According to Cravotoo et al.
, [
14], citral was rapidly absorbed from the gastro-intestinal tract of mouse and rat, and also much of an applied dermal dose was lost due to its extreme volatility, but the citral remaining on the skin was fairly well absorbed. Besides that, citral was rapidly metabolized and excreted as metabolites and urine is the major route of elimination. Acute toxicity of this chemical is low in rodents because the oral or dermal lethal dose (LD
50) values were more than 1000 mg/kg, this chemical is irritating to skin and not irritating to eyes in rabbits [
15]. Citral has been previously reported to exhibit cytotoxic activity against breast [
3] and hematopoietic [
16] cancer cell lines through the induction of apoptosis. Similarly, our data has shown that the IC
50 value on MDA-MB-231 cells is 10 μg/mL (Fig.
1a). However, the potential of citral to specifically target the drug resistant breast cancer cells has not yet been tested which was the focus of our current study. Ricardo et al. [
17] demonstrated that drug resistant breast cancer cells, which contained higher ALDH1 activity survived and formed spheroids when cultured in serum-free medium. Furthermore, a previous study has shown that the ability of spheroids to be consecutively passaged is an indirect marker of drug resistant cancer cell’s self-renewal capacity [
13]. Thus, MDA-MB-231 spheroids were used as an in vitro culture model (Figs.
2,
3 and
4) to evaluate the cytotoxicity of citral on drug resistant breast cancer cells in this study. The cultured MDA-MB-231 spheroids showed higher levels of ALDH1 activity (Fig.
6a), which underwent self-renewal (indicated by the capacity of sphere formation in subsequent passages (Fig.
4b), and also showed higher IC
50 value against tamoxifen (results not shown). The MDA-MB-231 spheroids treated with citral at different concentrations (2.5 μg/mL, 5.0 μg/mL and 10.0 μg/mL) showed more than 7 and 30 fold increase at early and late apoptotic populations, respectively when compared to the vehicle control. The connection between Wnt/β catenin signaling pathway, ALDH drug resistant population and apoptosis is been well established, but different studies have shown that Wnt signaling regulates early and late stages apoptosis through a selection of mechanisms. Moreover, the expression of cyclin D1 following DNA damage is essential for cell cycle re-entry and apoptosis [
18‐
21]. MDA-MB-231 spheroids treated with different concentrations of citral (Fig.
6a) demonstrated significantly lower ALDH+ population, poorer sphere formation efficiency (both primary and secondary passages of MDA-MB-231 spheroids), consistent with reduced volume of the treated spheroids in a dosage dependent manner.
As we observed that citral can effectively control the self-renewal of breast cancer spheroids, the effects of citral in regulating Wnt/β-catenin signaling pathway of MDA-MB-231 spheroids was further investigated with western blot and quantitative real time PCR analyses. β-catenin is the key effector of Wnt signaling pathway, which regulates multiple important biological processes such as cell proliferation and stem cell maintenance [
22]. Axin and APC are the tumor suppressor genes that bind β-catenin and recruit CK1 to facilitate destruction of β-catenin through phosphorylation [
23]. Moreover, cancer incidences are always found to be associated with dyregulation of Wnt signaling pathway [
24]. In addition to that, overexpression of β-catenin and Wnt pathway targeted gene, cyclin D1 were found to serve as poor prognostic markers in human cancer especially in breast cancer [
25]. Hence, hyperactive transcription of Wnt signaling and downregulation of Wnt signaling related tumor suppressor genes such as APC, indicate higher levels of self-renewal and dyregulated proliferation of these cancer cells [
22,
26]. A previous review has shown that drug targeting on aberrant Wnt signaling pathway can favour cancer treatment outcome [
27]. For example, Niclosamide that blocked Wnt co-receptor LRP6 suppressed the growth of Wnt-driven MDA-MB-231 and T-47D breast cancer cells [
28]. In this study, we found that mRNA expression of Wnt destruction complex Axin, APC and CK1 were upregulated, while Wnt co-receptor LRP6 was downregulated in MDA-MB-231 spheroids by citral treatment in a dosage dependent manner. These activities are may lead to the phosphorylation of β-catenin and target the phospho β-catenin to proteosomal degradation, which essentially suppressed the Wnt/β-catenin signaling pathway and expression of its target gene, cyclin D1. These results have proposed that citral treatment may regulate the growth of MDA-MB-231 spheroids through downregulation of Wnt signaling and its downstream cell cycle target gene, cyclin D1. However, further studies must be performed to validate the interaction of citral treatment with the components of the Wnt signaling pathway.