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WOGONIN



RESEARCH



Wogonin is one of the flavonoids isolated from Scutellaria baicalensis (huang qin), with its dry herb weight consisting of up to 0.39mg/100mg of wogonin (1). Wogonin has been widely used in the treatment of various inflammatory diseases owing to its inhibition of nitric oxide (NO), prostaglandin E2 and pro-inflammatory cytokines production, as well as its reduction of cyclooxygenase-2 (COX-2). In vitro studies (2-6) have shown wogonin to possess cytostatic and cytotoxic activities against several human tumor cell lines. Wogonin induces apoptosis through the mediation of Ca2+ and/or inhibition of NF-κB, shifting O2 - to H2O2 to some extent; H2O2, in turn, serves as a signaling molecule that activates phospholipase Cγ. Ca2+ efflux from the endoplasmic reticulum is then regulated, leading to the activation of Bcl-2-associated agonist of cell death (2).
Wogonin may also directly activate the mitochondrial Ca2+ channel uniporter and enhance Ca2+ uptake, resulting in Ca2+ overload and mitochondrial damage (2). Furthermore, wogonin induces cell type-dependent cell cycle inhibitions in cancer cells, such as those observed in human cervical carcinoma HeLa cells at the G1 phase (6) and in THP-1 cells at the G2/M phase (4) respectively. Unlike the inhibitory effect of baicalein and baicalin on normal human fetal lung diploid TIG-1 cells (4), wogonin imposes minor or almost no toxicity on normal peripheral T cells (2), TIG-1 cells (4) and human prostate epithelial cells (5). This selective inhibition of wogonin is due to a high expression of L-type voltage dependent Ca2+ channels in cancer cells (2). In addition, wogonin suppresses VEGF-stimulated migration and tube formation in HUVEC by inhibiting VEGF receptor 2 (VEGFR2) instead of VEGFR1 phosphorylation (7).
The synergistic effect of wogonin on chemotherapy drugs, such as etoposide, has also been investigated. Wogonin significantly improves etoposide-induced apoptosis in cancer cells in a similar capacity as the typical P-glycoprotein (P-gp) inhibitors verapamil and cyclosporine A (8-10). However, other P-gp substrates, such as doxorubicin and vinblastine, do not show any synergistic effect (10). Similar effect was also found when combination treatment with 5-FU in human gastric MGC-803 cells and in MGC-803 transplanted nude mice (11). The underlying mechanisms might be due to its pro-apoptotic effect and inhibition of NF-κB nuclear translocation activity (10). Anti-inflammatory and anti-viral activities of wogonin may also contribute to tumor prevention (11). Wogonin is a good anti-cancer candidate due to its broad toxicities to various types of tumor cell lines and the low toxicities to normal tissues, as well as the synergistic effects.
Source:
Tan et al. 2011 Anti-cancer natural products isolated from Chinese medicinal herbs. Chinese Medicine 2011, 6:27 doi:10.1186/1749-8546-6-27
References:
1. Li C, Zhou L, Lin G, Zuo Z: Contents of major bioactive flavones in proprietary traditional Chinese medicine products and reference herb of radix Scutellariae. J Pharm Biomed Anal 2009, 50:298-306.
2. Baumann S, Fas SC, Giaisi M, Müller WW, Merling A, Gülow K, Edler L, Krammer PH, Li-Weber M: Wogonin preferentially kills malignant lymphocytes and suppresses T-cell tumor growth by inducing PLCγ1- and Ca2+ -dependent apoptosis. Blood 2008, 111:2354-2363.
3. Yu JQ, Liu HB, Tian DZ, Liu YW, Lei JC, Zou GL: Changes in mitochondrial membrane potential and reactive oxygen species during wogonin-induced cell death in human hepatoma cells. Hepatol Res 2007, 37:68-76.
4. Himeji M, Ohtsuki T, Fukazawa H, Tanaka M, Yazaki S-i, Ui S, Nishio K, Yamamoto H, Tasaka K, Mimura A: Difference of growth-inhibitory effect of Scutellaria baicalensis-producing flavonoid wogonin among human cancer cells and normal diploid cell. Cancer Lett 2007, 245:269-274.
5. Lee D-H, Kim C, Zhang L, Lee YJ: Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells. Biochem Pharmacol 2008, 75:2020-2033.
6. Yang L, Zhang HW, Hu R, Yang Y, Qi Q, Lu N, Liu W, Chu YY, You QD, Guo QL: Wogonin induces G1 phase arrest through inhibiting Cdk4 and cyclin D1 concomitant with an elevation in p21Cip1 in human cervical carcinoma HeLa cells. Biochem Cell Biol 2009, 87:933-942.
7. Lu N, Gao Y, Ling Y, Chen Y, Yang Y, Gu H-Y, Qi Q, Liu W, Wang X-T, You Q-D, Guo Q-L: Wogonin suppresses tumor growth in vivo andVEGF-induced angiogenesis through inhibiting tyrosine phosphorylation of VEGFR2. Life Sci 2008, 82:956-963.
50. Lee E, Enomoto R, Koshiba C, Hirano H: Inhibition of P-glycoprotein by wogonin is involved with the potentiation of etoposide-induced apoptosis in cancer cells. Ann N Y Acad Sci 2009, 1171: 132-136.
8. Lee E, Enomoto R, Suzuki C, Ohno M, Ohashi T, Miyauchi A, Tanimoto E, Maeda K, Hirano H, Yokoi T, Sugahara C: Wogonin, a plant flavone, potentiates etoposide-induced apoptosis in cancer cells. Ann N Y Acad Sci 2007, 1095: 521-526.
9. Enomoto R, Koshiba C, Suzuki C, Lee E: Wogonin potentiates the antitumor action of etoposide and ameliorates its adverse effects. Cancer Chemother Pharmacol 2011,67:1063-1072.
10. Zhao Q, Wang J, Zou MJ, Hu R, Zhao L, Qiang L, Rong JJ, You QD, Guo QL: Wogonin potentiates the antitumor effects of low dose 5-fluorouracil against gastric cancer through induction of apoptosis by down-regulation of NF-kappaB and regulation of its metabolism. Toxicol Lett 2010, 197: 201-210.
11. Li-Weber M: New therapeutic aspects of flavones: The anticancer properties of Scutellaria and its main active constituents Wogonin, Baicalein and Baicalin. Cancer Treatment Reviews 2009, 35:57-68.

