Simone Cristine Semprebon

Antiproliferative activity of goniothalamin enantiomers involves DNA damage, cell cycle arrest and apoptosis induction in MCF-7 and HB4a cells.

(R)-goniothalamin (R-GNT) is a styryl lactone that exhibits antiproliferative property against several tumor cell lines. (S)-goniothalamin (S-GNT) is the synthetic enantiomer of R-GNT, and their biological properties are poorly understood. The aim of this study was to evaluate the antiproliferative mechanisms of (R)-goniothalamin and (S)-goniothalamin in MCF-7 breast cancer cells and HB4a epithelial mammary cells. To determine the mechanisms of cell growth inhibition, we analyzed the ability of R-GNT and S-GNT to induce DNA damage, cell cycle arrest and apoptosis. Moreover, the gene expression of cell cycle components, including cyclin, CDKs and CKIs, as well as of genes involved in apoptosis and the DNA damage response were evaluated. The natural enantiomer R-GNT proved more effective in both cell lines than did the synthetic enantiomer S-GNT, inhibiting cell proliferation via cell cycle arrest and apoptosis induction, likely in response to DNA damage. The cell cycle inhibition caused by R-GNT was mediated through the upregulation of CIP/KIP cyclin-kinase inhibitors and through the downregulation of cyclins and CDKs. S-GNT, in turn, was able to cause G0/G1 cell cycle arrest and DNA damage in MCF-7 cells and apoptosis induction only in HB4a cells. Therefore, goniothalamin presents potent antiproliferative activity to breast cancer cells MCF-7. However, exposure to goniothalamin brings some undesirable effects to non-tumor cells HB4a, including genotoxicity and apoptosis induction.

(S)-Goniothalamin induces DNA damage, apoptosis, and decrease in BIRC5 messenger RNA levels in NCI-H460 cells

(R)-Goniothalamin (R-GNT) is a secondary metabolite isolated from the plants of the genus Goniothalamus. This molecule has attracted the attention of researchers because of its selective cytotoxicity against tumor cells and its ability to induce apoptosis. (S)-Goniothalamin (S-GNT) is a synthetic enantiomer of R-GNT, and its mechanism of action is largely unknown. In this study, we investigated the activity of S-GNT in a human non-small cell lung cancer NCI-H460 cells. We observed that the cells exposed to this compound exhibited cytotoxicity in a concentration-dependent manner. Based on the data obtained through the assessment of apoptosis induction in situ and the comet assay, we suggest that this cytotoxicity occurs due to the potential ability of this molecule to induce DNA damage with the consequent induction of cell death via apoptosis. A significant reduction in the messenger RNA levels of baculoviral inhibitor of apoptosis repeat-containing 5 (BIRC5) gene that encodes the survivin protein was found. This novel finding may explain the inhibition of cell proliferation and induction of apoptosis in tumor cells caused by this compound.

Salinomycin efficiency assessment in non-tumor (HB4a) and tumor (MCF-7) human breast cells

The search for anticancer drugs has led researchers to study salinomycin, an ionophore antibiotic that selectively destroys cancer stem cells. In this study, salinomycin was assessed in two human cell lines, a breast adenocarcinoma (MCF-7) and a non-tumor breast cell line (HB4a), to verify its selective action against tumor cells. Real-time assessment of cell proliferation showed that HB4a cells are more resistant to salinomycin than MCF-7 tumor cell line, and these data were confirmed in a cytotoxicity assay. The half maximal inhibitory concentration (IC50) values show the increased sensitivity of MCF-7 cells to salinomycin. In the comet assay, only MCF-7 cells showed the induction of DNA damage. Flow cytometric analysis showed that cell death by apoptosis/necrosis was only induced in the MCF-7 cells. The increased expression of GADD45A and CDKN1A genes was observed in all cell lines. Decreased expression of CCNA2 and CCNB1 genes occurred only in tumor cells, suggesting G2/M cell cycle arrest. Consequently, cell death was activated in tumor cells through strong inhibition of the antiapoptotic genes BCL-2, BCL-XL, and BIRC5 genes in MCF-7 cells. These data demonstrate the selectivity of salinomycin in killing human mammary tumor cells. The cell death observed only in MCF-7 tumor cells was confirmed by gene expression analysis, where there was downregulation of antiapoptotic genes. These data contribute to clarifying the mechanism of action of salinomycin as a promising antitumor drug and, for the first time, we observed the higher resistance of HB4a non-tumor breast cells to salinomycin.

