Yuxin Zhou

Oroxylin A induces dissociation of hexokinase II from the mitochondria and inhibits glycolysis by SIRT3-mediated deacetylation of cyclophilin D in breast carcinoma

Oroxylin A is a major active component of the Chinese traditional medicinal plant Scutellaria baicalensis Georgi, which has been reported as a potential anticancer drug. We demonstrated that, Oroxylin A inhibited the glycolysis and the binding of hexokinase II (HK II) with mitochondria in human breast carcinoma cell lines, which was dependent on sirtuin-3 (SIRT3). The level of SIRT3 in mitochondria was increased by Oroxylin A. Then SIRT3 deacetylated cyclophilin D, diminished its peptidyl-prolyl cis-trans isomerase activity and induced its dissociation from the adenine nucleotide translocator. Finally, SIRT3-induced inactivation of cyclophilin D resulted in the detachment of mitochondrial HK II and the inhibition of glycolysis. These results have important implications for the metabolism reprogramming effect and the susceptibility to Oroxylin A-induced mitochondrial cytotoxicity through the regulation of SIRT3 in breast carcinoma.

Wogonin inhibits tumor angiogenesis via degradation of HIF-1α protein

Wogonin, a plant-derived flavone, has been shown recently to have antitumor effects. However, the mechanisms that wogonin inhibits tumor angiogenesis are not well known. In this study, we investigated the effects of wogonin on expression of hypoxia-inducible factor-1α (HIF-1α) and secretion of vascular endothelial growth factor (VEGF) in tumor cells. We found that wogonin decreased the expression of HIF-1α by affecting its stability and reduced the secretion of VEGF, which suppressed angiogenesis in cancer. Wogonin promoted the degradation of HIF-1α by increasing its prolyl hydroxylation, which depended on prolyl hydroxylase (PHD) and the von Hippel-Lindau tumor suppressor (VHL). Intriguingly, wogonin impeded the binding between heat-shock protein 90 (Hsp90) and HIF-1α. In addition, wogonin down-regulated the Hsp90 client proteins EGFR, Cdk4 and survivin, but did not affect the level of Hsp90. Wogonin also increased ubiquitination of HIF-1α and promoted its degradation in proteasome. We also found that wogonin could inhibit nuclear translocation of HIF-1α. Electrophoresis mobility shift assay (EMSA) showed that wogonin decreased the binding activity of exogenous consensus DNA oligonucleotide with HIF-1α in nuclear extracts from MCF-7 cells. Chromatin immunoprecipitation (ChIP) assay also revealed that HIF-1α directly binded to endogenous hypoxia-responsive element (HRE) and this binding was significantly decreased in MCF-7 cells treated with wogonin. Preliminary results indicated in vivo activity of wogonin against xenograft-induced angiogenesis in nude mice. Taken together, the results suggested that wogonin was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of wogonin against cancers.

Wogonin reverses multi-drug resistance of human myelogenous leukemia K562/A02 cells via downregulation of MRP1 expression by inhibiting Nrf2/ARE signaling pathway.

Constitutive NF-E2-related factor 2 (Nrf2) activation has been recently reported to play a pivotal role in enhancing cell survival and resistance to anticancer drugs in many tumors. Previously, much effort has been devoted to the investigation of blocking Nrf2 function in cultured cells and cancer tissues, but few researches have been undertaken to evaluate the precise mechanism of flavonoids-induced sensitivity by inhibiting Nrf2. In this study, we investigated the reversal effect of Wogonin, a flavonoid isolated from the root of Scutellaria baicalensis Georgi, in resistant human myelogenous leukemia. Data indicated that Wogonin had strong reversal potency by inhibiting functional activity and expression of MRP1 at both protein and mRNA in adriamycin (ADR)-induced resistant human myelogenous leukemia K562/A02 cells. Consequently, the inhibition of MRP1 by Wogonin was dependent on Nrf2 through the decreased binding ability of Nrf2 to antioxidant response element (ARE). Further research revealed Wogonin modulated Nrf2 through the reduction of Nrf2mRNA at transcriptional processes rather than RNA degradation, which is regulated by the PI3K/Akt pathway. Moreover, DNA-PKcs was found to be involved in the Wogonin-induced downregulation of Nrf2 mRNA at transcriptional levels. In summary, these results clearly demonstrated the effectiveness of using Wogonin via inhibiting Nrf2 to combat chemoresistance and suggested that Wogonin can be developed into an efficient natural sensitizer for resistant human myelogenous leukemia.

