Yen Chou Chen

Wogonin, baicalin, and baicalein inhibition of inducible nitric oxide synthase and cyclooxygenase-2 gene expressions induced by nitric oxide synthase inhibitors and lipopolysaccharide

We previously reported that oroxylin A, a polyphenolic compound, was a potent inhibitor of lipopolysaccharide (LPS)-induced expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). In the present study, three oroxylin A structurally related polyphenols isolated from the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, were examined for their effects on LPS-induced nitric oxide (NO) production and iNOS and COX-2 gene expressions in RAW 264.7 macrophages. The results indicated that these three polyphenolic compounds inhibited LPS-induced NO production in a concentration-dependent manner without a notable cytotoxic effect on these cells. The decrease in NO production was in parallel with the inhibition by these polyphenolic compounds of LPS-induced iNOS gene expression. However, these three compounds did not directly affect iNOS enzyme activity. In addition, wogonin, but not baicalin or baicalein, inhibited LPS-induced prostaglandin E2 (PGE2) production and COX-2 gene expression without affecting COX-2 enzyme activity. Furthermore, N-nitro-L-arginine (NLA) and N-nitro-L-arginine methyl ester (L-NAME) pretreatment enhanced LPS-induced iNOS (but not COX-2) protein expression, which was inhibited by these three polyphenolic compounds. Wogonin, but not baicalin or baicalein, similarly inhibited PGE2 production and COX-2 protein expression in NLA/LPS or L-NAME/LPS-co-treated RAW 264.7 cells. These results indicated that co-treatment with NOS inhibitors and polyphenolic compounds such as wogonin effectively blocks acute production of NO and, at the same time, inhibits expression of iNOS and COX-2 genes.

Wogonin and fisetin induce apoptosis in human promyeloleukemic cells, accompanied by a decrease of reactive oxygen species, and activation of caspase 3 and Ca2+-dependent endonuclease

Seven structurally related flavonoids including luteolin, nobiletin, wogonin, baicalein, apigenin, myricetin and fisetin were used to study their biological activities on the human leukemia cell line, HL-60. On MTT assay, wogonin, baicalein, apigenin, myricetin and fisetin showed obvious cytotoxic effects on HL-60 cells, with wogonin and fisetin being the most-potent apoptotic inducers among them. The cytotoxic effects of wogonin and fisetin were accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including DNA fragmentation, apoptotic bodies and the sub-G1 ratio. Treatment with an apoptosis-inducing concentration of wogonin or fisetin causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity. Further, cleavage of poly(ADP-ribose) polymerase (PARP) and decrease of pro-caspase 3 protein were detected in wogonin- and fisetin-treated HL-60 cells. An increase in the pro-apoptotic protein, bax, and a decrease in the anti-apoptotic protein, Mcl-1, were detected in fisetin- and wogonin-treated HL-60 cells. However, Bcl-2, Bcl-XL, and Bad all remained unchanged in wogonin- and fisetin-treated HL-60 cells. In vitro chromatin digestion revealed that endonuclease activity was profoundly enhanced in wogonin- and fisetin-treated HL-60 cells, and the addition of ethylenediaminetetraacetic acid (EDTA) or ethyleneglycoltetraacetic acid (EGTA) into the reaction blocked endonuclease activation and at an optimum pH of 7.5. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated wogonin- and fisetin-induced DNA ladders, PARP cleavage, and endonuclease activation. Pretreatment of HL-60 cells with N-acetyl-cysteine or catalase efficiently inhibited H(2)O(2) (200 microM)-induced apoptosis, but showed no inhibitory effect on wogonin- and fisetin-induced DNA ladders, caspase 3 activation, or bax protein induction. Decrease in endogenous ROS production was detected in wogonin- and fisetin-treated HL-60 cells by DCHF-DA assay. In conclusion, our experiments indicate that a decrease in intracellular peroxide level was involved in wogonin- and fisetin-induced apoptosis; activation of caspase 3 and endonuclease, induction of bax protein and suppression of Mcl-1 protein were detected in the process.

