SR140333 counteracts NK-1 mediated cell proliferation in human breast cancer cell line T47D

Background It has been demonstrated that certain NK-1 antagonists could reduce proliferation of several cancer cell lines, however, it is unknown whether SR140333 exerts proliferation inhibition in breast cancer cell line. Methods Immunohistochemical staining was carried out to investigate the immunolocation of NK-1 in breast cancer tissues and T47D cell line, thereafter, various concentrations of [Sar9, Met(O2)11]substance P and SR140333 were applied alone or combined. MTT assay was applied to detect cytoactivation and coulter counter was to detect growth curve. The Hoechst33258 staining was performed to detect apoptosis. Results We found that breast cancer and T47D cells bear positive expression of NK-1. SR140333 inhibited cell growth in a dose dependent manner. Furthermore, SR140333 could counteract [Sar9, Met(O2)11]substance P induced proliferation. Hoechst33258 staining revealed the presence of apoptosis after SR140333 treatment. Conclusions Our study demonstrated SR140333 exert proliferation inhibition in breast cancer cell line T47D and indicates NK-1 play a central role in the substance P related cell proliferation in breast cancer.


Background
Breast cancer is the most common cancer among women worldwide. It already is an urgent public health problem in high-resource regions, and is becoming an increasingly urgent problem in low-resource regions, where incidence rates have been increasing by up to 5% per year [1]. Despite earlier radiological examination, complete surgical resection and aggressive chemotherapy, it is still a social dilemma. Research studies have shown relevance of neuroendocrine molecules in breast cancer development, such as substance P and its receptor, NK-1, which belongs to G protein coupled receptor [2,3]. Substance P is a member of neurokinin family. Pharmacological studies have confirmed NK-1 as the high affinity receptor of substance P. It is well known that substance P and NK-1 are widely expressed in neural and non-neural sources [4][5][6][7][8][9][10][11]. Moreover, substance P could mediate cell mitogenesis through NK-1 activation [7], and using specific NK-1 antagonists (such as CP-96345, C-99994) in breast cancer cell lines could blunt the autocrine and/or paracrine cell proliferation [2,3].
Two forms of NK-1 are reported in humans, full-length (NK1-FL) and truncated (NK1-Tr). The cytoplasmic end of NK1-Tr lacks 100 residues, a region that functions as the substrate for G protein-receptor kinase [12]. By in situ hybridization, the existence of NK-1 mRNA has been demonstrated in malignant breast tissue but not detected in benign tissue [2]. Western blots showed coexpression of NK1-Tr and NK1-FL in several different breast cancer cell lines, including T47D [3]. Moreover, Previous RT-PCR study showed T47D cells contain more abundant NK-1 and substance P than others [3]. Both NK1-Tr and NK1-FL can activate PKC through incorporating G proteins, which has been suggested as a potential cancer target [13,14]. Recently, the expression of NK-1 in human tumors has been investigated using immunohistochemistry [8]. In several cell types, tumor cells bear more NK-1 than normal cells. These findings suggest that NK-1 may serve as a specific factor involved in the development of breast cancer. However, it is unknown the exact cellular location of NK-1 in breast cancer cells. Although earlier in vitro studies have demonstrated that NK-1 antagonists could inhibit the growth of certain tumor cells in presence or absence of apoptosis [2,3,[15][16][17][18][19][20][21][22], no study has been carried out on the antitumor action of specific NK-1 antagonist SR140333 in human breast cancer. Further-more, it is also unclear whether the NK-1 specific agonist SMSP exerts proliferation promoting action or not in breast cancer cells.
Therefore, in this study, we first generated an immunohistochemical study to investigate the immunolocation of NK-1 on breast cancer tissues and T47D cell line. Then we examined the effect of SMSP and SR140333 on in vitro growth of human breast cancer cell line T47D and further detected whether the NK-1 receptor antagonist SR140333 produce apoptosis in this cell line. Our study may enable us to develop a potential therapeutic target for breast cancer therapy.
All breast tissues were obtained from Qingdao Municipal Hospital. The patients providing the tissues did not receive prior treatment with anticancer agents. The study was approved by the institutional review board of Qingdao University. The following tumors were investigated: infiltrating ductal carcinoma (n = 89), infiltrating lobular carcinoma (n = 14). The human breast cancer cell line T47D was purchased from Chinese Type Culture Collection (Shanghai, China). The T47D cells were seeded in 25 cm 2 culture flakes and maintained with DMEM supplemented with 10% FBS. The medium was renewed every two days and the cells were passaged by treatment with trypsin-EDTA on the six day after seeding. On the third day T47D cells entered exponential phase. Cells were incubated at 37°C in CO 2 incubator (SHEL LAB, Oregon, USA) containing 5% CO 2 . All T47D cells were dissociated by treatment with trypsin-EDTA at 80-90% cell confluence and inoculated at a density of 10 5 cells/mL in 6-well plates which contained cover slips. The medium was renewed after two days and the cover slips were extracted on the fourth day, then the specimens were put into acetone (4°C) to fix for 15 minutes.

