Wogonin potentiates the antitumor action of etoposide and ameliorates its adverse effects.

Wogonin, a flavone in the roots of Scutellaria baicalensis, reduced etoposide-induced apoptotic cell death in normal cells, such as bone marrow cells and thymocytes. On the other hand, wogonin potentiated the proapoptotic or cytotoxic action of etoposide in tumor cells, such as Jurkat, HL-60, A549, and NCI-H226. These contradictory actions of wogonin on apoptosis are distinguished by normal or cancer cell types. Wogonin had no effect on apoptosis induced by other anticancer agents in the tumor cells. Thus, the potentiation effect of wogonin was observed only in etoposide-induced apoptosis in tumor cells. In a functional assay for P-glycoprotein (P-gp), wogonin suppressed excretion of calcein, a substrate for P-gp, in these tumor cells. Moreover, wogonin decreased the excretion of radiolabeled etoposide and accordingly increased intracellular content of this agent in the cells. P-gp inhibitors showed a similar potentiation effect on etoposide-induced apoptosis in these tumor cells. Thus, wogonin is likely to potentiate the anticancer action of etoposide due to P-gp inhibition and accumulation of this agent. These findings suggest that wogonin may be a useful chemotherapeutic adjuvant to potentiate the pharmacological action of etoposide and ameliorate its adverse effects.

[ARTCEREB irrigation and perfusion solution for cerebrospinal surgery: pharmacological assessment using human astrocytes exposed to test solutions].

ARTCEREB irrigation and perfusion solution (Artcereb) is a preparation intended for the irrigation and perfusion of the cerebral ventricles, and it is therefore important to evaluate the effects of Artcereb on brain cells. In vitro assessment of the effects of Artcereb in cell cultures of human fetal astrocytes was conducted in comparison with normal saline and lactated Ringer's solution. The effects of exposure to Artcereb were evaluated based on microscopic images of the mitochondria stained with rhodamine 123. The effects of exposure to Artcereb on cell function were also evaluated by quantitative analysis of mitochondrial activity based on rhodamine 123 and (3)H-thymidine incorporation. Morphological changes in nuclear structure were also evaluated. The results of the present study showed that cell function in cell cultures of human astrocytes was relatively unaffected by exposure to Artcereb as compared with normal saline or lactated Ringer's solution, suggesting that Artcereb has less effect on brain cells than normal saline or lactated Ringer's solution when used for the irrigation or perfusion of the cerebral ventricles.

Inhibition of P-glycoprotein by wogonin is involved with the potentiation of etoposide-induced apoptosis in cancer cells.

Etoposide induces apoptotic cell death in normal and cancer cells. This apoptosis plays a role not only in anticancer effects but also in adverse reactions, such as myelosuppression. Because we had previously found that wogonin, a flavone found in a plant, suppresses thymocyte apoptosis induced by etoposide, we examined the effect of this flavone in cancer cells. Wogonin significantly potentiated etoposide-induced apoptosis in HL-60 cells. This flavone impaired the function of P-glycoprotein and then increased cellular content of etoposide in the cells. Thus, this flavone is likely to act as an inhibitor of P-glycoprotein and potentiate the apoptotic action of etoposide. On the other hand, wogonin inhibited etoposide-induced apoptosis in thymocytes, one of the normal cells. The potentiation by wogonin is likely to be a specific action for cancer cells but not normal cells. Therefore, this flavone may be used to reduce the excretion of the anticancer agents via P-glycoprotein and increase the pharmacological action of it in cancer cells. These results suggest that wogonin may play a role in overcoming multidrug resistance.

Wogonin prevents glucocorticoid-induced thymocyte apoptosis without diminishing its anti-inflammatory action.