Catalase Suppression-mediated H2O2 Accumulation in Cancer Cells by Wogonin Effectively Blocks TNF-induced NF-κB Activation and Sensitizes Apoptosis
Tremendous efforts have been made to improve the anticancer value of tumor necrosis factor (TNF). In this study, we showed that wogonin, a flavonoid isolated from Huang-Qin (Scutellaria baicalensis), synergistically sensitized cancer cells derived from cervix, ovary, and lung to TNF-induced apoptosis, which was associated with inhibition of catalase activity and increase of cellular hydrogen peroxide (H2O2). Wogonin-induced ROS block TNF-induced NF-κB activation through inhibiting phosphorylation on the NF-κB p65 subunit and consequently the DNA binding of NF-κB. In addition, wogonin suppressed the expression of the antiapoptotic factor c-FLIP, which is accompanied with potentiation of TNF-induced caspase 8 activation that initiates apoptosis. Importantly, wogonin did not sensitize normal bronchial epithelial cells to TNF-induced cell death, which was associated with the defect in induction of H2O2. Thus, wogonin specifically sensitizes cancer cells to TNF-induced cytotoxicity through H2O2-mediated NF-κB suppression and apoptosis activation. Our data provide important insights of the molecular mechanism underlying wogonin’s anticancer activity, and suggest this common flavonoid as a TNF adjuvant for cancer therapy.
Yang L, et al. Catalase Suppression-mediated H2O2 Accumulation in Cancer Cells by Wogonin Effectively Blocks TNF-induced NF-κB Activation and Sensitizes Apoptosis. Cancer Science. DOI: 10.1111/j.1349-7006.2011.01874.x



