summarized in Table I. All patients approved the use of tumor tissues for clinical research and China Medical University Ethics Committee approved the research protocols.

Table I

The antibodies used in the western blot analysis.

Table I

The antibodies used in the western blot analysis.

ProteinProducerCatalog no.Dilution
NGX6Santa Cruz (Santa Cruz, CA, USA)sc-1020361:200
P-Smad2/3sc-117691:200
T-Smad2/3sc-1330981:200
T-Smad4sc-79661:200
p21cip1sc-62461:200
CyclinD1sc-7531:200
Caspase-9sc-735481:200
Caspase-3sc-72721:200
β-actinsc-477781:1,000
Immunohistochemistry (IHC)

NGX6 was detected immunohistochemically in 4% buffered-formalin-fixed, paraffin-embedded tissue samples. Thick sections (4-μm thick) were cut and mounted on microscope slides. Endogenous peroxidase activity was blocked by incubating sections in 3% hydrogen peroxide for 30 min. Antigen retrieval was performed in citrate buffer (10 mM, pH 6.0) for 30 min at 95°C in an induction cooker (CH2082, Galanz®, Beijing, China). Primary antibody was applied and sections were incubated at 4°C overnight. Negative controls were without primary antibodies. Sections were washed with PBS and then incubated with a biotinylated secondary antibody at 37°C for 2 h and then exposed to a streptavidin complex (HRP; Santa Cruz Biotechnology Santa Cruz, CA). Positive reactions were visualized using 3, 3′-diaminobenzidine tetrahydrochloride (DAB; Santa Cruz), followed by counterstaining with hematoxylin (Beyotime, Shanghai, China).

Cell culture

MCF-7 cell lines were obtained from ATCC (Rockville, MD). Cells were maintained at 37°C in a 5% CO2 incubator in RPMI-1640 medium (Sigma-Aldrich, Carlsbad, CA) containing 10% heat-inactivated FBS and 1% penicillin-streptomycin. Tamoxifen-resistant MCF-7 (TRM-7) cells were established according to Kim et al(17).

Construction of vectors and transfection

The NGX6 cDNA 2.1-kb fragment that was obtained from the plasmid pBSK including NGX6 (a gift of Wang Yan, China Medical University) was cloned into the EcoRI/XholI sites of the pcDNA3.1 vector. The directional cloning of NGX6 cDNA insert was confirmed by restriction mapping (data not shown). TRM-7 cells were transfected with the pcDNA3.1-NGX6 vector using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA) according to the manufacturer’s instructions. Twenty-four hours post-transfection with pcDNA3.1-NGX6, cells were selected with G418 (450 μg/ml) (Invitrogen) for 10–12 days. Drug-resistant clones were isolated and expanded. The three resulting NGX6-positive cell lines were named T1, T2 and T3.

RNA interference

T1, T2 and T3 cells were transfected at 70% confluency with 200 nM of Smad2/3-targeted siRNA (Santa Cruz, sc-37238) using Oligofectamine (Invitrogen). Twenty-four hours after transfection, the cells were collected and processed for western blotting and real-time PCR as well as for assessments of cell cycle and apoptosis.

Quantitative real-time PCR and RT-PCR

Total tissue and cellular RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer’s instructions. First-strand cDNAs were generated in reverse transcriptase reactions containing total RNA, poly(dT) oligonucleotides and SuperScript II reverse transcriptase (Invitrogen). The level of tissular NGX6 mRNA was then subjected to quantitative real-time PCR analysis. NGX6 primers used included 5′-TGACCTGTTCCAAAGAGTCCCTG-3′ (forward) and 5′-GCAGCTTCCAGCACATATCGACT-3′ (reverse). GAPDH primers used included 5′-AGAAGGCTGGGGCTCATTTG-3′ (forward) and 5′-AGGGGCCATCCACAGTCTTC-3′ (reverse). Amplification of NGX6 and GAPDH was performed with one cycle at 95°C for 10 min and 40 cycles of 95°C for 15 sec and 60°C for 60 sec. The fold changes between groups were determined by the comparative Ct method (2−ΔΔCT) (18).