The effect of wogonin, a flavone highly purified from the roots of Scutellaria baicalensis, on apoptotic cell death was re-evaluated in rat thymocytes. This flavone inhibited glucocorticoid-induced apoptotic changes such as DNA fragmentation, phosphatidylserine translocation, and nuclear condensation in rat thymocytes. Similar inhibition was also observed in apoptosis induced by other inducers such as etoposide. No significant changes of these apoptotic features were observed in rat thymocytes treated with wogonin alone, suggesting that this flavone protects against glucocorticoid-mediated immunosuppression caused by thymocyte apoptosis. Wogonin was reported to possess anti-inflammatory action in some previous studies, but this flavone had no effect on carrageenan-induced paw edema in this study. The simultaneous treatment of wogonin and glucocorticoid neither enhanced nor reduced the anti-inflammatory effect of glucocorticoid. These results indicate that wogonin is likely to prevent the immunosuppression of glucocorticoid without diminishing its drug efficacy as an anti-inflammatory agent.

Cationic surfactants induce apoptosis in normal and cancer cells.

Cationic surfactants, such as benzalkonium chloride and benzethonium chloride, possess quaternary ammonium salt. These surfactants have antimicrobial action and are used as a preservative and an antiseptic. The positively charged polar head of cationic surfactants seems to play a role in the antimicrobial action of these compounds. Recently, benzalkonium chloride in eye drops has been reported to induce apoptosis in conjunctival cells. Here, we examined whether various types of surfactants including anionic and amphoteric surfactants induce apoptosis or not in mammalian cells. Antimicrobial cationic surfactants induced apoptosis at lower concentration than its critical micelle concentration (CMC) in rat thymocytes. Other quaternary ammonium surfactants, such as cetyltrimethylammonium bromide, similarly increased biochemical and morphological features of apoptosis, whereas both anionic and amphoteric surfactants had no significant effect on these apoptotic features. These results suggest that the positive charge of quaternary ammonium surfactants is involved with onset of the apoptotic process. The treatment of benzethonium chloride also led to apoptotic cell death in Jurkat cells. These results indicate that cationic surfactants induce apoptosis in the normal and cancer cells.

Wogonin prevents immunosuppressive action but not anti-inflammatory effect induced by glucocorticoid.

Glucocorticoid, such as dexamethasone, has anti-inflammatory and immunosuppressive action as major pharmacological effects. The latter action caused by lymphocyte apoptosis is not only a therapeutic effect but also an adverse reaction. Wogonin, a plant flavone found in Scutellaria baicalensis Georgi, inhibited dexamethasone-induced apoptotic changes, such as DNA fragmentation, nuclear condensation, phosphatidylserine translocation, and caspase activation in rat thymocytes. Since wogonin inhibited dexamethasone-induced DNA fragmentation in a noncompetitive manner, a target of this flavone is unlikely to be an antagonist of glucocorticoid receptor. Wogonin did not only act as an inhibitor of caspases, but also protected apoptosis induced by other glucocorticoids. Since wogonin reduced one of the major pharmacological effects of dexamethasone, we examined whether this flavone diminishes the anti-inflammatory action, another pharmacological effect. The anti-inflammatory action of dexamethasone was evaluated by carrageenan-induced paw edema model. Although dexamethasone significantly suppressed paw edema induced by carrageenan, wogonin had no effect on the anti-inflammatory action of dexamethasone. These results suggest that wogonin may be a useful compound to reduce the immunosuppressive side effect of glucocorticoid.

Wogonin, a plant flavone, potentiates etoposide-induced apoptosis in cancer cells.

Etoposide, a podophylotoxin anticancer agent, induces apoptotic cell death in normal and cancer cells. Etoposide-induced apoptosis plays a role in not only anticancer effect but also adverse reaction, such as myelosuppression. Since we have found that wogonin, a flavone found in Scutellaria baicalensis Georgi, prevents thymocyte apoptosis induced by various compounds including etoposide, we examined the effect of this flavone on etoposide-induced apoptosis in cancer cells. Although 100 muM wogonin itself significantly increased DNA fragmentation in HL-60 cells, this change was not observed in Jurkat cells. On the other hand, this flavone significantly potentiated etoposide-induced apoptosis in Jurkat and HL-60 cells. Similarly, wogonin accelerated etoposide-induced cell death in lung cancer cells. Since wogonin had no effect on the action of other anticancer agents, such as 5-FU and cisplatin, this flavone seems to accelerate only etoposide-induced apoptotic cell death in cancer cells. These results suggest that the modification of etoposide-induced apoptosis by wogonin may be available to reduce the adverse reaction of this agent.

