Simultaneous knock-down of Bcl-xL and Mcl-1 induces apoptosis through Bax activation in pancreatic cancer cells

Anti-apoptotic Bcl-2 family proteins have been reported to play an important role in apoptotic cell death of human malignancies. The aim of this study was to delineate the mechanism of anti-apoptotic Bcl-2 family proteins in pancreatic cancer (PaCa) cell survival. We first analyzed the endogenous expression and subcellular localization of anti-apoptotic Bcl-2 family proteins in six PaCa cell lines by Western blot. To delineate the functional role of Bcl-2 family proteins, siRNA-mediated knock-down of protein expression was used. Apoptosis was measured by Cell Death ELISA and Hoechst 33258 staining. In the results, the expression of anti-apoptotic Bcl-2 family proteins varied between PaCa cell lines. Mcl-1 knock-down resulted in marked cleavage of PARP and induction of apoptosis. Down-regulation of Bcl-2 or Bcl-xL had a much weaker effect. Simultaneous knock-down of Bcl-xL and Mcl-1 strongly induced apoptosis, but simultaneous knock-down of Bcl-xL/Bcl-2 or Mcl-1/Bcl-2 had no additive effect. The apoptosis-inducing effect of simultaneous knock-down of Bcl-xL and Mcl-1 was associated with translocation of Bax from the cytosol to the mitochondrial membrane, cytochrome c release, and caspase activation. These results demonstrated that Bcl-xL and Mcl-1 play an important role in pancreatic cancer cell survival. Targeting both Bcl-xL and Mcl-1 may be an intriguing therapeutic strategy in PaCa.     

Free radical scavenging and antioxidant activities of flavonoids extracted from the radix of Scutellaria baicalensis Georgi

Free radical scavenging and antioxidant activities of baicalein, baicalin, wogonin and wogonoside, the four major flavonoids in the radix of Scutellaria baicalensis Georgi, were examined in different systems. ESR results showed that baicalein and baicalin scavenged hydroxyl radical, DPPH radical and alkyl radical in a dose-dependent manner, while wogonin and wogonoside showed subtle or no effect on these radicals. Ten micromol/l of baicalein and baicalin effectively inhibited lipid peroxidation of rat brain cortex mitochondria induced by Fe(2+)-ascorbic acid, AAPH or NADPH, while wogonin and wogonoside showed significant effects only on NADPH-induced lipid peroxidation. In a study on cultured human neuroblastoma SH-SY5Y cells system, it was found that 10 micromol/l of baicalein and baicalin significantly protected cells against H(2)O(2)-induced injury. Baicalein was the most effective antioxidant among the four tested compounds in every system due to its o-tri-hydroxyl structure in the A ring. Compared with a well-known flavonoid, quercetin, the antioxidant activity of baicalein was lower in DPPH or AAPH system, but a little higher in those systems which might associate with iron ion. These results suggest that flavonoids in the radix of Scutellaria baicalensis with o-di-hydroxyl group in A the ring, such as baicalein and baicalin, could be good free radical scavengers and might be used to cure head injury associated with free radical assault.     

Baicalin,baicalein and wogonin inhibits high glucose-induced vascular inflammation in vitro and in vivo

Vascular inflammatory process has been suggested to play a key role in initiation and progression of atherosclerosis, a major complication of diabetes mellitus. Thus, in this study, we attempted to determine whether three structurally related polyphenols found in the Chinese herb Huang Qui, namely baicalin, baicalein, and wogonin, can suppress vascular inflammatory processes induced by high glucose (HG) in human umbilical vein endothelial cells (HUVECs) and mice. Data showed that HG induced markedly increased vascular permeability, monocyte adhesion, expressions of cell adhesion molecules (CAMs), formation of reactive oxygen species (ROS) and activation of nuclear factor (NF)-κB. Remarkably, all of the above mentioned vascular inflammatory effects of HG were attenuated by pretreatment with baicalin, baicalein, and wogonin. Vascular inflammatory responses induced by HG are critical events underlying development of various diabetic complications, therefore, our results suggest that baicalin, baicalein, and wogonin may have significant therapeutic benefits against diabetic complications and atherosclerosis. [BMB Reports 2015; 48(9): 519-524]     

