Investigation of effect of tectorigenin (O-methylated isoflavone) on Ca2+ signal transduction and cytotoxic responses in canine renal tubular cells.

Tectorigenin, a traditional Chinese medicine, is isolated from the flower of plants such as Pueraria thomsonii Benth. It is an O-methylated isoflavone, a type of flavonoid. Previous studies have shown that tectorigenin evoked various physiological responses in different models, but the effect of tectorigenin on cytosolic-free Ca2+ levels ([Ca2+]i) and cytotoxicity in renal tubular cells is unknown. Our research explored if tectorigenin changed Ca2+ signal transduction and viability in Madin-Darby Canine Kidney (MDCK) renal tubular cells. [Ca2+]iin suspended cells were measured by applying the fluorescent Ca2+-sensitive probe fura-2. Viability was explored by using water-soluble tetrazolium-1 as a fluorescent dye. Tectorigenin at concentrations of 5-50 µM induced [Ca2+]irises. Ca2+ removal reduced the signal by approximately 20%. Tectorigenin (50 µM) induced Mn2+ influx suggesting of Ca2+ entry. Tectorigenin-induced Ca2+ entry was inhibited by 10% by three inhibitors of store-operated Ca2+ channels, namely, nifedipine, econazole, and SKF96365. In Ca2+-free medium, treatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin inhibited 83% of tectorigenin-evoked [Ca2+]irises. Conversely, treatment with tectorigenin abolished thapsigargin-evoked [Ca2+]irises. Inhibition of phospholipase C with U73122 inhibited 50% of tectorigenin-induced [Ca2+]irises. Tectorigenin at concentrations between 10 and 60 µM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca2+ with 1,2-bis (2-aminophenoxy)ethane-N, N, N', N'-tetraacetic acid/acetoxy methyl did not reverse tectorigenin's cytotoxicity. Our data suggest that, in MDCK cells, tectorigenin evoked [Ca2+]irises and induced cell death that was not associated with [Ca2+]irises. Therefore, tectorigenin may be a Ca2+-independent cytotoxic agent for kidney cells.

Cytotoxic effects of gastrodin extracted from the rhizome of Gastrodia elata Blume in glioblastoma cells, but not in normal astrocytes, via the induction of oxidative stress-associated apoptosis that involved cell cycle arrest and p53 activation.

Researches have been conducted to explore the biological effect of gastrodin, a natural compound extracted from the rhizome of Gastrodia elata Blume, in different models. However, the effects of gastrodin on cytotoxicity, cell cycle distribution and oxidative stress in glia cells have not been explored. The aim of this study was to investigate the cytotoxic effect of gastrodin and its mechanisms in DBTRG-05MG human glioblastoma cells and CTX TNA2 rat astrocytes. In DBTRG-05MG cells but not in CTX TNA2 cells, gastrodin (20-30 µM) induced cytotoxicity, G2/M phase cell cycle arrest and apoptosis. Regarding oxidative stress, gastrodin (20-30 µM) elevated intracellular ROS levels but reduced GSH levels. Treatment with the antioxidant NAC (10 µM) partially reversed gastrodin-altered antioxidant enzymes levels. Furthermore, gastrodin induced mitochondria-associated apoptosis. The apoptotic effects evoked by gastrodin were partially inhibited by the antioxidant NAC and the pancaspase inhibitor Z-VAD-FMK. Together, in DBTRG-05MG cells, but not in CTX TNA2 cells, gastrodin activated ROS-associated mitochondrial apoptotic pathways that involved cell cycle arrest. These data provide insight into the molecular mechanisms governing the ability of gastrodin to induce cytotoxicity in human glioblastoma cells and further suggest that gastrodin is a new potential agent for the treatment of human gliblasoma.

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.

