Herpes zoster mRNA vaccine induces superior vaccine immunity over licensed vaccine in mice and rhesus macaques.

Herpes zoster remains an important global health issue and mainly occurs in aged and immunocompromised individuals with an early exposure history to Varicella Zoster Virus (VZV). Although the licensed vaccine Shingrix has remarkably high efficacy, undesired reactogenicity and increasing global demand causing vaccine shortage urged the development of improved or novel VZV vaccines. In this study, we developed a novel VZV mRNA vaccine candidate (named as ZOSAL) containing sequence-optimized mRNAs encoding full-length glycoprotein E encapsulated in an ionizable lipid nanoparticle. In mice and rhesus macaques, ZOSAL demonstrated superior immunogenicity and safety in multiple aspects over Shingrix, especially in the induction of strong T-cell immunity. Transcriptomic analysis revealed that both ZOSAL and Shingrix could robustly activate innate immune compartments, especially Type-I IFN signalling and antigen processing/presentation. Multivariate correlation analysis further identified several early factors of innate compartments that can predict the magnitude of T-cell responses, which further increased our understanding of the mode of action of two different VZV vaccine modalities. Collectively, our data demonstrated the superiority of VZV mRNA vaccine over licensed subunit vaccine. The mRNA platform therefore holds prospects for further investigations in next-generation VZV vaccine development.

Selective anti-tumor activity of wogonin targeting the Warburg effect through stablizing p53.

Most cancer cells generate energy through aerobic glycolysis to enable their rapid growth and proliferation, which is a phenomenon known as Warburg effect. Inhibition of aerobic glycolysis reduces lactate and ATP generation in cancer cells, and ultimately kills tumor cells. Increasing evidence suggests that wogonin, a flavonoid isolated from Scutellaria baicalensis Georgi, exhibits potent anti-tumor effects in vivo and in vitro. However, the role of wogonin in the aerobic glycolysis of tumor cells has not yet been elucidated. In this study, the effect of wogonin on glucose uptake, lactate generation and ATP content is assessed in colon, ovarian and hepatocellular cancer cells. The results indicate that wogonin reduces glycolysis and cell proliferation in cancer cells expressing wild-type p53 but not mutated p53. Wogonin increases the expression of p53 and p53-inducible glycolysis and apoptosis regulator (TIGAR), while decreases glucose transporter 1 (GLUT1) and some key glycolytic enzymes. Expressing wild-type and mutant-type p53 in HCT116 p53-/- cells proved that the inhibitory effect of wogonin on glycolysis in cancer cells is dependent on wild type p53. Mechanistically, wogonin induced the phosphorylation and acetylation of p53 and inhibited the expression of MDM2 to enhance the stability of p53. Furthermore, wogonin suppressed the growth and glycolysis of transplanted wild-type p53 expressing A2780 cells on nude mice, but did not affect mutant-type p53 expressing HT-29 cells. In conclusion, these findings explain the broad anti-tumor effect of wogonin, and offer a novel avenue for the therapeutic strategy in cancer.

Aspafilioside B induces G2/M cell cycle arrest and apoptosis by up-regulating H-Ras and N-Ras via ERK and p38 MAPK signaling pathways in human hepatoma HepG2 cells.

