Romidepsin exhibits anti-esophageal squamous cell carcinoma activity through the DDIT4-mTORC1 pathway.

Esophageal squamous cell carcinoma (ESCC) is one of the most common human malignancies worldwide and is associated with high morbidity and mortality. Current treatment options are limited, highlighting the need for development of novel effective agents. Here, a high-throughput drug screening (HTS) was performed using ESCC cell lines in both two- and three-dimensional culture systems to screen compounds that have anti-ESCC activity. Our screen identified romidepsin, a histone deactylase inhibitor, as a potential anti-ESCC agent. Romidepsin treatment decreased cell viability, induced apoptosis and cell cycle arrest in ESCC cell lines, and these findings were confirmed in ESCC cell line-derived xenografted (CDX) mouse models. Mechanically, romidepsin induced transcriptional upregulation of DNA damage-inducible transcript 4 (DDIT4) gene by histone hyperacetylation at its promoter region, leading to the inhibition of mammalian target of rapamycin complex 1 (mTORC1) pathway. Furthermore, romidepsin exhibited better efficacy and safety compared to the conventional therapeutic drugs in ESCC patient-derived xenografted (PDX) mouse models. These data indicate that romidepsin may be a novel option for anti-ESCC therapy.

Overexpression of ABCB4 contributes to acquired doxorubicin resistance in breast cancer cells in vitro.

PURPOSE: Doxorubicin is one of the most active agents in the first-line therapy for metastatic breast cancer, but its utility is partially limited by the frequent emergence of doxorubicin resistance. In this study, we aimed to investigate the role of ATP-binding cassette sub-family B, member 4 (ABCB4) in acquired doxorubicin resistance in breast cancer cells, as well as its potential mechanism. METHODS: In doxorubicin-sensitive and -resistant breast cancer cell lines MCF-7 and MDA-MB-231, the expression levels of ABCB4 were detected using real-time quantitative PCR and Western blot analysis, the DNA methylation and histone acetylation status of ABCB4 gene were investigated by bisulfite-sequencing PCR (BSP) and chromatin immunoprecipitation (ChIP) assays, and the doxorubicin sensitivity and intracellular doxorubicin accumulation were observed using cell cytotoxicity assay and flow cytometry. In Madin-Darby Canine Kidney (MDCKII) cells, In vitro transport assay was used to assess the ABCB4-mediated transport of doxorubicin. RESULTS: ABCB4 was overexpressed in doxorubicin-resistant breast cancer cells compared to their doxorubicin-sensitive counterparts, which was associated with reduced DNA methylation as well as increased histone acetylation at the ABCB4 promoter. ABCB4 could actively pump doxorubicin out of the cells, and knockdown of ABCB4 increased doxorubicin sensitivity and intracellular accumulation in doxorubicin-resistant breast cancer cells. CONCLUSIONS: Our results indicate that ABCB4 is overexpressed in breast cancer cells with acquired doxorubicin resistance, which could be attributed, at least partially, to the epigenetic modifications of ABCB4 gene. ABCB4 mediates the efflux transport of doxorubicin, and contributes to the acquired resistance of doxorubicin in breast cancer cells.

Combined Influence of Genetic Polymorphism and DNA Methylation on ABCB1 Expression and Function in Healthy Chinese Males.

