Roles of histamine on the expression of aldehyde dehydrogenase 1 in endometrioid adenocarcinoma cell line

Cancer-initiating cells (CICs) are a limited number of cells that are essential for maintenance, recurrence, and metastasis of tumors. Aldehyde dehydrogenase 1 (ALDH1) has been recognized as a marker of CICs. We previously reported that ALDH1-high cases of uterine endometrioid adenocarcinoma showed poor prognosis, and that ALDH1 high population was more tumorigenic, invasive, and resistant to apoptosis than ALDH1 low population. Histamine plays a critical role in cancer cell proliferation, migration, and invasion. Here, we examined the effect of histamine on ALDH1 expression in endometrioid adenocarcinoma cell line. The addition of histamine increased ALDH1 high population, which was consistent with the result that histamine enhanced the invasive ability and the resistance to anticancer drug. Among 4 types of histamine receptors, histamine H1 and H2 receptor (H1R and H2R) were expressed in endometrioid adenocarcinoma cell line. The addition of H1R agonist but not H2R agonist increased ALDH1. The antagonist H1R but not H2R inhibited the effect of histamine on ALDH1 expression. These results indicated that histamine increased the expression of ALDH1 via H1R but not H2R. These findings may provide the evidence for exploring a new strategy to suppress CICs by inhibiting ALDH1 expression with histamine.


Cancer Medicine
Open Access coupled receptors [18,19]. Therefore, histamine receptors have been identified as important targets for the treatment of cancers [5,12].
Cancers consist of heterogeneous cell populations derived from a single clone. Recently, it has been demonstrated that cells with tumorigenic potential are limited to a small stem-cell population, called cancer-initiating cells (CICs) [20,21]. Thus, eradication of CICs is essential to cure cancers. Several markers have been reported in CICs [22][23][24][25][26][27][28]. Among these, aldehyde dehydrogenase 1 (ALDH1) is demonstrated to be a general CIC marker of various malignancies, such as leukemia, breast, brain, colon, and lung cancers [28][29][30][31][32][33][34]. ALDH1 is a cytosolic enzyme responsible for oxidizing a range of aldehydes to their corresponding carboxylic acids, and involves in the degradation of toxins and the self-protection of cells [35]. Interestingly, ALDH also involves in the degradation of histamine [36].
We found previously that ALDH1 could be a marker of CICs in uterine endometrioid adenocarcinoma. The population of ALDH1 high cells was more invasive, antiapoptotic, and tumorigenic than the population of ALDH1 low cells in endometrial adenocarcinoma. High activity of ALDH1 was shown to be associated with poor prognosis in these cells [37]. In this study, the effects of histamine and histamine receptors on ALDH1 expression were examined in endometrioid adenocarcinoma cell line. We found that histamine enhanced tumor cell invasion ability and resistance against anticancer drug. Meanwhile, histamine via H1R increased ALDH1 expression; while, the antagonist of histamine via H1R inhibited the ALDH1 expression in endometrial adenocarcinoma cell line. This finding suggested that histamine via H1R plays a stimulatory role on CICs in endometrial adenocarcinoma and H1R may be a potential therapeutic target to cure the cancer via eradicating CICs.

Reagents and cells
Histamine dihydrochloride was purchased from Sigma-Aldrich (St.louis, MO). The agonist of H1R, histamine trifluoromethyl toluidide dimaleate (HTM), the agonist of H2R, dimaprit dihydrochloride (DIM) and the antagonist of H2R, cimetidine (CIM) were purchased from Tocris Bioscience (Minneapolis, MN). The antagonist of H1R, pyrilamine maleate salt (PYR) was purchased from Sigma-Aldrich. HEC-1 endometrioid adenocarcinoma cell line and MCF-7 breast cancer cell line were obtained from the Health Science Research Resources Bank of Osaka, Japan. HMC-1 mastocytoma cell line was kindly provided from Dr. Kitamura (Osaka University, Osaka, Japan). The doses used in these experiments of these ag-onists and antagonists were adjusted to those in previous reports [8,18,19].

Matrigel invasion assay
Invasion of tumor cells into Matrigel was examined with a BD BioCoat Matrigel Invasion Chamber (BD Biosciences, Franklin Lakes, NJ). Briefly, HEC-1 cells were seeded in Dulbecco's modified eagle medium (DMEM) without fetal bovine serum (FBS) in the Matrigel invasion upper chamber and cultured for 72 h. The lower chamber contained DMEM supplemented with10% FBS. Invading cells were stained with a Diff quick staining kit (Siemens, Munich, Germany). The number of invading cells was counted in a blinded manner at four microscopic fields per well with a magnification of 209 and the extent of invasion was expressed as the average number of cells per square millimeter.