Effect of selenium-deficient diet on tubular epithelium in normal rats.

Selenium (Se) deficiency reduces glutathione peroxidase (GPx) activity, resulting in increased oxidative stress. We examined how Se deficiency induces renal injury via oxidative stress over time during the Se-deficient period. Seventy-two male Wistar rats were divided into two groups and fed either a control or Se-deficient diet. Rats were sacrificed on weeks 1, 2, 4, 6, 9, and 12. Blood and urine samples were collected, and the kidneys were removed. Urinalysis was performed, and creatinine clearance (Ccr) was calculated. Expressions of cellular GPx (cGPx) and phospholipid hydroperoxidase GPx (PHGPx) mRNA and GPx activity were measured. Histology was evaluated by light microscopy with immunohistochemistry for 4-hydroxy-2-nonenal (HNE) and vimentin. The Se-deficient diet caused significant decreases in GPx activity and cGPx mRNA expression but no change in PHGPx mRNA, together with significant proteinuria and glucosuria and slight decline in Ccr. The Se-deficient diet induced calcification in the kidney and increased the distribution of HNE and vimentin immunostaining in proximal tubuli, particularly around the outer medulla stripe. However, the histological damage did not progress after 6 weeks of deficiency. Se deficiency induces proteinuria and glucosuria with renal calcification, which may be primarily induced by injury of proximal tubuli via oxidative stress.

Effect of selenium on Cisplatin-induced nephrotoxicity in rats.

Cisplatin (CP), a commonly used antineoplastic drug, is nephrotoxic. CP-induced nephrotoxicity involves oxidative pathways. A deficiency of selenium (Se) reduces glutathione peroxidase (GPx) activity resulting in oxidative stress. We investigated how Se deficiency or oral Se administration influences CP-induced nephrotoxicity. Thirty male Wistar rats were fed a Se-deficient or control diet for 4 weeks. Then they were given intraperitoneal (i.p.) CP alone, i.p. saline alone, or Se by gavage 24 and 1 h prior to i.p. CP. Blood and urine samples were collected and the kidneys were removed 5 days after CP treatment. Urinalysis, renal function, GPx activity, and expression of cellular GPx mRNA were measured. Histology was evaluated by light microscopy with immunohistochemistry for 4-hydroxy-2-nonenal (HNE), vimentin, and heme oxygenase (HO)-1. CP induced renal tubular damage with increased expression of vimentin, HO-1 and HNE staining, which represents lipid peroxidation. Se deficiency exacerbated CP-induced nephrotoxicity as shown by deterioration of the above parameters and depressed GPx activity and expression of GPx mRNA. Se treatment ameliorated CP-induced nephrotoxicity, but did not significantly improve renal function. These findings suggest that Se deficiency increases oxidative stress and enhances CP-induced nephrotoxicity, whereas oral Se treatment partially protects against the nephrotoxicity in rats.

Expression of multidrug resistance proteins and accumulation of cisplatin in human non-small cell lung cancer cells.

In order to understand and overcome multidrug resistance (MDR) of human non-small cell lung cancer (NSCLC), mRNA and protein expression levels of P-glycoprotein (MDR1), multidrug resistance-associated protein 1 (MRP1), and lung resistance-related protein (LRP) were investigated and compared with the chemosensitivity and the intracellular/intranuclear cisplatin accumulation of three NSCLC cell lines (Ma-10, Ma-31, and Ma-46). Ma-31 was more resistant than Ma-10 and Ma-46 to cisplatin, carboplatin, etoposide, and paclitaxel. The mRNA level of MDR1 was extremely low, and MDR1 protein was not detected in all cell lines. MRP1 mRNA expression was highest in Ma-31 and lowest in Ma-10, but there was no notable difference between the MRP1 protein expression in three cell lines. LRP mRNA/protein was equally expressed in Ma-10 and Ma-31, but was nominal in Ma-46. The intracellular/intranuclear cisplatin accumulation of the cells was determined to be Ma-31>Ma-46>Ma-10. Thus, MDR1, MRP1, and LRP mRNA and protein expression levels were not correlated with the chemosensitivity or the intracellular/intranuclear cisplatin accumulation of each cell line. The present results indicate that MDR proteins (MDR1, MRP1, and LRP) may not play an important role in the chemoresistance and drug efflux of NSCLC cells.

