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.

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.

mRNAs biomarker related to the control of proliferation and cell death in HepG2/C3A spheroid and monolayer cultures treated with piperlongumine

Background: Cell culture (spheroid and 2D monolayer cultures) is an essential tool in drug discovery. Piperlongumine (PLN), a naturally occurring alkaloid present in the long pepper (Piper longum), has been implicated in the regulation of GSTP1 activity. In vitro treatment of cancer cells with PLN increases ROS (reactive oxygen species) levels and induces cell death, but its molecular mode of action has not been entirely elucidated. Methods: In this study, we correlated the antiproliferative effects (2D and 3D cultures) of PLN (CAS 20069­09-4, Sigma-Aldrich) with morphological and molecular analyses in HepG2/C3A cell line. We performed assays for cytotoxicity (MTT), comet assays for genotoxicity, induction of apoptosis, analysis of the cell cycle phase, and analysis of the membrane integrity by flow cytometry. Relative expression of mRNA of genes related to proliferation, apoptosis, cell cycle control, metabolism of xenobiotics, and reticulum endoplasmic stress. Results: PLN reduced the cell proliferation by the cell cycle arrest in G2/M. Changes in the mRNA expression for CDKN1A (4.9x) and CCNA2 (0.5x) of cell cycle control genes were observed. Cell death occurred due to apoptosis, which may have been induced by increased expression of proapoptotic mRNAs (BAK1, 3.1x; BBC3, 2.4x), and by an increase in 9 and 3/7 active caspases. PLN induced cellular injury by ROS generation and DNA damage. DNA damage induced MDM2 signaling (3.0x) associated with the appearance of the monastral spindle in mitosis. Genes associated with ROS degradation also showed increased mRNA expression (GSR, 2.0x; SOD1, 2.1x). PLN induce endoplasmic reticulum stress with the increase in the mRNA expression of ERN1 (4.5x) and HSPA14 (2.2x). The xenobiotic metabolism showed increased mRNA expression for CYP1A2 (2.2x) and CYP3A4 (3.4x). In addition to 2D culture, PLN treatment also inhibited the growth of 3D culture (spheroids). Conclusion: Thus, the findings of our study show that several gene expression biomarkers (mRNAs) and monastral spindle formation indicated the many pathways of damage induced by PLN treatment that contributes to its antiproliferative effects

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.

Up and down-regulation of mRNA in the cytotoxicity and genotoxicity of Plumbagin in HepG2/C3A.

Studies that evaluated the mechanisms of action of Plumbagin (PLB) and its toxicity may contribute to future therapeutic applications of this compound. We investigate biomarker important in the mechanisms of action correlate the expression of mRNA with the cytotoxic and genotoxic effects of PLB on HepG2/C3A. In the analysis of cytotoxicity, PLB decreased cell viability and membrane integrity at concentrations ≥ 15µM. Xenobiotic-metabolizing system showed strong mRNA induction of CYP1A1, CYP1A2, and CYP3A4, suggesting extensive metabolization. PLB induced apoptosis and an increase in the mRNA expression of genes BBC3, CASP3, and CASP8. At a concentration of 15µM, there was a reduction in the expression of PARP1 mRNA and an increase in the expression of BECN1 mRNA, suggesting that PLB may also induce cell death by autophagy. PLB induced an arrest at the G2/M phase due to DNA damage, as observed in the comet assay. This damage is associated with the increased mRNA expression of genes p21, GADD45A, and H2AFX and with changes in the expression of proteins H2AX, p21, p53, Chk1, and Chk2. These results allow a better understanding of the cellular action of PLB and of its toxicity, thereby contributing to the development of PLB-based drugs, with markers of mRNA expression possibly playing a role as indicators for monitoring toxicity in human cells.

Molecular pathways related to the control of proliferation and cell death in 786-O cells treated with plumbagin.