Apoptosis-based anticancer drug assay
Cisplatin, an anticancer drug, is commonly used for the treatment of endometrioid adenocarcinoma. Cells (2 9 10 5 ) were seeded into 6-well cell culture plates with DMEM-10% FBS and cultured for 18 h at 37°C in 5% CO 2 condition. Various concentrations of cisplatin (0, 4, 8 lg/mL, Sigma, St Louis, MO) were added, with or without the pretreatment of histamine for 2 h. After 24 h, the apoptosis of cells was assessed with APOPCYTO Annexin V-Azami-Green Apoptosis Detection Kit (MBL, Nagoya, Japan) according to the manufacturer's instruction using FACS Canto II (BD Biosciences).

Scratch assay
Confluent HEC-1 cells were scraped using sterilized tips, and incubated in culture medium for 24 h. Images were captured at 0 and 24 h after the scratch.

Aldefluor assay
Cells were cultured in DMEM (Wako, Osaka, Japan) supplemented with 10% FBS (Nippon Bio-Supply Center, Tokyo, Japan). The Aldefluor kit (StemCell Technologies, Vancouver, BC, Canada) was used to evaluate ALDH1 high population, according to the manufacturer's instruction. Briefly, cells were suspended in Aldefluor assay buffer containing the ALDH substrate BODIPY-aminoacetaldehyde and incubated for 45 min at 37°C. The brightly fluorescent ALDH1-expressing cells were detected with FACS Canto II (BD Biosciences). As a negative control, cells were treated with 50 lmol/L diethylaminobenzaldehyde (DEAB). ALDH1 high population was evaluated with or without histamine dihydrochloride and the agonist and antagonist of H1R and H2R. In some experiments, ALDH1 high and ALDH low populations were sorted with FACS Aria II (BD Biosciences).

Quantitative RT-PCR
The quantitative RT-PCR (qRT-PCR) was performed with StepOnePlus TM Real-Time PCR instrument (Applied Biosystems, Foster City, CA) using Taqman probe/primer sets specific for human ALDH1A1, H1R, H2R, H3R, and H4R. GAPDH was used as a reference for gene amplification (Applied Biosystems).

Statistical analysis
Statistical analysis for experimental studies was carried out using Student's t-tests. The values are shown as the mean AE standard error (SE) of at least three experiments. The P ≤ 0.05 was considered as statistical significance.

Results
Effect of histamine on HEC-1 endometrial adenocarcinoma cell line in the invasion ability, resistance against anticancer drug, and migration ability Histamine has been reported to be involved in cancer cell migration and invasion [5,12]. Here, to examine the effect of histamine on HEC-1 endometrial adenocarcinoma cell line in the invasion ability, matrigel invasion assay was performed. When HEC-1 cells were incubated with histamine for 2 h, the number of invading cells was higher than that of histamine-untreated cells. The result indicated that histamine-treated cells possessed stronger invasive ability than untreated cells (Fig. 1A and B).
To examine the effect of histamine on the resistance against anticancer drug, cisplatin was added to cells with or without the pretreatment of histamine for 2 h. Cells without the pretreatment of histamine were more vulnerable to cisplatin than those pretreated with histamine ( Fig. 1C and D). These results indicated that histamine enhanced the resistance to anticancer drug in HEC-1.
Moreover, the effect of histamine on migration ability was examined by scratch assay. In the presence of hista-mine, the number of migrating cells at 24 h after scratch was higher than that without histamine (Fig. 1E).

Effect of histamine on the expression of ALDH1
ALDH1 activity was examined with Aldefluor assay. The brightly fluorescent cells were detected in the absence of DEAB but disappeared in the presence of DEAB (Fig. 2A). When sorted, the brightly fluorescent cells expressed higher amount of ALDH1 mRNA and higher amount of ALDH protein as compared to the nonfluorescent cells ( Fig. 2B  and C). Then, the fluorescent cells were ALDH-high and the nonfluorescent cells ALDH-low. To examine the effect of histamine on ALDH1 activity, HEC-1 cells were treated with histamine for 2 h at two different concentrations. With the treatment of histamine, the proportion of ALDH1 high population increased in a dose-dependent manner ( Fig. 2D and E). The qRT-PCR was performed to detect whether histamine regulated ALDH1 expression in mRNA level. When histamine was added, the mRNA level of ALDH1 increased significantly already at 5 min, and its increase was relatively steady during the observed period (5 min to 24 h) ( Fig. 2F and G).

Expression of histamine receptors
As four kinds of histamine receptors have been reported [18][19][20], the effect of histamine on HEC-1 cells were thought to be mediated through those receptors. RT-PCR revealed that H1R and H2R were expressed in HEC-1 (Fig. 3A). In contrast, the expression of H3R and H4R was hardly detected. These results were confirmed by qRT-PCR ( Fig. 3A and B).