Baicalein and wogonin are activators of rat TREK-2 two-pore domain K+ channel and are significantly neuroprotective
Abstract
Aim: Earlier studies have shown that TREK-1 and TREK-2 (TREKs), members of the two-pore domain K+ (K2P) channel family that are highly expressed under pathological conditions, are activated by neuroprotective agents. Baicalein and wogonin, oriental flavonoids originating from the root of the medicinal herb Scutellaria baicalensis, are known to have beneficial effects for neuroprotection. However, little is known about the effects of baicalein and wogonin on ion channels including TREKs. We investigated whether baicalein and wogonin modulate the TREK-2 channel, which has been less studied than TREK-1.
Methods: Single-channel recordings were performed in COS-7 cells transfected with rat TREK-2 and analyzed baicalein- or wogonin-induced channel activity.
Results: We found that baicalein and wogonin activated the TREK-2 current by increasing the opening frequency (channel activity: from 0.05 ± 0.01 to 0.17 ± 0.06 in baicalein treatment and from 0.03 ± 0.01 to 0.29 ± 0.09 in wogonin treatment, P < 0.05), while leaving the single-channel conductance and mean open time unchanged. Baicalein continuously activated TREK-2, whereas wogonin transiently activated TREK-2. Application of baicalein and wogonin activated TREK-2 in both cell attached and excised patches, suggesting that baicalein and wogonin may modulate TREK-2 either directly or indirectly with different mechanisms.
Conclusion: These results suggest that baicalein- and wogonin-induced TREK-2 activation help set the resting membrane potential of cells exposed to pathological conditions and thus may give beneficial effects in neuroprotection.
Kim E-J, Kang D, Han J. Acta Physiologica. Volume 202, Issue 2, pages 185–192, June 2011. DOI: 10.1111/j.1748-1716.2011.02263.x


Wogonin, an active ingredient of Chinese herb medicine Scutellaria baicalensis, inhibits the mobility and invasion of human gallbladder carcinoma GBC-SD cells by inducing the expression of maspin
Dong P, Zhang Y, Gua J, et al. 2011 Journal of Ethnopharmacology. doi:10.1016/j.jep.2011.08.005

Abstract


Ethnopharmacological relevance
A traditional Chinese medicine Scutellaria baicalensis is prescribed for the treatment of a variety of inflammatory diseases and tumors in clinic in China. However, the detailed mechanism of anti-metastasis effect of wogonin, a main active ingredient of Scutellaria baicalensis, remains elusive.

Aim of the study
The present study was designed to investigate the action and mechanism of wogonin on the mobility and invasion of human gallbladder carcinoma GBC-SD cells.

Materials and methods
Viability, apoptosis, mRNA and protein expression of tumor cells were analyzed by MTT, Annexin V staining, real-time PCR and Western blot, respectively. The migration and invasion assay was used to evaluate the anti-metastasis effect of wogonin. Knockdown of maspin was performed by specific small interference RNA.

Results
Wogonin at the dose of 1–10 μM, which did not induce apoptosis, significantly inhibited the mobility and invasion activity of human gallbladder carcinoma GBC-SD cells. In addition, the expressions of matrix metalloproteinase (MMP)-2, MMP-9 and phosphorylated extracellular regulated protein kinase 1/2 (ERK1/2) but not phosphorylated Akt were dramatically suppressed by wogonin in a concentration-dependent manner. Furthermore, the metastasis suppressor maspin was confirmed as the downstream target of wogonin. Both maspin mRNA and protein were upregulated by wogonin. Interestingly, the knockdown of maspin resulted in almost completely blocking of wogonin-induced inhibition of MMP-2, MMP-9 and phosphorylated ERK1/2 as well as the mobility and invasion activity of GBC-SD cells.

Conclusions
These findings suggest that wogonin inhibits cell mobility and invasion by upregulating the metastasis suppressor maspin. Together, these data provide novel insights into the chemoprotective effect of wogonin, a main active ingredient of Chinese medicine Scutellaria baicalensis.

Discussion
The highlight of this work is the revelation that in the 1–10 μM range wogonin does not induce apoptosis but significantly inhibits the mobility and invasion activity of human gallbladder carcinoma GBC-SD cells by upregulating the metastasis suppressor maspin. Previously, wogonin has been reported to effectively inhibit the proliferation and induce the apoptosis at the dosages of more than 10 μM in several cancer cell lines ([Lee et al., 2008] and [Zhang et al., 2008]). We also found that wogonin at the dosages of more than 10 μM could significantly inhibit the viability of several human tumor cells including human gallbladder carcinoma GBC-SD cells, human prostate carcinoma LNCaP cells and human U-937 leukemia cells. However, the non-apoptosis-inducing action of wogonin on tumor cells at the dosages of less than 10 μM remains unclear. In the present study, 1–10 μM range wogonin did not inhibit the proliferation and induce the apoptosis in human gallbladder carcinoma GBC-SD cells. Whether wogonin at the sub-dose of apoptosis-inducing effect (1–10 μM) could influence other functions of tumor cells? Results showed that 1–10 μM wogonin significantly inhibited the mobility and invasion activity of human gallbladder carcinoma GBC-SD cells in a dose-dependent manner. Thus, the mechanisms of wogonin on the metastasis potentiality of tumor cells, at a sub-dose of apoptosis induction (1–10 μM) needed to be further investigated.

