BACKGROUND AND AIM: Osmotic changes represent a burden for the body and their limitation would be beneficial. We hypothesized that ubiquitous natural compounds could guard against cytotoxic effects of osmotic stress. We evaluated the anti-hypertonic mechanism of quercetin and 2,3-dehydrosilybin in H9c2 cells in vitro. EXPERIMENTAL PROCEDURE: Protective effect of both compounds was determined by neutral red assay, cell apoptosis was estimated by measuring caspase-3 activity and verified by western blot and annexin V assay. Phosphorylation level of selected proteins was also detected. Mitochondrial membrane potential was evaluated using dye JC-1. Ca2+ signals were evaluated using genetically encoded fluorescent Ca2+ biosensor GCaMP7f. Formation of reactive oxygen species was measured using an oxidant-sensing probe dihydrofluorescein diacetate. KEY RESULTS: Quercetin protected H9c2 cells against hypertonic stress-induced cell death. We observed a significant increase in intracellular Ca2+ levels ([Ca2+]cyto) when cells originally placed in a hypertonic solution were returned to a normotonic environment. Quercetin was found to prevent this increase in [Ca2+]cyto and also the depolarization of mitochondrial membrane potential. CONCLUSIONS AND IMPLICATIONS: Quercetin, but not 2,3-dehydrosilybin, reduced adverse effects of osmotic stress mainly by dampening the elevation of [Ca2+]cyto and mitochondrial Ca2+ overload. This may consequently prevent MPTP pore opening and activation of apoptosis.
Etoposide is a well-known and widely used anticancer drug that displays several side effects. In addition, tumors often acquire resistance to this drug. Our aim is to develop a combination therapy that would augment toxicity of etoposide in malignant cells. Based on literature and our experiments, we selected mifepristone (RU486) as a potential supporting molecule that is able to enhance etoposide toxicity against cancer cells. All experiments were performed with Hep G2 cells, a well-known and described human hepatocellular carcinoma cell line. By using xCELLigence system, we demonstrated that mifepristone enhances toxicity of etoposide in a dose dependent manner with concomitant caspase-3 activity. We evaluated upregulation of Bax because mifepristone was demonstrated to modulate proapoptotic Bax protein expression. Our data show only weak and not statistically significant increase of Bax expression. On the other hand, we show that mifepristone increases etoposide toxicity via inhibition of ABC transporters, coupled with significant increase of intracellular etoposide concentration. In conclusion, we demonstrate that mifepristone has a synergistic effect with etoposide treatment in the Hep G2 cells and that the effect is related to ABC transporters inhibition.
- MeSH
- ATP Binding Cassette Transporter, Subfamily G, Member 2 antagonists & inhibitors MeSH
- Biological Transport drug effects MeSH
- Hep G2 Cells MeSH
- K562 Cells MeSH
- Etoposide pharmacology MeSH
- Antineoplastic Agents, Phytogenic pharmacology MeSH
- Caspase 3 metabolism MeSH
- Humans MeSH
- Mifepristone pharmacology MeSH
- Neoplasm Proteins antagonists & inhibitors MeSH
- ATP Binding Cassette Transporter, Subfamily B antagonists & inhibitors MeSH
- bcl-2-Associated X Protein metabolism MeSH
- Drug Synergism MeSH
- Cell Survival drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Etoposide is commonly used as a monotherapy or in combination with other drugs for cancer treatments. In order to increase the drug efficacy, ceaseless search for novel combinations of drugs and supporting molecules is under way. MiRNAs are natural candidates for facilitating drug effect in various cell types. We used several systems to evaluate the effect of miR-29 family on etoposide toxicity in HeLa cells. We show that miR-29b significantly increases etoposide toxicity in HeLa cells. Because Mcl-1 protein has been recognized as a miR-29 family target, we evaluated downregulation of Mcl-1 protein splicing variant expression induced by miR-29 precursors and confirmed a key role of Mcl-1 protein in enhancing etoposide toxicity. Despite downregulation of Mcl-1 by all three miR-29 family members, only miR-29b significantly enhanced etoposide toxicity. We hypothesized that this difference may be linked to the change in Mcl-1L/Mcl-1S ratio induced by miR-29b. We hypothesized that the change could be due to miR-29b nuclear shuttling. Using specifically modified miR-29b sequences with enhanced cytosolic and nuclear localization we show that there is a difference, albeit statistically non-significant. In conclusion, we show that miR-29b has the synergistic effect with etoposide treatment in the HeLa cells and that this effect is linked to Mcl-1 protein expression and nuclear shuttling of miR-29b.
