Evolutionary Engineering of an Iron-Resistant Saccharomyces cerevisiae Mutant and Its Physiological and Molecular Characterization.

Iron plays an essential role in all organisms and is involved in the structure of many biomolecules. It also regulates the Fenton reaction where highly reactive hydroxyl radicals occur. Iron is also important for microbial biodiversity, health and nutrition. Excessive iron levels can cause oxidative damage in cells. Saccharomyces cerevisiae evolved mechanisms to regulate its iron levels. To study the iron stress resistance in S. cerevisiae, evolutionary engineering was employed. The evolved iron stress-resistant mutant "M8FE" was analysed physiologically, transcriptomically and by whole genome re-sequencing. M8FE showed cross-resistance to other transition metals: cobalt, chromium and nickel and seemed to cope with the iron stress by both avoidance and sequestration strategies. PHO84, encoding the high-affinity phosphate transporter, was the most down-regulated gene in the mutant, and may be crucial in iron-resistance. M8FE had upregulated many oxidative stress response, reserve carbohydrate metabolism and mitophagy genes, while ribosome biogenesis genes were downregulated. As a possible result of the induced oxidative stress response genes, lower intracellular oxidation levels were observed. M8FE also had high trehalose and glycerol production levels. Genome re-sequencing analyses revealed several mutations associated with diverse cellular and metabolic processes, like cell division, phosphate-mediated signalling, cell wall integrity and multidrug transporters.

Different propolis samples, phenolic content, and breast cancer cell lines: Variable cytotoxicity ranging from ineffective to potent.

Researchers have started focusing on investigating the anticarcinogenic effects of natural products with the slightest side effects possible, because current breast cancer treatment approaches are unable to achieve absolute success especially on aggressive subtypes. Propolis is among these products with its antimicrobial, antifungal, anti-inflammatory, and anticancer effects. Therefore, seven different samples were collected from different regions (Argentina, China, and Istanbul-Turkey) and applied on nonaggressive breast cancer cell line (BCCL) MCF-7 and aggressive cell lines SK-BR-3, and MDA-MB-231. Initially, the phenolic/flavonoid constituents of the propolis ethanol extracts were investigated by liquid chromatography-mass spectrometry-mass spectrometry (LS-MS/MS) and high-performance liquid chromatography (HPLC) analyses. Then, the anticarcinogenic effects of the propolis samples on MCF-7, SK-BR-3, MDA-MB-231 were evaluated by WST1 analysis and only selected ones on MCF-10A and hPdLF. According to the LS-MS/MS and HPLC analysis, Turkey originated propolis (Turkey3) were found to be richer than the other propolis samples in terms of phenolic/flavonoid compounds. Turkey propolis significantly inhibited cell proliferation in both nonaggressive and aggressive BCCL (P < 0.01). Therefore, Turkey3 propolis was selected for further evaluation using Annexin V-PI apoptosis detection assays. In addition, selected compounds among the propolis contents such as galangin, caffeic acid, apigenin, quercetin, and ferulic acid were applied to the MCF-7 cell line to detect cytotoxic and apoptotic effects. Galangin, caffeic acid, apigenin, and quercetin remarkably induced cell proliferation inhibition at all time intervals, whereas ferulic acid was found non efficient on the MCF-7 cell line. Annexin V-PI assay clarified that all cell proliferation inhibitions were markedly apoptotic. Our findings indicated that the inhibition effect of propolis on breast cancer cell proliferation was in a propolis type-, dose- and time-dependent fashion. Turkey3 propolis showed statistically significant cytotoxic effects on both the nonaggressive and aggressive BCCL. These findings were consistent with the effects of its rich phenolic and flavonoid contents, in terms of variety. © 2018 IUBMB Life, 71(5):619-631, 2019.

Anatolian honey is not only sweet but can also protect from breast cancer: Elixir for women from artemis to present.