Selective fraction of Scutellaria baicalensis and its chemopreventive effects on MCF-7 human breast cancer cells

Based on our previous observation, the whole Scutellaria baicalensis extract (SbE) did not show significant breast cancer cell inhibitory effect. In this study, we isolated a baicalin-deprived-fraction (SbF1) of Scutellaria baicalensis, and baicalin-fraction (SbF3), and evaluated their anti-breast cancer properties using MCF-7 cells. The content of four flavonoids in extract/fractions were determined using high performance liquid chromatography. Analytical data showed that in SbF1, the major constituents are baicalein and wogonin, while SbF3 only contains baicalin. The antiproliferative effects of fractions and SbE were assayed using modified trichrome stain method. SbF1 showed significant antiproliferative effect. Treated with 100 μg/ml of SbF1 for 72 h inhibited MCF-7 cell growth by 81.6%, while in the same treatment concentration, SbF3 increased cell growth by 22.6%. SbF1 was recognized as an active fraction of SbE. The effects of four flavonoids in SbE, scutellarin, baicalin, baicalein and wogonin, were determined, and data showed that baicalein and wogonin significantly inhibited MCF-7 cell growth. In contrast, in certain concentrations, scutellarin and baicalin increased cancer cell growth. The effects of SbF1 on cell cycle and apoptosis were assayed using flow cytometry. SbF1 arrested MCF-7 cells in S- and G2/M-phases, and significantly increased induction of cell apoptosis. These combined phytochemical and biological data provide evidence for further chemopreventive studies of the baicalin-deprived SbE on breast cancer.     

Wogonin and related natural flavones are inhibitors of CDK9 that induce apoptosis in cancer cells by transcriptional suppression of Mcl-1

The wogonin-containing herb Scutellaria baicalensis has successfully been used for curing various diseases in traditional Chinese medicine. Wogonin has been shown to induce apoptosis in different cancer cells and to suppress growth of human cancer xenografts in vivo. However, its direct targets remain unknown. In this study, we demonstrate for the first time that wogonin and structurally related natural flavones, for example, apigenin, chrysin and luteolin, are inhibitors of cyclin-dependent kinase 9 (CDK9) and block phosphorylation of the carboxy-terminal domain of RNA polymerase II at Ser². This effect leads to reduced RNA synthesis and subsequently rapid downregulation of the short-lived anti-apoptotic protein myeloid cell leukemia 1 (Mcl-1) resulting in apoptosis induction in cancer cells. We show that genetic inhibition of Mcl-1 or CDK9 expression by siRNA is sufficient to mimic flavone-induced apoptosis. Pull-down and in silico docking studies demonstrate that wogonin directly binds to CDK9, presumably to the ATP-binding pocket. In contrast, wogonin does not inhibit CDK2, CDK4 and CDK6 at doses that inhibit CDK9 activity. Furthermore, we show that wogonin preferentially inhibits CDK9 in malignant compared with normal lymphocytes. Thus, our study reveals a new mechanism of anti-cancer action of natural flavones and supports CDK9 as a therapeutic target in oncology.     

