Background: Tamoxifen (tam) is widely used to treat estrogen-positive breast cancer. However, cancer recurrence after chemotherapy remains a major obstacle to achieve good patient prognoses. In this study, we aimed to identify genes responsible for epigenetic regulation of tam resistance in breast cancer. Methods: Methylation microarray data were analyzed to screen highly hypomethylated genes in tam resistant (tamR) breast cancer cells. Quantitative RT-PCR, Western blot analysis, and immunohistochemical staining were used to quantify expression levels of genes in cultured cells and cancer tissues. Effects of matrix metalloproteinase-1 (MMP1) expression on cancer cell growth and drug resistance were examined through colony formation assays and flow cytometry. Xenografted mice were generated to investigate the effects of MMP1 on drug resistance in vivo. Results: MMP1 was found to be hypomethylated and overexpressed in tamR MCF-7 (MCF-7/tamR) cells and in tamR breast cancer tissues. Methylation was found to be inversely associated with MMP1 expression level in breast cancer tissues, and patients with lower MMP1 expression exhibited a better prognosis for survival. Downregulating MMP1 using shRNA induced tam sensitivity in MCF-7/tamR cells along with increased apoptosis. The xenografted MCF-7/tamR cells that stably expressed short hairpin RNA (shRNA) against MMP1 exhibited retarded tumor growth compared to that in cells expressing the control shRNA, which was further suppressed by tam. Conclusions: MMP1 can be upregulated through promoter hypomethylation in tamR breast cancer, functioning as a resistance driver gene. MMP1 can be a potential target to suppress tamR to achieve better prognoses of breast cancer patients.

Cold atmospheric plasma (CAP) has been recognized as a potential alternative or supplementary cancer treatment tool, which is attributed by its selective antiproliferation effect on cancer cells over normal cells. Standardization of the CAP treatment in terms of biological outputs such as cell growth inhibition and gene expression change is essential for its clinical application. This study aims at identifying genes that show consistent expression profiles at a specific CAP condition, which could be used to monitor whether CAP is an appropriate treatment to biological targets. To do this, genes showing differential expression by two different CAP treatment conditions were screened in the MCF-7 breast cancer cells. As a result, ZNRD1 was identified as a potential marker with being consistently upregulated by 600 s but downregulated by the 10×30s CAP treatment scheme. Expression of ZNRD1 was increased in breast cancer tissues compared to normal tissues, judged by cancer tissue database analysis, and supported by the antiproliferation after siRNA-induced downregulation in MCF-7. Interestingly, the antisense long noncoding RNA (lncRNA) of ZNRD1, ZNRD1-AS1, was regulated to the opposite direction of ZNRD1 by CAP. The siRNA-based qPCR analysis indicates that ZNRD1 downregulates ZNRD1-AS1, but not vice versa. ZNRD1-AS1 was shown to increase a few cis-genes such as HLA-A, HCG9, and PPP1R11 that were also regulated by CAP. Altogether, this study identified a pair of gene and its antisense lncRNA of which expression is precisely controlled by CAP in a dose-dependent manner. These genes could help elucidate the molecular mechanism how CAP regulates lncRNAs in cancer cells.

Gastric mucosal injury is associated with acute epithelial damage, inflammatory cytokine release, and disruption of the protective mucus barrier. NPKE6-FM is a fermented metabolite preparation produced from puffed soybean and rice inoculated with Bacillus amyloliquefaciens NPKE6, a strain isolated from Korean water kimchi. Although NPKE6-FM has shown antioxidant and anti-inflammatory potential in previous in vitro and in vivo studies, its gastroprotective efficacy against acute gastric injury has not been fully evaluated. This study investigated the protective effects of NPKE6-FM in an HCl/ethanol-induced acute gastritis model in ICR mice. Male ICR mice were randomly assigned to six groups after weight measurement (n = 5 per group): normal control (distilled water only), model group (HCl/ethanol plus distilled water), positive control (HCl/ethanol plus rebamipide, 50 mg/kg), and NPKE6-FM treatment groups receiving 10, 50, or 100 mg/kg. Test substances or vehicle were orally administered for 14 consecutive days. Following an approximately 18 h fast on Day 15, acute gastric mucosal injury was induced by oral administration of 200 mM HCl in 80% ethanol. Gastric tissues were collected approximately 1 h after injury induction for macroscopic lesion assessment, histopathological examination, cytokine analysis, and quantitative real-time PCR analysis of inflammation- and mucin-related genes. HCl/ethanol administration induced marked hemorrhagic and erosive gastric lesions, histopathological mucosal damage, and increased levels of TNF-α and IL-6. NPKE6-FM treatment reduced gross gastric lesion scores in a dose-dependent manner, with the 100 mg/kg group showing the greatest protective effect. Histological evaluation further indicated improved preservation of gastric mucosal architecture in NPKE6-FM-treated mice. In addition, NPKE6-FM significantly suppressed TNF-α and IL-6 levels and downregulated the mRNA expression of inflammatory mediators, including COX-2, iNOS, TNF-α, and IL-6. Conversely, the HCl/ethanol-induced reduction in Muc5ac expression was substantially restored by NPKE6-FM treatment. These findings demonstrate that NPKE6-FM protects against HCl/ethanol-induced acute gastric mucosal injury through anti-inflammatory activity and reinforcement of the gastric mucus barrier. NPKE6-FM may therefore represent a promising functional food ingredient or nutraceutical candidate for the prevention or attenuation of gastric mucosal injury.