PCR amplification of cellular cDNA was performed in 15-μl mixtures. The primers were used as described above. The RT-PCR conditions were: one cycle at 94°C for 5 min, followed by 35 cycles of 94°C for 45 sec, 72°C for 30 sec, 56°C for 45 sec and final extension at 72°C for 10 min. Finally, products were resolved by 1% agarose gel electrophoresis and visualized by ethidium bromide staining and a UV imaging system (UVP, Upland, CA).

Cell growth assay

Tamoxifen (T5648) was purchased from Sigma-Aldrich. Cells (1,500 cells/well) including TRM-7, TRM-7 transfected with pcDNA3.1-NGX6 (T1, T2 and T3), T1, T2 and T3 treated with tamoxifen (T1-tam, T2-tam and T3-tam) and T1, T2 and T3 treated with both tamoxifen and siRNA (T1-tam+siRNA, T2-tam+siRNA and T3-tam+siRNA) were plated in 96-well plates and incubated in normal conditions. Then cells were treated with 10 μl of MTT solution (5 mg/ml, Sigma). Finally, absorbance was measured at 550–560 nm. MCF-7 cells treated with tamoxifen were used as a control.

TUNEL assay

For apoptosis detection, cells were washed in PBS, fixed, permeabilized and subjected to TUNEL labeling using the in situ cell death detection kit (Keygen, no. KGA702, Nanjing, China) according to the manufacturer’s protocol. After counterstaining with DAPI (1 μg/ml), photographic images were taken using an Olympus CX71 fluorescence microscope (Olympus, Tokyo, Japan). TUNEL-positive nuclei were stained green and all other nuclei were stained blue (19,20).

Apoptosis was assessed by the Annexin V/propidium iodide (PI) double staining

In order to calculate the ratio of apoptotic cells quantitatively, cells were harvested and stained with Annexin V/PI according to the manufacturer’s instructions (Keygen, no. KGA107). Briefly, cells were collected, washed twice in ice-cold PBS and resuspended in binding buffer at a density of 1×106 cells/ml. The cells were simultaneously incubated with fluorescein-labeled Annexin V and propidium iodide (PI) for 20 min and flow cytometric (FCM) analysis performed using FACSCalibur (Model FACSC 420, BD Biosciences, Baltimore, MD). Annexin V-FITC generated signals were detected with a FITC signal detector (FL1, 525 nm). PI signals were monitored using a detector for phycoerythrin emission (FL2, 575 nm). Data were analyzed using CellQuest software (BD Biosciences).

PHYRE database was used to generate a predicted structural model

The protein sequence of NGX6 was obtained from the PubMed (http://www.ncbi.nlm.nih.gov/protein/NP_001036055.1) and submitted to Protein Homology/analogY Recognition Engine (PHYRE version 2). Based on homology sequence in PHYRE server, the three-dimensional structure of NGX6 protein was predicted. This method uses structural alignments of homologous proteins of similar three-dimensional structure in the structural classification of protein databases to obtain a structural equivalence of residues (21,22). Once a three-dimensional structure for NGX6 was predicted using PHYRE database, homology modeling was performed using Modeler (23).

Preparation of the proteins and ligand structures for docking

We applied our approach to the predicted structural model of NGX6. The molecular structure of tamoxifen (CID_2733526) was downloaded from Pubchem Compound (http://www.ncbi.nlm.nih.gov/pccompound). Data were imported into the modeling software SYBYL-X 1.3 (Tripos International, St. Louis, MO). All non-protein components such as water molecules, metal ions and lipids were deleted and hydrogen atoms were added to the protein structures. The interaction of tamoxifen and NGX6 protein was analyzed by SYBYL-X 1.3.

Network data and analysis

The complete set of protein-protein associations for NGX6 was extracted from the downloaded STRING database (http://string-db.org) (24); each association between a pair of proteins has a confidence score (S) ranging from 0.15 to 0.999 that was inferred from the evidence used to establish the association.