We recently establish that aspafilioside B, a steroidal saponin extracted from Asparagus filicinus, is an active cytotoxic component. However, its antitumor activity is till unknown. In this study, the anticancer effect of aspafilioside B against HCC cells and the underlying mechanisms were investigated. Our results showed that aspafilioside B inhibited the growth and proliferation of HCC cell lines. Further study revealed that aspafilioside B could significantly induce G2 phase cell cycle arrest and apoptosis, accompanying the accumulation of reactive oxygen species (ROS), but blocking ROS generation with N-acetyl-l-cysteine (NAC) could not prevent G2/M arrest and apoptosis. Additionally, treatment with aspafilioside B induced phosphorylation of extracellular signal-regulated kinase (ERK) and p38 MAP kinase. Moreover, both ERK inhibitor PD98059 and p38 inhibitor SB203580 almost abolished the G2/M phase arrest and apoptosis induced by aspafilioside B, and reversed the expression of cell cycle- and apoptosis-related proteins. We also found that aspafilioside B treatment increased both Ras and Raf activation, and transfection of cells with H-Ras and N-Ras shRNA almost attenuated aspafilioside B-induced G2 phase arrest and apoptosis as well as the ERK and p38 activation. Finally, in vivo, aspafilioside B suppressed tumor growth in mouse xenograft models, and the mechanism was the same as in vitro study. Collectively, these findings indicated that aspafilioside B may up-regulate H-Ras and N-Ras, causing c-Raf phosphorylation, and lead to ERK and p38 activation, which consequently induced the G2 phase arrest and apoptosis. This study provides the evidence that aspafilioside B is a promising therapeutic agent against HCC.

Oroxylin A regulates glucose metabolism in response to hypoxic stress with the involvement of Hypoxia-inducible factor-1 in human hepatoma HepG2 cells.

Metabolic alteration in cancer cells is one of the most conspicuous characteristics that distinguish cancer cells from normal cells. In this study, we investigated the influence and signaling ways of oroxylin A affecting cancer cell energy metabolism under hypoxia. The data showed that oroxylin A remarkably reduced the generation of lactate and glucose uptake under hypoxia in HepG2 cells. Moreover, oroxylin A inhibited HIF-1α expression and its stability. The downstream targets (PDK1, LDHA, and HK II), as well as their mRNA levels were also suppressed by oroxylin A under hypoxia. The silencing or the overexpression of HIF-1α assays suggested that HIF-1α is required for metabolic effect of oroxylin A in HepG2 cells during hypoxia. Furthermore, oroxylin A could reduce the expression of complex III in mitochondrial respiratory chain, and then decrease the accumulation of ROS at moderate concentrations (0-50 µM) under hypoxia, which was benefit for its inhibition on glycolytic activity by decreasing ROS-mediated HIF-1 expression. Besides, oroxylin A didn't cause the loss of MMP under hypoxia and had no obvious effects on the expression of OXPHOS complexes, suggesting that oroxylin A did not affect mitochondrial mass at the moderate stress of oroxylin A. The suppressive effect of oroxylin A on glycolysis led to a significantly repress of ATP generation, for ATP generation mostly depends on glycolysis in HepG2 cells. This study revealed a new aspect of glucose metabolism regulation of oroxylin A under hypoxia, which may contribute to its new anticancer mechanism. © 2015 Wiley Periodicals, Inc.

III-10, a newly synthesized flavonoid, induces cell apoptosis with the involvement of reactive oxygen species-mitochondria pathway in human hepatocellular carcinoma cells.

Study of the mechanisms of apoptosis in tumor cells is an important field of tumor therapy and cancer molecular biology. We recently established that III-10, a new flavonoid with a pyrrolidinyl and a benzyl group substitution, exerted its anti-tumor effect via inducing differentiation of human U937 leukemia cells. In this study, we demonstrated that III-10 induced cell apoptosis in human hepatocellular carcinoma cells. The activation of caspase-3, caspase-9, and the increased expression ratio of Bax/Bcl-2 were detected in III-10-induced apoptosis. Z-VAD-FMK, a pan-caspase inhibitor, partly attenuated the apoptotic induction of III-10 on both HepG2 and BEL-7402 cells. Furthermore, the increase of intracellular reactive oxygen species levels and the reduction of mitochondria ΔΨm were also observed in BEL-7402 and HepG2 cells after the treatment of III-10. Pretreatment with NAC, a reactive oxygen species production inhibitor, partly attenuated the apoptosis induced by III-10 via blocking the reactive oxygen species generation. Our data also showed that III-10 induced the release of cytochrome c and AIF to cytosol followed after the reactive oxygen species accumulation. Moreover, the GSH levels and ATP generation were both inhibited after III-10 treatment. Besides, the MAPK, the downstream effect of reactive oxygen species accumulation including JNK could be activated by III-10, as well as the inactivation of ERK. Collectively, the generation of reactive oxygen species might play an crucial role in III-10-induced mitochondrial apoptosis pathway, provided more stubborn evidence for III-10 as a potent anticancer therapeutic candidate.

