Wei-Zhe Liang

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract Resveratrol is a natural compound that affects cellular Ca2+ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells. The Ca2+-sensitive fluorescent dye fura-2 was used to measure [Ca2+]i and WST-1 was used to measure viability. Resveratrol-evoked [Ca2+]i rises concentration-dependently. The response was reduced by removing extracellular Ca2+. Resveratrol-evoked Ca2+ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca2+]i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca2+]i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca2+]i. Previous studies showed that resveratrol between 10 and 100 µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10 μM) increased cell viability, which was abolished by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10 µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca2+]i by evoking phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10 µM also caused Ca2+-dependent cell proliferation.

Effect of the pesticide, deltamethrin, on Ca2+ signaling and apoptosis in OC2 human oral cancer cells

Abstract Deltamethrin is a synthetic pyrethroid insecticide used extensively in pest control. Although deltamethrin has been shown to induce cytosolic free Ca2+ concentration ([Ca2+]i) rises and apoptosis in different cancer cells, there is no information concerning the effects of deltamethrin on oral cancer. This study explored the effects of deltamethrin on [Ca2+]i and viability in OC2 human oral cancer cells. Deltamethrin, at concentrations of 5–10 μM, increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Deltamethrin-induced [Ca2+]i rise was not inhibited by econazole, SK&F96365, phorbol 12-myristate 13 acetate (PMA) or GF109203X, but was inhibited by nifedipine. In Ca2+-free medium, 10-μM deltamethrin pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitor, 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with BHQ inhibited deltamethrin-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with phospholipase C (PLC) inhibitor U73122 did not suppress deltamethrin-induced Ca2+ release. At concentrations between 20 and 100 μM, deltamethrin killed cells in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid/acetoxymethyl. Deltamethrin-induced cell death was not caused by a preceding [Ca2+]i rise. Annexin V/propidium iodide staining data suggest that deltamethrin (40–60 μM) induced apoptosis in a concentration-dependent manner. To conclude, in OC2 cells, deltamethrin evoked a [Ca2+]i rise by inducing PLC-independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry by nifedipine-sensitive Ca2+ channels. Further, deltamethrin induced Ca2+-independent cell death might involve apoptosis.

Esculetin, a natural coumarin compound, evokes Ca(2+) movement and activation of Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest in ZR-75-1 human breast cancer cells.

AbstractEsculetin (6,7-dihydroxycoumarin), a derivative of coumarin compound, is found in traditional medicinal herbs. It has been shown that esculetin triggers diverse cellular signal transduction pathways leading to regulation of physiology in different models. However, whether esculetin affects Ca2+ homeostasis in breast cancer cells has not been explored. This study examined the underlying mechanism of cytotoxicity induced by esculetin and established the relationship between Ca2+ signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca2+ concentration ([Ca2+]i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca2+ signal response was reduced by removing extracellular Ca2+ and was inhibited by the store-operated Ca2+ channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca2+-free medium, pre-treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca2+]i rises. Conversely, incubation with esculetin abolished TG-induced [Ca2+]i rises. Esculetin induced cytotoxicity that involved apoptosis, as supported by the reduction of mitochondrial membrane potential and the release of cytochrome c and the proteolytic activation of caspase-9/caspase-3, which were partially reversed by pre-chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester (BAPTA-AM). Moreover, esculetin increased the percentage of cells in G2/M phase and regulated the expressions of p53, p21, CDK1, and cyclin B1. Together, in ZR-75-1 cells, esculetin induced [Ca2+]i rises by releasing Ca2+ from the ER and causing Ca2+ influx through 2-APB-sensitive store-operated Ca2+ entry. Furthermore, esculetin activated Ca2+-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest. Graphical abstractThe summary of esculetin-evoked [Ca2+]i rises and -activated Ca2+-associated mitochondrial apoptotic pathways that involved cell cycle arrest. The natural coumarin derivative esculetin caused Ca2+ influx via 2-APB-sensitive store-operated Ca2+ entry and induced Ca2+ release from the endoplasmic reticulum. Moreover, esculetin activated the mitochondrial pathway of apoptosis in a Ca2+-associated manner that involved G2/M arrest.

