Acevaltrate promotes apoptosis and inhibits proliferation by suppressing HIF-1α accumulation in cancer cells.

Acevaltrate is a natural product isolated from the roots of Valeriana glechomifolia F.G.Mey. (Valerianaceae) and has been shown to exhibit anti-cancer activity. However, the mechanism by which acevaltrate inhibits tumor growth is not fully understood. We here demonstrated the effect of acevaltrate on hypoxia-inducible factor-1α (HIF-1α) expression. Acevaltrate showed a potent inhibitory activity against HIF-1α induced by hypoxia in various cancer cells. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently. Further analysis revealed that acevaltrate inhibited HIF-1α protein synthesis and promoted degradation of HIF-1α protein, without affecting the expression level of HIF-1α mRNA. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by acevaltrate. In addition, acevaltrate promoted apoptosis and inhibited proliferation, which was potentially mediated by suppression of HIF-1α. We also found that acevaltrate administration inhibited tumor growth in mouse xenograft model. Taken together, these results suggested that acevaltrate was a potent inhibitor of HIF-1α and provided a new insight into the mechanisms of acevaltrate against cancers.

Digitoxin promotes apoptosis and inhibits proliferation and migration by reducing HIF-1α and STAT3 in KRAS mutant human colon cancer cells.

Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor. New treatment options are needed to improve survival in patients with KRAS mutated colorectal cancer. Digitoxin is a cardiotonic drug, which has been demonstrated to exhibit anticancer effects in a number of cancers. However, the anticancer mechanisms of digitoxin in KRAS mutant human colon cancer cells remain elusive. Our result demonstrated that digitoxin but not cetuximab markedly decreased the expression of hypoxia-inducible factor-1α (HIF-1α), signal transducer and activator of transcription 3 (STAT3) and p-STAT3 protein in KRAS mutant colon cancer cells. Further analysis revealed that digitoxin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. The phosphorylation levels of ribosomal protein S6 kinase (p70S6K) and eIF4E binding protein-1 (4E-BP1) were significantly suppressed by digitoxin. Digitoxin inhibited the expression and activation of STAT3 through upregulation of phosphatase and tensin homolog deleted on chromosome ten (PTEN), SHP1 and protein inhibitors of activated STAT3 (PIAS3) and direct binding to STAT3. Meanwhile, digitoxin inhibited HIF-1α in STAT3-independent manner in KRAS mutant colon cancer cells. Moreover, digitoxin promoted apoptosis and inhibited proliferation and migration, which was potentially mediated by suppression of HIF-1α and STAT3. We also found that digitoxin administration inhibited tumor growth in a mouse xenograft model. Taken together, our findings highlight the therapeutic potential of digitoxin for the treatment of cetuximab-resistant human colon cancer.

Progress of cationic gene delivery reagents for non-viral vector.

Gene delivery systems play a vital role in gene therapy and recombinant protein production. The advantages of using gene delivery reagents for non-viral vector include the capacity to accommodate a large packaging load and their low or absent immunogenicity. Furthermore, they are easy to produce at a large scale and preserve. Gene delivery reagents for non-viral vector are commonly used for transfecting a variety of cells and tissues. It is mainly composed of liposomes and non-liposome cationic polymers. According to the different head structures used, the non-viral cationic transfection reagents include a quaternary ammonium salt, amine, amino acid or polypeptide, guanidine salt, and a heterocyclic ring. This article summarizes these approaches and developments of types and components of transfection reagents and optimization of gene delivery. The optimization of mammalian cell transient recombinant protein expression system and cationic reagents for clinical or clinical trials are also discussed.

Effects of different 2A peptides on transgene expression mediated by tricistronic vectors in transfected CHO cells.

Multicistronic vectors can increase transgene expression and decrease the imbalance of gene expression in the Chinese hamster ovary (CHO) cell expression system. Small, self-cleaving 2A peptides have a high cleavage efficiency and are essential for constructing high-expression multicistronic vectors. In this study, we investigated the effects of two different 2A peptides on transgene expression in CHO cells via their mediating action on tricistronic vectors. The enhanced green fluorescent protein (eGFP) and red fluorescent protein (RFP) genes were linked by the porcine teschovirus-1 (P2A) and Thosea asigna virus (T2A) peptides in a multicistronic vector. We transfected CHO cells with these vectors and screened for the presence of blasticidin-resistant colonies. Flow cytometry and real-time quantitative PCR (qPCR) were used to detect the expression levels of eGFP and RFP and the copy numbers of stably transfected cells. The results showed that P2A could enhance eGFP and RFP expression by 1.48- and 1.47-fold, respectively, compared to T2A. The expression levels of the genes were not proportional to their copy numbers. In conclusion, we found that P2A can effectively drive transgene expression in CHO cells and a potent 2A peptide can be used for recombinant protein production in the CHO cell system.

