| BackgroundBreast cancer is the common malignancy and becomes the leading cause of cancer-related death in women worldwide. During the last decade, the global incidence of breast cancer shows an upward trend. In 2012,1.7 million women were diagnosed with breast cancer, among which 522,000 patients were died of this disease. The incidence of breast cancer in China has been increasing with a 3% annual increase and becomes the fastest growing tumor, and the numbers of breast cancer patients will reach 2.5 million in 2021, which not only seriously affects women’s health, but also increase enormous economic burden to family and community.Estrogen receptor (ER) expression levels are closely correlated to the progress of breast cancer. Estrogen can bind to ER, activating ER to identify estrogen responsive element (ERE), which initiates transcription of multiplegene that are involved in the proliferation and differentiation of breast cancer cells. According to the level of ER expression, breast cancer can be classified into ER-positive and ER-negative breast cancer, among which ER-positive breast cancer accounts for about 65%. ERa expression level determines the effect of chemotherapy or endocrine therapy. Tamoxifen as the classic drugs of endocrine treatment can competitively inhibited the binding of estrogen to the ER, which suppresses the proliferation of ER-positive breast cancer cells. Due to the lack of expression or low expression of ER, ER-negative breast cancer is insensitive to treatment with tamoxifen. In addition, with the treatment, part of ER-positive breast cancer may turn to ER-negative, leading to lose the effect of tamoxifen. Therefore, how to increase sensitivity of ER-negative breast cancer to tamoxifen is a hot field of breast cancer treatment.Curcumin is an acidic phenolic compounds extracted from turmeric, ginger and other root plants, which has many effects including antihypertensive effects, anticoagulation, anti-oxidation, benefit bile, anti-cancer. Recently, application of curcumin in the treatment of breast cancer has been widely concerned, for example, curcumin can activate autophagy-dependent degradation of Akt to inhibit proliferation and migration of ERa-negative MDA-MB-231 breast cancer cells. Curcumin inhibits fatty acid synthase to promote apoptosis of MDA-MB-231 cells. PARP inhibitor combined with curcumin can inhibit MDA-MB-231 cell proliferation. Curcumin can inhibit MDA-MB-231 cell proliferation and invasion by reducing NF-κB/AP-1 transcriptional activity. Curcumin have effectively reduced lung metastasis of breast cancer in vivo; Furthermore, curcumin also can inhibit the activity of other transcription factors, including TNFa, IL, STAT3 and PPARy, which plays a key role in regulating multiplegene transcription in breast cancer.SLUG (Snai2), is a zinc finger transcription factor and can inhibit gene transcription by binding E-box (CANNTG) in the promoter-region of gene. SLUG expression levels are closely related to poor prognosis of colon cancer, lung cancer, and ovarian cancer. Compared with other subtypes of breast cancer, triple negative breast cancer shows higher level of SLUG. ERa-negative breast cancer cells MDA-MB-231 overexpressing ERa significantly inhibits SLUG expression in the presence of estradiol. On the contrary, ERa-positive breast cancer MCF7 and T47D cells depleting ERa will significantly increase the expression of SLUG Recently, although SLUG was found involved in the transcriptional regulation of ERa, it was necessary to confirm whether SLUG was implicated in sensitivity of ERa negative breast cancer to tamoxifen. Moreover, whether curcumin targeted SLUG will be further studied.Methods and ResultsThis paper aims to investigate the functional mechanism of curcumin on sensitivity of ERa-negative breast cancer to tamoxifen, including the following contents:1. The effect of curcumin on ERa-negative breast cancer1.1 MethodsWe first examined the dose-dependent and time-dependent effects of curcumin on ERa-positive and ERa-negative breast cancer, and assayed the dose-dependent and time-dependent effects of 4-hydroxytamoxifen on ERa-positive and ERa-negative breast cancer. The cell viability, cytotoxicity and cell apoptosis were respectively determined by CCK8 assay, lactic dehydrogenase (LDH) release assay, which contributed to confirm the exact working dose and time to analyze the combined effect of curcumin and 4-hydroxytamoxifen on sensitivity of ERa-negative breast cancer to 4-hydroxytamoxifen.1.2 ResultsCCK8 assay showed that curcumin significantly inhibited the proliferation of ERa-negative breast cancer compared to ERa-positive breast cancer. In contrast, 4-hydroxytamoxifen significantly inhibited the proliferation of ERα-positive breast cancer in dose-dependent and time-dependent manners.5μM curcumin and 10μM 4-hydroxytamoxifen were used in this study. LDH assay and flow cytometry analysis demonstrated that Curcumin promoted the sensitivity of ERa-negative breast cancer to 4-hydroxytamoxifen.2. The functional mechanism of how curcumin promoted the sensitivity of ERa-negative breast cancer to 4-hydroxytamoxifen2.1 MethodsThe differential expression of SLUG in ERa-positive and ERa-negative breast cancer were determined by western blot analysis. The effects of curcumin and 4-hydroxytamoxifen on SLUG expression were determined by western blot analysis. The binding capacity and transcriptional activity of SLUG on hexokinase (HK2) were determined using luciferase reporter and chromatin immunoprecipitation (ChIP). To verify the effect of SLUG inhibition on the sensitivity of ERa-negative breast cancer to 4-hydroxytamoxifen and HK2 expression, SLUG silencing in ERa-negative breast cancer cells was performed