Canthin-6-one Isolated from Brucea javanica Root Blocks Cancer Cells in the G₂/M phase and Synergizes with Cisplatin

Abstract

Poor prognosis of most cancer patients is in part, due to limited therapeutic options. Furthermore, as chemotherapy remains the standard-of-care for several cancers, partial or lack of response remains a concern and compounding this are the adverse side effects of the treatment that severely impacts the quality of life and survival. In pursuit of improving treatment options, we have opted to investigate the unique chemical skeleton of natural compounds as anticancer therapies. In this study, from an initial screen of 31 crude methanol extracts from ~15 plant species using HL60 cells, the root extract of Brucea javanica (L.) Merr indicated the presence of bioactive compounds. Subsequent bioassay-guided purification on the root extract yielded two alkaloids canthin-6-one (1) and bruceolline J (2), which were further investigated for their bioactivity in representative human cancer lines and normal phenotypic counterparts. MTT assay demonstrated ED50 values from 34.7–72.9 μM for 1 and 16.0–54.0 μM for 2 for the cancer cell lines panel. NP69 cells also demonstrated sensitivity to both compounds (9.3 μM and 4.5 μM). As amount of 2 isolated were limiting, we focused on 1 to further identify novel anticancer properties in PC3 and HeLa cancer lines. We observed at 30 μM, 1 induced a G2/M phase arrest coinciding with decreased cell proliferation. Furthermore, 1 was able to synergize the cytotoxic effect of cisplatin when used in combination, suggesting the potential of combination therapy for those less responsive lesions to standard chemotherapy.

Canthin-6-One Inhibits Developmental and Tumour-Associated Angiogenesis in Zebrafish

“…In an effort to identify novel anti-angiogenic compounds from our in-house library of 70 natural compounds isolated from plants and marine species and 137 semi-synthetic compounds [ 24 , 25 ], we took advantage of the Tg(fli1a:EGFP) transgenic zebrafish, where the blood vessels are fluorescently labelled [ 28 ]. The embryos were treated at 16 hpf, just prior to primary sprouting of ISVs [ 18 ], to examine the effect of the test compounds on ISV development.…”

“…Overall, we identified 3 compounds from our library, including kaempferol, which had previously been shown to be anti-angiogenic [ 29 ], that inhibited ISV development at 48 hpf ( Figure S1E–L ). Of these, canthin-6-one, an alkaloid isolated from a plant named Brucea javanica (L.) Merr [ 24 ], was noted to have minimal developmental phenotypic defects such as body shortening and developmental delay, and therefore was chosen for further characterisation ( Figure S1E’–G’ ). As the amount of isolated canthin-6-one was limited in our library, we purchased commercially available canthin-6-one for all subsequent experiments.…”

“…In this current study, we used the zebrafish model to identify novel anti-angiogenic agents from our library of natural compounds isolated from Malaysian terrestrial plants (Brucea and Blumea genus) and marine species (soft coral and liverworts), as well as semi-synthetic compounds broadly consisting of curcumin derivatives, diarylpentanoids, flavonoids, and alkaloids [ 24 , 25 ]. To assess the anti-angiogenic potential of these compounds, we examined their ability in perturbing the development of ISVs in zebrafish embryos at 20 µM concentration.…”

“…To assess the anti-angiogenic potential of these compounds, we examined their ability in perturbing the development of ISVs in zebrafish embryos at 20 µM concentration. From this, we identified canthin-6-one, an alkaloid that we isolated from Brucea javanica (L.) Merr with anti-cancer properties [ 24 ] as a promising anti-angiogenic compound. Canthin-6-one is also present in other plants including Rutaceae, Simaroubacea, Amaranthaceae, Caryophyllacaea, Zygophyllacaea families, and fungi and marine organisms [ 26 ].…”

Cu(II) Coordination Polymer Inhibits Liver Cancer Development via Targeting BCL-2 Protein and Activating Apoptotic Pathway

“…Cytotoxicity assay was performed utilizing 96-well plates for MTT staining and culturing in accordance with the protocol [ 18 ]. Shortly, the normal and cancer cells were inoculated and collected in 96-well plates with 1 × 10 6 cells per well density, and the cells were then cultivated in 5 percent CO 2 wet air at 37°C.…”