Dictamnine promotes apoptosis and inhibits epithelial-mesenchymal transition, migration, invasion and proliferation by downregulating the HIF-1α and Slug signaling pathways.

Dictamnine (DTM) is a natural alkaloid isolated from the root of Dictamnus dasycarpus Turcz and has been shown to exhibit multiple biological functions, including anti-inflammatory, antifungal, anti-angiogenic and anticancer activity. However, the mechanisms by which dictamnine inhibits tumor growth are not fully understood. In this study, we investigated the effectiveness of dictamnine as a treatment for cancer and to identify the underlying mechanisms of its anticancer activity. Here, dictamnine showed the potent inhibitory activity against HIF-1α and Slug activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α and Slug protein in a dose-dependent manner. Further analysis revealed that dictamnine inhibited HIF-1α protein synthesis, without affecting its degradation. Our results demonstrated that dictamnine reduced HIF-1α protein synthesis by downregulating the mTOR/p70S6K/eIF4E and MAPK pathways, and reduced the expression of Slug by inhibiting the GSK-3ß/Slug signaling pathway. Moreover, epithelial-mesenchymal transition (EMT) was inhibited in dictamnine-treated tumors by downregulation of HIF-1α and Slug, as reflected by the upregulation of E-cadherin and Occludin, and the downregulation of N-cadherin and Vimentin. Phenomenological experiments showed that dictamnine reduced migration and invasion, inhibited HCT116 cell proliferation and promoted HCT116 cell apoptosis by downregulating HIF-1α and Slug. In vivo studies further confirmed that dictamnine treatment caused significant inhibition of tumor growth in a xenograft tumor model. These findings suggest that dictamnine is a potent cancer inhibitor, providing a rationale for anticancer pathway-targeted therapy.

Zinc finger protein 91 positively regulates the production of IL-1ß in macrophages by activation of MAPKs and non-canonical caspase-8 inflammasome.

BACKGROUND AND PURPOSE: IL-1ß is a cytokine of critical importance in inflammatory, infectious and autoimmune diseases. Zinc finger protein 91 (ZFP91) has been reported to be involved in multiple biological processes. Here, we identified a previously unknown role for ZFP91 in the production of biologically active IL-1ß and investigated the underlying mechanisms of its effects. EXPERIMENTAL APPROACH: In vitro, the underlying mechanisms of ZFP91 at inhibiting the expression of IL-1ß were investigated by ELISA, RT-PCR, Western blotting, immunoprecipitation and immunofluorescence assays. In vivo, colitis was induced by giving 4% dextran sulfate sodium (DSS) p.o. in drinking water for 5 days. Peritonitis was induced by injecting 700 µg alum i.p. for 12 h. KEY RESULTS: ZFP91 activated the non-canonical caspase-8 inflammasome, which resulted in robust IL-1ß secretion. Using an immunoprecipitation assay and immunofluorescence assay, we found that ZFP91 promoted the assembly of the non-canonical caspase-8 inflammasome complex. Moreover, ZFP91 enhanced the activation of ERK, p38 MAPK and JNK in macrophages. In addition, our data demonstrate that the synthesis of pro-IL-1ß is dependent on activation of these MAPK signalling pathways. In vivo experiments, the symptoms and colonic inflammation associated with DSS-induced colitis were ameliorated in mice deficient in ZFP91. Furthermore, the inflammation in alum-induced peritonitis was also attenuated in mice deficient in ZFP91. CONCLUSIONS AND IMPLICATIONS: Our research describes a mechanism by which ZFP91 promotes production of IL-1ß under physiological conditions and suggests that ZFP91 may be a promising therapeutic target for intervention in inflammatory, infectious and autoimmune-related diseases.

Fraxinellone has anticancer activity in vivo by inhibiting programmed cell death-ligand 1 expression by reducing hypoxia-inducible factor-1α and STAT3.

Dictamnus dasycarpus is a traditional Chinese medicine thathas been commonly used in the treatment of cancer. Fraxinellone is a natural product isolated from the D. dasycarpus plant, which has been shown to exhibit neuroprotective and anti-inflammatory activities. However, whether fraxinellone exerts anticancer effects and the mechanisms by which it may inhibit tumor growth remain unknown. Here, we found that fraxinellone, in a dose-dependented manner, inhibited the expression of programmed cell death ligand-1 (PD-L1), which plays a pivotal role in tumorigenesis. It was subsequently shown that fraxinellone reduced HIF-1α protein synthesis via the mTOR/p70S6K/eIF4E and MAPK pathways. It also inhibited activation of STAT3 via the JAK1, JAK2, and Src pathways. Immunoprecipitation and western blotting assays showed that fraxinellone inhibited PD-L1 expression by reducing STAT3 and HIF-1α cooperatively. Flow cytometry, colony formation, and EdU incorporation assays demonstrated that fraxinellone inhibited cell proliferation through suppression of PD-L1. Tube formation, migration, and invasion assays showed that fraxinellone inhibits angiogenesis by suppressing PD-L1. In vivo studies further supported anticancer role for fraxinellone, demonstrating that fraxinellone treatment inhibited the growth of tumor xenografts. We concluded that fraxinellone inhibits PD-L1 expression by downregulating the STAT3 and HIF-1α signaling pathways, subsequently inhibiting proliferation and angiogenesis in cancer cells. These studies reveal previously unknown characteristics of fraxinellone and provide new perspectives into the mechanism of cancer inhibition of the compound.

