Swietenolide isolated from Swietenia macrophylla King in Hook seeds shows in vitro anti-colorectal cancer activity through inhibition of mouse double minute 2 (MDM2) homolog

Abstract Swietenia macrophylla King in Hook (SM) is known to have several medicinal properties. Chloroform extracts of SM seeds (SMCE) as well as two isolated limonoids swietenine (1) and swietenolide (2) showed significant in vitro anti-CRC activity in human colon carcinoma (HCT116) cell line. 2 (IC50 = 5.6 µM) was found to be two times more potent than 1 (IC50 = 10 µM). Both compounds showed anti-CRC activity through inhibition of the Mouse Double Minute 2 homolog (MDM2) of the MDM2-p53 pathway. The Selectivity Index (S.I.) of isolated compounds 1 and 2 for cancer cells were about 6.6 and 12.8 fold respectively which was significantly better than the S.I. of the extract (S.I. ∼1.5). Graphic Abstract


Introduction
Swietenia macrophylla King in Hook (Sm) belongs to the family meliaceae, with 50 genera and 1400 species (Rastogi and mehrotra 1990).it is called 'sky fruit' , because of the upward trend of its fruits towards the sky. it is an evergreen, beautiful larger tropical tree with a height of 30-40 m and a girth of 3-4 m (arumugasamy et al. 2004).the skyward pointing fruits are egg-shaped, hard, woody, and brown, measuring about 8-10 cm in length and contain 35-45 winged seeds.Sm grows in west india, southern China, and malaysia as well as tropical regions of the americas, in particular, central and south american countries such as mexico and Bolivia (mulholland et al. 2000;pennington 2002;paiva et al. 2006;Goh and Kadir 2011).almost all of the plant's organs are utilized in traditional medicine to cure a variety of human ailments.the seeds of Sm, in particular, are a source of a large number of bioactive molecules (moghadamtousi et al. 2013) and are used traditionally in various countries (Segura-Correa et al. 1993;Graham et al. 2000;Goh et al. 2014).it is usually used in dry powder form (india), but the decoction of seeds is also reported to be used in indonesia (Kadota et al. 1990;moghadamtousi et al. 2013).it has been reported to be used as an abortion medicine and as an anti-leishmanial drug by an amazonian ethnic group in Bolivia (Bourdy et al. 2000).it has also been reported to be used as an anti-diabetic (maiti et al. 2009), anti-malarial (muñoz et al. 2000), antimicrobial, anti-hypertensive, anti-inflammatory, anti-nociceptive and anticancer drug (Goh and Kadir 2011;moghadamtousi et al. 2013;Goh et al. 2014).the main phytochemicals reported in this plant are limonoids (major) (Cheng et al. 2014;Chen et al. 2015;ma et al. 2018), lignin (Wu et al. 2012), and polyphenolic compounds (Falah et al. 2008).Limonoids have been reported to exhibit several biological activities such as anti-diabetic (dewanjee et al. 2009(dewanjee et al. ), anti-bacterial (Rahman et al. 2009)), anti-inflammatory (Chen et al. 2015), andanti-viral (Yuan-bin et al. 2014).
the crude ethyl acetate extract of mahogany seeds (SmEa) has earlier been reported to be active with iC 50 of 35 µg/mL against the human colorectal cancer (CRC) cell line HCt116 (Goh and Kadir 2011;Goh et al. 2014) but the active principles, responsible for its anti-CRC activity, have not yet been reported.Here we report that chloroform (CHCl 3 ) extract of Sm seeds (SmCE) as well as two isolated limonoids from it viz.swietenine (1) (CaS 4801-97-2) and swietenolide (2) (CaS 3776-48-5) are active against CRC and show their possible mechanism of anti-cancer activity.
SmCE was tested against three different cancer cell lines viz.HCt116 (colon cancer cell line), HepG-2 (Human hepatocellular carcinoma), and a549 (Human lung carcinoma). it was most active against HCt116 (CRC cell line) with an iC 50 value of 42.6 ± 3.8 µg/ mL.We wanted to identify the active principle of SmCE.Since the relative abundance of 1, 2, and 3 were 235:160:1 w/w, hence, only the two most abundant compounds 1 and 2 were tested against the above three cancer cell lines.3 was too low in abundance to be relevant for correlating its activity with that of the extract (SmCE) and hence was not tested.SmCE showed its highest activity with an iC 50 value of 50.2 ± 8.5 µg/mL against the HCt116 cell line as compared to an iC 50 value of 72.1 ± 7.5 µg/mL and 80.9 ± 7.9 µg/mL against HEpG2 and a549 cell lines respectively (Figure S28 (a-c)).Similarly, iC 50 of 1 and 2 were 10.5 ± 2.7 µm and 5.6 ± 1.4 µm (against HCt116); 16.1 ± 3.5 µm and 11.5 ± 4.2 µm (against HepG2); and 44.5 ± 4.3 µm and 37.5 ± 3.6 µm (against a549) respectively.Both 1 and 2 were significantly more active than SmCE and 2 showed higher activity than 1 in all the cell lines tested.SmCE as well as 1 and 2 showed better activity against HCt116 cell lines compared to other cancer cell lines.this could be due to multiple factors which may include a small amount of cell heterogeneity, differential expression of proteins in various pathways, differential drug permeability, and differential drug efflux from the cell, etc. the variety of molecular changes in cancer cells, which leads to so-called intrinsic resistance, is common.Cell line to cell line biochemical characteristics differ, leading to their differential sensitivity against drugs.