Western blotting

Cells (4×105 cells/ml) were lysed using lysis buffer (50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% Triton X-100, 0.1% sodium dodecyl sulfate (SDS), 1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM Na3VO4, 1 mM NaF). The extracts were incubated on ice for 20 min, centrifuged at 12,000 × g for 20 min at 4°C and supernatants collected. Protein concentrations were determined using Bradford assay (Bio-Rad, Hercules, CA) and proteins were resolved by 10% Bis-Tris gel electrophoresis transferred to a nitrocellulose membrane and western blot analysis performed. The cell signaling related proteins probed with primary antibodies are summarized in Table I.

Statistical analysis

All the data were analyzed with the software Statistical Package for Social Sciences (SPSS; V.16.0 for windows; Chicago, IL) and Student’s t-test was used. P<0.05 indicated a statistically significant difference.

Results

The levels of NGX6 protein and mRNA in breast cancer specimens

We classified the 24 patients according to tamoxifen sensitivity. The two groups of patients showed no differences in clinicopathologic background factors except hormone receptor status (Table II). NGX6 protein expression in cancer tissue was significantly lower than in normal tissue in all patients (P<0.05) (Fig. 1A). However, NGX6 expression was significantly lower in tumor tissues from tamoxifen-resistance patients (samples 1–4) compared to tumor tissues from tamoxifen-sensitive patients (samples 13–16) (P<0.05) (Fig. 1A). NGX6 transcription was also reduced by real-time PCR analysis. Results showed that the levels of NGX6 mRNA and protein were coincident with each other (P<0.05) (Fig. 1B). The immunohistochemical staining results were consistent with the western blot analysis results (Fig. 1C).

Table II

Clinicopathologic background factors.

Table II

Clinicopathologic background factors.

Clinicopathologic featuresTamoxifen-sensitive group (%)Tamoxifen-resistant group (%)P-value
No. of patients1212
  Age (years)54±856±7NS
Hormone receptor status
  ER<0.01
    Positive11 (91.7)2 (16.7)
    Negative1 (8.3)10 (83.3)
  PR<0.01
    Positive9 (75.0)2 (16.7)
    Negative3 (25.0)10 (83.3)
StageNS
  15 (41.7)6 (50.0)
  2A3 (25.0)3 (25.0)
  2B4 (33.3)3 (25.0)
GradeNS
  I3 (25.0)4 (33.3)
  II6 (50.0)3 (25.0)
  III3 (25.0)5 (41.7)
Lymph node metastasisNS
  Negative7 (58.3)6 (50.0)
  Positive5 (41.7)6 (50.0)
Tumor histologyNS
  Invasive ductal carcinoma5 (41.7)6 (50.0)
  Invasive lobular carcinoma3 (25.0)3 (25.0)
  Medullary carcinoma4 (33.3)3 (25.0)

[i] NS, no significant (P>0.05).

The levels of NGX6 protein and mRNA in breast cancer cells

TRM-7 cells were transfected with pcDNA3.1-NGX6. Stable cell lines were generated and levels of NGX6 protein and mRNA were detected using western blotting and RT-PCR assays. In RT-PCR assays, levels of NGX6 mRNA were lower in TRM-7 cells than in transfected cells (Fig. 2A). To examine the relationship between levels of TRM-7 and transcription of TRM-7, western blotting of TRM-7 was performed. In western blot assays, levels of NGX6 were lower in untransfected TRM-7 cells compared with transfected TRM-7 cells (Fig. 2B).

Growth inhibitory effects of tamoxifen in NGX6-expressing breast cancer cells

We first determined the proper dose of tamoxifen necessary to induce growth inhibition in MCF-7 cells. The IC50 value for tamoxifen was determined by MTT assay (P<0.05) (Fig. 3A). The proliferation ratio of MCF-7 cells was significantly inhibited and showed a dose-dependent effect (IC50=5.03 μM). Growth inhibition was not found in TRM-7 cells after tamoxifen treatment (P>0.05) (Fig. 3A). T1 cells showed lower viability than TRM-7 cells (P<0.05) (Fig. 3A), however, the proliferation ratio of cells in the T1 group with tamoxifen treatment is significantly lower than cells in the T1 group or TRM-7 group (P<0.05) (Fig. 3A). Tamoxifen showed no effects on T1 group with Smad2/3-targeted siRNA treatment (P>0.05) (Fig. 3A).