ERK1/2 activation has been shown to be the major mechanism for promoting the production of MMP-2 and MMP-9 (Beshir et al., 2010), which are important for promoting tumor cellular invasion. We presented evidence indicating that wogonin was able to inhibit human gallbladder carcinoma cell migration and invasion in vitro in parallel with down-regulation of MMP-2 and MMP-9 as well as ERK1/2 activation. Previously, Lee et al. also reported that wogonin significantly suppressed tumor necrosis factor-α-induced MMP-9 expression in human aortic smooth muscle cells (Lee et al., 2006). Together, these findings suggest wogonin has potential anti-metastatic effect in vitro and shed light on the investigation of wogonin on gallbladder carcinoma metastasis in vivo.

To further dissect the molecular mechanisms underlying the anti-invasive activity of wogonin, we focused on the role of maspin, a tumor metastasis suppressor. First, we found that both maspin mRNA and protein were remarkably upregulated by wogonin. Then, to validate the functional significance of maspin upregulation in wogonin-treated cells, we knocked down maspin by siRNA and examined the invasive activity. As shown knockdown of maspin restored the inhibition of cell invasion induced by wogonin. Moreover, knockdown of maspin blocked wogonin-induced inhibition of MMP-2, MMP-9 and phosphorylated ERK1/2 expression. Thus, maspin may be responsible for the anti-invasive activity of wogonin. Accumulated functional studies have demonstrated that maspin reduces tumor metastasis in vivo and tumor cell motility and invasion in vitro ([Hong et al., 2009], [Lonardo et al., 2010] and [Yeom et al., 2010]). Thus, in addition to the function of suppression of cancer cell growth at the dose of 25–100 μM, wogonin may be involved in the inhibition of metastasis through upregulating maspin at the dose of 1–10 μM. Furthermore, maspin has also been shown to be involved in cell apoptosis as well as tumor growth and metastasis. Jiang et al. have been reported that endogenous maspin expression sensitizes breast carcinoma cells to staurosporine-induced apoptosis in vitro (Jiang et al., 2002). In the present study, we found that wogonin could induce significant apoptosis in GBC-SD cells at the higher dose

Anxiolytic effect of wogonin, a benzodiazepine receptor ligand isolated from Scutellaria baicalensis
Hui KM, Huen MSY, Wang HY, et al. Biochemical Pharmacology. Volume 64, Issue 9, 1 November 2002, Pages 1415-1424 doi:10.1016/S0006-2952(02)01347-3

The search for novel anxiolytics devoid of undesirable side-effects typical of classical benzodiazepines (BDZs) has been intense, and flavonoids, as a relative new class of ligands, have been shown to possess anxiolytic effects in vivo. The present study evaluated the pharmacological properties of a naturally occurring monoflavonoid, 5,7-dihydroxy-8-methoxyflavone or wogonin. The affinity (Ki) of wogonin for the benzodiazepine site (BZD-S) on the γ-aminobutyric acidA (GABAA) receptor complex was 0.92 μM. Using electrophysiological techniques, we showed that wogonin enhanced the GABA-activated current in rat dorsal root ganglion neurons, and in Xenopus laevis oocytes expressing recombinant rat GABAA receptors, the enhancement was partially reversed by the co-application of a 1 μM concentration of the BZD-S antagonist anexate (Ro15-1788). Acute toxicity and behavioral effects were examined in mice. Acute lethal activity was low, with an Image 50 of 3.9 g/kg. Oral administration of wogonin (7.5 to 30 mg/kg) elicited an anxiolytic response that was similar to that elicited by diazepam in the elevated plus-maze; a dose-dependent increase in open arm entries and time spent in open arms was observed. More importantly, its anxiolytic effect was blocked by the co-administration of Ro15-1788. In the holeboard test, not only did wogonin-treated mice experience an increased number of head-dips but they also spent more time at it, showing no signs of sedation. Furthermore, wogonin did not cause myorelaxant effects in the horizontal wire test. Taken together, these data suggest that wogonin exerts its anxiolytic effect through positive allosteric modulation of the GABAA receptor complex via interaction at the BZD-S. Its anxiolytic effect was not accompanied by sedative and myorelaxant side-effects typical of BDZs.

