Piperlongumine inhibits antioxidant enzymes, increases ROS levels, induces DNA damage and G2/M cell cycle arrest in breast cell lines.

Piperlongumine (PLN) is a biologically active alkaloid/amide derived from Piper longum, with known promising anticancer activity. The aim of this study was to compare the antiproliferative activity of PLN in human breast MCF-7 adenocarcinoma cell line with effects in HB4a normal mammary epithelial non-tumor cell line. The parameters examined were cell growth, viability, reactive oxygen species (ROS) levels and DNA damage, as well as the effects on the modulating targets responsible through regulation of these pathways. PLN increased ROS levels and expression of the SOD1 antioxidant enzyme. PLN inhibited the expression of the antioxidant enzymes catalase, TRx1, and PRx2. The ability of PLN to inhibit antioxidant enzyme expression was associated with the oxidative stress response. PLN induced genotoxicity in both cell lines and upregulated the levels of GADD45A mRNA and p21 protein. The DNA damage response ATR protein was downregulated in both cell lines and contributed to an enhanced PLN genotoxicity. In HB4a cells, Chk1 protein, and mRNA levels were also decreased. In response to elevated ROS levels and DNA damage induction, the cells were arrested at the G2/M phase, probably in an attempt to promote cell survival. Although cell viability was reduced in both cell lines, only HB4a cells underwent apoptotic cell death, whereas other types of cellular death may be involved in MCF-7 cells. Taken together, these data provide insight into the anticancer mechanisms attributed to PLN effects, which acts as an inhibitor of DNA damage response (DDR) proteins and antioxidant enzymes.

Cellular and molecular antiproliferative effects in 2D monolayer and 3D-cultivated HT-29 cells treated with zerumbone.

Zerumbone (ZER) is a phytochemical isolated from plants of the Zingiberaceae family. Numerous studies have demonstrated its diverse pharmacological properties, particularly its potent antitumorigenic activity. This study aimed to assess the antiproliferative effects of ZER on HT-29 cells cultivated in both two-dimensional (2D) monolayer and three-dimensional (3D) spheroid culture systems. The evaluation of growth (size), cell death, and cell cycle arrest in 3D spheroid HT-29 cells was correlated with mRNA expression data. Treatment of 2D cells revealed that ZER exhibited cytotoxicity at concentrations above 30 µM, and an IC50 of 83.54 µM (24-h post-ZER treatment) effectively suppressed cell migration. In the 3D model, ZER induced an increase in spheroid volume over a 72-h period attributed to disaggregation and reconfiguration of characteristic zones. Analysis of cell death demonstrated a significant rise in apoptotic cells after 24 h of ZER treatment, along with cell cycle arrest in the G1 phase. Furthermore, ZER treatment resulted in alterations in mRNA expression, affecting key signaling pathways involved in cell death (BCL2 and BBC3), endoplasmic reticulum stress (ERN1), DNA damage (GADD45A), cell cycle regulation (CDKN1A, NFKB1, MYC, and TP53), and autophagy (BECN1 and SQSTM1). These findings suggested that ZER holds promise as a potential candidate for the development of novel anticancer agents that can modulate crucial cell signaling pathways. Additionally, the use of the 3D culture system proved to be a valuable tool in our investigation.

Regulation of cytokinesis and necroptosis pathways by diosgenin inhibits the proliferation of NCI-H460 lung cancer cells.

Aim Overcoming resistance to apoptosis and antimitotic chemotherapy is crucial for effective treatment of lung cancer. Diosgenin (DG), a promising phytochemical, can regulate various molecular pathways implicated in tumor formation and progression. However, the precise biological activity of DG in lung cancer remains unclear. This study aimed to investigate the antiproliferative activity of DG in NCI-H460 lung carcinoma cells to explore the underlying antimitotic mechanisms and alternative cell death pathways. MATERIALS AND METHODS: In a 2D culture system, we analyzed cell viability, multinucleated cell frequency, cell concentration, cell cycle changes, cell death induction, intracellular reactive oxygen species (ROS) production, and nuclear DNA damage, particularly in relation to target gene expression. We also evaluated the antiproliferative activity of DG in a 3D culture system of spheroids, assessing volume changes, cell death induction, and inhibition of proliferation recovery and clonogenic growth. KEY FINDINGS: DG reduced cell viability and concentration while increasing the frequency of cells with multiple nuclei, particularly binucleated cells resulting from daughter cell fusion. This effect was associated with genes involved in cytokinesis regulation (RAB35, OCRL, BIRC5, and AURKB). Additionally, DG-induced cell death was linked to necroptosis, as evidenced by increased intracellular ROS production and RIPK3, MLKL, TRAF2, and HSPA5 gene expression. In tumor spheroids, DG increased spheroid volume, induced cell death, and inhibited proliferation recovery and clonogenic growth. SIGNIFICANCE: Our study provides new insights into the biological activities of DG in lung cancer cells, contributing to the development of novel oncological therapies.

