Curcumol Inhibit Breast Cancer Growth Via NCL/ERα36 And PI3K/AKT Pathway

Purpose: This study is to investigate the effect and mechanism of curcumol on ERα36 positive breast cancer cells, and the relationship between curcumol’s target protein nucleolin (NCL) and ERα36. Methods: The anti-tumor effect of curcumol were quantified via MTT assay, colony formation and cycle arrest respectively. The expression of ERα36, NCL and the proteins involved in PI3K/AKT signaling were evaluated by western blotting. The interaction between two proteins were detected using co-immunoprecipitation (Co-IP) and immunofluorescence assay. Mouse xenograft model was 2 established to verify the role of ERα36 in breast cancer cells and curcumol’s effect on ERα36 positive cancer cells. Results: Curcumol inhibited the cell growth, caused cell cycle arrest, decreased cell cycle related-proteins and inactivated PI3K/AKT pathway in ERα36 positive breast cancer cells. There is a positive correlation between NCL and ERα36 in breast cancer cells. In addition, ERα36 bound to NCL, the two proteins were distributed in the nucleus, cytoplasm and on the plasma membrane, where their expression were obviously decreased by curcumol. Moreover, NCL silenced by NCL siRNA blocked the cell cycle progress and inhibited the activation of PI3K/AKT in MDA-MB-231 cells, while overexpressed ERα36 increased the expression of NCL, promoted cell cycle progress and enhanced the activity of PI3K/AKT in MCF-7 cells. NCL knockdown or ERα36 overexpressed all attenuated the effect of curcumol on breast cancer cells. Conclusion: Curcumol reduced the proliferation of breast cancer cells by targeting NCL/ERα36 and inactivated PI3K/AKT pathway.


Introduction
Breast cancer is the most common malignancy in women worldwide (https://www.who.int/publications/i/item/who-report-on-cancer-setting-priorities-investing-wisely-andproviding-care-for-all). Nearly 70% breast cancers express estrogen receptors(ERs) [1]. For ER-positive breast cancer, endocrine therapy is a common treatment for blocking estrogen signaling pathway [2].
Tamoxifen (TAM) is a classic ER-positive breast cancer treated drugs [3]. Unfortunately, it usually develops into TAM-resistance. For triple-negative breast cancer (TNBC), which always showed high metastasis, high invasiveness and poor prognosis [4]. There has no effective therapy for the lack of receptors expression. So, finding more effective therapeutic strategies for the treatment of drug-resistance and triple-negative breast cancer is urgent.
2005, Wang and his colleagues identified a ERα66 variant named ERα36[5], It is expressed in many breast cancer cells, especially highly expressed in TAM-resistant cell lines and triple-negative breast cancer [6]. Studies have shown that expression of ERα36 is related to the prognosis, metastasis and drug resistance of breast cancer [7,8]. It also stimulates the activation of PI3K/AKT and MAPK/ERK pathway [9]. More and more researches showed that ERα36 is a novel biomarker to overcome drug resistance and enhance the effectivity of triple negative breast cancer [10]. Therefore, novel and effective agents target of ERα36 are urgently needed.
Curcumol, an effective ingredient extracted from the volatile oil of traditional Chinese medicine Curcuma zedoaria (Christm.) Rosc. Studies have shown that curcumol can inhibit cell proliferation of various cancers [11][12][13][14][15]. For breast cancer, curcumol can induce cell apoptosis by inhibiting cancer cell metastasis and epithelial-mesenchymal transition, induce cell cycle arrest and act as an adjuvant therapy to increase the sensitivity of cells to drugs [15]. In our previous experiment, we found that NCL is a protein target of curcumol [16]. NCL is associated with poor prognosis in multitudinous cancer, it affects DNA repair, cell survival, angiogenesis, epithelial-mesenchymal transition and stemness [17].
And anti-NCL is a promising strategy for the treatment of breast cancer [18]. Recently, we found that ERα36 and NCL could interact with each other, so we want to explore whether curcumol inhibits breast cancer growth is related with the expression of NCL and ERα36, and whether the PI3K/AKT pathway is involved in the anti-breast cancer effect of curcumol.