Overexpression of constitutive signal transducer and activator of transcription 3 mRNA in cisplatin-resistant human non-small cell lung cancer cells.

Non-small cell lung cancer (NSCLC) often shows intrinsic multidrug resistance, which is one of the most serious problems in cisplatin-based adjuvant chemotherapy. Recently, the constitutive activation of signal transducer and activator of transcription (STAT) factors has been found in a variety of human cancers. In the present study, the mRNA expression of STATs in various human NSCLC cell lines was investigated by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR) to determine whether STATs can be implicated in cisplatin resistance and apoptosis inducibility. Cisplatin triggered apoptosis in Ma-46 based on biochemical and morphological findings, but not in Ma-31. The mRNA expression of STAT3 was highest in cisplatin-resistant Ma-31 and lowest in cisplatin-sensitive Ma-46. A 6-hour exposure of cancer cells to cisplatin failed to stimulate STAT3 mRNA expression. Therefore, an increased transcriptional level of constitutive STAT3 may be related to the suppressive regulation of the apoptotic pathway in intrinsically chemo-resistant NSCLC cells.

The structure-activity relationship of various YO compounds, novel plasmin inhibitors, in the apoptosis induction.

We have previously reported that YO-2, a selective plasmin inhibitor, induces thymocyte apoptosis. To elucidate the mechanism of YO-2-induced apoptosis, other YO compounds with different plasmin inhibitory action were tested for the pro-apoptotic activity in this study. The treatment of rat thymocytes with the YO compounds which had the hydrophobic but not the hydrophilic moiety at the C-terminal increased DNA fragmentation, the number of condensed nuclei and caspase-3-like activity. All pro-apoptotic YO compounds not only were potent plasmin inhibitors but also had the hydrophobic C-terminal as the common structure. Therefore, the target molecule of the YO compounds may be located not on the cell surface but rather inside the cells.

Terfenadine induces thymocyte apoptosis via mitochondrial pathway.

The treatment of rat thymocytes with 10 microM terfenadine resulted in a significant increase in DNA fragmentation. The DNA fragmentation induced by terfenadine was dependent on its concentration and incubation time. In terfenadine-treated cells, the translocation of phosphatidylserine from the inside of plasma membrane to the outside, an early event of the apoptotic process, and chromatin condensation, the morphological characterization of apoptotic cell death, were observed. Terfenadine stimulated caspase-8, -9 and -3-like activities in an incubation time-dependent manner in thymocytes. The active forms of caspase-3 and -9 were detected in the extract from terfenadine-treated cells by immunoblotting analysis using specific antibodies to caspases, but active caspase-8 was not found in this fraction. Decrease in mitochondrial membrane potential and the release of cytochrome c from mitochondria to cytosol were observed in terfenadine-treated thymocytes. These results suggest that terfenadine induces apoptosis in rat thymocytes via mitochondrial pathway.

Involvement of histone phosphorylation in apoptosis of human astrocytes after exposure to saline solution.