Influence of temperature variation on gene expression and cocoon production in Bombyx mori Linnaeus, 1758 (Lepidoptera: Bombycidae).

Silkworms (Bombyx mori) are lepidopterans of economic importance for global silk production. However, factors that directly affect the yield and quality of silkworm cocoon production, such as diseases and temperature fluctuations, cause great economic losses. Knowing how they respond to rearing temperature during the most critical stage of their life cycle (i.e., fifth instar) could provide information on their adaptation and improve silk production. In the current work, we analyzed transcriptional data from two groups of B. mori that were reared at 26 °C and 34 °C throughout the fifth instar. The silkworms and cocoons were weighed. In total, 3115 transcripts were differentially expressed (DE; including 1696 down-regulated and 1419 up-regulated) among the 29,157 sequences found by transcriptome assembly. We emphasize the genes associated with immunological response, transcription factors, silk biosynthesis, and heat shock proteins, among the DE transcripts in response to the temperature conditions. Silkworms reared at 34 °C presented a reduced mean body weight (-0.944 g in comparison to the 26 °C group), which had a direct impact on the weight of cocoons formed and the silk conversion rate. These changes were statistically significant when compared to silkworms reared at 26 °C. Mortality rates (6 and 9 %, at 26 °C and 34 °C, respectively) were similar to those obtained in breeding fields. The findings provide information on the biological processes involved in the temperature response mechanism of silkworms, as well as information that may be used in future climatization processes at rearing facilities and in breeding for improved thermotolerance.

Vitamin D3 and Salinomycin synergy in MCF-7 cells cause cell death via endoplasmic reticulum stress in monolayer and 3D cell culture.

1α, 25, dihydroxyvitamin D3 (1,25D), the active form of vitamin D3, has antitumor properties in several cancer cell lines in vitro. Salinomycin (Sal) has anticancer activity against cancer cell lines. This study aims to examine the cytotoxic and antiproliferative effect of Sal associated with 1,25D on MCF-7 breast carcinoma cell line cultured in monolayer (2D) and three-dimensional models (mammospheres). We also aim to evaluate the molecular mechanism of Sal and 1,25D-mediated effects. We report that Sal and 1,25D act synergistically in MCF-7 mammospheres and monolayer causing G1 cell cycle arrest, reduction of mitochondrial membrane potential (MMP), and reactive oxygen species (ROS) overproduction with a long-lasting cytotoxic response represented by clonogenic and mammosphere assay. We observed the induction of cell death by apoptosis with upregulation in mRNA levels of apoptosis-related genes (CASP7, CASP9, and BBC3). Extensive cytoplasmic vacuolization, a morphological characteristic found in paraptosis, was also seen and could be triggered by endoplasmic reticulum stress (ER) as we found transcriptional upregulation of genes related to ER stress (ATF6, GADD153, GADD45G, EIF2AK3, and HSPA5). Overall, Sal and 1,25D act synergistically, inhibiting cell proliferation by activating simultaneously multiple death pathways and may be a novel and promising luminal A breast cancer therapy strategy.

Salinomycin induces cell cycle arrest and apoptosis and modulates hepatic cytochrome P450 mRNA expression in HepG2/C3a cells.

Salinomycin (SAL) is a monocarboxylic polyether ionophore antibiotic isolated from Streptomyces albus. It exhibits an effective antitumor potential against numerous human cancer cells. This study aimed to assess the antiproliferative effects of SAL in human hepatocellular carcinoma HepG2/C3a cell line. We investigated the effects of SAL on cell growth, DNA damage induction, cell cycle changes and apoptosis; and relative changes in expression of cell cycle-related, apoptosis-related, and CYP450 genes. SAL induced cell cycle arrest in the G2/M phase, upregulation of CDKN1A and GADD45A and downregulation of cyclin genes including CCNB1 and CCNA2. SAL effectively suppressed mRNA levels of CTNNB1 gene, an important oncogene that promotes tumorigenesis. The decrease of HepG2/C3A cells' survival can also be due to downregulation of antiapoptotic BCL-2 expression, thus promoting the induction of apoptosis by SAL. This study also demonstrated the ability of SAL in modulating hepatic cytochrome P450 (CYP) mRNA expression, such that SAL caused the upregulation of CYP1A members and CYP3A5; and downregulation of CYP3A4. Taken together, these data contribute to the understanding of the mechanism of action of SAL, highlighting that metabolizing enzymes modulated by SAL can interfere with chemotherapy treatment and it must be considered in associated treatments.

Carnosic acid exhibits antiproliferative and proapoptotic effects in tumoral NCI-H460 and nontumoral IMR-90 lung cells.