Effect of agonists and antagonists of H1R and H2R on ALDH1
To examine whether the effect of histamine on ALDH1 expression was mediated by H1R and H2R,  the agonists and antagonists were used. HTM and DIM are agonists of H1R and H2R, respectively. PYR and CIM are antagonists of H1R and H2R, respectively. The addition of HTM but not DIM increased the proportion of ALDH1 high population and the ALDH1 mRNA level (Fig. 4A, B, and C). The addition of PYR but not CIM inhibited the effect of histamine on ALDH1 activity (Fig. 4D, E, and F). These results indicated that H1R but not H2R mediated the effect of histamine on ALDH1. We confirmed that no expression of HDC was detected in HEC-1 cells under any conditions (Fig. 4G).
Effect of H1R agonist HTM on HEC-1 endometrial adenocarcinoma cell line in the invasion ability, resistance against anticancer drug, and migration ability When HTM was added, the number of invading cells was higher than that of untreated cells (Fig. 5A). The addition of HTM reduced the apoptotic cells induced by cisplatin ( Fig. 5B), indicating that the resistance to cisplatin was mediated by the H1 receptor. Moreover, the migrating cell number increased when HTM was added (Fig. 5C).

Discussion
Targeting CICs are important for the treatment of cancers, because CICs are considered to be responsible for the maintenance, recurrence, and metastasis [38,39]. Considerable evidence indicates that ALDH1 activity serves as a valuable functional marker for the identification of CICs. ALDH1-high expressing tumor cells are tumorigenic and resistant to chemotherapy [40][41][42]. In the clinical studies, increased ALDH1 activity is correlated with metastasis and poor prognosis in several types of tumors [28][29][30][31][32][33][34]. Based on these reports, we previously demonstrated that ALDH1 expression was correlated to size of tumor, lymphatic invasion, recurrence, and prognosis of patients in uterine endometrioid adenocarcinoma. The population of ALDH1 high cells was more invasive, antiapoptotic, and tumorigenic than the population of ALDH1 low cells in HEC-1 endometrioid adenocarcinoma cell line, in which abundant ALDH high cells were detected by Aldefluor assay as compared to other endometrioid adenocarcinoma cell lines [37]. Here, we focused on potential factors regulating ALDH1 expression. Histamine enhances migration in several normal cells, such as eosinophil, dendritic cells, and lung fibroblast [43][44][45]. In addition, histamine induces the proliferation and differentiation of hematopoietic and neural stem cells [7][8][9]. Since the first report in 1984 showing that the inhibition of the histamine-synthesizing enzyme, HDC which catalyzes the biosynthesis of histamine from histidine, resulted in antitumoral effects on experimental tumors in rodents [46], a large body of experimental evidence has supported the critical role of HDC and histamine in tumor development and progression [5,6,[12][13][14][15][16][17]. In diverse human tumors, histamine concentration is higher than that of surrounding normal tissue [15,17,47]. Moreover, histamine has been reported to be involved in cancer cell proliferation, migration and invasion, tumor microenvironment, and immune system responses in diverse human tumors including melanoma, lymphomas, leukemia, colon, gynecologic and breast cancer, as well as in experimental tumors [12][13][14][15][16][17]. Similar with these reports, our findings suggested that histamine appeared to play a tumorigenic role in endometrioid adenocarcinoma cell line. These batteries of evidence prompted us to investigate the correlation of histamine and ALDH1 expression. We showed that histamine increased the proportion of ALDH1 high cells in endometrioid adenocarcinoma cell line. Since ALDH1 expression is supposed to be correlated to CIC function, [28][29][30][31][32][33][34][35][36][37] histamine might possess stimulatory effect on CICs. It is known that histamine exerts its effect through the activation of four kinds of specific histamine receptors designated H1R-H4R. These four receptors exhibit various effects [48]. Recent studies have shown that the four subtypes of histamine receptors have varying degrees of expression in different forms of cancers [5]. Here, we found that H1R and H2R were highly expressed in HEC-1 endometrioid adenocarcinoma cell line. Moreover, only H1R mediated the effect of histamine on the proportion of ALDH1 high population and ALDH1 mRNA expression. Therefore, H1R was suggested to be an effective and prompt "switch" to the regulation of ALDH1 expression and the proportion of CICs in endometrioid adenocarcinoma cell line. Various antagonists of H1R might be effective adjuvant therapeutics to eradicate CICs.
Taken together, the present study is the first to demonstrate the effect of histamine and H1R on ALDH1 expression in endometrioid adenocarcinoma. As ALDH1 activity is supposed to be a general CIC marker of various tumors [28][29][30][31][32][33][34]37], this finding provides an adjuvant therapeutic approach to eradicate CICs and cure the cancer. Further studies are necessary to clarify the effect of histamine receptors on the other types of CICs and the precise mechanism of histamine activity.