DOK1/PPARgamma pathway mediates anti-tumor ability of all-trans retinoic acid in breast cancer MCF-7 cells
Introduction
All trans-retinoic acid (ATRA) is a kind of vitamin A metabolic intermediate with extensive biological activities in vivo. Its antitumor effect has been widely studied in the field of basic and clinical medicine. Due to its characteristics such as inhibiting tumor cell differentiation, inducing tumor cell apoptosis, promoting antitumor effect of immune system, enhancing the sensitivity of tumor cells to radiotherapy and chemotherapy, ATRA has been used in clinical treatment of some types of tumors including acute promyelocytic leukemia (APL) [1], [2], [3]. Recent studies have shown that ATRA may also be used in all forms of breast cancer treatment [4]. However, the mechanism underlying anti-breast cancer ability of ATRA remains poorly understood.
Doking protein (DOK) family plays an important role in the regulatory of tumorigenesis, insulin resistance, immune regulation and etc [5], [6], [7]. Wherein, DOK1-6 mRNAs have been demonstrated to express in normal and breast cancer tissue [8], [9]. Previous studies have shown that the molecular weight of 62 kDa protein DOK1 could bind with p120 rasGAP, thereby inhibiting the Ras-Raf-MEK-ERK signaling pathway [10], [11]. Because of this feature, DOK1 is considered to be one of the major tumor suppressor gene in vivo [5], [7], [12]. Indeed, mice lacking of DOK1, DOK2, DOK3 predisposed to cancer [6]. Additionally, the decrease in the expression level of DOK2 and DOK6 is closely related to the progression of breast cancer [9]. However, the functional role of DOK1 in the development and treatment of breast cancer has not been reported.
PPARγ belongs to the family of nuclear hormone receptors (NHRs) which directly regulate transcription of target genes [13]. Accumulating evidence exists on the overexpression of PPARγ in many tumors including breast cancer. Yet, the biological significance of its role in cancer remains controversial. Previous studies have demonstrated that naturally-occurring and synthetic PPARγ agonists promote growth inhibition, apoptosis, and differentiation of tumor cells [13], [14], [15], [16]. Moreover, PPARγ ligands can counteract leptin stimulatory effects on breast cancer growth in either in vivo or in vitro models [17]. However, Zhou et al. demonstrated that PPARγ ligands induced autophagy in MDA-MB-231 breast cancer cells by up-regulating the expression of HIF1α and BNIP3 leading to maintaining cell viability [18]. In short, PPARγ’s specific role in breast cancer is uncertain.
We here reported that ATRA can activate PPARγ by increasing DOK1 expression, resulting in inhibition of cell proliferation and promotion of and promotion of cell apoptosis in human breast cancer MCF-7 cell. Our results reveal that DOK1-mediated activation of PPARγ contribute to anti-tumor ability of ARTA.
Section snippets
Reagents
All trans-retinoic acid (ATRA),3-(4,5)-dimethylthiahiazo (-2-y1)-2,5-diphenytetrazo-lium-bromide (MTT) and anti-tubulin antibody were purchased from Sigma-Aldrich (St Louis, MO, USA); Anti-Caspase 3 and anti-PPARγ antibodies were obtained from Cell Signaling Technology (Beverly, MA, USA); Anti-DOK1 antibody and anti–antibody phospha-tase labered secondary antibody were from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA). DMEM (Dulbeccos Modified Eagles Medium) and fetal bovine serum (FBS)
ATRA inhibited survival of MCF-7 cells
To observe the effect of ATRA on cell proliferation, MCF-7 cells incubated in medium with 2% FBS and 1% P/S were treated with 3.33 μmol/L (1 μg/ml) ATRA or the same dose of control solvent for 5 days. The cell proliferation was determined by MTT and BrdU assays, respectively. As show in Fig. 1A and B, ATRA treatment significantly decreased cell proliferation. To investigate the impact of ATRA on cell apoptosis, MCF-7 cells were incubated in medium with 1% FBS, 1% P/S, and 3.33 μmol/L (1 μg/ml)
Discussion
Generally, ATRA functions its anti-tumor ability through binding with its nuclear receptors including retinoic acid receptor (RAR) and retinoid X receptor (RXR) [20]. The association of ATRA with its receptor would activate the transcription of target genes, then its function is achieved [1], [3]. In addition, previous studies have also shown that some signaling pathways may mediate ATRA anti-tumor effects, such as PI3K/AKT and ERK pathway [21], mTOR signaling pathway [22], and Wnt/β-catenin
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Contributed equally to this work.