Induction of apoptosis in human hepatocellular carcinoma cells by extracts of Lannea coromandelica (Houtt.) Merr. and Diospyros castanea (Craib) Fletcher

The five plants (LC, CT, ES, DC leaves, DC twig, and BA) used in this study are found in northeastern Thailand and were selected based on their ethnopharmacology. All the crude extracts showed significantly lower cytotoxicity than melphalan (P  0.001, P  0.001, P  0.001, P = 0.002, P = 0.023 and P  0.001, respectively). Based on their cytotoxicity and selectivity classification, the extracts could be categorized as follows: (a) potentially cytotoxic (IC₅₀  10 ?g/mL) with high selectivity (SI  3); (b) moderately cytotoxic (IC₅₀  100 ?g/mL) with high selectivity (SI  3); (c) moderately cytotoxic (IC₅₀  100 ?g/mL) with less selectivity (SI  3); (d) cytotoxic to only normal cells with less selectivity; and (e) nontoxic [32]. Only the crude extract of DC (twig) could be classified as highly cytotoxic toward HepG2 cells and highly selective (SI = 4.5). The SI of this extract was not significantly different from melphalan (P = 0.959). DC (leaf) and other crude plant extracts had moderate cytotoxicity and less selectivity (SI  3). The LC showed a cytotoxic effect against normal cells with low selectivity (SI  3). To our knowledge, this study demonstrates for the first time the anticancer potential of LC extract against human HCC (HepG2) cancer cells. The extracts of CT, ES, and BA showed moderate cytotoxicity on HepG2 cells but with less selectivity.

Several chemotherapeutic compounds have been reported to induce apoptosis [36]. In apoptotic cells, specific DNA cleavage is evident on electrophoretic analysis as a characteristic ladder pattern resulting from the multiple DNA fragments of oligonucleosomal size (180–200 bp) [37]. The DNA ladder assay has a lower sensitivity for apoptosis detection [38] because ladder formation can only be clearly observed when the extent of oligonucleosomal cleavage is extensive. Although more than 50 % of the apoptotic cells were observed with the DAPI staining assay in all of the ethanolic plant extracts, only LC and DC were positive according to the DNA ladder assay.

It was necessary to standardize the two potential crude extracts for quality control experiments as 50 % ethanol–water crude extracts of DC (twig) and LC (twig) were used in the current study. We then determined which phytochemicals in the crude extracts were associated with the apoptosis effects using GC–MS analyses.

2-Palmitoylglycerol was the major compound common to the LC (twig) and DC (twig) crude extracts. 2-Palmitoylglycerol is involved in the regulation of endogenous cannabinoid receptor activity by increasing the biological activity of 2-arachidonylglycerol, and inhibition of adenyl cyclase 2-palmitoylglycerol significantly inhibits the inactivation of 2-arachidonylglycerol by neuronal and basophilic cells [39].

2-Arachidonylglycerol inhibits the production of TNF-alpha by mouse macrophages in vitro and the effect is enhanced in the presence of 2-palmitoylglycerol [40]. 2-Palmitoylglycerol is also a key component in modulating pain sensitivity as a result of its ability to interact with endocannabinoids [41]. In the present study, 2-palmitoylglycerol was the one compound found in both DC and LC extracts; however, the 2-palmitoylglycerol content was twice as low in the DC (twig) (7.5 %) than in the LC (twig) (14.0 %) and the DC extracts were more cytotoxic. It was not possible to specifically identify 2-palmitoylglycerol as the key compound; therefore, more detailed chemical identification of the plant extracts is required.

Pyrogallol was predominantly found (18.7 %) in the DC (twig) crude extract, which possessed high toxicity and selectivity against the HepG2 cells. Pyrogallol is a superoxide anion generator because it potently increases intracellular superoxide anion levels and decreases glutathione content in HeLa cells [42]. Research shows that pyrogallol-induced apoptosis resulting from the loss of mitochondrial membrane potential in calf pulmonary artery endothelial cells is accompanied by glutathione depletion [43]. In one study, the apoptosis induction of pyrogallol was indicated by a pyrogallol-type structure in a B-ring of catechins. DNA fragmentation activity was exhibited in a concentration-dependent manner in histiocytic lymphoma U937 cells after treatment with catechins containing a pyrogallol-type structure in a ?-ring. Notwithstanding, catechins without a pyrogallol-type structure in any position did not show apoptosis-inducing activity [44]. A 3-O-gallate residue with cis-relationship to the ?-ring enhanced the activity and the destruction of a pyrogallol-type structure by its methylation reduced this effect. By contrast, a 3-O-gallate residue in trans-relationship to the ?-ring had little effect [44].

DC (twig) crude extract also contained lupeol as the third major compound (5.8 %). In a previous study, two hepatoma cell lines (SMMC7721 and HepG2) treated with lupeol decreased in a concentration-dependent manner (a) cell viability, (b) the induction of active caspase-3 and poly (ADP-ribose) polymerase cleavage, (c) cell accumulation in the S phase, and (d) apoptosis. Treatment with lupeol three times a week also resulted in chemosensitization of hepatoma cells and significantly inhibited tumor growth in nude mice implanted with SMMC7721 cells [45]. Lupeol chemosensitized hepatoma cells synergistically with a PI3-kinase inhibitor (S14161) in both in vitro and in vivo models [46]. In the present study, the lupeol content of the DC (twig) extract (5.8 %) was higher than that of LC (twig) extract (1.0 %). Pyrogallol was the major compound in the DC (twig) extract but not in the LC (twig) extract. These two major compounds might contribute to the high cytotoxicity and high SI of DC (twig) extract as well as the high toxicity with lesser selectivity of LC (twig) extract.

Hexadecanoic acid and octadecenoic acid were also identified as the major compounds in LC (twig) crude extract in this study. The crystal structure (at a resolution of 2.5 Ã…) and kinetics studies have shown that n-hexadecanoic acid can act as an anti-inflammatory compound by inhibiting phospholipase A₂ in a competitive manner [47]. This fatty acid possibly contributes as the antihepatoma cell potential in the 50 % hydroethanolic herb extracts [13]. Pereira et al. [48] showed that the lipophilic extract from Marthasterias glacialis L. contained mainly palmitic acid and the sterol ergosta-7,22-dien-3-ol: these affected DNA synthesis, lipid droplets, and chromatin condensation, compatible with apoptosis in human breast cancer (MCF-7) and human neuroblastoma (SH-SY5Y) cell lines.

n-Hexadecanoic acid and 9,12-octadecadienoic acid (Z, Z) are fatty acids commonly found in the CO₂ supercritical fluid extraction that mediates apoptosis in human hepatoma SMMC-7721 cells, involving a reactive oxygen species-mediated mitochondrial signaling pathway [49]. The mixture of an octadecenoic acid extract—comprising mainly oleic and linoleic acids—from Euphorbia kansui resulted in a concentration-dependent reduction in the number of human gastric (SGC-7901), HCC (BEL-7402), and leukemia (HL-60) cells and significantly inhibited cell proliferation, with induced apoptosis and G₀/G₁ phase cell cycle arrest [50]. The octadecenoic acids not only caused cell apoptosis/necrosis but also functional and structural damage to the tumor cell membrane and cell ultrastructures [50]. A fraction of the dichloromethane extract of Protaetia brevitarsis larva (composed of at least three free fatty acids: palmitic acid, (Z)-9-octadecenoic acid, and octadecenoic acid) expresses apoptosis-inducing activity as shown by DNA laddering and caspase-3 activation in colon 26 tumor cells [51].