Ambrosin exerts strong anticancer effects on human breast cancer cells via activation of caspase and inhibition of the Wnt/β-catenin pathway

Purpose: To investigate the antitumor effects of ambrosin sesquiterpene lactone on drug-resistant MDA-MB-231 breast cancer cells. Methods: The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used for cell viability studies. Apoptotic effects were determined using 4′,6-diamidino2-phenylindole (DAPI) staining assay, while Annexin V/PI was used for the quantification of apoptosis. Levels of reactive oxygen species (ROS) were determined by flow cytometry using DCFH-DA fluorescence staining while the effects of ambrosin on apoptosis and Wnt/beta-catenin signaling pathway-allied proteins were evaluated by Western blotting assay. Results: Ambrosin significantly suppressed the viability of MDA-MB-231 cells in a doseand timedependent manner (p < 0.05). Results from Annexin V/PI staining revealed that ambrosin induced production of apoptotic crops and blebbing of plasma membrane in MDA-MB-231 cells. Furthermore, annexin V/PI assay results showed increases in percentage of cells in different stages of apoptosis. Thus, ambrosin promoted caspase-dependent-apoptosis in MDA-MB-231 cells. Moreover, ambrosin enhanced the formation of ROS in MDA-MB-231 cells, as was evident from increased DCFfluorescence (p < 0.05). The results also showed dose-dependent inhibition of Wnt/beta-Catenin signaling pathway by ambrosin. Conclusion: Ambrosin exerts a chemo-preventive effect on breast cancer cells via induction of programmed cell death, enhanced ROS production, caspase activation and suppression of Wnt/betacatenin signaling. These findings have potential benefits for breast cancer research and drug discovery.

Thus, SLs are alkylating in nature, an effect which is enhanced by the presence of αmethylene-γ-lactone ring. This property enables them to alkylate nucleophiles, including cellular proteins which bear sulfhydryl groups.
Specifically, SLs target certain proteins e.g. p-65, an associate of heterodimeric transcription factor NF-κB which is involved in regulation of several intracellular activities such as invasion, metastasis, angiogenesis, immune responses, survival and proliferation [2,3]. Ambrosin ( Figure  1) molecule is a pseudo guanolide SL which occurs naturally and abundantly in ragweed species [4,5]. Structurally, ambrosin bears no phenolic moiety and is non-polar in nature. Studies have revealed that ambrosin possesses numerous biological as well as pharmacological properties, including cancer suppression and NF-κB inhibition [6,7]. In addition, immunohistochemical, behavioural and molecular studies have shown that ambrosin exerts suppressive effects on neuro-inflammation, amyloidogenesis and neuronal death [8]. Breast cancer (BC) is a neoplasm prevalent in females [9]. It is associated with high mortality and morbidity, and its prevalence is on the increase. For example, in the last three decades China, United States, Malaysia and Asian subcontinent experienced nearly 3.1% increase in overall BC cases, a situation which is very disturbing. Nearly 1.6 million new BC patients were documented globally between 2010 and 2012 [10,11]. The disease is heterogeneous in nature, with respect to histopathology and genetics. Till date, the pathogenesis of BC has not been fully understood [12]. However, it is known that hormone receptors and HER2 activity influence different BC sub-types such as luminal, HER2 and triple-negative [13]. Genetic mutations in luminal or basal progenitor cells give rise to distinct sub-types which accentuate the lethality of breast cancer. Breast cancer sub-types are also associated with differences in metastasis, biology and management strategies [14].
The treatment options for BC are limited. Moreover, apart from having low efficacy, the available treatment options cause life-threating side-effects. Natural products are of immense pharmacological potential. They have remarkable bioactivity profiles. Indeed, approximately 70 % of current chemotherapeutics are either natural products or compounds based on them, and many more are yet to be discovered [15][16][17][18][19][20]. Thus, with a knowledge of the potential of ambrosin sesquiterpene, the current study was designed to investigate its effect on breast cancer cells, and its effects on programmed cell death, ROS production, caspases and the Wnt/beta-catenin signaling pathway.

Determination of effect of ambrosin on viability of MDA-MB-3231 BC cells
The effect of ambrosin on the proliferation of MDA-MB-231 BC cells was determined with MTT assay. The MDA-MB-231 cells at logarithmic growth phase were seeded in 96-well plates at a density of 4 × 10 4 cells per well. Each well contained 100 μL of cells in DMEM culture medium. The cells were incubated for 24 h at 37 o C, followed by treatment with different doses of ambrosin viz 8, 16, 32 and 64 μM for 24 and 48 h. Untreated wells served as control. Thereafter, 20 μL of MTT working solution (5 mg/mL) was added to the wells which were then further incubated for 4 h. Then, the culture medium in each well was discarded and replaced with 200 μL of dimethyl sulfoxide (DMSO) to dissolve the resultant formazan crystals. The absorbance of each formazan solution was read at 490 nm in a microplate reader (BioRad, Segrate, Italy).

Analysis of apoptosis in MDA-MB-231 cells
Following seeding of MDA-MB-231 BC cells at a density of 2 x 10 5 cells/well in 96-well plates, the cells were exposed to varying doses of ambrosin viz 8, 32 and 64 μM. The plates were then incubated for 24 h. Thereafter, cells in each well were subjected to DAPI staining, followed by rinsing in PBS and fixation in 10 % formaldehyde. Finally, using a fluorescence microscope, the DAPI-stained MDA-MB-231 BC cells were analysed for apoptosis. A similar procedure was followed in annexin V/PI staining assay, except that staining was performed with annexin V/PI (Beyotime, China), and the stained cells were examined via flow cytometry.