The ratio of apoptotic cells in each group was determined by TUNEL assay. Results indicate that there was slight apoptosis in NGX6-expressing cells. In contrast, there was a significant increase in the number of apoptotic cells (bright green) when NGX6-expressing cells underwent tamoxifen treatment (Fig. 3B). The results were quantified by using Annexin V and PI double-staining. T1 cells (3.4±0.37%) exhibited increased apoptosis compared to TRM-7 (1.3±0.28%) (P<0.05) (Fig. 3C). The number of apoptotic cells in the T1-tam group (23.7±1.25%) is significantly higher than that in the T1 group. Furthermore, the apoptotic cells in T1-tam-siRNA group showed no significantly changes compared to T1 group (P>0.05) (Fig. 3C).

The effect of NGX6-expression and tamoxifen on the cell cycle of breast cancer cells was determined by PI staining. The percentage of the T1 cells in G1 phase (45.27%) was increased when compared to the TRM-7 (24.55%) cells. More increased cells of T1-tam group (64.42%) in G1 phase were found than that in T1 group (P<0.05) (Fig. 3D). No changes were found in T1-tam-siRNA group compared to T1 group (P>0.05) (Fig. 3D). Results of all above experiments using T2 and T3 clones were comparable to results using the T1 clone (Fig. 3).

The mechanism(s) of tamoxifen in NGX6-expressing breast cancer cells

In addition, we detected the mechanism of NGX6 in breast cancer cells. Firstly, PHYRE analysis predicted reelin [a large glycoprotein secreted from Cajal-Retzius cells of developing cerebral cortex (25)] as a factor with significant structural homology to NGX6 (Fig. 4A). The homology modeling for NGX6 protein was performed using PHYRE server with template that generated with highest similarity percentage (Fig. 4B). Secondly, we found the interaction of NGX6 protein and tamoxifen. As shown in Fig. 4C, the results of the computer modeling showed that tamoxifen was bound to the active sites of NGX6 protein. Lastly, a raw network consisting of the highest scoring interaction partners of NGX6 was arranged by using STRING database (Fig. 4D). Based on the results, we focused on cyclin D1 (CCND1), a regulator of cell cycle, for G1 arrest was found in NGX6 expressing cells in our studies.

NGX6 activates Smad4-dependent signaling

To confirm activation of Smad4 following exogenous expression of NGX6 in T1-tam cells, we performed western blot analysis using antibodies that recognize phosphorylated, active Smad family members. Western blot analysis showed that phosphorylated levels of Smad2/3 significantly increased in T1-tam cells compared to T1 cells. Total protein levels of Smad2/3 remained unchanged (Fig. 5). In contrast, the total protein level of Smad4 significantly increased in T1-tam cells (Fig. 5). Upregulation of p21 and downregulation of cyclin D1 were found accompanied with activation of Smad4. After Smad2/3 target siRNA treatment, both the proteins of the Smad signaling pathway (Fig. 5) and the viability and apoptosis of T1 cells showed no changes (Fig. 3). These results indicated that Smad2/3 knockdown T1 cells showed no reaction to tamoxifen. The changes of all above proteins in T2 and T3 clones were similar to the T1 clone (Fig. 5).