Esculetin (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 Ca(2+) 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 Ca(2+) signaling and cytotoxicity in human breast cancer cells. The results showed that esculetin induced concentration-dependent rises in the intracellular Ca(2+) concentration ([Ca(2+)]i) in ZR-75-1 (but not in MCF-7 and MDA-MB-231) human breast cancer cells. In ZR-75-1 cells, this Ca(2+) signal response was reduced by removing extracellular Ca(2+) and was inhibited by the store-operated Ca(2+) channel blocker 2-aminoethoxydiphenyl borate (2-APB). In Ca(2+)-free medium, pre-treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished esculetin-induced [Ca(2+)]i rises. Conversely, incubation with esculetin abolished TG-induced [Ca(2+)]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 Ca(2+) 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 [Ca(2+)]i rises by releasing Ca(2+) from the ER and causing Ca(2+) influx through 2-APB-sensitive store-operated Ca(2+) entry. Furthermore, esculetin activated Ca(2+)-associated mitochondrial apoptotic pathways that involved G2/M cell cycle arrest. Graphical abstract The summary of esculetin-evoked [Ca(2+)]i rises and -activated Ca(2+)-associated mitochondrial apoptotic pathways that involved cell cycle arrest. The natural coumarin derivative esculetin caused Ca(2+) influx via 2-APB-sensitive store-operated Ca(2+) entry and induced Ca(2+) release from the endoplasmic reticulum. Moreover, esculetin activated the mitochondrial pathway of apoptosis in a Ca(2+)-associated manner that involved G2/M arrest.

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

BACKGROUND AND OBJECTIVES: 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. MATERIALS AND METHODS: LPLI at 810 nm with an energy density from 10 to 60 J/cm(2) was used to irradiate human oral cancer OC2 cells and normal HGF cells. RESULTS: We found that LPLI significantly diminished cell viability of human oral cancer OC2 cells due to cell cycle arrest at the G1 phase and the induction of cell death but that it had no or little effects on cell cycle progression and death in normal HGF cells. Moreover, the production of reactive oxygen species (ROS) and the loss of mitochondrial membrane potential (MMP) were elevated in human oral cancer OC2 cells compared with the un-irradiated cells. In contrast, these effects remained unchanged in normal HGF cells after exposure to LPLI. LPLI also induced apoptosis in caspase-3 dependent manner in human oral cancer OC2 cells, a mode of action that could be mediated by ROS and mitochondrial damage. CONCLUSION: Our findings imply LPLI might be a potential therapy for oral cancers.

Effect of NPC-14686 (Fmoc-l-Homophenylalanine) on Ca²âº Homeostasis and Viability in OC2 Human Oral Cancer Cells.

The effect of the anti-inflammatory compound NPC-14686 on intracellular Ca²âº concentration ([Ca²âº](i)) and viability in OC2 human oral cancer cells was investigated. The Ca²âº-sensitive fluorescent probe fura-2 was used to examine [Ca²âº](i). NPC-14686 induced [Ca²âº](i) rises in a concentration-dependent fashion. The effect was reduced approximately by 10% by removing extracellular Ca²âº. NPC-14686- elicited Ca²âº signal was decreased by nifedipine, econazole, SKF96365, and GF109203X. In Ca²âº-free medium, incubation with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished NPC-14686-induced [Ca²âº](i) rises. Conversely, pretreatment with NPC-14686 abolished thapsigargin or BHQ-induced [Ca²âº](i) rises. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²âº](i) rises. At 20-100 µM, NPC-14686 inhibited cell viability, which was not reversed by chelating cytosolic Ca²âº with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-acetoxymethyl ester (BAPTA/AM). NPC-14686 between 20 µM and 40 µM also induced apoptosis. Collectively, in OC2 cells, NPC-14686 induced [Ca²âº](i) rises by evoking phospholipase C-dependent Ca²âº release from the endoplasmic reticulum and Ca²âº entry via protein kinase C-regulated store-operated Ca²âº channels. NPC-14686 also caused Ca²âº-independent apoptosis.

The mechanism of honokiol-induced intracellular Ca(2+) rises and apoptosis in human glioblastoma cells.

Honokiol, an active constituent of oriental medicinal herb Magnolia officinalis, caused Ca(2+) mobilization and apoptosis in different cancer cells. In vivo, honokiol crossed the blood-brain or -cerebrospinal fluid barrier, suggesting that it may be an effective drug for the treatment of brain tumors, including glioblastoma. This study examined the effect of honokiol on intracellular Ca(2+) concentration ([Ca(2+)]i) and apoptosis in DBTRG-05MG human glioblastoma cells. Honokiol concentration-dependently induced a [Ca(2+)]i rise. The signal was decreased partially by removal of extracellular Ca(2+). Honokiol-triggered [Ca(2+)]i rise was not suppressed by store-operated Ca(2+) channel blockers (nifedipine, econazole, SK&F96365) and the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA), but was inhibited by the PKC inhibitor GF109203X. GF109203X-induced inhibition was not altered by removal of extracellular Ca(2+). In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) abolished honokiol-induced [Ca(2+)]i rise. Conversely, incubation with honokiol abolished TG or BHQ-induced [Ca(2+)]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished honokiol-induced [Ca(2+)]i rise. Honokiol (20-80µM) reduced the cell viability, which was not reversed by prechelating cytosolic Ca(2+) with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Honokiol (20-60µM) enhanced reactive oxygen species (ROS) production, decreased mitochondrial membrane potential, released cytochrome c, and activated caspase-9/caspase-3. Together, honokiol induced a [Ca(2+)]i rise by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-dependent, non store-operated Ca(2+) channels. Moreover, honokiol activated the mitochondrial pathway of apoptosis in DBTRG-05MG human glioblastoma cells.