Carnosic acid (CA) is a phenolic diterpene with many important biological activities including antimicrobial, antioxidant, anti-inflammatory properties, and anti-proliferative properties. The aim of the present study was to investigate cytotoxic activity, cell cycle, apoptotic, and molecular effects attributed to CA in non-tumoral IMR-90 (human fetal lung fibroblasts), as well as tumoral NCI-H460 (human non-small-cell lung cancer) cell lines. Cell proliferation was evaluated by Real-Time Cell Analysis system, while apoptosis and cell cycle were assessed using flow cytometry. RT-qPCR was used to estimate the relative expression of genes involved in cell cycle regulation, DNA damage and repair, and apoptosis induction. CA inhibited proliferation of IMR-90 and NCI-H460 cells via cell cycle arrest at G0/G1 and G2/M phases, according to the treatment concentration. The mRNA levels of genes encoding cyclins A2, B1, and B2 were downregulated in response to CA treatment of IMR-90 cells. Apoptosis was induced and proapoptotic gene PUMA was upregulated in both cell lines. mRNA levels of genes ATR, CCND1, CHK1, CHK2, MYC, GADD45A, H2AFX, MTOR, TP53, and BCL2, CASP3 were not markedly changed following CA treatments. Although CA exerted antiproliferative activity against NCI-H460 tumor cells, this phytochemical induced toxic effects in non-tumoral cells, and thus needs to be considered carefully prior to pharmacological use therapeutically.

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.

Role of 1α,25-Dihydroxyvitamin D3 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.

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.

Comparison of the Effects of Monastrol and Oxomonastrol on Human Hepatoma Cell Line HepG2/C3A.

Monastrol and its analog oxomonastrol differ by replacement of the sulfur atom present in monastrol to an oxygen atom in oxomonastrol. Monastrol inhibits the mitotic kinesin family member 11 (EG5), which has been studied for its potential use in cancer therapy. The aim of this study was to investigate the effect of monastrol and oxomonastrol on HepG2/C3A cells. Our results showed that monastrol induced DNA damage, reduced cell proliferation, and up-regulated the cytochrome P450 family 1 subfamily A member 1 (CYP1A1) mRNA levels. However, oxomonastrol was cytotoxic only at the highest concentrations used, without reducing cell proliferation and viability. Moreover, no genotoxic damage or alteration of levels of mRNA were found. Our results suggest that monastrol has greater antiproliferative activity compared to oxomonastrol, and this effect is probably related to the DNA damage induced by monastrol and its possible bioactivation demonstrated by the increase in CYP1A1 mRNA expression. Moreover, these effects appear to be related to the presence of the sulfur atom in its structure.

Antiproliferative activity of monastrol in human adenocarcinoma (MCF-7) and non-tumor (HB4a) breast cells.

Monastrol is an allosteric inhibitor of the mitotic kinesin Eg5 that exhibits an antiproliferative effect against several cell lines. We investigated the antiproliferative effect of monastrol on human breast adenocarcinoma cells (MCF-7) and mammary epithelial cells (HB4a, non-tumoral). Monastrol treatment decreased cell viability only in MCF-7 tumor cells. Real-time cell growth kinetic analysis showed a decrease in the proliferation of MCF-7 cells exposed to monastrol, while in the HB4a cells, only a concentration of 100 µM was able to induce this effect. In a cell cycle analysis, exposure of MCF-7 cells to monastrol led to an increased population of cells in both the G1 and G2/M phases. In HB4a cells, the proportion of cells in the G2/M phase was increased. Monastrol led to an increased mitotic index in both cell lines. Monastrol was not able to induce cell death by apoptosis in any of the cell lines studied. Gene expression analysis was performed to measure the mRNA levels of cell cycle genes, DNA damage indicator gene, and apoptotic related genes. Treatment with monastrol induced in MCF-7 cells a 5-fold increase in the mRNA levels of the CDKN1A gene, an inhibitor of CDKs related with cell cycle arrest in response a stress stimulus, and a 2-fold decrease in CDKN1C mRNA levels in HB4a cells. These results provide evidence that monastrol has a greater antiproliferative effect on MCF-7 tumor cells compared with non-tumor HB4a cells; however, no selective is observed.

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

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.

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.

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.

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

The ß-glucans (ß-G) are polysaccharides produced by various organisms, and sulfation of ß-G renders them more soluble. With the objective to assess the effects of sulfated and non-sulfated ß-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 ß-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 ß-G, with no genotoxicity for either ß-G in comet assay. Proliferation was decreased only at 72 h at a concentration of 100 µg/mL of sulfated ß-G. Treatment with 5 µg/mL of sulfated ß-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 ß-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 ß-G have greater cytotoxicity compared to non-sulfated ß-G.