Plumbagin (PLB) is a phytochemical being used for centuries in traditional medicines. Recently, its capacity to inhibit the development of human tumors has been observed, through the induction of apoptosis, cell cycle arrest, and inhibition of angiogenesis and metastasis. Here we evaluated the mechanism of action of PLB in the kidney adenocarcinoma 786-O cell line, which are metabolizing cells important for toxicology studies. After the treatment with PLB, we observed increased apoptosis and cell cycle arrest in S and G2/M phases, starting at 5 µM. In addition, PLB was cytotoxic, genotoxic and induced loss of cell membrane integrity. Regarding gene expression, treatment with 7.5 µM PLB reduced the amount of MTOR, BCL2 and ATM transcripts, and increased CDKN1A (p21) transcripts. Phosphorylation levels of yH2AX was increased and MDM2 protein level was reduced following the treatment with PLB, demonstrating its genotoxic effect. Our results suggest that PLB acts in molecular pathways related to the control of proliferation and cell death in 786-O cells.

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.

Cis-Nerolidol Induces Endoplasmic Reticulum Stress and Cell Death in Human Hepatocellular Carcinoma Cells through Extensive CYP2C19 and CYP1A2 Oxidation.

Of late, many studies are attempting to find new molecules with anticancer properties, especially those with the capability to inhibit cell growth. The aim of this work was to evaluate nerolidol, a plant-based compound, as its cytotoxicity, genotoxicity, antiproliferative and apoptotic induction, cell cycle, mitochondrial membrane potential and RT-qPCR of transcripts related to those pathways in the human hepatocellular carcinoma cell line (HepG2/C3A). Only cis-nerolidol (C-NER) demonstrated cytotoxicity (100-250 µM) activity and was selected to conduct the following experiments. C-NER did not show genotoxic activity, but altered the mitochondrial membrane potential, reduced cell proliferation by arresting cell cycle in G1 phase and induced cell death. RT-qPCR showed that C-NER down-regulated genes related to apoptosis (BAK1, BAX, CAPN1, CASP8, CASP9, PARP1 and TP53), cell cycle (CCND1, CCNE1, CDK1 and CDK2), xenobiotic metabolism (CYP2D6 and CYP3A4) and paraptosis (IGF1R receptor). Up-regulation was seen in case of genes related to cell survival (BBC3 and MYC) and reticulum stress protein response (EIF2AK3 and ERN1) and xenobiotic metabolism (CYP1A2 and CYP2C19). We deduced that the antiproliferative activity of C-NER is attributable to its modulation of the cyclins and cyclin-dependent kinases as these proteins are necessary for G1/S phase transition. EIF2AK3, ERN1, CYP2C19 and CYP1A2 up-regulation suggests that endoplasmic reticulum stress was induced owing to the increased activity of cytochrome P450 enzymes. Caspase-independent cell death was also observed, indicating that another type of cell death, paraptosis, was triggered. Our results indicate that C-NER has considerable potential in anticancer therapy because it modulates important molecular targets of cell survival and proliferation.

Risk Assessment via Metabolism and Cell Growth Inhibition in a HepG2/C3A Cell Line Upon Treatment with Arpadol and its Active Component Harpagoside.

Harpagophytum procumbens (Hp) has been used as antiinflammatory and analgesic agent for the treatment of rheumatic diseases. The principal active component of Hp is harpagoside (HA). We tested the toxicity of this new therapeutic agent in a hepatic cell line (HepG2/C3A). Hp was found to be cytotoxic, and HA was found to decrease the number of cells in S phase, increase the number of cells in G2/M phase and induce apoptosis. Neither Hp nor HA was genotoxic. The expression of CDK6 and CTP3A4 was reduced by Hp, and both HA and Hp caused a significant reduction of CYP1A2 and CYP3A4 expression. It is possible that the cytotoxicity caused by HA and Hp does not involve transcriptional regulation of the cyclins and CDKs tested but is instead related to the inhibition of metabolism. This is evidenced by the results of an MTT assay and changes in the expression of genes related to drug metabolism, leading to cell death. Indeed, the cells exhibited decreased proliferation upon exposure to Hp and HA. The data show that treatment with either Hp or HA can be cytotoxic, and this should be taken into consideration when balancing the risks and benefits of treatments for rheumatic diseases. Copyright © 2016 John Wiley & Sons, Ltd.