Two p53-related metabolic regulators, TIGAR and SCO2, contribute to oroxylin A-mediated glucose metabolism in human hepatoma HepG2 cells

Metabolic alteration in cancer cells is one of the most conspicuous characteristics that distinguish cancer cells from normal cells. Many studies suggest that several underlying mechanisms lead to the Warburg effect (increased aerobic glycolysis) during cancer development. Here, we explored how oroxylin A affected the glycolytic metabolism in cancer cells and the underlying mechanism involved in this process. Our data revealed that both oroxylin A and adriamycin could inhibit lactate generation and glucose uptake in HepG2 cells at mild concentrations, without causing robust cell apoptosis. Oroxylin A has exerted little influence on the oxygen consumption, whereas adriamycin decreased oxygen consumption in a concentration-dependent manner. Moreover, oroxylin A could increase protein and mRNA expression of TP53-induced glycolysis and apoptosis regulator (TIGAR) and synthesis of cytochrome c oxidase 2 (SCO2), which are the key metabolic modulators regulated by p53. Meanwhile adriamycin could increase protein and mRNA expression of TIGAR and SCO2, but decrease that of phosphoglycerate mutase (PGM). Oroxylin A and adriamycin also modulated the stability and activity of p53 through inducing phosphorylation of p53 at Ser15 and suppressing the expression of MDM2. Furthermore, p53 siRNA and p53 inhibitor assay in wild-type p53 HepG2 cells both revealed the key role of p53 in oroxylin A and adriamycin-mediated glycolytic metabolism regulation. Transfecting wt p53 plasmid to p53-deficient H1299 cells could inverse some of the metabolic characteristics regulated by oroxylin A. This study revealed a new aspect of glucose metabolism regulation of oroxylin A, which may contribute to its new anticancer mechanism.

Wogonin suppresses human alveolar adenocarcinoma cell A549 migration in inflammatory microenvironment by modulating the IL-6/STAT3 signaling pathway.

Increasing evidence from various clinical and experimental studies has demonstrated that the inflammatory microenvironment facilitates tumor metastasis. Clinically, it will be a promising choice to suppress tumor metastasis by targeting inflammatory microenvironment. Our previous studies have demonstrated that wogonin (a bioflavonoid isolated from the traditional Chinese medicine of Huang‐Qin) possesses the anti‐metastatic and anti‐inflammatory activity, but we have little idea about its efficacy on inflammatory‐induced tumor metastasis and the mechanism underlying it. In this study, we focused on epithelial mesenchymal transition (EMT), the first step of tumor metastasis, to evaluate the effects of wogonin on tumor metastasis in inflammatory microenvironment. We found that wogonin inhibited THP‐1 conditioned‐medium‐ (CM‐) and IL‐6‐induced EMT by inactivating STAT3 signal. And in wogonin‐treated A549 cells which pretreated with THP‐1 CM or IL‐6, the expression level of E‐cadherin, an EMT negative biomarker, increased while that of N‐cadherin, Vimentin, and EMT‐related transcription factors including Snail and Twist decreased. Moreover, wogonin inhibited IL‐6‐induced phosphorylation of STAT3, prevented p‐STAT3 dimer translocation into the nucleus, and suppressed the DNA‐binding activity of p‐STAT3. Interestingly, similar results were obtained in the tumor xenografts mice, including downregulation of p‐STAT3, N‐cadherin, and Vimentin while up‐regulation of E‐cadherin. Wogonin also inhibit the metastasis of A549 cells in vivo. Taken all data together, we concluded that wogonin suppresses tumor cells migration in inflammatory microenvironment by inactivating STAT3 signal.

