IntroductionLoss of histone H4 lysine 20 trimethylation (H4K20me3) is associated with multiple cancers, but its role in breast tumors is unclear. In addition, the pathological effects of global reduction in H4K20me3 remain mostly unknown. Therefore, a major goal of this study was to elucidate the global H4K20me3 level in breast cancer tissue and investigate its pathological functions.MethodsLevels of H4K20me3 and an associated histone modification, H3 lysine 9 trimethylation (H3K9me3), were evaluated by immunohistochemistry in a series of breast cancer tissues. Univariate and multivariate clinicopathological and survival analyses were performed. We also examined the effect of overexpression or knockdown of the histone H4K20 methyltransferases, SUV420H1 and SUV420H2, on cancer-cell invasion activity in vitro.ResultsH4K20me3, but not H3K9me3, was clearly reduced in breast cancer tissue. A reduced level of H4K20me3 was correlated with several aspects of clinicopathological status, including luminal subtypes, but not with HER2 expression. Multivariate analysis showed that reduced levels of H4K20me3 independently associated with lower disease-free survival. Moreover, ectopic expression of SUV420H1 and SUV420H2 in breast cancer cells suppressed cell invasiveness, whereas knockdown of SUV420H2 activated normal mammary epithelial-cell invasion in vitro.ConclusionsH4K20me3 was reduced in cancerous regions of breast-tumor tissue, as in other types of tumor. Reduced H4K20me3 level can be used as an independent marker of poor prognosis in breast cancer patients. Most importantly, this study suggests that a reduced level of H4K20me3 increases the invasiveness of breast cancer cells in a HER2-independent manner.

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Nine phenolic compounds, including two phenolic carboxylic acids, 1 and 2, seven hydrolyzable tannins, 3-9, eight triterpenoids, including four oleanane-type triterpene acids, 10-13, and four of their glucosides, 14-17, isolated from a MeOH extract of the gall of Terminalia chebula Retz. (myrobalan tree; Combretaceae), were evaluated for their inhibitory activities against melanogenesis in B16 melanoma cells induced by α-melanocyte-stimulating hormone (α-MSH), against the Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol 13-acetate (TPA) in Raji cells, and against TPA-induced inflammation in mice. Their 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical-scavenging activities and cytotoxic activities against four human cancer cell lines were also evaluated. Compounds 6-9 and 12 exhibited potent inhibitory activities against melanogenesis (39.3-66.3% melanin content) with low toxicity to the cells (74.5-105.9% cell viability) at a concentration of 10 μM. Western-blot analysis revealed that isoterchebulin (8) reduced the protein levels of MITF (=microphtalmia-associated transcription factor), tyrosinase, and TRP-1 (=tyrosine-related protein 1), mostly in a concentration-dependent manner. Eight triterpenoids, 10-17, showed potent inhibitory effects on EBV-EA induction with the IC50 values in the range of 269-363 mol ratio/32 pmol TPA, while these compounds exhibited no DPPH scavenging activities (IC50 >100 μM). On the other hand, the nine phenolic compounds, 1-9, exhibited potent radical-scavenging activities (IC50 1.4-10.9 μM) with weak inhibitory effects on EBV-EA induction (IC50 460-518 mol ratio/32 pmol TPA). The tannin 6 and seven triterpenoids, 10-16, have been shown to inhibit TPA-induced inflammation (1 μg/ear) in mice with the ID50 values in the range of 0.06-0.33 μmol/ear. Arjungenin (10) exhibited inhibitory effect on skin-tumor promotion in an in vivo two-stage mouse-skin carcinogenesis test based on 7,12-dimethylbenz[a]anthracene (DMBA) as initiator and with TPA as promoter. Compounds 1, 2, 4, 5, 7-9, 12, and 13, against HL60 cell line, compounds 1 and 4, against AZ521 cell line, and compounds 1, 11, and 12, against SK-BR-3 cell line, showed moderate cytotoxic activities (IC50 13.9-73.2 μM).

Nine limonoids, 1-9, one apocarotenoid, 11, one alkaloid, 12, and one steroid, 13, from the leaf extract; and one triterpenoid, 10, five steroids, 14-18, and two flavonoids, 19 and 20, from the bark extract of Melia azedarach L. (Chinaberry tree; Meliaceae) were isolated. Among these compounds, three compounds, 4-6, were new, and their structures were established as 3-deacetyl-28-oxosalannolactone, 3-deacetyl-28-oxosalanninolide, and 3-deacetyl-17-defurano-17,28-dioxosalannin, respectively, on the basis of extensive spectroscopic analyses and comparison with literature data. All of the isolated compounds were evaluated for their cytotoxic activities against leukemia (HL60), lung (A549), stomach (AZ521), and breast (SK-BR-3) cancer cell lines. 3-Deacetyl-4'-demethyl-28-oxosalannin (3) against HL60 and AZ521 cells, and methyl kulonate (10) against HL60 cells exhibited potent cytotoxicities with IC50 values in the range of 2.8-5.8 μM. In addition, upon evaluation of compounds 1-13 against production of nitric oxide (NO) in mouse macrophage RAW 264.7 cells induced by lipopolysaccharide (LPS), seven, i.e., trichilinin B (1), 4, ohchinin (7), 23-hydroxyohchininolide (8), 21-hydroxyisoohchininolide (9), 10, and methyl indole 3-carboxylate (12), inhibited production of NO with IC50 values in the range of 4.6-87.3 μM with no, or almost no, toxicity to the cells (IC50 93.2-100 μM). Western blot analysis revealed that compound 7 reduced the expression levels of the inducible NO synthase (iNOS) and COX-2 proteins in a concentration-dependent manner. Furthermore, compounds 5, 6, 13, and 18-20 exhibited potent inhibitory effects (IC50 299-381 molar ratio/32 pmol TPA) against Epstein-Barr virus early antigen (EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cell line.

Seven triterpenoids, 1 - 7, two diarylheptanoids, 8 and 9, four phenolic compounds, 10 - 13, and three other compounds, 14 - 16, were isolated from the hexane and MeOH extracts of the bark of Myrica cerifera L. (Myricaceae). Among these compounds, betulin (1), ursolic acid (3), and myricanol (8) exhibited cytotoxic activities against HL60 (leukemia), A549 (lung), and SK-BR-3 (breast) human cancer cell lines (IC 3.1 - 24.2 μm). Compound 8 induced apoptotic cell death in HL60 cells (IC 5.3 μm) upon evaluation of the apoptosis-inducing activity by flow cytometric analysis and by Hoechst 33342 staining method. Western blot analysis on HL60 cells revealed that 8 activated caspases-3, -8, and -9 suggesting that 8 induced apoptosis via both mitochondrial and death receptor pathways in HL60. Upon evaluation of the melanogenesis-inhibitory activity in B16 melanoma cells induced with α-melanocyte-stimulating hormone (α-MSH), erythrodiol (7), 4-hydroxy-2-methoxyphenyl β-d-glucopyranoside (13), and butyl quinate (15) exhibited inhibitory effects (65.4 - 86.0% melanin content) with no, or almost no, toxicity to the cells (85.9 - 107.4% cell viability) at 100 μm concentration. In addition, 8, myricanone (9), myricitrin (10), protocatechuic acid (11), and gallic acid (12) revealed potent DPPH radical-scavenging activities (IC 6.9 - 20.5 μm).