Effect of allyl sulfides from garlic essential oil on intracellular ca2+ levels in renal tubular cells.

Diallyl sulfide (1), diallyl disulfide (2), and diallyl trisulfide (3), which are major organosulfur compounds of garlic (Allium sativum), are recognized as a group of potential chemopreventive compounds. In this study, the early signaling effects of 3 were examined on Madin-Darby canine kidney (MDCK) cells loaded with the Ca(2+)-sensitive dye fura-2. It was found that 3 caused an immediate and sustained increase of [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 40 µM). Compound 3 also induced a [Ca(2+)](i) elevation when extracellular Ca(2+) was removed, but the magnitude was reduced by 45%. In Ca(2+)-free medium, the 3-induced [Ca(2+)](i) level was abolished by depleting stored Ca(2+) with 1 µM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor). Elevation of [Ca(2+)](i) caused by 3 in the Ca(2+)-containing medium was not affected by modulation of protein kinase C activity. The 3-induced Ca(2+) influx was inhibited by nifedipine and nicardipine (1 µM). U73122, an inhibitor of phospholipase C, abolished ATP (but not the 3-induced [Ca(2+)](i) level). These findings suggest that 3 induced a significant [Ca(2+)](i) elevation in MDCK renal tubular cells by stimulating both extracellular Ca(2+) influx and thapsigargin-sensitive intracellular Ca(2+) release via as yet unidentified mechanisms. Furthermore, the order of the allyl sulfide-induced [Ca(2+)](i) elevation and cell viability was 1 < 2 < 3. The differential effect of allyl sulfides on Ca(2+) signaling and cell death appears to correlate with the number of sulfur atoms in the structure of these allyl sulfides.

Effects of Antrodia camphorata extracts on the viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation of OC2 human oral cancer cells.

The effect of Antrodia camphorata (AC) on human oral cancer cells has not been explored. This study examined the effect of AC on the viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ regulation of OC2 human oral cancer cells. AC at a concentration of 25 microM induced an increase in cell viability, but AC at concentrations > or = 50 microg/ml decreased viability in a concentration-dependent manner. AC at concentrations of 100-200 microg/ml induced apoptosis in a concentration-dependent manner as demonstrated by propidium iodide staining. AC (25 microg/ml) did not alter basal [Ca2+]i, but decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin, and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment failed to alter ATP-induced increase in viability, potentiated bradykinin-induced increase in viability, decreased histamine-induced increase in viability and reversed thapsigargin-induced decrease in viability. Immunoblotting suggested that AC induced phosphorylation of ERK and JNK MAPKs, but not p38 MAPK. Collectively, for OC2 cells, AC exerted multiple effects on their viability and [Ca2+]i, induced their ERK and JNK MAPK phosphorylation, and probably evoked their apoptosis.

Effects of antrodia camphorata on viability, apoptosis, and [Ca2+]i in PC3 human prostate cancer cells.

Antrodia camphorata (AC) has been used as a health supplement in Asia to control different cancers; however, the cellular mechanisms of its effects are unclear. The effect of AC on cultured human prostate cancer cells (PC3) has not been explored. This study examined the effect of AC on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ handling in PC3 cells. AC at concentrations of 5-50 microg/ml did not affect cell viability, but at 100-200 microg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25-200 microg/ml did not alter basal [Ca2+]i, but at a concentration of 25 microg/ml decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment inhibited ATP-, bradykinin-, and histamine-induced enhancement on viability, but reversed thapsigargin-induced cytotoxicity. Immunoblotting showed that AC (200 microg/ml) did not induce the phosphorylation of ERK, JNK, and p38 MAPKs. Collectively, in PC3 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis via pathways unrelated to [Ca2+]i signal and phosphorylation of ERK, JNK and p38 MAPKs.