FV-429 Induced Apoptosis Through ROS-Mediated ERK2 Nuclear Translocation and p53 Activation in Gastric Cancer Cells.

Following our previous finding which revealed that FV-429 induces apoptosis in human hepatocellular carcinoma HepG2 cells, in this study, we found that FV-429 could also induce apoptosis in human gastric cancer cells. Firstly, FV-429 inhibited the viability of BGC-823 and MGC-803 cells with IC50 values in the range of 38.10 ± 6.28 and 31.53 ± 6.84 µM for 24 h treatment by MTT-assay. Secondly, FV-429 induced apoptosis in BGC-823 and MGC-803 cells through the mitochondrial-mediated pathway, showing an increase in Bax/Bcl-2 ratios, and caspase-9 activation, without change in caspase-8. Further research revealed that the mitogen-activated protein kinases, including c-Jun N-terminal kinase, extracellular regulated kinase, and p38 mitogen-activated protein kinase, could be activated by FV-429-induced high level ROS. Moreover, FV-429 also promoted the ERK2 nuclear translocation, resulting in the co-translocation of p53 to the nucleus and increased transcription of p53-regulated proapoptotic genes. FV-429 significantly inhibited the nude mice xenograft tumors growth of BGC-823 or MGC-803 cells in vivo.

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.

Wogonin suppresses melanoma cell B16-F10 invasion and migration by inhibiting Ras-medicated pathways.

The patients diagnosed with melanoma have a bad prognosis for early regional invasion and distant metastases. Wogonin (5,7-dihydroxy-8-methoxyflavone) is one of the active components of flavonoids that extracts from Scutellariae radix. Several previous studies reported that wogonin possesses antitumor effect against leukemia, gastrointestinal cancer and breast cancer. In this study, we used melanoma cell B16-F10 to further investigate the anti-invasive and anti-migratory activity of wogonin. Our date showed that wogonin caused suppression of cell migration, adhesion, invasion and actin remodeling by inhibiting the expression of matrix metalloproteinase-2 and Rac1 in vitro. Wogonin also reduced the number of the tumor nodules on the whole surface of the lung in vivo. Furthermore, the examination of mechanism revealed that wogonin inhibited Extracellular Regulated protein Kinases and Protein Kinase B pathways, which are both medicated by Ras. Insulin-like growth factor-1-induced or tumor necrosis factor-α-induced invasion was also inhibited by wogonin. Therefore, the inhibitory mechanism of melanoma cell invasion by wogonin might be elucidated.

Oroxylin A inhibits ATRA-induced IL-6 expression involved in retinoic acid syndrome by down-regulating CHOP.

Production of IL-6 constituted the major cause of death in the ATRA trial called retinoic acid syndrome (RAS). LAP and LIP are active and inactive isoforms of C/EBPß, respectively. Inactive LIP dimerized with LAP to eliminate its activity. Following treatment with ATRA, CHOP expression was increased and dimerized with LIP more preferentially than LAP to rescue function of LAP. Oroxylin A has been reported to activate CHOP, a key mediator of unfolded protein response (UPR) pathway, and resulted in apoptosis. Interestingly, we found that low concentration of oroxylin A (≦ 40 µM) showed no apoptosis effect on NB4 and HL-60 cells and decreased the CHOP protein level via promoting its degradation. MG132 was utilized to conform the effect of oroxylin A on degrading CHOP. Our results showed that oroxylin A decreased the level of IL-6 secretion of NB4 cells with or without ATRA treatment while the effect was eliminated by C/EBPß siRNA. We conclude that oroxylin A possessed abilities of inhibiting the ATRA-induced IL-6 production via modulation of LAP/LIP/CHOP in leukemia cell lines, which could providing a therapeutic strategy for RAS.

