Antiproliferative effects of pomegranate extract in MCF-7 breast cancer cells are associated with reduced DNA repair gene expression and induction of double strand breaks.

Pomegranate extract (PE) inhibits the proliferation of breast cancer cells and stimulates apoptosis in MCF-7 breast cancer cells. While PE is a potent antioxidant, the present studies were conducted to examine the mechanisms of action of PE beyond antioxidation by studying cellular and molecular mechanisms underlying breast tumorigenesis. PE inhibited cell growth by inducing cell cycle arrest in G2 /M followed by the induction of apoptosis. In contrast, antioxidants N-acetylcysteine and Trolox did not affect cell growth at doses containing equivalent antioxidant capacity as PE, suggesting that growth inhibition by PE cannot solely be attributed to its high antioxidant potential. DNA microarray analysis revealed that PE downregulated genes associated with mitosis, chromosome organization, RNA processing, DNA replication and DNA repair, and upregulated genes involved in regulation of apoptosis and cell proliferation. Both microarray and quantitative RT-PCR indicated that PE downregulated important genes involved in DNA double strand break (DSB) repair by homologous recombination (HR), such as MRE11, RAD50, NBS1, RAD51, BRCA1, BRCA2, and BRCC3. Downregulation of HR genes correlated with increased levels of their predicted microRNAs (miRNAs), miR-183 (predicted target RAD50) and miR-24 (predicted target BRCA1), suggesting that PE may regulate miRNAs involved in DNA repair processes. Further, PE treatment increased the frequency of DSBs. These data suggest that PE downregulates HR which sensitizes cells to DSBs, growth inhibition and apoptosis. Because HR represents a novel target for cancer therapy, downregulation of HR by PE may be exploited for sensitization of tumors to anticancer drugs.

MicroRNA-9 inhibition of cell proliferation and identification of novel miR-9 targets by transcriptome profiling in breast cancer cells.

Although underexpression of miR-9 in cancer cells is reported in many cancer types, it is currently difficult to classify miR-9 as a tumor suppressor or an oncomir. We demonstrate that miR-9 expression is down-regulated in MCF-7 and MDA-MB-231 breast cancer cells compared with MCF-10-2A normal breast cell line. Increasing miR-9 expression levels in breast cancer cells induced anti-proliferative, anti-invasive, and pro-apoptotic activity. In addition, microarray profiling of the transcriptome of MCF-7 cells overexpressing miR-9 identified six novel direct miR-9 targets (AP3B1, CCNG1, LARP1, MTHFD1L, MTHFD2, and SRPK1). Among these, MTHFD2 was identified as a miR-9 target gene that affects cell proliferation. Knockdown of MTHFD2 mimicked the effect observed when miR-9 was overexpressed by decreasing cell viability and increasing apoptotic activity. Despite variable effects on different cell lines, proliferative and anti-apoptotic activity of MTHFD2 was demonstrated whereby it could escape from miR-9-directed suppression (by overexpression of MTHFD2 with mutated miR-9 binding sites). Furthermore, endogenous expression levels of miR-9 and MTHFD2 displayed inverse expression profiles in primary breast tumor samples compared with normal breast samples; miR-9 was down-regulated, and MTHFD2 was up-regulated. These results indicate anti-proliferative and pro-apoptotic activity of miR-9 and that direct targeting of MTHFD2 can contribute to tumor suppressor-like activity of miR-9 in breast cancer cells.

Oral ingestion of silver nanoparticles induces genomic instability and DNA damage in multiple tissues.

Silver nanoparticles (AgNPs) are widely used in consumer and medical products. However, most AgNP toxicity data are based on in vitro studies. Only a few studies were performed in mammals and no studies systematically assessed cancer risk of AgNPs. In this study, we examined whether oral exposure to polyvinylpyrrolidone (PVP)-coated AgNPs induces DNA damage and permanent genome alterations, and modulates DNA repair gene expression in vivo in mice. We found that AgNPs induced large DNA deletions in developing embryos, irreversible chromosomal damage in bone marrow, and double strand breaks and oxidative DNA damage in peripheral blood and/or bone marrow. DNA Repair RT Profiler PCR Array showed that AgNPs altered expression of 36 of the 84 genes from which 24 genes were downregulated and 12 genes were upregulated. In particular, AgNPs downregulated a significant proportion of base excision repair (BER) genes. We hypothesized that downregulation of BER by AgNPs contributes to oxidative DNA damage and subsequent genomic instability, which predicts that BER defects enhance sensitivity to AgNPs. We tested this hypothesis in mice deficient in MutY homologue (Myh). Myh excises adenine mispaired with 8-oxoguanine to counteract its promutagenic activity and also has a role in cell cycle check points and apoptosis. MYH mutations are common in humans and predispose to colorectal and other types of cancer. Myh deficient mice were hypersensitive to AgNP-induced chromosomal damage. In summary, oral ingestion of AgNPs induces permanent genome alterations and may therefore cause cancer. In addition, BER defects, especially, Myh mutations, enhance sensitivity to AgNPs.