Liquid chromatography with tandem mass spectrometry for the simultaneous determination of baicalein, baicalin, oroxylin A and wogonin in rat plasma

A rapid, sensitive and selective liquid chromatography-tandem mass spectrometric (LC-MS/MS) method for the determination of baicalein, baicalin, oroxylin A and wogonin, Scutellaria baicalensis active components in rat plasma was developed. After liquid-liquid extraction with 2-(3,4-dimethoxy-phenyl)-5,7-dihydroxy-chromen-4-one as internal standard, baicalein, baicalin, oroxylin A and wogonin were eluted from an Atlantis C(18) column within 7 min with isocratic mobile phase consisting of methanol and 0.1% formic acid (60:40, v/v). The analytes were detected using an electrospray ionization tandem mass spectrometry in the multiple reaction monitoring (MRM) mode. The standard curves were linear (r=1.000) over the concentration ranges of 5-500 ng/ml for baicalein, wogonin and oroxylin A and 5-5000 ng/ml for baicalin. The coefficients of variation and relative errors of baicalein, wogonin, oroxylin A and baicalin for intra- and inter-assay at three or four quality control (QC) levels were 0.8-6.1% and -4.0 to 5.8%, respectively. The lower limits of quantification for baicalein, wogonin, oroxylin A and baicalin were 5ng/ml using 50 microl of plasma sample. This method was successfully applied to the pharmacokinetic study of baicalein, baicalin, wogonin and oroxylin A after an intravenous administration of Scutellariae radix extract to male Sprague-Dawley rats.     

Baicalein Inhibits Progression of Gallbladder Cancer Cells by Downregulating ZFX

Baicalein, a widely used Chinese herbal medicine, has multiple pharmacological activities. However, the precise mechanisms of the anti-proliferation and anti-metastatic effects of baicalein on gallbladder cancer (GBC) remain poorly understood. Therefore, the aim of this study was to assess the anti-proliferation and anti-metastatic effects of baicalein and the related mechanism(s) on GBC. In the present study, we found that treatment with baicalein induced a significant inhibitory effect on proliferation and promoted apoptosis in GBC-SD and SGC996 cells, two widely used gallbladder cancer cell lines. Additionally, treatment with baicalein inhibited the metastasis of GBC cells. Moreover, we demonstrated for the first time that baicalein inhibited GBC cell growth and metastasis via down-regulation of the expression level of Zinc finger protein X-linked (ZFX). In conclusion, our studies suggest that baicalein may be a potential phytochemical flavonoid for therapeutics of GBC and ZFX may serve as a molecular marker or predictive target for GBC.     

The mechanisms of lipoxygenase inhibitor-induced apoptosis in human breast cancer cells

Previous experimental studies have shown that high dietary fat intake is associated with mammary carcinogenesis. In the current study, the effect of 5-LOX or 12-LOX inhibitors on human breast cancer cell proliferation and apoptosis, as well as the possible mechanisms were investigated. The LOX inhibitors, NDGA, Rev-5901, and baicalein all inhibited proliferation and induced apoptosis in MCF-7 (ER+) and MDA-MB-231 (ER-) breast cancer cell in vitro. In contrast, the LOX products, 5-HETE and 12-HETE had mitogenic effects, stimulating the proliferation of both cell lines. These inhibitors also induced cytochrome c release, caspase-9 activation, as well as downstream caspase-3, caspase-7 activation, and PARP cleavage. LOX inhibitor treatment also reduced the levels of anti-apoptotic proteins Bcl-2 and Mcl-1 and increased the levels of the pro-apoptotic protein bax. In conclusion, blockade of both 5-LOX and 12-LOX pathways induces apoptosis in breast cancer cells through the cytochrome c release and caspase-9 activation, with changes in the levels of Bcl-2 family proteins.     

Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress

Oxidative stress induces JNK activation, which leads to apoptosis through mitochondria-dependent caspase activation. However, little is known about the mechanism by which JNK alters mitochondrial function. In this study, we investigated the role of phosphorylation of myeloid cell leukemia 1 (Mcl-1), an anti-apoptotic member of the Bcl-2 family, in oxidative stress-induced apoptosis. We found that JNK phosphorylated Ser-121 and Thr-163 of Mcl-1 in response to stimulation with H(2)O(2) and that transfection of unphosphorylatable Mcl-1 resulted in an enhanced anti-apoptotic activity in response to stimulation with H(2)O(2). JNK-dependent phosphorylation and thus inactivation of Mcl-1 may be one of the mechanisms through which oxidative stress induces cellular damage.     