Effect of Methoxychlor on Ca2+ Handling and Viability in OC2 Human Oral Cancer Cells

Abstract:  The effect of the insecticide methoxychlor on the physiology of oral cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca2+ concentrations ([Ca2+]i) in human oral cancer cells (OC2) by using the Ca2+‐sensitive fluorescent dye fura‐2. Methoxychlor at 5–20 μM increased [Ca2+]i in a concentration‐dependent manner. The signal was reduced by 70% by removing extracellular Ca2+. Methoxychlor‐induced Ca2+ entry was not affected by nifedipine, econazole, SK&F96365 and protein kinase C modulators but was inhibited by the phospholipase A2 inhibitor aristolochic acid. In Ca2+‐free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited or abolished methoxychlor‐induced [Ca2+]i rise. Incubation with methoxychlor also inhibited thapsigargin‐ or BHQ‐induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter methoxychlor‐induced [Ca2+]i rise. At 5–20 μM, methoxychlor killed cells in a concentration‐dependent manner. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca2+ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N’,N’‐tetraacetic acid/AM (BAPTA/AM). Annexin V‐FITC data suggest that methoxychlor (10 and 20 μM) evoked apoptosis in a concentration‐dependent manner. Together, in human OC2, methoxychlor induced a [Ca2+]i rise probably by inducing phospholipase C‐independent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via phospholipase A2‐sensitive channels. Methoxychlor induced cell death that may involve apoptosis.

Effect of clotrimazole on cytosolic Ca2+ rise and viability in HA59T human hepatoma cells

Abstract Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca2+ homeostasis. This study examined the effect of clotrimazole on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in HA59T human hepatoma cells. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Clotrimazole induced [Ca2+]i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca2+. Clotrimazole-evoked Ca2+ entry was suppressed by store-operated channel inhibitors (nifedipine, econazole and SK&F96365) and protein kinase C modulators (GF109203X and phorbol, 12-myristate, 13-acetate). In Ca2+-free medium, incubation with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-tert-butylhydroquinone abolished clotrimazole-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished clotrimazole-induced [Ca2+]i rise. At 10–40 µM, clotrimazole inhibited cell viability, which was not reversed by chelating cytosolic Ca2+. Clotrimazole at 10 and 30 µM also induced apoptosis. Collectively, in HA59T cells, clotrimazole-induced [Ca2+]i rises by evoking phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via store-operated Ca2+ channels. Clotrimazole also caused apoptosis.

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells was explored. The Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Diindolylmethane at concentrations of 20–50 µM induced [Ca2+]i rise in a concentration-dependent manner. The response was reduced partly by removing Ca2+. Diindolylmethane-evoked Ca2+ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca2+, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca2+]i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca2+]i rise. At concentrations of 50–100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca2+]i rise in PC3 cells by evoking phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via phospholipase A2-sensitive store-operated Ca2+ channels. Diindolylmethane caused cell death in which apoptosis may participate.

Mechanisms underlying effect of the mycotoxin cytochalasin B on induction of cytotoxicity, modulation of cell cycle, Ca2+ homeostasis and ROS production in human breast cells

Cytochalasin B, a cell-permeable mycotoxin isolated from the fungus Phoma spp., shows a wide range of biological effects, among which its potent antitumor activity has raised great interests in different models. However, the cytotoxic activity of cytochalasin B and its underlying mechanisms have not been elucidated in breast cells. This study examined the effect of cytochalasin B on MCF 10A human breast epithelial cells and ZR-75-1 human breast cancer cells. Cytochalasin B (10-20μM) concentration-dependently induced cytotoxicity, cell cycle arrest, and [Ca2+]i rises in ZR-75-1 cells but not in MCF 10A cells. In ZR-75-1 cells, cytochalasin B triggered G2/M phase arrest through the modulation of CDK1, cyclin B1, p53, p27 and p21 expressions. The Ca2+ signal response induced by cytochalasin B was reduced by removing extracellular Ca2+ and was inhibited by the store-operated Ca2+ channel blocker 2-APB and SKF96365. In Ca2+-free medium, cytochalasin B induced Ca2+ release through thapsigargin-sensitive endoplasmic reticulum stores. Moreover, cytochalasin B increased H2O2 levels but reduced GSH levels. The apoptotic effects evoked by cytochalasin B were partially inhibited by prechelating cytosolic Ca2+ with BAPTA-AM and the antioxidant NAC. Together, in ZR-75-1 cells but not in MCF 10A cells, cytochalasin B activated Ca2+-associated mitochondrial apoptotic pathways that involved G2/M phase arrest and ROS signaling. Furthermore, cytochalasin B induced [Ca2+]i rises by releasing Ca2+ from the endoplasmic reticulum and causing Ca2+ influx through 2-APB or SKF96365-sensitive store-operated Ca2+ entry. Our findings provide new insights into the possible application of cytochalasin B in human breast cancer therapy.