Discussion

In an attempt to provide a novel biotherapy to tamoxifen-resistant breast tumors, we carried out this study. A number of independent studies have evaluated the expression levels of NGX6 gene in various malignancies (2628). Eskandari-Nasab et al(29) found that the relative level of NGX6 mRNA was significantly lower in cancer than that in non-cancer breast tissue specimens by real-time PCR. Consistent with previous studies, we found the levels of NGX6 mRNA and protein in breast cancer tissues were lower than in normal tissue. Interesting, NGX6 expressed at lower level in cancer tissues from tamoxifen-resistant patients than tamoxifen-sensitive patients. Based on this result, we predicted that NGX6 may affect the sensitivity of breast cancer to tamoxifen. So we upregulated NGX6 expression in tamoxifen-resistant breast cancer cells TRM-7 and detected the antitumor activities of NGX6. We confirmed that NGX6 could inhibit viability, induce cell cycle arrest and apoptosis. Previous studies have showed that NGX6 inhibits proliferation (9) and arrests cell cycle progression at G0/G1 phase of colon cancer cells in vitro(30). However, the main purpose of our study was to identify whether NGX6 could improve the sensitivity of tamoxifen-resistant breast cancer cells. Furthermore, we confirmed a higher apoptotic ratio in NGX6-expressing breast cancer cells after tamoxifen treatment. The results indicated NGX6 may be as a receptor to tamoxifen. In other words, tamoxifen may play its function through NGX6. Interesting, we found tamoxifen could dock into the active centers of NGX6 by using computer simulation tool. Guo et al(31) found that indomethacin and NGX6 inhibit the proliferation and invasiveness of HT-29 and SW620 colon cancer cells by attenuating the WNT/β-catenin signaling pathway. Overexpression of NGX6 led to the inhibition of nuclear translocation of JNK1/2 proteins and caused the accumulation of p-JNK1 protein in cytoplasm (32). However, the mechanism of NGX6 in breast cancer cells is not clear. We predicted the potential signaling pathway of NGX6 by using STRING database. According to both the simulation and the experimental results, we performed a preliminary screen of signaling pathways that have tumor suppressor function and are not confirmed by previous studies to determine which pathways are affected by NGX6 expression in TRM-7 cells. We found that of all the pathways that we screened, only Smad4-dependent signaling changed with the combination of NGX6 and tamoxifen. Interesting, we identified that NGX6 and tamoxifen activated downstream proteins in the Smad signaling pathway. In addition, the results were confirmed by using Smad2/3 siRNA. The Smad family of proteins are classified according to their different functions; Smad2 and -3 are the ‘receptor-regulated SMADs’, Smad4 is termed the ‘common-mediator SMAD’ (33). Phosphorylated Smad2/3 form complexes with Smad4, translocate to the nucleus and regulate the transcription of target genes by direct binding to specific DNA sequences (34). We also confirmed the levels of p-Smad2/3 and Smad4 were significantly increased after NGX6 and tamoxifen treatment. The effects were reversed by Smad2/3 siRNA. Consistent with the results of cell experiments, knockdown of Smad2/3 expression by RNAi could also reduce the antitumor activities of NGX6 and tamoxifen.

In conclusion, our data showed the distinct content of NGX6 in tamoxifen-resistant and tamoxifen-sensitive patients. NGX6 may play a role in inhibiting proliferation, inducing apoptosis and arresting cell cycle of breast cancer cells. However, in combination with tamoxifen, the effects of NGX6 were significantly strengthened. Tamoxifen could dock into the active centers of NGX6. Furthermore, we confirmed NGX6 and tamoxifen activated the Smad signaling pathway (Fig. 6). These data provide experimental basis for biotherapy of tamoxifen-resistant breast cancer.

Acknowledgements

The authors wish to thank Miss Yu Miao for technical support.

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June 2013
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Zhao W and Zhao W: NGX6 expression improves the sensitivity of tamoxifen-resistant MCF-7 cells through modulation of the Smad signaling pathway. Int J Oncol 42: 2060-2068, 2013
APA
Zhao, W., & Zhao, W. (2013). NGX6 expression improves the sensitivity of tamoxifen-resistant MCF-7 cells through modulation of the Smad signaling pathway. International Journal of Oncology, 42, 2060-2068. https://doi.org/10.3892/ijo.2013.1886
MLA
Zhao, W., Wang, K."NGX6 expression improves the sensitivity of tamoxifen-resistant MCF-7 cells through modulation of the Smad signaling pathway". International Journal of Oncology 42.6 (2013): 2060-2068.
Chicago
Zhao, W., Wang, K."NGX6 expression improves the sensitivity of tamoxifen-resistant MCF-7 cells through modulation of the Smad signaling pathway". International Journal of Oncology 42, no. 6 (2013): 2060-2068. https://doi.org/10.3892/ijo.2013.1886