Novel action of lignans isolated from Hernandia nymphaeifolia on Ca(2+) signaling in renal tubular cells.

The effect of five lignans, epi-aschantin, epi-magnolin, epi-yangambin, deoxypodophyllotoxin and yatein, isolated from Hernandia nymphaeifolia on Ca(2+) signaling in Madin-Darby canine kidney cells was examined using fura-2 as a Ca(2+) indicator. These lignans at concentrations between 10 and 100 microM increased [Ca(2+)](i) in a concentration-dependent manner. Removal of extracellular Ca(2+) abolished the Ca(2+) signals evoked by 50 microM of the lignans. La(3+)(50 microM) abolished the Ca(2+) signals induced by 100 microM of epi-aschantin, epi-magnolin and epi-yangambin, and 20 microM deoxypodophyllotoxin, but inhibited by 60% 50 microM yatein-induced responses. All five lignans (50-100 microM) inhibited by 42-65% thapsigargin-induced capacitative Ca(2+) entry, and inhibited by 23-61% thapsigargin-induced intracellular Ca(2+) release. Epi-yangambin (100 microM), epi-magnolin (100 microM), and epi-aschantin (100 microM) inhibited by 8-38% 10 microM ATP-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with deoxypodophyllotoxin or yatein (but not the other lignans) decreased cell viability in a concentration-dependent manner. Together, the results suggest that, in renal tubular cells, these lignans exert multiple actions on Ca(2+) signaling. They caused Ca(2+) influx but reduced thapsigargin-induced capacitative Ca(2+) entry and also thapsigargin- and ATP-induced Ca(2+) release. Additionally, deoxypodophyllotoxin and yatein may be cytotoxic.

Effect of lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils.

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17beta-estradiol, tamoxifen and clomiphene)-induced Ca(2+) mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca(2+)-containing medium, the lignans (50-100 microM) inhibited 10 microM 17beta-estradiol- and 5 microM tamoxifen-induced increases in intracellular free Ca(2+) levels ([Ca(2+)](i)) without changing 25 microM clomiphene-induced [Ca(2+)](i) increase. 17beta-estradiol and tamoxifen increased [Ca(2+)](i) by causing Ca(2+) influx and Ca(2+) release because their responses were partly reduced by removing extracellular Ca(2+). In contrast, clomiphene solely induced Ca(2+) release. The effect of the lignans on these two Ca(2+) movement pathways underlying 17beta-estradiol- and tamoxifen-induced [Ca(2+)](i) increases was explored. All the lignans (50-100 microM) inhibited 10 microM 17beta-estradiol-and 5 microM tamoxifen-induced Ca(2+) release, and 17beta-estradiol-induced Ca(2+) influx. However, only 100 microM epi-aschantin was able to reduce tamoxifen-induced Ca(2+) influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca(2+) signaling in human neutrophils in a multiple manner.

Novel action of lignans isolated from Hernandia nymphaeifolia on Ca2+ signaling in human neutrophils.

The effects of five lignans (epi-aschantin, epi-magnolin, epi-yangambin, deoxypodophyllotoxin, yatein) isolated from Hernandia nymphaeifolia (Presl.) Kubitzki (Hernandiaceae) on intracellular Ca2+ levels ([Ca2+]i) in human neutrophils were investigated by using fura-2 as a fluorescent probe. In both Ca2+-containing and Ca2+-free media, the lignans (50-100 microM) did not alter basal [Ca2+]i but inhibited the [Ca2+]i increase induced by platelet activating factor (PAF, 10 microM), leukotriene B4 (LTB4, 0.2 microM), and thapsigargin (1 microM) to different extents. In Ca2+-free medium, after depleting stores of Ca2+ with PAF, LTB4 or thapsigargin, addition of 3 mM Ca2+ induced Ca2+ influx. Each of the lignans (50-100 microM) caused 39-89% inhibition of PAF-induced Ca2+ influx; whereas only epi-aschantin was able to inhibit LTB4- and thapsigargin-induced Ca2+ influx by 54-79%. Together, the results suggest that in human neutrophils, these lignans did not alter basal [Ca2+]i but inhibited Ca2+ movement induced by Ca2+ mobilizing agents.