Selective cytotoxic effects of low-power laser irradiation on human oral cancer cells

Low‐power laser irradiation (LPLI) is known to regulate cell proliferation and migration in clinical use. Recent studies have shown that LPLI induces cell death in some certain types of cancer cell lines. However, the cytotoxic selectivity of LPLI for cancer cells is not fully understood. The aim of this study was to compare the cytotoxic effects of LPLI in both human oral cancer OC2 cells and normal human gingival fibroblast (HGF) cells.

Ca2+ movement and apoptosis induced by deltamethrin in Madin–Darby canine kidney canine renal tubular cells

This study explored the effect of deltamethrin, a pesticide, on free Ca2+ concentration [Ca2+]i, viability, and apoptosis in Madin–Darby canine kidney (MDCK) canine renal tubular cells. Deltamethrin at concentrations between 10μM and 40μM evoked [Ca2+]i rises in a concentration‐dependent manner. The Ca2+ entry was inhibited by nifedipine, econazole, phorbol 12‐myristate 13‐acetate, and SKF96365. Treatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) in a Ca2+‐free medium abolished deltamethrin‐induced [Ca2+]i rise. Treatment with deltamethrin also abolished BHQ‐induced [Ca2+]i rise. Inhibition of phospholipase C (PLC) activity with U73122 abolished deltamethrin‐evoked [Ca2+]i rise. Deltamethrin killed cells at 30–60μM in a concentration‐dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca2+ with the acetoxymethyl ester of 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid. Annexin V/propidium iodide staining data suggest that 30–50μM deltamethrin induced apoptosis. Together, in MDCK renal tubular cells, deltamethrin induced [Ca2+]i rises that involved Ca2+ entry through protein kinase C‐mediated store‐operated Ca2+ channels, and PLC‐dependent Ca2+ release from the endoplasmic reticulum. Deltamethrin also induced Ca2+‐independent cell death that might involve apoptosis.

The effect of oleuropein from olive leaf (Olea europaea) extract on Ca2+ homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in HepG2 human hepatoma cells

Oleuropein, a phenolic compound found in the olive leaf (Olea europaea), has been shown to have biological activities in different models. However, the effects of oleuropein on Ca(2+) homeostasis, cytotoxicity, cell cycle distribution and ROS signaling in liver cells have not been analyzed. Oleuropein induced [Ca(2+)]i rises only in HepG2 cells but not in AML12, HA22T or HA59T cells due to the different status of 3-hydroxy-3-methylglutaryl-CoA reductase expression. In HepG2 cells, this Ca(2+) signaling response was reduced by removing extracellular Ca(2+), and was inhibited by the store-operated Ca(2+) channel blockers 2-APB and SKF96365. In Ca(2+)-free medium, pretreatment with the ER Ca(2+) pump inhibitor thapsigargin abolished oleuropein-induced [Ca(2+)]i rises. Oleuropein induced cell cycle arrest which was associated with the regulation of p53, p21, CDK1 and cyclin B1 levels. Furthermore, oleuropein elevated intracellular ROS levels but reduced GSH levels. Treatment with the intracellular Ca(2+) chelator BAPTA-AM or the antioxidant NAC partially reversed oleuropein-induced cytotoxicity. Together, in HepG2 cells, oleuropein induced [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through store-operated Ca(2+) channels. Moreover, oleuropein induced Ca(2+)-associated cytotoxicity that involved ROS signaling and cell cycle arrest. This compound may offer a potential therapy for treatment of human hepatoma.