Natural products with bioactive components are widely studied on various cancer cell lines for their possible cytotoxic effects, recently. Among these products, honey stands out as a valuable bee product containing many active phenolic compounds and flavonoids. Numerous types of multifloral honey and honeydew honey are produced in Turkey owing to its abundant vegetation. Therefore, in this study, we investigated the cytotoxic effects of particular tree-originated honeys from chestnut, cedar, pine, and multifloral honey on cell lines representing different types of the most common cancer of women, breast cancer, MCF7, SKBR3, and MDAMB-231, and fibrocystic breast epithelial cell line, MCF10A as a control. All honey samples were analyzed biochemically. The dose- (1, 2.5, 5, 7.5, and 10 µg/mL) and time (24th, 48th, and 72nd hours)-dependent effects of ethanol/water solutions of the honey samples were scrutinized. Cell viability/cytotoxicity was evaluated by the water soluble tetrazolium Salt-1 (WST-1) method. Apoptotic status was detected by Annexin V-PI assay using FACSCalibur. The statistical analysis was performed using GraphPad Prism 6 and the clustering data analysis with the R programming language. The biochemical analyses of the honey samples showed that the tree-originated honey samples contained more total phenolic compounds than the multifloral honey. Phenolic content of the honey types increases in order of multifloral, pine, cedar, and chestnut, respectively, which is compatible with their cytotoxic affectivity and dark color. In addition, the antioxidant capacity of the studied honey types was observed to increase in order of multifloral < pine < cedar ≅ chestnut. According to the WST-1 data, chestnut honey induced cytotoxicity over 50% on all the cell lines, including the control MCF10A cells, even with low doses (honey concentrations starting from 1 µg/mL) (P < 0.0001). Similarly, Cedar honey was observed to be the second most effective honey in this study. Cedar honey, with the dose of 1 µg/mL, was detected statistically highly significant on MCF10A, MCF7, and SKBR3. In contrast, pine honey showed dramatically significant cytotoxicity only on the MDAMB 231 cells with a 1 µg/mL dose at the same time point (P = 0.018). While pine honey caused an anticancer effect on the MCF-7 and SKBR3 cancer cell lines with a 2.5-5 µg/mL dose (P < 0.0001), like cedar and chestnut honeys, it increased the viability of the MCF10A control cells with the doses of 2.5-5 µg/mL. It only showed cytotoxicity with higher doses (10 µg/mL) on the MCF10A cell line (P < 0.0001). Moreover, we have observed that the multifloral and artificial honey samples were mostly ineffective or increased cell viability with the doses of 1-5 µg/mL. Apoptotic effects of the other honey samples on the MCF-7 cell line were found as chestnut> pine> cedar> multifloral in the Annexin V-propidium iodide (PI) analysis. Chestnut, cedar, and pine honey displayed a remarkably cytotoxic effect on breast cancer cell lines, MCF7, SKBR3, and even on the most aggressive MDAMB 231, representing the triple negative breast cancer, which lacks of targeted anticancer therapy. The chestnut and cedar honeys stand out to be the most cytotoxic on all cell lines, while pine honey was found to be the least toxic on control cells with appropriate toxicity on the cancer cells. © 2017 IUBMB Life, 69(9):677-688, 2017.

Evaluation of Local CYP17A1 and CYP19A1 Expression Levels as Prognostic Factors in Postmenopausal Invasive Ductal Breast Cancer Cases.

