Tetraarsenic oxide affects non-coding RNA transcriptome through deregulating polycomb complexes in MCF7 cells.

Non-coding RNAs (ncRNAs) play important and diverse roles in mammalian cell biology and pathology. Although the functions of an increasing number of ncRNAs have been identified, the mechanisms underlying ncRNA gene expression remain elusive and are incompletely understood. Here, we investigated ncRNA gene expression in Michigan cancer foundation 7 (MCF7), a malignant breast cancer cell line, on treatment of tetraarsenic oxide (TAO), a potential anti-cancer drug. Our genomic analyses found that TAO up- or down-regulated ncRNA genes genome-wide. A subset of identified ncRNAs with critical biological and clinical functions were validated by real-time quantitative polymerase chain reaction. Intriguingly, these TAO-regulated genes included CDKN2B-AS, HOXA11-AS, SHH, and DUSP5 that are known to interact with or be targeted by polycomb repressive complexes (PRCs). In addition, the PRC subunits were enriched in these TAO-regulated ncRNA genes and TAO treatment deregulated the expression of PRC subunits. Strikingly, TAO decreased the cellular and gene-specific levels of EZH2 expression and H3K27me3. In particular, TAO reduced EZH2 and H3K27me3 and increased transcription at MALAT1 gene. Inhibiting the catalytic activity of EZH2 using GSK343 increased representative TAO-inducible ncRNA genes. Together, our findings suggest that the expression of a subset of ncRNA genes is regulated by PRC2 and that TAO could be a potent epigenetic regulator through PRCs to modulate the ncRNA gene expression in MCF7 cells.

Arsenic hexoxide has differential effects on cell proliferation and genome-wide gene expression in human primary mammary epithelial and MCF7 cells.

Arsenic is reportedly a biphasic inorganic compound for its toxicity and anticancer effects in humans. Recent studies have shown that certain arsenic compounds including arsenic hexoxide (AS4O6; hereafter, AS6) induce programmed cell death and cell cycle arrest in human cancer cells and murine cancer models. However, the mechanisms by which AS6 suppresses cancer cells are incompletely understood. In this study, we report the mechanisms of AS6 through transcriptome analyses. In particular, the cytotoxicity and global gene expression regulation by AS6 were compared in human normal and cancer breast epithelial cells. Using RNA-sequencing and bioinformatics analyses, differentially expressed genes in significantly affected biological pathways in these cell types were validated by real-time quantitative polymerase chain reaction and immunoblotting assays. Our data show markedly differential effects of AS6 on cytotoxicity and gene expression in human mammary epithelial normal cells (HUMEC) and Michigan Cancer Foundation 7 (MCF7), a human mammary epithelial cancer cell line. AS6 selectively arrests cell growth and induces cell death in MCF7 cells without affecting the growth of HUMEC in a dose-dependent manner. AS6 alters the transcription of a large number of genes in MCF7 cells, but much fewer genes in HUMEC. Importantly, we found that the cell proliferation, cell cycle, and DNA repair pathways are significantly suppressed whereas cellular stress response and apoptotic pathways increase in AS6-treated MCF7 cells. Together, we provide the first evidence of differential effects of AS6 on normal and cancerous breast epithelial cells, suggesting that AS6 at moderate concentrations induces cell cycle arrest and apoptosis through modulating genome-wide gene expression, leading to compromised DNA repair and increased genome instability selectively in human breast cancer cells.

Inhibition of STAT3/VEGF/CDK2 axis signaling is critically involved in the antiangiogenic and apoptotic effects of arsenic herbal mixture PROS in non-small lung cancer cells.

Despite the antitumor effects of asrsenic trioxide (As2O3), tetraarsenic hexoxide (As4O6 or PR) and tetraarsenic tetrasulfide (As4S4) in several cancers, their adverse poisoning, toxicity and resistance are still hot issues for effective cancer therapy. Here, antitumor mechanism of arsenic herbal mixture PROS including PR and OS (Oldenlandia diffusa and Salvia miltiorrhiza extract) was elucidated in non-small cell lung cancer cells (NSCLCs), since PR alone showed resistant cytotoxicity in NSCLCs compared to other cancers. PROS exerted significant cytotoxicity, induced sub-G1 phase and S phase arrest, increased apoptotic bodies, and attenuated the expression of pro-PARP, Bcl-2, Cyclin E, Cyclin A, CDK2, E2F1, p-Src, p-STAT3, p-ERK, p-AKT, COX-2 and SOCS-1 in A549 and H460 cells along with disrupted binding of STAT3 with CDK2 or VEGF. Notably, PROS inhibited VEGF induced proliferation, migration and tube formation in HUVECs and suppressed angiogenesis in chorioallantoic membrane (CAM) assay via reduced phosphorylation of VEGFR2, Src and STAT3. Consistently, PROS reduced the growth of H460 cells implanted in BALB/c athymic nude mice via inhibition of STAT3, and VEGF and activation of caspase 3. Overall, these findings suggest that PROS exerts antiangiogenic and apoptotic effects via inhibition of STAT3/ VEGF/ CDK2 axis signaling as a potent anticancer agent for lung cancer treatment.