Oroxylin A sensitizes non-small cell lung cancer cells to anoikis via glucose-deprivation-like mechanisms: c-Src and hexokinase II

BACKGROUND Cellular metabolism, particularly glycolysis, is altered during the metastatic process and is highly associated with tumor progression and apoptosis resistance. Oroxylin A, a natural plant flavonoid, exhibits chemopreventive and therapeutic anti-inflammatory and anticancer potential. However, the anticancer effects of oroxylin A on non-small cell lung carcinoma (NSCLC) remain poorly understood. METHODS In vitro studies were performed using 2D and 3D conditions. The effects on anoikis-sensitization and glycolysis-inhibition of oroxylin A in human non-small cell lung cancer A549 cells were examined. In vivo murine lung metastasis experiments were utilized to assess the anti-metastatic capacity of oroxylin A. RESULTS ROS-mediated activation of c-Src following detachment caused anoikis resistance in A549 cells. Oroxylin A sensitized A549 cells to anoikis by inactivating the c-Src/AKT/HK II pathway in addition to inducing the dissociation of HK II from mitochondria. Prior to sensitizing A549 cells to anoikis, oroxylin A decreased the ATP level and inhibited glycolysis. Furthermore, oroxylin A inhibited lung metastasis of A549 cells in vivo in nude mice. CONCLUSIONS Oroxylin A sensitized anoikis, which underlies distinct glucose-deprivation-like mechanisms that involved c-Src and HK II. GENERAL SIGNIFICANCE The findings in this study indicated that oroxylin A could potentially be utilized in the development of improved metastatic cancer treatments.

Oroxylin A inhibits glycolysis-dependent proliferation of human breast cancer via promoting SIRT3-mediated SOD2 transcription and HIF1α destabilization.

Alterations of cellular metabolism play a central role in the development and progression of cancer. Oroxylin A, an active flavonoid of a Chinese traditional medicinal plant, was previously shown to modulate glycolysis in cancer cells. However, the mechanism by which oroxylin A regulates glycolysis is still not well defined. Here, we show that oroxylin A inhibits glycolysis in breast cancer cells via the Sirtuin 3 (SIRT3)-mediated destabilization of hypoxia-inducible factor 1α (HIF1α), which controls glycolytic gene expression. Oroxylin A promotes superoxide dismutase (SOD2) gene expression through SIRT3-regulated DNA-binding activity of FOXO3a and increases the activity of SOD2 by promoting SIRT3-mediated deacetylation. In vivo, oroxylin A inhibits the growth of transplanted human breast tumors associated with glycolytic suppression. These data indicate that oroxylin A inhibits glycolysis-dependent proliferation of breast cancer cells, through the suppression of HIF1α stabilization via SIRT3 activation, providing preclinical information for the cancer therapies of SIRT3 stimulation.

Oroxylin A promotes PTEN-mediated negative regulation of MDM2 transcription via SIRT3-mediated deacetylation to stabilize p53 and inhibit glycolysis in wt-p53 cancer cells

Introductionp53 plays important roles in regulating the metabolic reprogramming of cancer, such as aerobic glycolysis. Oroxylin A is a natural active flavonoid with strong anticancer effects both in vitro and in vivo.Methodswt-p53 (MCF-7 and HCT116 cells) cancer cells and p53-null H1299 cancer cells were used. The glucose uptake and lactate production were analyzed using Lactic Acid production Detection kit and the Amplex Red Glucose Assay Kit. Then, the protein levels and RNA levels of p53, mouse double minute 2 (MDM2), and p53-targeted glycolytic enzymes were quantified using Western blotting and quantitative polymerase chain reaction (PCR), respectively. Immunoprecipitation were performed to assess the binding between p53, MDM2, and sirtuin-3 (SIRT3), and the deacetylation of phosphatase and tensin homolog (PTEN). Reporter assays were performed to assess the transcriptional activity of PTEN. In vivo, effects of oroxylin A was investigated in nude mice xenograft tumor-inoculated MCF-7 or HCT116 cells.ResultsHere, we analyzed the underlying mechanisms that oroxylin A regulated p53 level and glycolytic metabolism in wt-p53 cancer cells, and found that oroxylin A inhibited glycolysis through upregulating p53 level. Oroxylin A did not directly affect the transcription of wt-p53, but suppressed the MDM2-mediated degradation of p53 via downregulating MDM2 transcription in wt-p53 cancer cells. In further studies, we found that oroxylin A induced a reduction in MDM2 transcription by promoting the lipid phosphatase activity of phosphatase and tensin homolog, which was upregulated via sirtuin3-mediated deacetylation. In vivo, oroxylin A inhibited the tumor growth of nude mice-inoculated MCF-7 or HCT116 cells. The expression of MDM2 protein in tumor tissue was downregulated by oroxylin A as well.ConclusionsThese results provide a p53-independent mechanism of MDM2 transcription and reveal the potential of oroxylin A on glycolytic regulation in both wt-p53 and mut-p53 cancer cells. The studies have important implications for the investigation on anticancer effects of oroxylin A, and provide the academic basis for the clinical trial of oroxylin A in cancer patients.