using small interfering RNA (siRNA). To verify the effect of SLUG up-regulation on the sensitivity of ERa-negative breast cancer to 4-hydroxytamoxifen and HK2 expression, SLUG overexpression in ERa-positive breast cancer cells was performed by gene transfection. To confirm how curcumin combined with 4-hydroxytamoxifen promoted cell apoptosis of ERa-negative breast cancer, mTOR phosphorylation, release of cytochrome C from mitochondria to cytoplasma and Capase 3 activation were determined by western blot analysis.2.2 ResultsOur results showed that SLUG expression in ERa negative breast cancer cells MDA-MB-231 was significantly higher than that in ERa negative MCF7 breast cancer cells. Curcumin inhibited the expression of SLUG in a dose-dependent manner in MDA-MB-231 cells while 4-hydroxy-tamoxifen promoted the expression of SLUG in dose-dependent manner.5 uM curcumin blocked the effect of 4-hydroxytamoxifen on SLUG expression. To clarify how SLUG functioned in ERa negative breast cancer cells, we analyzed the transcriptional activity of SLUG on HK2 by luciferase reporter assay. The results showed that SLUG could identify the promoter region of HK2 to promote the expression of luciferase. ChIP assay further confirmed the binding of SLUG to HK2 promoter, suggesting that SLUG directly regulated transcription of HK2. The results from RT-PCR indicated that curcumin reduced HK2 mRNA levels while 4-hydroxytamoxifen promotes HK2 mRNA levels, which may be due to their effects on SLUG expression. Western blot and flow cytometry analysis revealed that HK2 expression in MDA-MB-231 cells was significantly higher than that in MCF7 cells. SLUG silencing in MDA-MB-231 cells inhibited expression of HK2 and increased cell sensibility to 4-hydroxytamoxifen. Interestingly, curcumin and 4-hydroxy tamoxifen had no significant effect on the expression of HK2 in MCF7 cells and MDA-MB-231 cells depleting SLUG In contrast, overexpression of SLUG in MCF7 cells promoted HK2, and reduced cell sensibility to 4-hydroxytamoxifen. Curcumin and 4-hydroxytamoxifen suppressed and enhanced the expression of HK2 in MDA-MB-231 cells or MCF7 cells overexpressing SLUG, respectively. These results suggest that curcumin promoted sensitive of cells to 4-hydroxytamoxifen by inhibiting SLUG espression, and overexpression of SLUG conferred resistance of breast cancer cells to 4-hydroxytamoxifen, which seggested that curcumin might inhibit the expression of HK2 by targeting SLUG, promoting sensitivity of ERa negative breast cancer cells to tamoxifen. Curcumin combined with 4-hydroxytamoxifen could inhibit mTOR phosphorylation, promote release of cytochrome C to cytoplasma, and activate Caspase 3, suggesting that curcumin combined with 4-hydroxytamoxifen led to the mitochondria-mediated apoptosis.3. Analysis of differential expression of SLUG and HK2 in ERa positive and ERa negative breast cancers.3.1 MethodsThe expression of SLUG and HK2 in ERa positive and ERa negative breast cancers were determined using RT-PCR, Western blot and Immunohistochemistry.3.2 ResultsWe respectively selected 34 cases of ERa-positive and negative breast cancer for the followed analysis. We first tested the differential expression of SLUG by quantitative PCR. The results showed that expression of SLUG in ERa-negative breast cancer tissues was significantly higher than that in ERa-positive breast cancer tissue. Immunohistochemistry and Western blot also confirmed the higher expression of SLUG in ERa-negative breast cancer tissues. We further examined the differential expression of HK2 between ERa positive and negative breast cancer. Quantitative PCR showed that expression of HK2 in ERa-negative breast cancer is higher than that in ERa-positive breast cancer, which was further confirmed by immunohistochemical staining. Results from western blot also demonstrated that expression of HK2 in ERa-negative breast cancer is higher than that in ERa-positive breast cancer. Combined with the information of clinical specimens, we find 76.47% ERα-positive breast cancer express lower SLUG and 14.71% ERα-negative breast cancer express lower SLUG. SLUG expression is negatively correlated with Era. Regardless of ERa-positive or ERa-negative breast cancer, the expression of SLUG is positively correlated with tumor size, tumor stage, lymph node metastasis. The higher expression of SLUG may conducive to tumor growth and lymph node metastasis and the SLUG may be a potential target for breast cancer treatment.Conclusion1. Curcumin significantly inhibited the proliferation of ERa-negative breast cancer MDA-MB-231 cells compared to ERa-positive breast cancer MCF7 cells.2. Curcumin increased the sensitivity of ERa-negative breast cancer cells to 4-hydroxy tamoxifen.3. Curcumin promoted the sensitivity of ERa-negative breast cancer cells to 4-hydroxytamoxifen by inhibiting SLUG/hexokinase-2 pathway.4. SLUG directly bound to the promoter region of hexokinase-2 and activate transcription of hexokinase-2.5. SLUG contributed to attenuate the sensitivity of ERa-negative breast cancer cells to 4-hydroxytamoxifen, and curcumin promoted the sensitivity of ERa-negative breast cancer cells to 4-hydroxytamoxifen by targeting SLUG.6. The expression of SLUG 2 and hexokinase-2 in ERa-negative breast cancer tissues were significantly higher than ERa-positive breast cancer tissues.Significance1. We confirmed that curcumin increased the sensitivity of ERa-negative breast cancer to 4-hydroxytamoxifen and curcumin was a potential cofactors of tamoxifen in clinical.2. We provided a novel insight of that curcumin promoted the sensitivity of ERa-negative breast cancer cells to 4-hydroxytamoxifen.3. We identified SLUG as a new target of curcumin. |
PDF Full Text Request