BACKGROUND AND OBJECTIVES: It is well known that the expression and function of ATP-binding cassette transporter B1 (ABCB1) show high interindividual variability, but the reasons have not yet been fully elucidated. In this study, combined influence of genetic polymorphism and DNA methylation on ABCB1 mRNA expression and digoxin pharmacokinetics in healthy Chinese males was analyzed. METHODS: A total of 93 subjects who were homozygous for the ABCB1 1236-2677-3435 TTT or CGC haplotype were enrolled in this study. DNA methylation status of the ABCB1 promoter and ABCB1 mRNA expression level in exfoliated intestinal epithelial cells were analyzed using bisulfite sequencing PCR and real-time PCR. The pharmacokinetics of digoxin in subjects were investigated after administration of a single oral dose of digoxin 0.5 mg. RESULTS: The DNA methylation levels of ABCB1 promoter showed no significant difference between TTT/TTT and CGC/CGC carriers (P = 0.54). Subjects with TTT/TTT haplotype pair and high methylation status (TTT/TTT-HM) showed a significantly lower ABCB1 mRNA level compared to other subjects. Compared with TTT/TTT-HM subgroup, the area under the plasma concentration-time curve from time zero to 72 h (AUC0-72) of digoxin was decreased by 26.9 %, the maximum plasma concentration (C max) was decreased by 25 % and the apparent oral clearance (CL/F) was increased by 21.2 % in CGC/CGC-LM subgroup. The values of time to maximum concentration (t max) and terminal elimination half-life (t 1/2) showed no significant difference. CONCLUSIONS: Both genetic polymorphism and DNA methylation variation should be taken into consideration to explain the interindividual variability in ABCB1 expression and function more clearly.

Interindividual epigenetic variation in ABCB1 promoter and its relationship with ABCB1 expression and function in healthy Chinese subjects.

AIM: Interindividual epigenetic variation is likely to be an important mechanism contributing to the interindividual variability in the expression and function of ATP-binding cassette, sub-family B, member 1 (ABCB1). The aim of the present study was to explore the effect of interindividual epigenetic variability in the ABCB1 promoter on ABCB1 expression and function in healthy Chinese subjects. METHODS: Using bisulfite sequencing polymerase chain reaction (PCR) and chromatin immunoprecipitation assays, the DNA methylation and histone acetylation status of the ABCB1 promoter in stool DNA and exfoliated colonic epithelial cells of 157 healthy Chinese male volunteers was analysed. ABCB1 mRNA levels in colonic epithelial cells were detected by real-time PCR. The digoxin pharmacokinetics in subjects with different epigenetic profiles was investigated after a single oral administration of digoxin (0.5 mg). RESULTS: The methylation levels of ABCB1 promoter in stool DNA showed a significant interindividual variation, from 0.84% to 18.05%. A high methylation level of the ABCB1 promoter was closely related to the low levels of acetylated histone H3 and ABCB1 mRNA expression. In the high methylation group, the area under the concentration-time curves (AUC(0-4 h) and AUC(0-10 h) ) of digoxin was increased by 19% [95% confidence interval (CI) 10%, 31%; P = 0.024] and 13% (95% CI 8%, 26%; P = 0.026), respectively, and the peak concentration (Cmax ) of digoxin was increased by 30% (95% CI 12%, 41%; P = 0.021) compared with the low methylation group. CONCLUSIONS: The epigenetic modifications of the ABCB1 promoter show high interindividual variability in healthy Chinese subjects, and are closely related to the interindividual variation in ABCB1 mRNA expression and digoxin 0-4 h plasma concentrations in vivo.

Advances in molecular signaling mechanisms of ß-phenethyl isothiocyanate antitumor effects.

ß-Phenethyl isothiocyanate (PEITC) is an important phytochemical from cruciferous vegetables and is being evaluated for chemotherapeutic activity in early phase clinical trials. Moreover, studies in cell culture and in animals found that the anticarcinogenic activities of PEITC involved all the major stages of tumor growth: initiation, promotion, and progression. A number of mechanisms have been proposed for the chemopreventive activities of this compound. Here, we focus on the major molecular signaling pathways for the anticancer activities of PEITC. These include (1) activation of apoptosis pathways; (2) induction of cell cycle arrest; and (3) inhibition of the survival pathways. Furthermore, we also discussed the regulation of drug-metabolizing enzymes, including cytochrome P450s, metabolizing enzymes, and multidrug resistance.

The Effects of Lycopene on the Methylation of the GSTP1 Promoter and Global Methylation in Prostatic Cancer Cell Lines PC3 and LNCaP.