Bisphenol A reduces testosterone production in TM3 Leydig cells independently of its effects on cell death and mitochondrial membrane potential

Leydig cells are the major testosterone-producing cells of the male reproductive system, and damage to these cells can impair fertility of men. Bisphenol A (BPA) is one of the chemicals with the highest volume of production worldwide. The aim of this study was to evaluate the effects of BPA on the growth, viability, and testosterone production of TM3 murine Leydig cells after exposure to BPA for 24 or 48 h. BPA reduced testosterone production, cell viability and cell growth in a concentration-dependent manner. The highest tested concentration of BPA (100 μM) increased cellular death, as indicated by an increased sub-G1 phase population and a larger number of cells labeled with Hoechst 3342. This concentration of BPA also decreased the number of metabolically active mitochondria as revealed by rhodamine staining. Therefore, our data show that BPA is toxic to Leydig TM3 cells and impairs their steroidogenic function.

Effects of sulfated and non-sulfated β-glucan extracted from Agaricus brasiliensis in breast adenocarcinoma cells – MCF-7

Abstract The β-glucans (β-G) are polysaccharides produced by various organisms, and sulfation of β-G renders them more soluble. With the objective to assess the effects of sulfated and non-sulfated β-G extracted from Agaricus brasiliensis in MCF-7 cells, assays were used to evaluate cytotoxicity, genotoxicity, cell proliferation and mRNA expression. The sulfated and non-sulfated β-G showed dose-dependent cytotoxicity at concentrations of 5 and 10 μg/mL, by the MTT assay. However, only cytotoxicity was observed after 24 h by the Red Neutral test for sulfated β-G, with no genotoxicity for either β-G in comet assay. Proliferation was decreased only at 72 h at a concentration of 100 μg/mL of sulfated β-G. Treatment with 5 μg/mL of sulfated β-G for 6 h reduced the expression of pro-apoptotic genes and stress signaling genes, cell cycle arrest, damage and cell migration. The 5 μg/mL of non-sulfated β-G for 6 h reduced the expression of the stress response gene and signaling damage. These results indicate that the cytotoxicity in the MTT is not cell death, and that, in general, sulfated β-G have greater cytotoxicity compared to non-sulfated β-G.

Response of HepG2/C3A cells supplemented with sodium selenite to hydrogen peroxide-induced oxidative stress

Oxidative stress (OS) is involved in the onset of various pathological processes, and sodium selenite (Na2SeO3) is known to have antioxidant activity. This study evaluated the cellular response of human HepG2/C3A cells supplemented with Na2SeO3 when exposed to hydrogen peroxide (H2O2)-induced OS. We analyzed cytotoxicity, cell proliferation, and genotoxicity in comparison with molecular data of mRNA and protein expression. The MTT and comet assays revealed that Na2SeO3 conferred cytoprotective and anti-genotoxic effects. In contrast, RTCA (Real-Time Cell Analysis) and flow cytometry analysis revealed that Na2SeO3 did not inhibit H2O2-induced anti-proliferative effects or cell cycle arrest (G2/M). Cells exposed simultaneously to Na2SeO3 and H2O2 showed overexpression of GPX1 mRNA, indicating that Na2SeO3 influenced the cellular antioxidant system. Furthermore, downregulation of CAT mRNA and SOD1 and PRX2 proteins induced by H2O2, was minimal after the Na2SeO3+H2O2 treatment. Although normalization of CCN2B mRNA expression by Na2SeO3 was observed after the Na2SeO3+H2O2 treatment, this was not observed for other genes such as CDKN1A, CDKN1C, and CDKN2B, which are related to cell cycle control, nor for GADD45A, which is involved in the cellular response to DNA damage. Furthermore, both CDKN1B and CDKN1C expression were downregulated in HepG2/C3A cells treated with Na2SeO3 only. Our results indicate that cellular response to Na2SeO3 involved the modulation of the antioxidant system. Na2SeO3 was unable completely recover HepG2/C3A cells from H2O2-induced oxidative damage, as evidenced by analysis of cell proliferation kinetics, cell cycle assay, and expression of key genes involved in cell cycle progression and response to DNA damage.

Genistein affects expression of cytochrome P450 (CYP450) genes in hepatocellular carcinoma (HEPG2/C3A) cell line

