Cycloart-24-ene-26-ol-3-one, a New Cycloartane Isolated from Leaves of Aglaia exima Triggers Tumour Necrosis Factor-Receptor 1-Mediated Caspase-Dependent Apoptosis in Colon Cancer Cell Line

Abstract

Plants in the Meliaceae family are known to possess interesting biological activities, such as antimalaral, antihypertensive and antitumour activities. Previously, our group reported the plant-derived compound cycloart-24-ene-26-ol-3-one isolated from the hexane extracts of Aglaia exima leaves, which shows cytotoxicity towards various cancer cell lines, in particular, colon cancer cell lines. In this report, we further demonstrate that cycloart-24-ene-26-ol-3-one, from here forth known as cycloartane, reduces the viability of the colon cancer cell lines HT-29 and CaCO-2 in a dose- and time-dependent manner. Further elucidation of the compound's mechanism showed that it binds to tumour necrosis factor-receptor 1 (TNF-R1) leading to the initiation of caspase-8 and, through the activation of Bid, in the activation of caspase-9. This activity causes a reduction in mitochondrial membrane potential (MMP) and the release of cytochrome-C. The activation of caspase-8 and -9 both act to commit the cancer cells to apoptosis through downstream caspase-3/7 activation, PARP cleavage and the lack of NFkB translocation into the nucleus. A molecular docking study showed that the cycloartane binds to the receptor through a hydrophobic interaction with cysteine-96 and hydrogen bonds with lysine-75 and -132. The results show that further development of the cycloartane as an anti-cancer drug is worthwhile.

Fig 1. Chemical structure of cycloart-24-ene-26-ol-3-one.

The compound has a cyclopentano per hydro phenanthrene scaffold with a cyclopropane ring between C-9 and -10. The side chain attached to C-17 has a hydroxyl group substituent at C-26.

Fig 2. Dose- and time-response curves.

For cycloartane (0.39–200 μM) on HT-29 (A) and CaCO-2 (B) cell lines, cisplatin (3.9–2000 μM) on HT-29 (C) and CaCO-2 (D) cell lines at 24, 48 and 72 hours, 5-fluorouracil (200–8 x 10⁻⁶ mM) on HT-29 and CaCO-2 cell lines at 24 (E), 48 (F) and 72 (G) hours. Significant differences are indicated: *p < 0.05; **p < 0.01.

Fig 3. Effect of the cycloartane on HT-29 cell death (apoptosis and necrosis) and cell cycle progression.

(A) Percentage of cells showing phosphatidylserine translocation, as measured by cell-surface annexin V binding and free PI: cells negative for annexin V and positive for PI are necrotic (Q1); cells positive for both annexin V and PI are in late apoptosis (Q2); cells negative for both annexin V and PI (Q3) are viable cells; and cells positive for annexin V and negative for PI are in early apoptosis (Q4). Cells were incubated for 24 hours with 0.1% v/v DMSO₄ (vehicle control) or the cycloartane at concentrations of 12.5, 25, or 50 μM, as indicated. (B) Flow cytometry histograms showing the distribution of cells in different phases of the cell cycle (G₁, S and G₂/M) at the beginning of the experiment (0 hr) and at 12, 24 and 48 hours of treatment with 2.5 μM of the cycloartane. The result is representative of one of three replicates that had essentially similar results.

Fig 4. Fold changes in caspase activities and cell viability of treated HT-29 cells with respect to untreated control.

(A) Time course (0–30 hours) fold increase in caspase 3/7, 8 and 9 activities of HT-29 cells post-treatment with the cycloartane (50 μM). (B) Effects of caspase inhibitors on treated HT-29 cells. Changes in caspase 3/7, 8 and 9 activities, and cell viability of cells pre-treated with caspase 3 inhibitor (Z-DEVD-FMK), caspase 8 inhibitor (Z-IETD-FMK), caspase 9 inhibitor (Z-LEHD-FMK) or general caspase inhibitor (Z-VAD-FMK), followed by incubation with the cycloartane (50 μM). At 18 hours of treatment, caspase activities were determined, and at 24 hours of treatment cell viability was measured. All results are three independent determinations with fold increases and percent cell viabilities calculated based on the untreated control. Symbols indicate significantly higher compared to 0 hour or no inhibitor pre-treatment: *p < 0.05; **p < 0.01.

Fig 5. Expression of apoptosis signalling proteins in cycloartane-treated HT-29 cells over various time intervals (6, 12, 18 and 24 hours).

(A) Upregulated proteins detected in human apoptosis antibody array. (B) Fold changes of apoptosis signalling molecules in comparison to control with a cut off limit of 1.5 fold. (C) Western blot analysis of apoptotic signalling proteins in cycloartane treated HT-29 cells and TNF-α-treated as positive control for NFkB translocation over various time intervals (6, 12, 18 and 24 hours).

Fig 6. High content cell screening analysis.

(A) Images and bar chart of mitochondrial membrane potential (MMP) decrease. (B) Cytochrome c localization (red arrows) in control cells or release from cycloartane-treated HT29 colon cancer cells. (C) NFkB fluorescence intensity in the nucleus region are similar between control and cycloartane-treated cells, reduce intensity in combination treatment of cycloartane and TNF-α or increase intensity in TNF-α alone. Images were captured at 100X magnification and significant differences are indicated: *p < 0.05; **p < 0.01.

Fig 7. Molecular docking of the cycloartane (circle) onto TNF-R1.

Magnification showing hydrogen bonding (green dashed lines) between hydroxyl and carbonyl groups of the compound with lysine-75 and -132 and hydrophobic interactions (purple dashed lines) between the cyclohexane and cyclopentane of the compound with cysteine-96 of the receptor.

Fig 8. The apoptotic effects of the cycloartane in HT-29 cells.

The diagram illustrates the mechanism of apoptotic death when the cycloartane binds to tumour necrosis factor receptor 1 (TNF-R1). This action causes recruitment of TRADD, FADD and pro-caspase-8 to form the death-inducing signalling-complex (DISC). The release of caspase 8 signals Bid to activate Bax, Bad, cytochrome C and caspase 9, leading to mitochondrial membrane potential decrease. Both caspase 8 and 9 activate caspase 3/7, which inhibits PARP and causes DNA degradation, resulting in apoptotic death. No translocation of NKĸB from the cytoplasm to the nucleus was observed. Thick upward and downward arrows indicate increases or decreases, respectively, of expression levels or activities. Thick cross indicates that the signalling protein (NFĸB) did not migrate to the nucleus.