References
Beshir et al., 2010 A.B. Beshir, G. Ren, A.N. Magpusao, L.M. Barone, K.C. Yeung and G. Fenteany, Raf kinase inhibitor protein suppresses nuclear factor-kappaB-dependent cancer cell invasion through negative regulation of matrix metalloproteinase expression, Cancer Letter 299 (2010), pp. 137–149

Jiang et al., 2002 N. Jiang, Y. Meng, S. Zhang, E. Mensah-Osman and S. Sheng, Maspin sensitizes breast carcinoma cells to induced apoptosis, Oncogene 21 (2002), pp. 4089–4098.

Lee et al., 2006 S.O. Lee, Y.J. Jeong, M.H. Yu, J.W. Lee, M.H. Hwangbo, C.H. Kim and I.S. Lee, Wogonin suppresses TNF-alpha-induced MMP-9 expression by blocking the NF-kappaB activation via MAPK signaling pathways in human aortic smooth muscle cells, Biochemical and Biophysical Research Communications 351 (2006), pp. 118–125.

Lee et al., 2008 D.H. Lee, C. Kim, L. Zhang and Y.J. Lee, Role of p53, PUMA, and Bax in wogonin-induced apoptosis in human cancer cells, Biochemical Pharmacology 75 (2008), pp. 2020–2033.

Lonardo et al., 2010 F. Lonardo, X. Li, A. Kaplun, A. Soubani, S. Sethi, S. Gadgeel and S. Sheng, The natural tumor suppressor protein maspin and potential application in non small cell lung cancer, Current Pharmaceutical Design 16 (2010), pp. 1877–1881.

Yeom et al., 2010 S.Y. Yeom, H.L. Jang, S.J. Lee, E. Kim, H.J. Son, B.G. Kim and C. Park, Interaction of testisin with maspin and its impact on invasion and cell death resistance of cervical cancer cells, FEBS Letters 584 (2010), pp. 1469–1475.

Zhang et al., 2008 H.W. Zhang, Y. Yang, K. Zhang, L. Qiang, L. Yang, Y. Hu, X.T. Wang, Q.D. You and Q.L. Guo, Wogonin induced differentiation and G1 phase arrest of human U-937 leukemia cells via PKCdelta phosphorylation, European Journal of Pharmacology 591 (2008), pp. 7–12.

Anti-tumour and anti-metastatic actions of wogonin isolated from Scutellaria baicalensis roots through anti-lymphangiogenesis
Tumour growth and metastasis are associated with angiogenesis and lymphangiogenesis through the production of vascular endothelial growth factor (VEGF) or VEGF-C in tumours, and the phosphorylation of VEGF receptor (VEGFR)-2 or VEGFR-3 in vascular endothelial cells or lymphatic endothelial cells (LECs). Tumour-associated macrophages (TAMs) play an important role in tumour lymphangiogenesis, and consequently stimulate metastasis through the lymphatic system to lymph nodes.
Yoshiyuki Kimura & Maho Sumiyoshi examined the effects of wogonin isolated from Scutellaria baicalensis roots on tumour growth and metastasis using a highly metastatic model in osteosarcoma LM8-bearing mice. Wogonin (25 and 50mg/kg, twice daily) reduced tumour growth and metastasis to the lung, liver and kidney, angiogenesis (CD31-positive cells), lymphangiogenesis (LYVE-1-positive cells), and TAM (F4/80-positive cell) numbers in the tumors of LM8-bearing mice.
Wogonin (10–100μM) also inhibited increases in IL-1β production and cyclooxygenase (COX)-2 expression induced by lipopolysaccharide in THP-1 macrophages. Wogonin had no effect on VEGF-C production in LM8 cells, or VEGFR-3 expression in human lymphatic endothelial cells (HLECs), however, it inhibited VEGF-C-induced VEGFR-3 phosphorylation in HLECs. The anti-tumour and anti-metastatic actions of wogonin may be associated with the inhibition of VEGF-C-induced lymphangiogenesis through a reduction in VEGF-C-induced VEGFR-3 phosphorylation by the inhibition of COX-2 expression and IL-1β production in TAMs.
Kimura Y & Sumiyoshi M. Phytomedicine. Dec 07 2012. doi:10.1016/j.phymed.2012.10.016

 
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