Emodin induces apoptosis in human promyeloleukemic HL-60 cells accompanied by activation of caspase 3 cascade but independent of reactive oxygen species production

Emodin (1,3,8-trihydroxy-6-methylanthraquinone) is an active constituent of Rheum palmatum, and showed inhibitory activity on lipopolysaccharide-induced NO production in our previous study. However, the apoptosis-inducing activity of emodin has remained undefined. Among three structurally related anthraquinones, including emodin, physcion, and chrysophanol, emodin showed the most potent cytotoxic effects on HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells. Emodin at apoptosis-inducing concentrations causes rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase 3 fragments being detected in emodin- but not physcion- or chrysophanol-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in emodin-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bad remained unchanged. The caspase 3 inhibitor, Ac-DEVD-CHO, but not the caspase 1 inhibitor, Ac-YVAD-CHO, attenuated emodin-induced DNA ladders, associated with the blockage of PARP and D4-GDI cleavage. Free radical scavenging agents including NAC, catalase, SOD, ALL, DPI, L-NAME and PDTC showed no preventive effect on emodin-induced apoptotic responses, whereas NAC, CAT and PDTC prevented HL-60 cells from ROS (H(2)O(2))-induced apoptosis through inhibition of caspase 3 cascades. Induction of catalase, but not SOD, activity was detected in emodin-treated HL-60 cells by in gel activity assays, and H(2)O(2)-induced intracellular peroxide level was significantly reduced by prior treatment of emodin in HL-60 cells. Our experiments provide evidence that emodin is an effective apoptosis inducer in HL-60 cells through activation of the caspase 3 cascade, but that it is independent of ROS production.

In vitro and in vivo inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide and prostaglandin E2 production

Flavonoids are widely distributed in plants, but their biological functions are still unclear. In the present study, in vitro and in vivo experiments were performed to demonstrate the inhibitory activities of rutin, wogonin, and quercetin on lipopolysaccharide-induced nitric oxide (NO) and prostaglandin E(2) production in RAW 264.7 macrophages, primary peritoneal macrophages, and Balb/c mice, respectively. In vitro results showed that wogonin and quercetin dose-dependently suppressed lipopolysaccharide-induced NO production in RAW 264.7 macrophages and primary peritoneal macrophages without a notable cytotoxic effect on either cell types associated with a decrease in inducible nitric oxide synthase (iNOS) protein expression in both cells. Rutin, at 80 microM only, had a slight but obvious inhibitory effect on lipopolysaccharide-induced NO production in primary peritoneal macrophages. Both wogonin and quercetin attenuated lipopolysaccharide-induced prostaglandin E(2) production in vitro. Intravenous injection of lipopolysaccharide (10 mg/kg, i.v.) resulted in a time-dependent induction of NO production in serum, and pretreatment with the L-arginine analog N-nitro-L-arginine methyl ester (L-NAME) blocked this induction. Intravenous pretreatment of Balb/c mice with rutin, wogonin or quercetin for 1 h followed by lipopolysaccharide treatment significantly inhibited lipopolysaccharide-induced NO production, but no inhibition of prostaglandin E(2) production was found. A decrease in iNOS protein, but not cyclooxygenase-2 protein, was detected in liver and lung specimens of lipopolysaccharide-treated Balb/c mice in the presence of rutin, wogonin or quercetin. In conclusion, data obtained both in vitro and in vivo suggest that wogonin and quercetin exert inhibitory activity on lipopolysaccharide-induced NO production through suppression of iNOS expression.

Inhibition of nitric oxide synthase inhibitors and lipopolysaccharide induced inducible NOS and cyclooxygenase-2 gene expressions by rutin, quercetin, and quercetin pentaacetate in RAW 264.7 macrophages.