Tetra-arsenic oxide (Tetras) enhances radiation sensitivity of solid tumors by anti-vascular effect.

Tetras (tetra-arsenic oxide, As(4)O(6)) is a derivative of arsenic used in Korean traditional medicine for the treatment of cancer, but its mechanism remains largely undefined. Recently, a similar arsenic derivative, diarsenic trioxide (As(2)O(3), ATO), has been shown to mediate anti-tumor activity, therefore reigniting interest in the therapeutic effect of arsenic compounds. Here we report that Tetras can effectively mediate an anti-vascular effect on tumors, leading to delay in tumor growth and increased survival. Our study demonstrates for the first time the potential use of Tetras as a radiation therapy enhancement agent for solid tumors. These findings reveal an unappreciated role of Tetras in cancer therapy and its potential application to radiotherapy in achieving local tumor control.

Reverse effects of tetraarsenic oxide on the angiogenesis induced by nerve growth factor in the rat cornea.

To compare the antiangiogenic effects of tetraarsenic oxide (As4O6) with those of diarsenic oxide (As2O3) in the rat cornea, rat cornea micropocket assay was conducted to induce angiogenesis by implantation of the pellet contained 1.0 ng of nerve growth factor (NGF). Ten of thirty eyes of Sprague-Dawley rats were randomly assigned to one of three groups, namely, control group (no medication), As2O3 group (50 mg/kg As2O3, PO, s.i.d.), and As4O6 group (50 mg/kg As4O6, PO, s.i.d.). After implantation, the number of new vessels, vessel length and clock hour of neovascularization were examined under the microscope from day 3 to day 7. The area of neovascularization was calculated using a mathematical formula. Although new vessels in control and As2O3 groups were first noticed at day 3, whereas those of As4O6 group were first observed on day 5. The number, length, clock hour of neovascularization and areas of the vessels in As4O6 group showed more significant inhibition than those of control and As2O3 groups from day 5 (P<0.05). However, there were no differences in all parameters between control group and As2O3 group during the entire study period. These results showed that As4O6 had antiangiogenic effects on the new vessels induced by NGF in the rat cornea.

Tetraarsenic oxide induces apoptosis in U937 leukemic cells through a reactive oxygen species-dependent pathway.

In the present study, we investigated the effect of tetraarsenic oxide (As4O6, 2,4,6,8,9,10-Hexaoxa-1,3,5,7-tetraarsatricyclo[3.3.1.13,7]decane) upon induction of apoptosis in arsenic trioxide (diarsenic oxide, As2O3) resistant U937 leukemic cells. As4O6 induced apoptosis in U937 leukemic cells at much lower concentrations than As2O3 via an early increase of cellular reactive oxygen species (ROS), and a decrease in cellular mitochondrial membrane potential, followed by cytochrome c release and caspase-3 activation. As4O6 generated ROS and induced caspase-3 activation more potently than As2O3 in U937 cells. Incubation of the cells with N-acetyl-L-cysteine and catalase resulted in significant suppression of As4O6-induced apoptotic cell death. These results show that the generation of ROS leads to the consequences associated with apoptosis induced by As4O6. In conclusion, As4O6 might be a new arsenic compound which may induce apoptosis in U937 leukemic cells by activating unique apoptotic signaling mediated by ROS more potently than As2O3, and deserves further evaluation.

Tetraarsenic oxide, a novel orally administrable angiogenesis inhibitor.

Arsenic compounds have been used to treat angiogenic diseases such as cancer, psoriasis, and rheumatoid arthritis in traditional oriental medicine. In recent years, arsenic trioxide (As2O3, diarsenic oxide) has been successfully used to treat acute promyelocytic leukemia. We investigated the antiangiogenic properties of tetraarsenic oxide (As4O6), another trivalent arsenic compound. In in vitro studies, tetraarsenic oxide inhibited the proliferation (IC50 = 99.7 nM), migration into the denuded area (IC50 = 27.4 nM), and invasion through a layer of Matrigel (IC50 = 73.5 nM) of basic fibroblast growth factor (bFGF)-stimulated bovine capillary endothelial (BCE) cells in a dose-dependent manner. Tetraarsenic oxide also inhibited the tube formation of human umbilical vein endothelial cells. Tetraarsenic oxide induced cell cycle arrest of bFGF-stimulated BCE cells in the G2/M phase and inhibited the secretion of matrix metalloproteinase-2 from BCE cells. Orally administered tetraarsenic oxide (50 mg/kg/day) inhibited bFGF-induced new-vessel formation in a rat corneal micropocket assay, and reduced by about 54% the number of experimental pulmonary metastatic nodules in mice implanted with B16F10 melanoma cells. Thus, we provide evidence that tetraarsenic oxide has effective antiangiogenic activities.