Immunohistochemistry
All tissue specimens were fixed in formalin and embedded in paraffin. Seven-μm paraffin sections were cut and floated onto polylysine adhered slides. The sections were dewaxed in xylene and rinsed in alcohol and graded alcohol/water mixtures. The immunohistochemical staning was performed using Elivision™ plus two-step System. Briefly, all sections were incubated with 3% hydrogen peroxide for 15 minutes to block endogenous peroxidase activity at first. The sections were subsequently treated in a microwave oven twice for 6 minutes in citrate buffer at 600W to undergo antigen repairing. After blocking with goat serum for 30 minutes, rabbit anti-human NK-1 was applied on the sections at the dilution of 1: 700 for 90 minutes at room temperature. After rinsing, staining was performed with Reagent A and Reagent B subsequently. The color was developed by reacting with DAB. Sections were then counterstained with hematoxylin, dehydrated, cleared and coverslipped. The T47D cell specimens were also stained by the above procedures except antigen repairing. The specificity of immunolabelling was demonstrated by the absence of labelling for NK-1 receptors when the primary antibody was omitted. The benign breast tumors (fibroadenoma: n = 5 and adenosis: n = 6) are used for negative control. Pancreatic adenocarcinoma was used as positive control for the immunohistochemical study [23].
All specimens were observed by two investigators using an Olympus BX-51 microscope (Tokyo, Japan) Only the brown particles that were easily visible with a low power objective was categorized positive staining.

Drug treatment
SMSP and SR140333 were dissolved in culture medium respectively to obtain experimental concentration. Different concentrations of SR140333 were evaluated in preliminary experiment to determine the 50% inhibition concentration (IC50) (unpublished data). In present study we performed various concentrations of SR140333 ranging from 10 -9 M to 10 -5 M to examine. In order to determine SMSP induced cell proliferation, different concentrations of SMSP (10 -10 M-10 -6 M) were evaluated. Furthermore, to learn whether SR140333 could counteract SMSP induced effect or not and at which concentration the counteract function would occur, we carried out competition experiments in which all T47D cells were treated using SMSP combined with various concentrations of SR140333. The most effective concentration of SMSP for this cell line was incubated 1 hour before the addition of SR140333.

Proliferation assay
Cell proliferation was assessed using MTT assay. Cells were cultured in 96-well plates and the cell numbers were quantified using a coulter counter (Coulter Electronics, Inc., Hialeah, FL). Each well contained 2 × 10 4 cells in a total volume of 200 μL. The plate included blank wells (0 cells/mL), control wells (2 × 10 4 cells/0.2 Ml, untreated group), control wells with DMSO (no cells), control wells treated with SR140333 (10 -9 M-10 -5 M), control wells treated with SMSP (10 -10 M-10 -6 M) and control wells treated with SMSP (most effective concentration) combined with different concentrations of SR140333 (10 -9 M-10 -5 M). Drugs were added on day 3 (at exponential phase) and the assay was performed after 24 hours. For the proliferation assay, 20 μL MTT was added in each well. After 4 hour at 37°C supernatant was removed and 100 μL DMSO was added in each well. The optical density (OD) was detected in the microplate reader at 570 nm wavelength (Biotech Instruments, New York, USA). Each experimental condition (blank wells, control wells, and control wells treated with drugs) was assayed in duplicate and each study was repeated on at least three separate occasions. Representative data from each experiment are shown in this article.

Growth study
T47D cells (2 × 10 5 cells/mL) were grown in 24-well tissue culture plates and each well containing 500 μL DMEM with 10% FBS. After 24 hours, medium was removed and fresh medium containing SMSP (the most effective concentration) or SR140333 (10 -5 M) was added at the same time (day 1). Control cells received only DMEM contained 10% FBS. On the subsequent five days, total cell counts were performed by a Coulter counter. Cell numbers determined by a Coulter counter were similar (less than 5% difference) to viable cell numbers determined by a dye (trypan blue) exclusion method using a hemocytometer.