Bladder cancer cell viability inhibition and apoptosis induction by baicalein through targeting the expression of anti-apoptotic genes

The study was aimed to investigate the effect of baicalein, a flavonoid molecule isolated from the plant Oroxylum indicum on bladder cancer cell viability. The results revealed that baicalein treatment of T24 and 253J bladder cancer cells targeted the expression of mRNA and proteins corresponding to the anti-apoptotic genes. RT-PCR assay showed that anti-apoptotic genes were markedly over-expressed in the bladder cancer cells. Exposure of the bladder cancer cells to baicalein at 5 mg/mL doses for 72 h led to reduction in the expression of mRNA levels of antiapoptotic genes. In T24 cells, the levels of BCL2, Bcl-xL, XIAP and surviving was reduced by 65, 69, 58 and 72%, respectively. In T24 and 253J cells exposure to baicalein for 72 h resulted respectively in 39 and 46% reduction in cell viability. Baicalein treatment also induced apoptosis in the bladder cancer cells. In T24 and 253J cells baicalein treatment at 5 mg/mL for 72 h induced apoptosis in 79 and 86% cells respectively. Thus, baicalein mediated reduction in antiapoptotic gene expression inhibits viability and induces apoptosis in bladder cancer cells. Therefore, baicalein is of therapeutic importance for the development of bladder cancer treatment strategy.     

Iron-binding and anti-Fenton properties of baicalein and baicalin

Baicalein and baicalin, the major bioactive compounds found in the Chinese herb Scutellaria baicalensis, have been shown to be effective against cancer, bacterial infections and oxidative stress diseases. However, little is known about their mechanisms of action. To probe whether iron homeostasis modulation may play a role in their bioactivity, we have investigated their iron binding characteristics under physiologically relevant conditions. A 2:1 baicalein-ferrous complex was readily formed in 20 mM phosphate buffer, pH 7.2, with a binding constant ∼2-9 × 10¹¹ M⁻², whereas a 1:1 baicalein-ferric complex was formed, under the same conditions, with an apparent binding constant ∼1-3 × 10⁶ M⁻¹. Baicalein appears to bind the ferrous ion more strongly than ferrozine, a well known iron(II) chelator. Using ¹ H NMR and Zn²⁺ and Ga³⁺ as probes, the iron-binding site on baicalein was elucidated to be at the O6/O7 oxygen atoms of the A-ring. No binding was observed for baicalin under the same NMR conditions. Furthermore, baicalein strongly inhibits the Fe-promoted Fenton chemistry via a combination of chelation and radical scavenging mechanism while baicalin can provide only partial protection against radical damage. These results indicate that baicalein is a strong iron chelator under physiological conditions and hence may play a vital role in modulating the body’s iron homeostasis. Modulation of metal homeostasis and the inhibition of Fenton chemistry may be one of the possible mechanisms for herbal medicine.     

Molecular characterization of flavonoid biosynthetic genes and accumulation of baicalin,baicalein, and wogonin in plant and hairy root of Scutellaria lateriflora