the ability of 1 and 2 to specifically target cancer cells than normal cells was also studied. it was found that in the normal human epithelial kidney (HEK293t) cell line 50% of cells dead (CC 50 ) happens at 73.5 ± 5.9 µg/mL with SmCE, 69.2 ± 3.1 µm with 1 and 71.9 ± 7.5 µm with 2 (Figure S28 (d)).thus, for HCt116 cell lines 1 and 2 offer a selectivity index [Si = (CC 50 )/iC 50 ] of about 6.6 fold and 12.8 fold respectively compared to S.i. of only about 1.5 for SmCE.Hence, both 1 and 2 may be more useful agents against CRC than SmCE due to their much higher selectivity indices than that of SmCE.morphological and nuclear changes upon treatment with 1 and 2 suggested that both may be working by causing late-stage apoptosis of cancer cells which was observed to be significantly higher for 2 than 1 (Figure S29). in a.o./Et.Br staining the colour of cells turns from green to yellow to reddish-orange when progressing from live cells to early apoptosis and late apoptosis.in Figure S29(d), more cells showed the orange colour, and fewer cells were yellow as compared to Figure S29(c).Hence, upon treatment with 2, more number of cells were in the late apoptotic stage as compared to treatment with 1.We couldn't assay cell cycle analysis upon treatment with 1 and 2 in the present study, which may form part of a more elaborate future study.overall results suggest that 2 is a better candidate for inducing apoptosis in the HCt116 cell line than 1.
the mechanism of cell death was studied using 1 and 2 on the HCt116 cell line with 5-fluorouracil (5-Fu) (a marketed colon cancer drug, +ve control), at an iC 50 value of 5 µm (Shakibaei et al. 2013) for 24h (Figure S30 (a-d)).With escalating doses, 1 and 2 showed a concomitant significant dose-dependent increase of p53 and p21 expression levels compared to the untreated cancer cells.the effect was more marked for 1 than 2 with the latter showing an almost flat dose-response. 2 also shows a slight reduction in mdm2 expression level, which is significantly more than that shown by 1. this coupled with the inhibition of mdm2 by both 1 and 2, suggests probable saturation of mdm2 activity of 2 at the given concentrations but not of 1, which shows dose dependency for its activity.
the probable modes of binding of 1 and 2 with mdm2 were studied by molecular docking using the 3d structure of mdm2 (pdB id 4oGn) as shown in (Figure S32). 2 showed a slightly better binding affinity with mdm2 than 1 (-8.5 kcal/mol and −8.2 kcal/ mol respectively).the overall results suggested that both 1 and 2 may bind in the active site pocket of mdm2 by interacting with residue His96 similar to that observed for crystal structure ligand in pdB id 4oGn, which can cause inhibition of its activity.
Since the docking study is carried out without solvents, we carried out further molecular dynamic (md) Simulation studies where the entire system is solvated to mimic the actual protein environment, which may provide a more realistic picture.md studies showed that in the mdm2-1 complex, the RmSd of the protein and the ligand were decoupled and they showed different values over simulation time while in the mdm2-2 complex, there was a strong correlation between the RmSd of the protein and the bound ligand where both showed similar RmSd of about 0.2 nm as they moved together (Figure S33 (a, b)).during the production run (60-100 ns) the average no. of H-bonds in the mdm2-1 complex was only about 0.60 while for the mdm2-2 complex, it was significantly higher (0.98) as shown in Figure S33(d, e), suggesting a better binding affinity for 2. the overall md simulation studies clearly showed that 2 has a much better H-bonding interaction with mdm2 than 1. the oH group at the C3 position in 2 strongly H-bonds with residue tyr67 of mdm2, while the bulky ester substitution at this position in 1 doesn't form any H-bond with it rather it provides steric hindrance for overall fitting of 1 in the deeper binding pocket which may be responsible for better inhibitory activity of 2 than 1 against mdm2.additionally, the inherent differences in 3d structures of 1 and 2 due to differences in positions of double bonds in the four 6-membered ring core structures of these limonoids (Figure S17) may also be playing some role in their different interactions with mdm2, as observed during md studies.

Experimental
See supplementary material

Conclusion
1 and 2 isolated from SmCE show potent in vitro inhibitory activity against CRC HCt116 cell line having iC 50 values of 10.5 µm and 5.6 µm respectively.their anti-CRC activity is through the inhibition of mdm2 in the mdm2-p53 apoptotic pathway.Hence, due to their higher selectivity for cancer cells than that of SmCE, these two compounds may be useful for further development as potential candidates for the treatment of human CRC.