Cinnamomum cassia Suppresses Caspase-9 through Stimulation of AKT1 in MCF-7 Cells but Not in MDA-MB-231 Cells.

BACKGROUND:Cinnamomum cassia bark is a popular culinary spice used for flavoring and in traditional medicine. C. cassia extract (CE) induces apoptosis in many cell lines. In the present study, particular differences in the mechanism of the anti-proliferative property of C. cassia on two breast cancer cell lines, MCF-7 and MDA-MB-231, were elucidated. METHODOLOGY/PRINCIPAL FINDINGS:The hexane extract of C. cassia demonstrated high anti-proliferative activity against MCF-7 and MDA-MB-231 cells (IC50, 34 ± 3.52 and 32.42 ± 0.37 μg/ml, respectively). Oxidative stress due to disruption of antioxidant enzyme (SOD, GPx and CAT) activity is suggested as the probable cause for apoptosis initiation. Though the main apoptosis pathway in both cell lines was found to be through caspase-8 activation, caspase-9 was also activated in MDA-MB-231 cells but suppressed in MCF-7 cells. Gene expression studies revealed that AKT1, the caspase-9 suppressor, was up-regulated in MCF-7 cells while down-regulated in MDA-MB-231 cells. Although, AKT1 protein expression in both cell lines was down-regulated, a steady increase in MCF-7 cells was observed after a sharp decrease of suppression of AKT1. Trans-cinnamaldehyde and coumarin were isolated and identified and found to be mainly responsible for the observed anti-proliferative activity of CE (Cinnamomum cassia). CONCLUSION:Activation of caspase-8 is reported for the first time to be involved as the main apoptosis pathway in breast cancer cell lines upon treatment with C. cassia. The double effects of C. cassia on AKT1 gene expression in MCF-7 cells is reported for the first time in this study

CE induces apoptosis in breast cancer cells.

<p>(A) MCF-7, MDA-MB-231 cells were stained with Annexin V/PI and subjected to flow cytometric analysis. The four quadrants represent living cells (Annexin V-PI-), early apoptotic (Annexin V+PI-), late apoptotic (Annexin+PI+) or necrotic (Annexin V-PI+) stages. (B) Percentage level of apoptotic cells when the cells were treated with different concentrations of CE. Values shown are percentages of each quadrant. *P<0.05, in comparison to control. The development of color was examined by bright field microscopy. (Magnification 200X). Z-VAD-FMK (caspase-8 inhibitor) was used as a negative control.</p

The effect of CE on gene expression in MCF-7 and MDA-MB-231 cells.

<p>Both cell lines were treated with the IC₅₀ concentration (35 μg/ml) of CE for 24 h. Total RNA was extracted and cDNA was synthesized using commercial kits. Real-time RT-PCR was performed and the relative expression of the genes was calculated using the delta-delta Ct method and normalized with 18S eukaryotic rRNA. Results are expressed as fold variation over carrier control (blank). Results are expressed as mean ± standard deviation. Statistical significance was calculated based on the mean ΔCt values by the Student’s <i>t</i> test. *Indicates significant differences from untreated cells (p < 0.05).</p

The Caspase-Glo luminoscence of MCF-7 and MDA-MB-231 cells treated with CE, without treatment, and treated with caspase inhibitor.

<p>Cells were treated with the IC₅₀ concentration (35 μg/ml) of CE at varying time points (2, 8, 16, 24, 28 h). (A) The Caspase-Glo luminoscence of MCF-7 and MDA-MB-231cells treated with CE, without treatment and treated with caspase inhibitor. (B) The fold change in caspase activity of MCF-7 and (C) MDA-MB-231cells treated with CE. Results are expressed as mean ± standard deviation. P < 0.05 compared to the control (without extract) as tested by the Student’s <i>t</i>-test.</p

Antiproliferative activity of CE, <i>trans</i>-cinnamaldehyde and coumarin in MDA-MB-231 and MCF-7 cells.

<p>(A) The % inhibition when MDA-MB-231 and MCF-7 cells were exposed to various concentrations of the CE. (B) The % inhibition when MDA-MB-231 cells were exposed to various concentrations of <i>trans</i>-cinnamaldehyde and coumarin. (C) The % inhibition when MCF-7 cells were exposed to the various concentrations of <i>trans</i>-cinnamaldehyde and coumarin. Results are expressed as mean ± std. dev. (n = 3). IC₅₀ is defined as the concentration of extract that inhibited 50% of cell proliferation. ND = not detected.</p

Intracellular ROS generated in MCF-7 cells and MDA-MB-231cells treated with CE at varying time points.

<p>Cells were treated with the IC₅₀ concentration (35 μg/ml) of CE and incubated for 6, 9, 12, 24, and 48 h. Results are expressed as mean ± standard deviation. P < 0.05 compared to the control (without extract) as tested by the Student’s <i>t</i>-test.</p

The effect of CE on AKT1 protein expression in MCF-7 and MDA-MB-231 cells using Western blot analysis.

<p>(A) Western blot analysis of the expression level of AKT1 in MDA-MB-231 and MCF-7 cells treated with different concentrations of CE, and caspase-8 inhibitor. AKT1 is down-regulated in all the treatments, except with the caspase-8 inhibitor. (B) Western blot analysis of the expression level of AKT1 in MDA-MB-231 and MCF-7 cells treated with the IC₅₀ (35 μg/ml) of CE with time was carried. It was demonstrated that in MDA-MB-231, the expression of AKT1 was reduced in a time-dependent manner. (C) In MCF-7 cells, the expression level of AKT1 decreased dramatically from 0–4 h and then gradually increased by 31% from 4–16 h. In MDA-MB-231 the expression level of AKT1 decreased from control to 16 h by 76%. The blots were scanned and analyzed using ImageJ software.</p

Semi-preparative HPLC chromatogram of CE and GCMS total ion chromatogram profile.

<p>(A) The peaks were detected at UV wavelength 254 nm. The two isolated peaks were identified as coumarin (1) and <i>trans</i>-cinnamaldehyde (2) through mass spectral library. (B) The two major peaks were identified as coumarin (1) and <i>trans</i>-cinnamaldehyde (2) (Wiley 9^th edition NIST11 Mass Spectral Library, USA).</p

Morphological assessment of CE-treated and untreated MCF-7 and MDA-MB231 cells.

<p>A double fluorescent dye (AO/PI) staining method was used on cells after 24 h incubation with the IC₅₀ concentration of CE. Apoptosis characteristics such as, early apoptosis (ap) nuclear compaction (nc), chromatin condensation (cc), the membrane plasma blebbing (mb) and the mitotic cells (mt) were observed in CE-treated (C) MCF-7 and (D) MDA-MB231 cells using an Olympus BX50 fluorescence microscope. Untreated controls (A) MCF-7 and (B) MDA-MB-231 were included. (200 X) magnification morphology.</p