Background: Genistein (5,7-Dihydroxy-3-(4-hydroxyphenyl)-4H-1-benzopyran-4-one) is the most abundant isoflavone in soybean, which has been associated with a lower risk of development of cancer and cardiovascular diseases. Of particular interest regarding cancer preventive properties of flavo-noids is their interaction with cytochrome P450 enzymes (CYPs). However, contradictory data report the effect of genistein on expression of СYPs enzymes. Objective: The aim of this study was to investigate the effects of genistein on cytochrome P450 (CYP) gene expression levels in human hepatocellular carcinoma (HepG2/C3A) and colon adenocarcinoma (HT29) cells. Methods: Real-time RT–PCR was used to examine the expression of genes families involved in xenobi-otic metabolism, such as CYP1 (CYP1A1, CYP1B1), CYP2 (CYP2E1, CYP2D6), CYP3 (CYP3A4); and of a family involved in the catabolism of the all-trans-retinoic acid (ATRA), CYP26 (CYP26A1, CYP26B1). Results: RT-qPCR data analysis showed that after 12 h of exposure of HepG2/C3A cells to genistein (5 and 50 µM) there was an upregulation of CYP1A1 and CYP1B1 and downregulation of CYP2D6, CYP26A1 and CYP26B1 mRNA levels. There was no change in the mRNA levels of CYP P450 genes in HT29 cells. Conclusion: Our results suggest that treatment with genistein in non-toxic concentrations may impact the expression level of CYPs involved in the biotransformation of xenobiotics and drug metabolizing en-zymes. Moreover, the downregulation of ATRA metabolism-related genes opens a new research path for the study of genistein as retinoic acid metabolism blocking agent for treating cancer and other patholo-gies.

Role of 1α,25-Dihydroxyvitamin D 3 in Adipogenesis of SGBS Cells: New Insights into Human Preadipocyte Proliferation

Background/Aims: Compared with non-obese individuals, obese individuals commonly store more vitamin D in adipose tissue. VDR expression in adipose tissue can influence adipogenesis and is therefore a target pathway deserving further study. This study aims to assess the role of 1,25(OH)2D3 in human preadipocyte proliferation and differentiation. Methods: RTCA, MTT, and trypan blue assays were used to assess the effects of 1,25(OH)2D3 on the viability, proliferation, and adipogenic differentiation of SGBS cells. Cell cycle and apoptosis analyses were performed with flow cytometry, triglycerides were quantified, and RT-qPCR was used to assess gene expression. Results: We confirmed that the SGBS cell model is suitable for studying adipogenesis and demonstrated that the differentiation protocol induces cell maturation, thereby increasing the lipid content of cells independently of treatment. 1,25(OH)2D3 treatment had different effects according to the cell stage, indicating different modes of action driving proliferation and differentiation. In preadipocytes, 1,25(OH)2D3 induced G1 growth arrest at both tested concentrations without altering CDKN1A gene expression. Treatment with 100 nM 1,25(OH)2D3 also decreased MTT absorbance and the lipid concentration. Moreover, increased normalized cell index values and decreased metabolic activity were not induced by proliferation or apoptosis. Exposure to 100 nM 1,25(OH)2D3 induced VDR, CEBPA, and CEBPB expression, even in the preadipocyte stage. During adipogenesis, 1,25(OH)2D3 had limited effects on processes such as VDR and PPARG gene expression, but it upregulated CEBPA expression. Conclusions: We demonstrated for the first time that 1,25(OH)2D3 induces changes in preadipocytes, including VDR expression and growth arrest, and increases the lipid content in adipocytes treated for 16 days. Preadipocytes are important cells in adipose tissue homeostasis, and understanding the role of 1,25(OH)2D3 in adipogenesis is a crucial step in ensuring adequate vitamin D supplementation, especially for obese individuals.

Roles of chlorophyllin in cell proliferation and the expression of apoptotic and cell cycle genes in HB4a non-tumor breast cells.

Abstract Chlorophyllin (Chl) has attracted interest in the scientific community due to its chemopreventive and antimutagenic properties. However, the molecular mechanisms of action of Chl remain unclear. This study assesses the effects on cell proliferation and the expression of genes involved in apoptosis, and the cell cycle in HB4a cells treated with Chl. Chl was cytotoxic and induced apoptosis to HB4a cells at 400 μg/mL. Analysis of gene expression showed that there was a decrease in the mRNA level of BIRC5 and CCNA2 genes involved in apoptosis and cell cycle progression, respectively. The proapoptotic gene BAX and the antiapoptotic genes BCL-2 and BCL-XL were upregulated. The cytotoxicity of Chl has been attributed to increases in the expression of BAX and decreases in the expression of genes involved in the cell cycle, but increases in the expression of anti-apoptotic genes suggests a mechanism for protection from cell death induced by Chl. This study provides important information about mechanisms that protect against or trigger damaging processes in non-tumor cells.

BIO045 Chlorophyllin changes the expression of apoptosis and cell cycle genes in non-tumor breast HB4a cells

The molecule of chlorophyllin has been the subject of the scientific community by presenting chemopreventive, antimutagenic and anticarcinogenic properties. However, the molecular mechanisms of action of this compound remain unclear. Thus, the study of genes involved in apoptosis and cell cycle in non-tumor cells treated with chlorophyllin can provide important information about the mechanisms that protect or trigger damaging processes, aiding in the development of therapies.