Hoechest33258 staining
In order to determine whether apoptosis is induced by the specific NK-1 antagonist SR140333, Hoechst33258 staining was performed. T47D cells were cultured in a 6well plate using the cover slip culture method. On the third day SR140333 (10 -5 M) was added and 24 hours later all the cover slips were taken out. Control cells were treated only with culture medium. The cell samples were washed twice with PBS and fixed by incubation with glacial acetic acid/methanol mixture (glacial acetic acid: methanol = 1:3) for 30 minutes. Following washing in PBS, cells were incubated in 1 μg/mL Hoechst33258 solution for 10 minutes in the dark at 37°C. The cells were analyzed by a fluorescence microscope (Olympus BX-51, Tokyo, Japan).

Statistical analysis
Statistical analysis was performed with SPSS 10.0 statistical software for Microsoft Windows. Values of proliferation assay and growth study were expressed as means ± SD. Data from the proliferation assay were analyzed using one-way ANOVA. The homogeneity of the variance was tested using the Levene test. If the variances were homogeneous, Fischer's least significant difference procedure for multiple comparisons with Bonferroni adjustment and Dunnett t tests were used. For data sets with nonhomogeneous variances, the ANOVA test with T3 Dunnett post hoc analysis was applied. Data from growth study were analyzed using Dunnett t tests. We only considered the variances among different treating factors at the same day. The criterion for significance was p < 0.05 for all comparisons.

Expression of NK-1 in breast cancer tissues and T47D cells
Prominent NK-1 immunostaining was detected in most malignant breast cancer tissues (infiltrating ductal carcinoma was 78/89 and infiltrating lobular carcinoma was 12/14, respectively) and T47D cells. The positively stained cells were widely distributed, and NK-1 receptors were present on nearly all breast cancer cells. The brown particles were frequently observed in plasma membrane and/or cytoplasma (Figure 1). The benign tumor tissues bear negative expression of NK-1.

Effects of SMSP and SR140333 on the proliferation of T47D cells
On the third day T47D cells entered exponential phase and different concentrations of SMSP was added. The results of MTT assay revealed that SMSP showed stimulatory effect at concentrations from 10 -10 M to 10 -7 M. Furthermore, at 10 -8 M SMSP exhibited the most effective stimulation manner. Instead, 10 -6 M of SMSP showed inhibitory effect as compared to the untreated group (Figure 2).
Proliferation inhibition of T47D cells by SR140333 was detected after the addition of increasing concentrations of the specific NK-1 antagonist. SR140333 showed the inhibitory effect in a dose dependent fashion at concentrations ranged from 10 -8 M to 10 -5 M, but 10 -9 M of SR140333 did not inhibit cell proliferation as compared to the untreated group ( Figure 2).
As 10 -8 M of SMSP exhibited the most effective stimulation manner, we took 10 -8 M as the most effective concentration to investigate. As compared with controls with SMSP alone, all cells showed proliferation inhibitory effect after administration of SMSP combined with various concentrations of SR140333. SR140333 inhibited the stimulatory effect of SMSP in a dose-dependent fashion. As compared with the untreated group, at 10 -6 M and 10 - Compared with untreated group (control), cells treated with SMSP showed growth stimulatory effect from the third day while SR140333 showed growth inhibitory effect from the fourth day. In the successive five days after the administration of SR140333, growth rates of T47D cells were not reduced to zero, though (Figure 4).

SR140333 induces apoptosis of T47D cells
Hoechst33258 staining revealed the presence of a great number of apoptotic cells after administration of SR140333 ( Figure 5). Typical morphological change of apoptotic cells was easily observed, which showed characteristic of chromatin condensation and nuclear fragmentation. In fact, we observed a 25.58 ± 3.86 (SD) % of apoptotic cells after administration of SR140333 while only 7.85 ± 1.53 (SD) % in the untreated cells (p < 0.01).