DNA (cytosine-5-) methylation silencing of GSTP1 function occurs in prostate adenocarcinoma (PCa). Previous studies have shown that there is an inverse relationship between dietary lycopene intake and the risk of PCa. However, it is unknown whether lycopene reactivates the tumor suppressor gene glutathioneS-transferase-π (GSTP1) by demethylation of the hypermethylated CpGs that act to silence the GSTP1 promoter. Here, we demonstrated that lycopene treatment significantly decreased the methylation levels of the GSTP1 promoter and increased the mRNA and protein levels of GSTP1 in an androgen-independent PC-3 cell line. In contrast, lycopene treatment did not demethylate the GSTP1 promoter or increase GSTP1 expression in the androgen-dependent LNCaP cell line. DNA methyltransferase (DNMT) 3A protein levels were downregulated in PC-3 cells following lycopene treatment; however, DNMT1 and DNMT3B levels were unchanged. Furthermore, the long interspersed element (LINE-1) and short interspersed element ALU were not demethylated when treated by lycopene. In LNCaP cells, lycopene treatment did not affect any detected DNMT protein expression, and the methylation levels of LINE-1 and ALU were decreased. These results indicated that the protective effect of lycopene on the prostate is different between androgen-dependent and androgen-independent derived PCa cells. Further, in vivo studies should be conducted to confirm these promising results and to evaluate the potential role of lycopene in the protection of the prostate.

Preferential induction of CYP1A1 over CYP1B1 in human breast cancer MCF-7 cells after exposure to berberine.

Estrogens are considered the major breast cancer risk factor, and the carcinogenic potential of estrogens might be attributed to the DNA modification caused by derivatives formed during metabolism. 17ß-estradiol (E2), the main steroidal estrogen present in women, is metabolized via two major pathways: formation of the 2-hydroxyestradiol (2-OH E2) and 4-hydroxyestradiol (4-OH E2) through the action of Cytochrome P450 (CYP) 1A1 and 1B1, respectively. Previous reports suggested that 2-OH E2 have putative protective effects, while 4-OH E2 is genotoxic and has potent carcinogenic activity. Thus, the ratio of 2-OH E2/4-OH E2 is a critical determinant of the toxicity of E2 in mammary cells. In the present study, we investigated the effects of the berberine on the expression profile of the estrogen metabolizing enzymes CYP1A1 and CYP1B1 in breast cancer MCF-7 cells. Berberine treatment produced significant induction of both forms at the level of mRNA expression, but with increased doses produced 16~ to 52~fold greater inductions of CYP1A1 mRNA over CYP1B1 mRNA. Furthermore, berberine dramatically increased CYP1A1 protein levels but did not influence CYP1B1 protein levels in MCF-7 cells. In conclusion, we present the first report to show that berberine may provide protection against breast cancer by altering the ratio of CYP1A1/CYP1B1, could redirect E2 metabolism in a more protective pathway in the breast cancer MCF-7 cells.

Genomic screening for targets regulated by berberine in breast cancer cells.

Berberine, a common isoquinoline alkaloid, has been shown to possess anti-cancer activities. However, the underlying molecular mechanisms are still not completely understood. In the current study, we investigated the effects of berberine on cell growth, colony formation, cell cycle distribution, and whether it improved the anticancer efficiency of cisplatin and doxorubicin in human breast cancer estrogen receptor positive (ER+) MCF-7 cells and estrogen receptor negative (ER-) MDA-MB-231 cells. Notably, berberine treatment significantly inhibited cell growth and colony formation in the two cell lines, berberine in combination with cisplatin exerting synergistic growth inhibitory effects. Accompanied by decreased growth, berberine induced G1 phase arrest in MCF-7 but not MDA-MB-231 cells. To provide a more detailed understanding of the mechanisms of action of berberine, we performed genome-wide expression profiling of berberine-treated cells using cDNA microarrays. This revealed that there were 3,397 and 2,706 genes regulated by berberine in MCF-7 and MDA-MB-231 cells, respectively. Fene oncology (GO) analysis identified that many of the target genes were involved in regulation of the cell cycle, cell migration, apoptosis, and drug responses. To confirm the microarray data, qPCR analysis was conducted for 10 selected genes based on previously reported associations with breast cancer and GO analysis. In conclusion, berberine exhibits inhibitory effects on breast cancer cells proliferation, which is likely mediated by alteration of gene expression profiles.