There is growing attention focused on local estrogen production in the breast tissue and its possible role in breast cancer initiation and progression. Understanding the underlying mechanisms for estrogen synthesis and the microenvironment consisting of tumor and its surrounding adipose tissue might open new avenues in breast cancer prevention, prognosis and treatment. In order to obtain insight, we compared peritumoral and tumor tissue expressions of CYP17A1 and CYP19A1 genes, which play an important role in estrogen biosynthesis. The paired tissue samples of 20 postmenopausal ER+/PR+ patients diagnosed with invasive ductal breast cancer were studied. In addition, 12 breast tissue samples obtained from premenopausal women without a history of breast cancer were also investigated as representative of normal conditions. Peritumoral adipose tissues expressed CYP19A1 approximately threefold higher than tumor itself (p = 0.001). A nonsignificant trend toward low expression of CYP17A1 was observed in peritumoral compared to tumor tissue (p = 0.687). Clinicopathological parameters and patient characteristics which are accepted as risk factors for breast cancer were also associated with individual and combined expressions of CYP17A1 and CYP19A1. This study offers that evaluation of CYP17A1 and CYP19A1 local expression levels might be useful for deciding on personalized treatment approaches and more accurate diagnosis, when evaluated together with several clinicopathological and disease risk factors. Considering the key role of these CYPs in estrogen synthesis, determining their expression levels may be useful as a postdiagnostic marker and for choosing the right treatment method in addition to the conventional approach.

Preliminary Study: Prominent miRNAs of Breast Malignant Tissues Compared to Normal Tissues in Turkish Patients with Breast Cancer.

miRNA involvement has been observed in almost every type of cancer, including breast cancer. The etiology of abnormal expression of miRNAs in cancer is still not clearly understood. In order to obtain insight into miRNA de-regulation in breast cancer, we analyzed expression levels of five breast cancer-related miRNAs, miRNA21, miRNA155, miRNA19a, miRNA17-5p and let7a miRNA, in both malignant and neighboring non-tumoral paraffin-embedded tissues of 47 patients with invasive ductal breast cancer. The targeted miRNAs, and a reference snRNA, U6, were analyzed by real-time polymerase chain reaction. let7a Levels were significantly lower in patients with lymphatic invasion than in those without (p=0.047). miR21 was down-regulated in 93.3% of patients with necrosis [p=0.017 (Fisher's exact test (FE))], while at least one oncogenic miRNA was up-regulated in 87.3% of the patients with invasive ductal carcinoma [p=0.009 (FE)]. In addition, tumor-suppressor miRNA was down-regulated or unaltered in 65.8% of the patients with tumor grade 2 or 3 and in all with grade 1 [p=0.047 (FE)]. Based on this preliminary study, we suggest that these miRNAs, especially let7a and miRNA21, might be useful markers in follow-up of breast cancer and in prognosis.

The effect of CYP1A1 and GSTM1 gene polymorphisms in bladder cancer development in a Turkish population.

BACKGROUND: The aim of this study was to investigate a possible association of the CYP1A1 Ile462Val and GSTM1 null polymorphisms with the risk of developing bladder cancer in a Turkish population. PATIENTS AND METHODS: The study constituted 176 patients with bladder cancer and 97 healthy individuals. Evaluation of CYP1A1 Ile462Val gene polymorphism was performed by polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). GSTM1 null gene polymorphism was exclusively determined by PCR. Our results were examined by statistical analyses. RESULTS: There were no significant differences in CYP1A1 genotype frequencies between patients and controls. Furthermore, the frequency of GSTM1 null genotype was higher in patients compared to controls, but it did not reach significance (p=0.622 χ(2)=0.243 OR=0.94 95% CI=0.75-1.18). Significance was discovered in combined analysis of CYP1A1 and GSTM1 genotypes. In the present study, GSTM1 null genotype with CYP1A1 Ile/Ile genotype combination was significantly more frequent in the patient group than in controls (p=0,04, χ(2)=4.217). At the same time, possessing both GSTM1 null genotype and CYP1A1 Val variants (Ile/Val+Val/Val) were significantly higher in control group than in patients (p=0.017, χ(2)=5.468). When the pathological tumor grades were assessed, the frequency of CYP1A1 Val mutant variant with GSTM1 null genotype combination was higher in patients with medium and high-grade tumors than in those with low-grade tumors (p=0.06, χ(2)=3.527, OR=1.36 95% CI=1.03-1.78). CONCLUSION: We suggest that the CYP1A1 Ile/Ile genotype with GSTM1 null genotype combination may contribute to the development of bladder cancer in this Turkish population.