Discussion
Our present study has clearly demonstrated expression of NK-1 in breast cancer tissues and T47D cell line using immunohistochemical study. This result is in agreement with the previous study which demonstrated that NK-1 is increased in breast biopsies by in situ hybridization [2]. Moreover, previous study has shown that malignant breast tissues bear over-expression of substance P [2], indicating involvement of neuroendocrine mechanism in breast cancer development. NK-1 receptors in tumor cells increase the amount of mitotic signals for the tumor cell, counteracting the different apoptotic and/or prosenescent pathways activated in the neoplastic cell population [24]. In breast cancers, increasing substance P could enhance the message transmitting through increasing NK-1; this may accelerate the proliferation process.
The increasing number of NK-1 in T47D cells leads us to investigate the role of NK-1 in tumor cell proliferation and growth. Therefore, we performed an in vitro study in which NK-1 receptors were activated or blocked by specific agonist SMSP or specific antagonist SR140333. The data of this study have shown, for the first time, that SMSP could stimulate the proliferation of breast cancer cell line T47D while SR140333 showed growth inhibitory effect. Further study by MTT assay has shown that SR140333 counteracted SMSP induced proliferation of T47D cells in vitro. These results suggest that the downstream signal transduction following NK-1 activation is significant for breast cancer development. It is known that substance P stimulates mitogenesis by activating NK-1 receptors in several neoplastic cell types [25,18,[4][5][6][7][8][9][10][11]. Since we merely exerted SMSP not exogenous substance P in this study, the exact effect of substance P on breast cancer cell line is still unclear. As endogenous substance P exhibits high affinity to NK-1 in vivo [10,11], the present study suggests that NK-1 plays a central role in substance P related cell proliferation in T47D cells. Thus, we conclude that blocking NK-1 by SR140333 also could inhibit substance P induced cell proliferation in T47D cells. An observation cannot be explained was 10 -6 M of SMSP showed inhibitory effect, and the detail needs to be further studied.
The other finding in this study was the presence of visible apoptosis after administration of SR140333. This is consistent with earlier studies, in which the use of certain NK-1 antagonists inhibited the growth of other human breast cancer cell lines such as MDA-MB-231 and MDA-MB-468 [26,27]. It was speculated that this finding was induced by a signal transduction pathway for apoptosis  . T47D cells were cultured in DMEM contained 10%FBS and SR140333 was added at logarithmic growth phase (on day 3, at about 30% cell confluences). We carried out Hoechst33258 staining on specimens obtained by the cover slip culture method. After treated with SR140333 for 24 h, T47D cells showed slower proliferation profile and visible apoptosis was detected by Hoechst33258. [7,20,28,29]. In addition, the blockade of NK-1 could inhibit both DNA synthesis and cell proliferation by the mitogen-activated protein kinase (MAPK) pathway [25]. However, in the presence of CP-96345 or C-99994, which belongs to NK-1 antagonist, no apoptotic cells but only inhibitory effect was observed in human breast cancer cell line T47D [2,3]. The authors think the reason is that the cell cycle remained in the G 2 phase [2]. Probably this different power action could be related with the different affinity for the NK-1 and with the expression of the amount of NK-1 receptors in the different tumor cells [30]. Moreover, previous studies have demonstrated that in the great majority of malignant tumors, NK-1 receptors were found on intra-and peritumoral blood vessels [6,23]. This finding indicated that NK-1 may serve as a preferred target for cancer therapy, which could mediate vasodilatation and mitogenesis. In fact, our unpublished immunohistochemical study has demonstrated the expression of NK-1 on both intratumoral and peritumoral blood vessels. Therefore, targeting NK-1 using SR140333 could decrease both nutrition supply and signal transduction.
It is well known that cell growth is regulated by various growth factors through their specific receptor linked various signal-transduction pathways [31]. A peptide growth factor may act through different receptors coupled to different post-receptor signal-transduction pathways [32] or the same receptor for a given peptide growth factor may be coupled to different post-receptor signal-transduction pathways by crosstalk [33]. T47D cells contain estrogen receptors (ER), and the ER dimer binds either directly to DNA at an estrogen response element or tethers to other bound transcription factors, thereby altering the transcription of estrogen sensitive genes [34] Although most ER is in the nucleus, a population resides in the cytoplasm and/or membrane, available for cross talk with other cytoplasmic/membrane-associated signaling molecules, such as shc. Because ER itself has no kinase activity, phosphorylation must occur through another molecule that associates with ER or is activated by the receptor. The activation of NK-1 induces releasing of G-protein βγ subunits, and the latter recruit components of the rasdependent cascade, such as shc, grb2, and src, leading to the activation of raf-1 and MAPK [35]. Shc binds to ER and src in T47D cells, leading to estradiol-induced activation of the MAPK pathway [36]. Therefore, the estradiolinduced nongenomic signaling pathway can also be activated by downstream of NK-1 pathway. As most ER is in nucleus, genomic signaling pathway is more important than nongenomic pathway. We speculate blockade of NK-1 only cut estradiol-mediated MAPK pathway. At present, it is still unclear whether SR140333 could counteract estradiol induced T47D's proliferation or not. The blockade of NK-1 by SR140333 could only break off one of many kinds of receptor related cell proliferation. Thus, only slower growth rate was observed and the growth rate was not reduced to zero (Figure 2) after administration of antagonist SR140333.

Conclusions
We have demonstrated the presence of NK-1 in breast cancer using immunohistochemistry. We also demonstrated the stimulatory effect of SMSP and inhibitory effect of SR1403333 on human breast cell line T47D. As only T47D cell line was bring into the present study, the effect of SR140333 on other cell lines is still not clear. Our observations indicate NK-1 may serve as a novel marker and target of breast cancer to study in the future.