LL-202, a newly synthesized flavonoid, inhibits tumor growth via inducing G(2)/M phase arrest and cell apoptosis in MCF-7 human breast cancer cells in vitro and in vivo.

We recently established that LL-202, a newly synthesized flavonoid, exhibited obvious anticancer effects against human breast cells in vivo and in vitro. The underlying mechanism of its anticancer activity remains to be elucidated. In this study, we demonstrated that LL-202 inhibited the growth and proliferation of human breast cancer MCF-7 cells in a concentration and time-dependent manner. We reported that LL-202 induced both mitochondrial- and death-receptor-mediated apoptosis, which were characterized by the dissipation of mitochondrial membrane potential (ΔΨm), cytochrome c (Cyt c) release from mitochondria to cytosol, the activation of several caspases and induction of poly (ADP-ribose) polymerase (PARP) and Bid cleavage. N-acetylcysteine (NAC), a general ROS scavenger, partly blocked the LL-202-induced ROS levels and apoptosis. In addition, LL-202 induced arrest in cell cycle progression at G2/M phase in MCF-7 cells. After the treatment with LL-202, the expression of cell cycle-related proteins, such as cyclin B1, cyclin A, and p-CDK1 (Thr161) were down-regulated, whereas the expression of p21(WAF1/Cip1) and p-CDK1 (Thr14/Tyr15) were up-regulated. Finally, in vivo studies, LL-202 significantly suppressed the growth of MCF-7 breast cancer xenograft tumors in a dose-dependent manner with low systemic toxicity. In conclusion, the results showed that LL-202 had significant anticancer effects against human breast cells via the induction of apoptosis and G2/M phase arrest and it may be a novel anticancer agent for treatment of breast cancer.

Oroxylin A inhibits hypoxia-induced invasion and migration of MCF-7 cells by suppressing the Notch pathway.

Tumor invasion and migration obstructs the treatment and prognosis of cancer. In this research, we investigated the effect of oroxylin A, a natural compound extracted from Scutellaria radix, the root of Scutellaria baicalensis, on inhibition of the invasion and migration of three different tumor cell lines: MCF-7, DU145, and HepG2. The results suggested that oroxylin A could inhibit hypoxia-induced migration and invasion of the three cell lines mentioned above. To study the detailed mechanisms, studies were carried out on MCF-7 cells and it was found that oroxylin A could regulate the expression of related markers in MCF-7 cells including E-cadherin, N-cadherin, and Vimentin. It was also found that oroxylin A inhibited the hypoxia-induced invasion and migration of MCF-7 cells by suppressing the Notch pathway. Oroxylin A inhibited N1ICD translocating to the nucleus and binding to epithelial-mesenchymal transition-related transcription factor Snail, thus suppressing the invasion and migration of MCF-7 cells. Therefore, oroxylin A is expected to be a promising candidate for antimetastasis treatment through suppression of the hypoxia-induced Notch pathway.

Wogonin induced G1 cell cycle arrest by regulating Wnt/ß-catenin signaling pathway and inactivating CDK8 in human colorectal cancer carcinoma cells.

Wogonin, a naturally occurring mono-flavonoid, has been reported to have tumor therapeutic potential and good selectivity both in vitro and in vivo. Herein, we investigated the anti-proliferation effects and associated mechanisms of wogonin in human colorectal cancer in vitro. The flow-cytometric analysis showed that wogonin induced a G1 phase cell cycle arrest in HCT116 cells in a concentration- and time-dependent manner. Meanwhile, the cell cycle-related proteins, such as cyclin A, E, D1, and CDK2, 4 were down-regulated in wogonin-induced G1 cell cycle arrest. Furthermore, we showed that the anti-proliferation and G1 arrest effect of wogonin on HCT116 cells was associated with deregulation of Wnt/ß-catenin signaling pathway. Wogonin-treated cells showed decreased intracellular levels of Wnt proteins, and activated degradation complex to phosphorylated and targeted ß-catenin for proteasomal degradation. Wogonin inhibited ß-catenin-mediated transcription by interfering in the transcriptional activity of TCF/Lef, and repressing the kinase activity of CDK8 which has been considered as an oncogene involving in the development of colorectal cancers. Moreover, CDK8 siRNA-transfected HCT116 cells showed similar results to wogonin treated cells. Thus, our data suggested that wogonin induced anti-proliferation and G1 arrest via Wnt/ß-catenin signaling pathway and it can be developed as a therapeutic agent against human colorectal cancer.