Measurement of ROS production
The MDA-MB-231 cells were cultured in 6-well plates. Post-culture, the cells were treated with ambrosin for 24 h at doses of 8, 32 and 64 μM. Untreated cells were used as control. The cells in each well were treated with serum-free 10 μM dichloro-dihydro-fluorescein diacetate (DCFHDA). Thereafter, the cells were incubated for 30 min at 37 °C. For full contact between the probe and cells, the mixture was re-treated every 4 -5 min. Finally, the cells were rinsed twice in PBS, and levels of ROS were calculated via flow cytometry.

Western blotting assay
Total protein was extracted from cells using lysis with RIPA buffer after the cells were treated with varying doses of ambrosin viz 8, 32 and 64 μM. The protein content of each lysate was quantified with BCA assay. Then, 40 μg of protein from each lysate was separated using SDS-polyacrylamide gel electrophoresis, and transferred to PVDF membranes. The membranes were blocked by incubation with trisbuffered saline Tween (TBST) containing 5 % skimmed milk powder solution for 1 h. Thereafter, the membranes were incubated overnight at 4 o C with primary antibodies against BAX, BCL-2, cleaved caspase-3 and caspase-9, Wnt3a, and β-catenin. Then, the membranes were washed three times with TBST before incubating them with horse radish peroxidasecoupled secondary antibody at room temperature for 1 h. Finally, the membranes were re-washed three times with TBST, and the protein bands were developed using enhancedchemiluminescent reagent.

Statistical analysis
All data are presented as mean ± SEM. Oneway ANOVA was used for determination of significant differences amongst multiple groups, in addition to Bonferroni and Dunnet post hoc tests (GraphPad Software, USA). Values of p < 0.05 were considered as indicative of statistically significant differences, in relation to control.

Inhibition of cellular proliferation by ambrosin in MDA-MB-231 cells
The

Ambrosin induced caspase-dependent apoptotic cell death in MDA-MB231 cells
The effect of ambrosin on pro-apoptotic cell death in MDA-MB-231 cells was investigated using DAPI and annexin V/PI staining assays. Results from DAPI staining revealed marked dose-dependent increases in the number of apoptotic cells, formation of apoptotic crops, and membrane blebbing. These results are shown in Figure 3.

Suppressive effect of ambrosin on Wnt/betacatenin signaling
After ambrosin exposure, MDA-MB-231 BC cells were subjected to western blotting assay for the expressions of Wnt/beta-Catenin signaling pathway proteins. The results indicated dosereliant inhibition of Wnt/beta-Catenin signaling pathway-allied proteins. There were significant reductions in the levels of Wnt3a and β-Catenin on exposure of MDA-MB-231 BC cells to ambrosin (Figure 8). Three distinctive mechanisms are involved in down-regulation of apoptosis during carcinogenesis: disparity in apoptosis-linked proteins, malfunctioning of death receptor signals, and loss of caspase activity. Therefore, targeting of the caspase cascade, Bcl-2 and other apoptosis-allied signaling pathways serves as a key strategy in cancer management. In the current study, the anticancer activity effect of ambrosin sesquiterpene lactone was investigated in drug-resistant human breast cancer cells (MDA-MB-231). In addition, the effect of ambrosin on programmed cell death (apoptosis), ROS production, caspase activation and Wnt/betaCatenin signaling pathway, were studied. It is already known that ambrosin exerts anti-proliferative effects on different human cancers, including breast cancer cell lines HCC1937, JIMT-1 and MCF-7 [6]. In this study, results from MTT assay revealed significant dose-and time-dependent suppression of proliferation in ambrosin-treated MDA-MB-231 BC cells.
Furthermore, DAPI staining assay revealed production of apoptotic crops and membrane blebbing, indicating that ambrosin induced apoptosis-related cell death in MDAMB-231 cells. Annexin V/PI staining assay showed dosereliant enhancement of annexin V+/PI-and annexin V+/PI+ cells. Western blotting revealed that the apoptosis-inducing effect of ambrosin was caspase-dependent and associated with upregulated expression of Bax. It has been previously reported that ambrosin interfered with key signalling processes in cancer cells, thereby promoting apoptosis. A similar study carried out recently showed promising inhibitory potential of ambrosin on the proliferation of breast cancer cells and its pro-apoptotic effects [21]. The findings in this study are similar to previous reports where it was shown that ambrosin inhibited proliferation and enhanced apoptosis.
Ambrosin induced marked and dose-dependent increases in ROS level, as revealed using DCFH-DA staining. Similarly, it has been demonstrated that ambrosin-induced accumulation of ROS in MDA-MB-231 cells enhanced its anti-apoptotic potential [21]. The effects of ambrosin on Wnt/beta-catenin signalling were determined for the first time via western blotting. The results indicated that ambrosin suppressed the expressions of the Wnt/beta-Catenin signaling-associated proteins in a dose-dependent manner.

CONCLUSION
The results obtained in this study suggest that ambrosin significantly inhibited the proliferation of MDA-MB231 cells. Moreover, ambrosin exerted anti-proliferative effects on MDA-MB231 cells via induction of apoptosis, enhancement of ROS production, caspase activation and downregulation of the Wnt/beta-Catenin signaling pathway. Thus, ambrosin may be a lead molecule in breast cancer research. However, more clinical studies are required to validate this conclusion.

Conflict of interest
No conflict of interest is associated with this work.

Contribution of authors
We declare that this work was done by the authors named in this article and all liabilities pertaining to claims relating to the content of this article will be borne by the authors.

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