Several natural flavonoids have been demonstrated to perform some beneficial biological activities, however, higher‐effective concentrations and poor‐absorptive efficacy in body of flavonoids blocked their practical applications. In the present study, we provided evidences to demonstrate that flavonoids rutin, quercetin, and its acetylated product quercetin pentaacetate were able to be used with nitric oxide synthase (NOS) inhibitors (N‐nitro‐L‐arginine (NLA) or N‐nitro‐L‐arginine methyl ester (L‐NAME)) in treatment of lipopolysaccharide (LPS) induced nitric oxide (NO) and prostaglandin E2 (PGE2) productions, inducible nitric oxide synthase (iNOS) and cyclooxygenase‐2 (COX‐2) gene expressions in a mouse macrophage cell line (RAW 264.7). The results showed that rutin, quercetin, and quercetin pentaacetate‐inhibited LPS‐induced NO production in a concentration‐dependent manner without obvious cytotoxic effect on cells by MTT assay using 3‐[4,5‐dimethylthiazol‐2‐yl]‐2,5‐diphenyltetrazolium bromide as an indicator. Decrease of NO production by flavonoids was consistent with the inhibition on LPS‐induced iNOS gene expression by western blotting. However, these compounds were unable to block iNOS enzyme activity by direct and indirect measurement on iNOS enzyme activity. Quercetin pentaacetate showed the obvious inhibition on LPS‐induced PGE2 production and COX‐2 gene expression and the inhibition was not result of suppression on COX‐2 enzyme activity. Previous study demonstrated that decrease of NO production by L‐arginine analogs effectively stimulated LPS‐induced iNOS gene expression, and proposed that stimulatory effects on iNOS protein by NOS inhibitors might be harmful in treating sepsis. In this study, NLA or L‐NAME treatment stimulated significantly on LPS‐induced iNOS (but not COX‐2) protein in RAW 264.7 cells which was inhibited by these three compounds. Quercetin pentaacetate, but not quercetin and rutin, showed the strong inhibitory activity on PGE2 production and COX‐2 protein expression in NLA/LPS or L‐NAME/LPS co‐treated RAW 264.7 cells. These results indicated that combinatorial treatment of L‐arginine analogs and flavonoid derivates, such as quercetin pentaacetate, effectively inhibited LPS‐induced NO and PGE2 productions, at the same time, inhibited enhanced expressions of iNOS and COX‐2 genes. J. Cell. Biochem. 82: 537–548, 2001.

Differential apoptosis‐inducing effect of quercetin and its glycosides in human promyeloleukemic HL‐60 cells by alternative activation of the caspase 3 cascade

Flavonoids were demonstrated to possess several biological effects including antitumor, antioxidant, and anti‐inflammatory activities in our previous studies. However, the effect of glycosylation on their biological functions is still undefined. In the present study, the apoptosis‐inducing activities of three structure‐related flavonoids including aglycone quercetin (QUE), and glycone rutin (RUT; QUE‐3‐O‐rutinoside), and glycone quercitrin (QUI; QUE‐3‐O‐rhamnoside) were studied. Both RUT and QUI are QUE glycosides, and possess rutinose and rhamnose at the C3 position of QUE, respectively. Results of the MTT assay showed that QUE, but not RUT and QUI, exhibits significant cytotoxic effect on HL‐60 cells, accompanied by the dose‐ and time‐dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, apoptotic bodies, and an increase in hypodiploid cells by flow cytometry analysis. QUE, but not RUT or QUI, caused rapid and transient induction of caspase 3/CPP32 activity, but not caspase 1 activity, according to cleavage of caspase 3 substrates poly(ADP‐ribose) polymerase (PARP) and D4‐GDI proteins, and the appearance of cleaved caspase 3 fragments being detected in QUE‐ but not RUT‐ or QUI‐treated HL‐60 cells. A decrease in the anti‐apoptotic protein, Mcl‐1, was detected in QUE‐treated HL‐60 cells, whereas other Bcl‐2 family proteins including Bax, Bcl‐2, Bcl‐XL, and Bag remained unchanged. The caspase 3 inhibitor, Ac‐DEVD‐FMK, but not the caspase 1 inhibitor, Ac‐YVAD‐FMK, attenuated QUE‐induced cell death. Results of DCHF‐DA assay indicate that no significant increase in intracellular peroxide level was found in QUE‐treated cells, and QUE inhibited the H2O2‐induced intracellular peroxide level. Free radical scavengers N‐acetyl‐cysteine (NAC) and catalase showed no prevention of QUE‐induced apoptosis. In addition, QUE did not induce apoptosis in an mature monocytic cell line THP‐1, as characterized by a lack of DNA ladders, caspase 3 activation, PARP cleavage, and an Mcl‐1 decrease, compared with those in HL‐60 cells. Our experiments provide evidence to indicate that the addition of rutinose or rhamnose attenuates the apoptosis‐inducing activity of QUE, and that the caspase 3 cascade but not free radical production is involved. J. Cell. Biochem. 89: 1044–1055, 2003.