We have previously found using inhibitors of protein phosphatase that phosphorylation of histones may be involved in thymocyte apoptosis. In this study, we examined whether histone modification occurs in astrocyte apoptosis induced by a pathological condition in the absence of drug. Incubation of cultured human astrocytes with growth medium for 24 h after exposure to saline solution for 30 min induced an increase in terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL)-positive cells and nuclear condensation, biochemical and morphological hallmarks of apoptotic cell death. Acetic acid-urea-Triton X-100 (AUT) gel electrophoresis of the nuclear histone fraction and N-terminal peptide analysis showed that the treatment with saline solution caused rapid changes in phosphorylation of H2A subfamilies, but not in histone acetylation. The phosphorylation of the two subtypes increased markedly, whereas the phosphorylation of one subtype decreased. In contrast, exposure to ACF-95, an artificial cerebrospinal fluid (CSF), was associated with little induction of apoptotic cell death and induced less changes in histone phosphorylation. These results support the previous idea that chemical modification of histones is involved in the DNA fragmentation in astrocytes undergoing apoptosis.

Thymocyte apoptosis induced by various compounds including YO-2 is accompanied by a change in chromatin structure.

To elucidate the role of chromatin structure in DNA fragmentation during apoptosis, we have examined whether chromatin structural change is observed after treatment with proapoptotic compounds. Analysis of the circular dichroism (CD) spectrum of the soluble chromatin from dexamethasone-treated thymocytes revealed a decrease in alpha-helical content. Mifepristone, an antagonist of glucocorticoid receptor, prevented both the change in chromatin structure and DNA fragmentation induced by dexamethasone. The effect of YO-2 [trans-4-aminomethylcyclohexanecarbonyl-l-(O-picolyl)tyrosine-n-octylamide], which possesses proapoptotic action, on chromatin structure was also examined. Judging from the CD spectrum of the soluble chromatin from YO-2-treated thymocytes, the structure was changed by this compound as well as by dexamethasone. A decrease in alpha-helical content was also observed in cells treated with etoposide, which is used clinically as an anticancer agent. These results suggest that the change in chromatin structure is likely to be an important process in DNA fragmentation of cells undergoing apoptosis.

Roles of cathepsins in reperfusion-induced apoptosis in cultured astrocytes.

Astrocytic apoptosis may play a role in the central nervous system injury. We previously showed that reperfusion of cultured astrocytes with normal medium after exposure to hydrogen peroxide (H(2)O(2))-containing medium causes apoptosis. This study examines the involvement of the lysosomal enzymes cathepsins B and D in the astrocytic apoptosis. Reperfusion after exposure to H(2)O(2) caused a marked increase in caspase-3 and cathepsin D activities and a marked decrease in cathepsin B activity. Pepstatin A, an inhibitor of cathepsin D, and acetyl-L-aspartyl-L-methionyl-L-glutaminyl-L-aspart-1-aldehyde (Ac-DMQD-CHO), a specific inhibitor of caspase-3, blocked the H(2)O(2)-induced decrease in cell viability and DNA ladder formation in cultured rat astrocytes. The (L-3-trans-(propylcarbamoyl)oxirane-2-carbonyl)-L-isoleucyl-L-proline methyl ester (CA074 Me), a specific inhibitor of cathepsin B, did not affect the H(2)O(2)-induced cell injury. On the other hand, CA074 Me decreased cell viability with DNA ladder formation when cultured in the presence of Ac-DMQD-CHO. This caspase-independent apoptosis was attenuated by the addition of the cathepsin D inhibitor pepstatin A. Caspase-3 like activity was markedly inhibited by Ac-DMQD-CHO and partially by pepstatin A. Pepstatin A and CA074 Me inhibited cathepsin B and cathepsin D activities, respectively, in the presence and absence of Ac-DMQD-CHO. These results suggest that cathepsins B and D are involved in astrocytic apoptosis: cathepsin D acts as a death-inducing factor upstream of caspase-3 and the caspase-independent apoptosis is regulated antagonistically by cathepsins B and D.

Involvement of the change in chromatin structure in thymocyte apoptosis induced by phosphorylation of histones.

The inhibitors of protein phosphatase such as calyculin A and okadaic acid induced thymocyte apoptosis. DNA fragmentation was increased in the nuclei from thymocytes treated with calyculin A, which accelerated phosphorylation of histones. Judging from the circular dichroism analysis, the structure of soluble chromatin was changed by the treatment with calyculin A. These results suggest that the change in chromatin structure may be one of the molecular mechanisms of internucleosomal DNA fragmentation.