Animal experiments
Four-weeks-old female nude mice were purchased from SJA Experimental Animal Co., Ltd.
(Changsha, Hunan). All animals were randomly divided into two groups. Every mouse was inoculated 1 × 10 7 cells suspending in 0.2 mL PBS. The cell MCF-7 ERα36 and MCF-7 NC were respectively injected into the flank of the mice. When tumor volume reached 100 mm 3 , the mice were randomly assigned to control group or experimental group. The experimental group received intragastric administration of 80 mg/kg curcumol every day for 3 weeks, control group treated with solvent. The growth of tumor was monitored every 3 days. Then, animals were sacrificed, the xenograft tumors were harvested. the volume of tumors was calculated according to the formula: volume = width 2 × length × 1/2.

Statistical analysis
The data were analyzed by SPSS version 21.0(SPSS, Inc.), All results were presented as the mean ± standard deviation (SD). Student's t-test and one-way ANOVA were conducted to analyze the statistical significance between different groups. P < 0.05 was considered statistically significant.

Curcumol inhibits breast cancer cells proliferation
As shown in Figure 1 a, MCF-7 and MCF-7 TAM-R were treated with 5, 10 μM TAM simultaneously, the viability of MCF-7 cells was decreased, while the cell viability was increased in MCF-7 TAM-R cells. Our data showed that MCF-7 TAM-R really resistance to TAM. As shown in

Curcumol induced breast cancer cells cycle arrest
The cell cycle distribution of curcumol intervention were examined by flow cytometry. As were significantly decreased by curcumol in a dose-dependent manner in cells. In MCF-7 TAM-R cells, curcumol regulated additionally the expression of P53 and P21, which two factors were also related to G1 phase arrest.

Curcumol inhibited the expression of ERα36 was related with its protein target NCL
As a high-malignant biomarker, NCL is highly expressed in breast cancer and is closely related

Curcumol inhibition on tumor growth was reversed by ERα36 in vivo
In order to investigate whether the expression of ERα36 affects curcumol's inhibition on the growth of breast cancer cells in vivo, we orthotopically grafted MCF-7 and MCF-7 ERα36-LV cell lines. As figure 7 a-c showed that ERα36 markedly increased the tumor growth of MCF-7 cells in vivo.
Curcumol obviously inhibited the proliferation of breast cancer whether ERα36 overexpressed or not.
However, compared with MCF-7 NC, the inhibitory effect of curcumol on tumor growth was sightly attenuated when ERα36 overexpression (Figure 7 c). And the body weight of nude mice showed no obviously difference between MCF-7 NC and MCF-7 ERα36 group (Figure 7 d). In addition, ERα36 expression in MCF-7 ERα36 xenograft tissues were markedly higher than in MCF-7 NC tissues, and curcumol's treatment decreased the expression of ERα36 more obviously in control than in ERα36 overexpressed tissues (Figure 7 e). Besides ERα36, the expression of NCL, P110, P85, P-AKT, PCNA, CDKS, Cyclin D1 were down-regulated not so remarkably in ERα36 overexpressed than in control group. These results suggested that curcumol still has anti-cancer effect on malignant tumors with high expression of ERα36, but ERα36 slightly reduce curcumol's sensitivity in vivo.

Discussion
In recent years, tamoxifen resistance presents a great clinical challenge for the ER-positive breast cancer therapy. Though there has been developed a number of regimens with single or comprehensive agents for the treatment of TNBC, few of them is satisfied and most of the clinical results is also                (e) The expression proteins were detected by western blotting. The intensity of bands was quanti ed by Image J. Data are presented as means ± SD (n= 3), *P < 0.05, **P < 0.01 compared with MCF-7 NC, ##P< 0.01, #P< 0.05 compared with MCF-7 ERα36-LV group Figure 8 Schematic representation of the action mechanisms of curcumol.

Supplementary Files
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