HQS-3, a newly synthesized flavonoid, possesses potent anti-tumor effect in vivo and in vitro.

HQS-3 is a newly baicalein derivative with a benzene substitution. We investigated the anticancer effect of HQS-3 in vivo and in vitro. HQS-3 significantly decreased tumor growth in mice inoculated with Heps and HepG2 cells; and had little influence on the state and weight of animals. After treatment with 20 mg/kg HQS-3, the inhibitory rate of tumor weight in mice inoculated with Heps and HepG2 cells were 63.62% and 68.03%, respectively. Meanwhile, HQS-3 inhibited the viability of various kinds of tumor cells with IC50 values in the range of 22.98-54.32 µM after 48 h treatment measured by MTT-assay. HQS-3 remarkably inhibited viability of hepatoma cells in a concentration- and time-dependent manner and induced apoptosis in HepG2 cells by DAPI staining and Annexin V/PI double staining. The apoptosis-induction effect of HQS-3 was attributed to its ability to modulate the activity of caspase-9, caspase-3 and PARP. Moreover, the expression of bax protein was increased while the bcl-2 protein was decreased, leading to an increase in Bax/Bcl-2 ratio. The accumulation of ROS induced by HQS-3 in HepG2 cells was also observed. The further results suggested that HQS-3 induced mitochondrial-mediated apoptosis by increasing ROS level and inhibiting the expression of anti-oxidative protein SOD2. HQS-3 exerted anti-tumor activity both in vitro and in vivo via inducing tumor cells apoptosis, and these results suggested that it deserves further investigation as a novel chemotherapy for human tumors.

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.

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.

Wogonin induced cytotoxicity in human hepatocellular carcinoma cells by activation of unfolded protein response and inactivation of AKT.

AIM: To investigate the potential anticancer effects of the natural flavonoid wogonin on human hepatocellular carcinoma (HCC) cells and tumor xenografts and the contribution of the unfolded protein response (UPR) and AKT pathways to the cytotoxicity of wogonin. METHODS: The HCC cell lines HepG2, SMMC-7721 and Hep3B were treated with wogonin. 3-(4 5-Dimethylthiazol-2-yl)-2 5-diphenyltetrazolium bromide assays were used to evaluate the cell viability. Flow cytometry assays were used to identify the cell death types and measure the concentrations of intracellular H2 O2 and Ca(2+) . Western blotting assays were used to detect the protein expression levels of members in the UPR and AKT pathways. Relative quantitative real-time polymerase chain reaction assays were used to analysis the mRNA expression levels of chop and trb3. Furthermore, the male BALB/c nude mice with SMMC-7721 xenografts were treated with wogonin. The tumor volume, tumor weight and bodyweight were monitored during the tumorigenicity assays. RESULTS: Wogonin significantly inhibited the viability of HCC cells by inducing apoptosis and necrosis. This cytotoxicity was at least partially attributed to the activation of the UPR pathway and consequent inactivation of AKT signaling, which resulted from the production of intracellular H2 O2 and causal release of endoplasmic reticulum Ca(2+) . Moreover, wogonin evidently repressed the growth of xenografts but slightly influenced the bodyweight of mice. CONCLUSION: Wogonin is a prospect for improving the systemic chemotherapy strategy on HCC by concurrently rectifying the aberrant UPR and AKT signaling pathways, which are crucial to the biology of HCC.