Thymocyte apoptosis induced by phosphorylation of histones is associated with the change in chromatin structure to allow easy accessibility of DNase.

The inhibitors of protein phosphatase such as calyculin A and okadaic acid induce the apoptotic cell death in rat thymocytes. To clarify the molecular mechanism of these inhibitor-induced apoptosis, the effect of calyculin A on DNA fragmentation in the isolated nuclei were studied. A significant increase in DNA fragmentation was observed in the nuclei prepared from the cells treated with calyculin A that caused histone hyperphosphorylation. No changes of the activities of caspase-8 and -3 were observed in the extract from the cells treated with calyculin A. The circular dichroism analysis of soluble chromatin from calyculin A-treated thymocyte nuclei indicated that phosphorylation of histones decreased its alpha-helical content. Thus, the change in the chromatin structure may be due to the chemical modification of histones. Moreover, the structural change in chromatin preceded DNA fragmentation in the nuclei. Therefore, these results suggest that the change of chromatin structure allow easy accessibility of nuclear DNase to chromosomal DNA.

Heat shock inhibits hydrogen peroxide-induced apoptosis in cultured astrocytes.

Heat shock proteins (HSPs) have been shown to act as inhibitors of apoptosis, but this anti-apoptotic effect is not known in the central nervous system. Prior heat shock has been demonstrated to protect astrocytes from cell death in a model of reperfusion injury (Brain Res. 735 (1996) 265). The present study examines the mechanism underlying the protective effect of the heat shock. Preincubation of astrocytes at 40 degrees C for 10 min attenuated the hydrogen peroxide (H(2)O(2))-induced decrease in cell viability, DNA ladder formation and nuclear condensation, and these effects were blocked by the protein synthesis inhibitor cycloheximide. The thermal stress inhibited the H(2)O(2)-induced increase in caspase-3 like protease activity, but it did not affect the H(2)O(2)-induced loss of mitochondrial membrane potential. The cytosol prepared from preheated cells did not affect Ca(2+)-induced swelling of mitochondria, a marker of the permeable transition pore. The protective effect of the thermal stress on the H(2)O(2)-induced decrease in cell viability was not affected by the mitogen-activated protein/extracellular signal-regulated kinase kinase inhibitor 2'-amino-3'-methoxyflavone, the phosphatidylinositol-3 kinase inhibitor wortmannin and the NF-kappaB inhibitor pyrrolidinedithiocarbamate. These findings suggest that HSPs inhibit apoptosis via an inhibition of caspase-3 activation without effect on mitochondrial dysfunction.

The nitric oxide donor NOC12 protects cultured astrocytes against apoptosis via a cGMP-dependent mechanism.

We examined the effect of 3-ethyl-3-(ethylaminoethyl)-1-hydroxy-2-oxo-1-triazene (NOC12), a nitric oxide (NO) donor, on apoptosis in cultured astrocytes. Reperfusion after hydrogen peroxide (H2O2) exposure caused a decrease in cell viability, loss of mitochondrial membrane potential, caspase-3 activation, DNA ladder formation, and nuclear condensation. NOC12 at 10-100 microM significantly attenuated these apoptotic changes, while the NO donor at 1 mM caused cell injury and exacerbated the H202-induced cell injury. NOC12 increased intracellular cGMP levels in a dose dependent manner with the maximal effect at 100 microM. The protective effect of NOC12 was mimicked by the NO-independent guanylate cyclase activator 3-(5'-hydroxymethyl-2'-furyl)-1-benzyl indazole, and was attenuated by the guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) and the cGMP-dependent protein kinase inhibitor KT5823. ODQ and KT5823 did not block but rather exacerbated the cytotoxic effect of NOC12 at 1 mM. These findings demonstrate that lower concentrations of NOC12 inhibit the H2O2-induced apoptosis of astrocytes in a cGMP-dependent way, but higher concentrations of NOC12 show a toxic effect on astrocytes in a cGMP-independent way.