UCP2-Related Mitochondrial Pathway Participates in Oroxylin A-Induced Apoptosis in Human Colon Cancer Cells

Oroxylin A is a flavonoid extracted from the root of Scutellaria baicalensis Georgi. Our previous research demonstrated that oroxylin A have various anti‐tumor effects including apoptosis, cell cycle arrest, drug‐resistant reversion, and others. This paper explores the mechanism how oroxylin A induce apoptosis by regulating uncoupling protein 2 (UCP2) in human colon cancer cells. We found that the inhibition of UCP2 by UCP2 siRNA significantly increased the sensitivity of cells to drugs, reactive oxygen species (ROS) generation and the opening of mitochondrial permeability transition pore (MPTP) of CaCo‐2 cells. We also found that UCP2 inhibition could lead to ROS‐mediated MPTP activation. Furthermore, we demonstrated that oroxylin A triggered MPTP‐dependent pro‐apoptotic protein release from mitochondria to matrix and then induced apoptotic cascade by inhibiting UCP2. Intriguingly, the inhibition of UCP2 by oroxylin A was able to block Bcl‐2 translocation to the mitochondria, keeping MPTP at open‐state. In conclusion, we have demonstrated that UCP2 plays a key role in mitochondrial apoptotic pathway; UCP2s inhibition by oroxylin A triggers the MPTP opening, and promotes the apoptosis in CaCo‐2 cells. J. Cell. Physiol. 230: 1054–1063, 2015.

GL-V9, a new synthetic flavonoid derivative, ameliorates DSS-induced colitis against oxidative stress by up-regulating Trx-1 expression via activation of AMPK/FOXO3a pathway

GL-V9, a new synthesized flavonoid derivative, has been reported to possess anti-cancer properties in our previous studies. Uncontrolled overproduction of reactive oxygen species (ROS) has been implicated in oxidative damage of inflammatory bowel disease (IBD). In this study, we aimed to investigate the protective effect of GL-V9 against dextran sulfate sodium (DSS)-induced colitis. GL-V9 attenuated DSS-induced body weight loss, colon length shortening and colonic pathological damage. GL-V9 also inhibited inflammatory cells infiltration and decreased myeloperoxidase (MPO) and inducible nitric oxide synthase (iNOS) activities. Moreover, GL-V9 inhibited ROS and malondialdehyde (MDA) generation, but enhanced superoxide dismutase (SOD), glutathione (GSH) and total antioxidant capacity. GL-V9 reduced pro-inflammatory cytokines production in serum and colon as well. Mechanically, GL-V9 could increase Trx-1 via activation of AMPK/FOXO3a to suppress DSS-induced colonic oxidative stress. Furthermore, GL-V9 decreased pro-inflammatory cytokines and ROS production and increased the antioxidant defenses in the mouse macrophage cells RAW264.7 by promoting Trx-1 expression. In conclusion, our study demonstrated that GL-V9 attenuated DSS-induced colitis against oxidative stress by up-regulating Trx-1 via activation of AMPK/FOXO3a pathway, suggesting that GL-V9 might be a potential effective drug for colitis.