Mitochondria-mediated caspase-independent apoptosis induced by cadmium in normal human lung cells.

Cadmium, a well‐known environmental hazard, has caused serious health problems in humans and animals. Accumulating evidence suggests the cadmium toxicity is mediated by oxidative stress‐induced cell death. However, the molecular signaling underlying cadmium‐induced apoptosis remains unclear. In this study, we demonstrate here that cadmium induced mixed types of cell death including primary apoptosis (early apoptosis), secondary necrosis (late apoptosis), and necrosis in normal human lung cells, MRC‐5, as revealed by chromatin condensation, phosphatidylserine (PS) externalization, and hypodiploid DNA content. The total apoptotic cells reached a plateau of around 40.0% after 24 h exposure of 100 μM cadmium. Pretreatment with Z‐Val–Ala–Asp–fluoromethylketone (Z‐VAD–fmk), a broad spectrum of caspase inhibitor, could not rescue apoptotic cells from cadmium toxicity. Coincidently, we failed to detect the activation of pro‐caspase‐3 and cleavage of PARP by immunoblot, which implies the apoptogenic activity of cadmium in MRC‐5 cells is caspase‐independent. JC‐1 staining also indicated that mitochondrial depolarization is a prelude to cadmium‐induced apoptosis, which was accompanied by a translocation of caspase‐independent pro‐apoptotic factor apoptosis‐inducing factor (AIF) into the nucleus as revealed by the immunofluorescence assay. In summary, this study demonstrated for the first time that cadmium induced a caspase‐independent apoptotic pathway through mitochondria‐mediated AIF translocation into the nucleus. J. Cell. Biochem. 89: 335–347, 2003.

Anti-inflammatory effect of heme oxygenase 1: Glycosylation and nitric oxide inhibition in macrophages

Flavonoids including the aglycones, hesperetin (HT; 5,7,3′‐trihydroxy‐4′‐methoxy‐flavanone), and naringenin (NE; 5,7,4′‐trihydroxy flavanone) and glycones, hesperidin (HD; 5,7,3′‐trihydroxy‐4′‐methoxy‐flavanone 7‐rhamnoglucoside) and naringin (NI; 5,7,4′‐trihydroxy flavanone 7‐rhamno glucoside), were used to examine the importance of rutinose at C7 on the inhibitory effects of flavonoids on lipopolysaccharide (LPS)‐induced nitric oxide production in macrophages. Both HT and NE, but not their respective glycosides HD and NI, induced heme oxygenase 1 (HO‐1) protein expression in the presence or absence of LPS and showed time and dose‐dependent inhibition of LPS‐induced nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) expression in RAW264.7, J774A.1, and thioglycolate‐elicited peritoneal macrophages. Additive inhibitory effect of an HO‐1 inducer hemin and NE or NI on LPS‐induced NO production and iNOS expression was identified, and HO enzyme inhibitor tin protoporphyrin (SnPP) attenuated the inhibitory effects of HT, NE, and hemin on LPS‐induced NO production. Both NE and HT showed no effect on iNOS mRNA and protein stability in RAW264.7 cells. Removal of rutinose at C7 of HD and NI by enzymatic digestion using hesperidinase (HDase) and naringinase (NIase) produce inhibitory activity on LPS‐induced NO production, according to the production of the aglycones, HT and NE, by high‐performance liquid chromatography (HPLC) analysis. Furthermore, the amount of NO produced by LPS or lipoteichoic acid (LTA) was significantly reduced in HO‐1‐overexpressing cells (HO‐1/RAW264.7) compared to that in parental cells (RAW264.7). Results of the present study provide scientific evidence to suggest that rutinose at C7 is a negative moiety in flavonoid inhibition of LPS‐induced NO production, and that HO‐1 is involved in the inhibitory mechanism of flavonoids on LPS‐induced iNOS and NO production.