Scutellaria lateriflora is well known for its medical applications because of the presence of flavanoids and alkaloids. The present study aimed to explore the molecular aspects and regulations of flavanoids. Five partial cDNAs encoding genes that are involved in the flavonoid biosynthetic pathway: phenylalanine ammonia lyase (SlPAL), cinnamate 4-hydroxylase (SlC4H), 4-coumaroyl CoA ligase (Sl4CL), chalcone synthase (SlCHS), and chalcone isomerase (SlCHI) were isolated from S. lateriflora. Organ expression analysis showed that these genes were expressed in all organs analyzed with the highest levels correlating with the richest accumulation of wogonin in the roots. Baicalin and baicalein differentially accumulated in S. lateriflora plants, with the highest concentration of baicalin and baicalein detected in the leaves and stems, respectively. Exogenous methyl jasmonate (MeJA) significantly enhanced the expression of SlCHS and SlCHI, and accumulation of baicalin (22.54 mg/g), baicalein (1.24 mg/g), and wogonin (5.39 mg/g) in S. lateriflora hairy roots. In addition, maximum production of baicalin, baicalein, and wogonin in hairy roots treated with MeJA was approximately 7.44-, 2.38-, and 2.12-fold, respectively. Light condition increased the expression level of SlCHS, the first committed step in flavonoid biosynthesis in hairy roots of S. lateriflora after 3 and 4 weeks of development compared to the dark condition. Dark-grown hairy roots contained a higher content of baicalin and baicalein than light-grown hairy roots, while light-grown hairy roots accumulated more wogonin than dark-grown hairy roots. These results may helpful for the metabolic engineering of flavonoids biosynthesis in S. lateriflora.     

HSP70 desensitizes osteosarcoma cells to baicalein and protects cells from undergoing apoptosis

Baicalein is a new drug that has shown promising anti-cancer effects against a broad spectrum of tumors. However, the potential effect on osteosarcoma cells and the mechanisms involved are still largely unknown. Resistance to chemotherapy remains a major obstacle in cancer therapy. Therefore, the aim of the present study was to investigate the anti-tumor effect of baicalein on human osteosarcoma cancer cells and the molecular mechanism involved, as well as identify possible mechanisms of drug resistance. Our results revealed that baicalein-induced apoptosis in osteosarcoma cells was via a mitochondrial pathway involving both caspase-dependent and independent mechanisms. Notably, baicalein treatment upregulated the expression of HSP70, which partially prevented human osteosarcoma cells from undergoing apoptosis. Moreover, it was revealed that HSP70 expression decreased the sensitivity of osteosarcoma cells to baicalein via activation of PI3K/AKT and MAPK/ERK pathways. These results suggest that targeting HSP70-mediated drug resistance, in combination with chemotherapy drugs, may provide novel therapeutic opportunities.     

Activation of two distinct anti-proliferative pathways, apoptosis and p38 MAP kinase-dependent cell cycle arrest, by tumor necrosis factor in human melanoma cell line A375

The proliferation of human melanoma cell line A375-6 cells is inhibited by several cytokines, including interleukin-1 (IL-1). A375-R8 cells, a subclone of A375-6, are resistant to IL-1-induced growth inhibition. The proliferation of both cell lines is inhibitable by tumor necrosis factor (TNF). In this study, we characterized the mechanisms of TNF-induced growth inhibition. TNF-induced growth inhibition in both cell lines was partially suppressed by a selective p38 mitogen-activated protein kinase (MAPK) inhibitor (SB203580), whereas a combination of SB203580 and Z-VAD-fmk, an inhibitor for a wide range of caspases, completely blocked TNF-induced growth inhibition, indicating that TNF-induced growth inhibition is mediated by both p38 MAPK and caspases. However, Z-VAD-fmk alone suppressed TNF-induced growth inhibition in A375-R8, but not A375-6, cells, suggesting that there may exist a TNF-induced anti-apoptotic mechanism in A375-6 cells which is lost or mutated in A375-R8 cells. Evidence in support of this notion includes (1) TNF-induced apoptosis only in A375-R8, but not A375-6 cells; (2) cycloheximide enabled TNF to induce apoptosis even in A375-6 cells; and (3) somatic hybrid cells between A375-6 and A375-R8 cells are resistant to TNF-induced apoptosis. Since TNF-induced NF-kappa B activation, cell cycle arrest, RB dephosphorylation, and E2F downregulation are indistinguishable in both cell lines, none of these factors is likely to be involved in the TNF-induced anti-apoptotic mechanism in A375-6 cells. Our results indicate that TNF activates two distinct anti-proliferative pathways including p38 MAPK-dependent cell cycle arrest and caspase-mediated apoptosis, as well as an anti-apoptotic mechanism in melanoma cells.