- MeSH
- Cell Cycle drug effects MeSH
- Down-Regulation MeSH
- Etoposide toxicity MeSH
- Antineoplastic Agents, Phytogenic toxicity MeSH
- HeLa Cells MeSH
- Humans MeSH
- MicroRNAs metabolism MeSH
- Myeloid Cell Leukemia Sequence 1 Protein genetics metabolism MeSH
- Cell Survival drug effects MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
A recent study showed that silymarin, a standardized extract of S. marianum might be used in the prevention of equine laminitis. We investigated the effects of quercetin and some compounds found in silymarin (silybin, taxifolin and dehydrosilybin) on reactive oxygen species (ROS) production and myeloperoxidase (MPO) release by stimulated equine neutrophils (PMNs) and on MPO activity. All compounds (tested between 100 nm and 100 μm) inhibited superoxide anion production by stimulated PMNs in a dose-dependent manner. Dehydrosilybin and quercetin inhibited superoxide production and MPO release from 10 μm. Classical MPO assay showed quercetin as the most potent inhibitor, followed by taxifolin, dehydrosilybin and silybin. SIEFED MPO assay highlighting the binding of tested compounds to MPO showed that only quercetin and taxifolin maintained an efficient inhibition above 90% at 10 μm. Altogether, our results showed a strong inhibition of PMN activation by planar compounds such as quercetin and dehydrosilybin and a strong inhibition of MPO activity by the smallest molecules, quercetin and taxifolin. In conclusion, the compounds from silymarin may be useful for modulating the oxidative response of PMNs, involved in the pathogenesis of laminitis, but further in vivo studies are needed.
- MeSH
- Antioxidants chemistry pharmacology MeSH
- Horses * MeSH
- Cells, Cultured MeSH
- Molecular Structure MeSH
- Neutrophils drug effects MeSH
- Silybum marianum chemistry MeSH
- Oxidative Stress MeSH
- Peroxidase antagonists & inhibitors MeSH
- Polyphenols chemistry pharmacology MeSH
- Reactive Oxygen Species metabolism MeSH
- Dose-Response Relationship, Drug MeSH
- Animals MeSH
- Check Tag
- Animals MeSH
- Publication type
- Journal Article MeSH
Silymarin, a standardised extract of Silybum marianum (milk thistle), comprises mainly of silybin, with dehydrosilybin (DHSB), quercetin, taxifolin, silychristin and a number of other compounds which are known to possess a range of salutary effects. Indeed, there is evidence for their role in reducing tumour growth, preventing liver toxicity, and protecting a number of organs against ischemic damage. The hepatoprotective effects of silymarin, especially in preventing Amanita and alcohol intoxication induced damage to the liver, are a well established fact. Likewise, there is weighty evidence that silymarin possesses antimicrobial and anticancer activities. Additionally, it has emerged that in animal models, silymarin can protect the heart, brain, liver and kidneys against ischemia reperfusion injury, probably by preconditioning. The mechanisms of preconditioning are, in general, well studied, especially in the heart. On the other hand, the mechanism by which silymarin protects the heart from ischemia remains largely unexplored. This review, therefore, focuses on evaluating existing studies on silymarin induced cardioprotection in the context of the established mechanisms of preconditioning.