Rutinoside at C7 attenuates the apoptosis-inducing activity of flavonoids

Rutinoside (rhamnoglucoside; rhamnose+glucose) addition has been examined extensively in the metabolism of flavonoids, however the effect of rutinoside on apoptosis-inducing activity of flavonoids is still unknown. In the present study, the two pairs of flavonoids of hesperetin (HT) and hesperidin (HD; HT-7-rutinose), and naringenin (NE) and naringin (NE-7-rutinose), were used to study their apoptosis-inducing activities in HL-60 cells. Both HD and NI are flavonoids which contain a rutinoside at the C7 of HT and NE, respectively. Results of the MTT assay showed that HT and NE, but not HD and NI, exhibited significant cytotoxic effect in HL-60 cells, accompanied by the dose- and time-dependent appearance of characteristics of apoptosis including an increase in DNA ladder intensity, morphological changes, appearance of apoptotic bodies, and an increase in hypodiploid cells by flow cytometry analysis. HT and NE, but not HD and NI, caused rapid and transient induction of caspase-3/CPP32 activity, but not caspase-1 activity, according to the cleavage of caspase-3 substrates poly(ADP-ribose) polymerase and D4-GDI proteins, the appearance of cleaved caspase-3 fragments detected in HT- or NE-, but not in HD- or NI-treated HL-60 cells. A decrease in the anti-apoptotic protein, Mcl-1, was detected in HT- and NE-treated HL-60 cells, whereas other Bcl-2 family proteins including Bax, Bcl-2, Bcl-XL, and Bag remained unchanged. The caspase-3 inhibitor, Ac-DEVD-FMK, but not the caspase-1 inhibitor, Ac-YVAD-FMK, attenuated HT- and NE-induced cell death. Interestingly, neither HT nor NE induced apoptosis in the mature monocytic cell line THP-1 and primary human polymorphonuclear cells, as characterized by a lack of DNA ladders, caspase-3 activation, poly(ADP-ribose) polymerase cleavage, and Mcl-1 decrease, compared with those in HL-60 cells. In addition, the rutinoside group in HD and NI was removed by hesperidinase and naringinase, accompanied by the production of HT and NE, respectively, according to HPLC analysis. Accordingly, hesperidinase and naringinase digestion recovered the apoptosis-inducing activity of HD and NI in HL-60 cells. Our experiments provide the first evidence to suggest that rutinoside in flavonoids prevents the induction of apoptosis, and that activation of the traditional caspase-3 cascade participates in HT- and NE-induced apoptosis.

Heme oxygenase-1 inhibits breast cancer invasion via suppressing the expression of matrix metalloproteinase-9

In the present study, we investigated the antitumor effects of the invasiveness and migration of heme oxygenase 1 (HO-1) in human breast carcinoma cells. 12-O-tetradecanoylphorbol-13-acetate (TPA)–induced matrix metalloproteinase-9 (MMP-9) enzyme activity and gene expression at both protein and mRNA levels were examined in human breast carcinoma cells (MCF-7 and MDA-MB-231), and the addition of the MMP-9 inhibitor, SB3CT, significantly suppressed TPA-induced invasion and migration according to the in vitro Transwell assay. Elevation of HO-1 gene expression by ferric protoporphyrin IX inhibited TPA-induced invasion of MCF-7 cells, which was blocked by adding the heme oxygenase inhibitor, tin protoporphyrin IX, or transfection of cells with HO-1 short hairpin RNA. MCF-7 cells overexpressing HO-1 (MCF-7/HO-1) were established in the present study, and TPA-induced MMP-9 gene expression, tumor invasion, and colony formation were significantly reduced in MCF-7/HO-1 cells, compared with those in Neo-transfected cells. Activation of protein kinase Cα/extracellular signal-regulated kinases/AP-1 with stimulation of reactive oxygen species production was involved in TPA-induced invasion of MCF-7 cells, which was attenuated by HO-1 protein induced by ferric protoporphyrin IX or transfection of HO-1 expression vectors. Additionally, the addition of carbon monoxide, but not ferric ions, biliverdin, or bilirubin, inhibited TPA-induced invasion through suppressing MMP-9, extracellular signal-regulated kinases, and AP-1 activation stimulated by TPA. The beneficial role of HO-1 in blocking tumor invasion was first identified in this study. [Mol Cancer Ther 2008;7(5):1195–1206]