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Molecular response mechanisms of encapsulated Tamoxifen (N-TAM) on DNA damage regulatory pathways

ABSTRACT: Background: We have applied an integrative approach, including classical genomic, microarray, and refined computational systems biology to study the regulatory pathways that involve DNA damage associated with typical drug preparations of Tamoxifen (TAM). We compared the effects of newly formulated Nano-emulsions of TAM (NanoTAM or N-TAM) on cultured HTB-20 cells to those of liquid preparations of TAM. Phenotypic screening was used to identify changes induced. Implicated regulatory pathways were systematically investigated by chromatin immunoprecipitation (ChIP) and DNA microarray analyses to identify alterations in protein-DNA interactions as well as genome-wide expression patterns. While liquid solutions of clinically used TAM can have a Z-score average of thousands of nm, after encapsulation and application of high pressure particle size was ~41 nm. ChIP-chip technology was used to classify promoter regions for target genes bound by E2F4 in human primary HTB-20 cells upon treatment with TAM or N-TAM. Specific drug effects were defined by comparing results obtained for Control/Control (CC) Control/Nano (CN), TAM (Drug)/Control (TC), and N-TAM (Drug)/Nano (TN). Functional effects noted included changes in apoptosis, cell proliferation, RNA processing, DNA damage, repair and replication, lipid and protein metabolism, transport, and transcriptional regulators. Cells treated with clinical TAM only expressed the ataxia telangiectasia mutated protein ATM as well as several histone proteins important for DNA damage sensing and activation of repair proteins, while N-TAM did not illicit this response.. In contrast N-TAM could not only enter the cells it could cross the inner mitochondrial membrane, induced more apoptosis and affecting mitochondrial DNA.

ORGANISM(S): Homo sapiens

PROVIDER: GSE178880 | GEO | 2021/06/25

REPOSITORIES: GEO

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Project description:Tamoxifen is a non-steroidal anti-estrogenic drug widely used for the treatment and prevention of breast cancer in women; however, there is evidence that tamoxifen is hepatocarcinogenic in rats, but not in mice. Additionally, it has been reported that tamoxifen may cause non-alcoholic fatty liver disease (NAFLD) in humans and experimental animals. The goals of the present study were to (i) investigate the mechanisms of the resistance of mice to tamoxifen-induced hepatocarcinogenesis, and (ii) clarify effects of tamoxifen on NAFLD-associated liver injury. Feeding female WSB/EiJ mice a 420 p.p.m. tamoxifen-containing diet for 12 weeks resulted in an accumulation of tamoxifen-DNA adducts, (E)-M-NM-1-(deoxyguanosin-N2-yl)-tamoxifen (dG-TAM) and (E)-M-NM-1-(deoxyguanosin-N2-yl)-N-desmethyltamoxifen (dG-DesMeTAM), in the livers. The levels of hepatic dG-TAM and dG-DesMeTAM DNA adducts in tamoxifen-treated mice were 578 and 340 adducts/108 nucleotides, respectively, while the extent of global DNA and repetitive elements methylation and histone modifications did not differ from the values in control mice. Additionally, there was no biochemical or histopathological evidence of NAFLD-associated liver injury in mice treated with tamoxifen. A transcriptomic analysis of differentially expressed genes demonstrated that tamoxifen caused predominantly down-regulation of hepatic lipid metabolism genes accompanied by a distinct over-expression of the lipocalin 13 (Lcn13) and peroxisome proliferator receptor gamma (PparM-NM-3), which may prevent the development of NAFLD. The results of the present study demonstrate that the resistance of mice to tamoxifen-induced liver carcinogenesis may be associated with its ability to induce genotoxic alterations only without affecting the cellular epigenome and an inability of tamoxifen to induce the development of NAFLD. Female and male WSB/EiJ mice were fed diet containing 420 p.p.m. tamoxifen for 12 weeks, and then tamoxifen-DNA adduct formation and gene expression profiles in the livers from four control and four tamoxifen-treated mice were investigated.

Project description:Tumor-associated macrophages (TAMs) represent alternatively activated (M2) macrophages that support tumor growth. Previously, we have described a special LYVE-1(+) M2 TAM subset in vitro and in vivo; gene profiling of this TAM subset identified MS4A8A as a novel TAM molecule expressed in vivo by TAM in mammary carcinoma and malignant melanoma. In vitro, Ms4a8a mRNA and MS4A8A protein expression was strongly induced in bone marrow-derived macrophages (BMDMs) by combining M2 mediators (IL-4, glucocorticoids) and tumor-conditioned media (TCM). Admixture of MS4A8A(+) TCM/IL-4/GC-treated BMDM significantly enhanced the tumor growth rate of subcutaneously transplanted TS/A mammary carcinomas. Upon forced overexpression of MS4A8A, Raw 264.7 macrophage-like cells displayed a special gene signature. Admixture of these MS4A8A(+) Raw 264.7 cells also significantly enhanced the tumor growth rate of subcutaneously transplanted mammary carcinomas. To identify the signaling pathways involved in synergistic induction of MS4A8A, the major signaling cascades with known functions in TAM were analyzed. Although inhibitors of NF-M-NM-:B activation and of the MAPK JNK and ERK did not show relevant effects, the p38M-NM-1/M-NM-2 MAPK inhibitor SB203580 strongly and highly significantly (p > 0.001) inhibited MS4A8A expression on mRNA and protein level. In addition, MS4A8A expression was restricted in M2 BMDM from mice with defective GC receptor (GR) dimerization indicating that classical GR gene regulation is mandatory for MS4A8A induction. In conclusion, expression of MS4A8A within the complex signal integration during macrophage immune responses may act to fine tune gene regulation. Furthermore, MS4A8A(+) TAM may serve as a novel cellular target for selective cancer therapy. A recombinant Ms4a8a cDNA was amplified by PCR (primer: Ms4a8a-SpeI-fw 5M-bM-^@M-^YATCGAATTCACTAGTAGCAAAGAGTTGGGAACCGGAGCAAGA3M-bM-^@M-^Y and Ms4a8a-NotI-rv: 5M-bM-^@M-^YATATGCGGCCGCTAGAGCATCTTTAT3M-bM-^@M-^Y) from Ms4a8a cDNA RZPDp981B0530D (IMAGE ID 905005), purified on agarose gel and subcloned after digestion with SpeI and NotI restriction enzymes into the expression vector pEF6/V5-His Topo (Invitrogen) according to standard molecular biology protocols. After confirming sequence identity, we transfected RAW264.7 cells with Ms4a8a vector DNA using Lipofectamine 2000 (Invitrogen) transfection reagent. Transfectants were selected by resistance to blasticidin (Invitrogen). Raw264.7Ms4a8a clone K8 with recombinant Ms4a4a expression were propagated. As a negative control, a vector-transfected RAW264.7mock (clone K3) was selected under parallel culture conditions.

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Molecular response mechanisms of encapsulated Tamoxifen (N-TAM) on DNA damage regulatory pathways

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