Chelidonine inhibits TNF-α-induced inflammation by suppressing the NF-κB pathways in HCT116 cells.

Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) is a complex that regulates several hundreds of genes, including those involved in immunity and inflammation, survival, proliferation, and the negative feedback of NF-κB signaling. Chelidonine, a major bioactive, isoquinoline alkaloid ingredient in Chelidonium majus, exhibits antiinflammatory pharmacological properties. However, its antiinflammatory molecular mechanisms remain unclear. In this work, we explored the effect of chelidonine on TNF-induced NF-κB activation in HCT116 cells. We found chelidonine inhibited the phosphorylation and degradation of the inhibitor of NF-κB alpha and nuclear translocation of RELA. Furthermore, by inhibiting the activation of NF-κB, chelidonine downregulated target genes involved in inflammation, proliferation, and apoptosis. Chelidonine also inhibited mitogen-activated protein kinase pathway activation by blocking c-Jun N-terminal kinase and p38 phosphorylation. These results suggest that chelidonine may be a potential therapeutic agent against inflammatory diseases in which inhibition of NF-κB activity plays an important role.

Amorfrutin A inhibits TNF-α induced JAK/STAT signaling, cell survival and proliferation of human cancer cells.

CONTEXT: Amorfrutin A is a natural product isolated from the fruits of Amorpha fruticosa L. and has been shown to exhibit multiple bioeffector functions. In the present study, we investigated whether amorfrutin A exerts anticancer effects by inhibiting STAT3 activation in cervical cancer cells. OBJECTIVE: To investigate the effectiveness of amorfrutin A as a treatment of cancer, and determine the underlying pharmacological mechanism of action. MATERIALS AND METHODS: HeLa, SK-Hep1, MDA-MB-231 and HCT116 cells were used in this study. Major assays were luciferase reporter assay, MTT, Western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, EdU labeling and immunofluorescence, xenografted assay. RESULTS: Amorfrutin A significantly inhibited tumor necrosis factor-α (TNF-α)-induced phosphorylation and nuclear translocation of STAT3 in human cervical carcinoma cells. Amorfrutin A also inhibited activation of the upstream kinases Janus-activated kinase 1 (JAK1), JAK2 and Src signaling pathways. Furthermore, amorfrutin A increased the expression of p53, p21, p27, induced cell cycle arrest in the G1 phase as well as decreased levels of various oncogene protein products. In vivo studies further confirmed the inhibitory effect of amorfrutin A on the expression of STAT3 proteins, leading to a decrease growth of HeLa cells in a xenograft tumor model. DISCUSSION AND CONCLUSIONS: The results indicated that amorfrutin A is a potent inhibitor of STAT3 and provide new perspectives into the mechanism of its anticancer activity.

Mollugin Has an Anti-Cancer Therapeutic Effect by Inhibiting TNF-α-Induced NF-κB Activation.

The NF-κB signaling pathway plays a pivotal role in regulating the immune response and inflammation. However, it has been shown that NF-κB also has a major role in oncogenesis. Therefore, NF-κB inhibitors have been considered as potential drugs against cancer. Herein, we searched for NF-κB inhibitors from natural sources and identified mollugin from the roots of Rubia cordifolia L. as an inhibitor of NF-κB activation. We found that mollugin significantly inhibited the expression of an NF-κB reporter gene induced by tumor necrosis factor (TNF)-α in a dose-dependent manner. Moreover, mollugin inhibited TNF-α-induced phosphorylation and nuclear translocation of p65, phosphorylation and degradation of inhibitor of κB (IκBα), and IκB kinase (IKK) phosphorylation. Furthermore, we discovered that pretreatment of cells with mollugin prevented the TNF-α-induced expression of NF-κB target genes, such as genes related to proliferation (COX-2, Cyclin D1 and c-Myc), anti-apoptosis (Bcl-2, cIAP-1 and survivin), invasion (MMP-9 and ICAM-1), and angiogenesis (VEGF). We also demonstrated that mollugin potentiated TNF-α-induced apoptosis and inhibited proliferation of HeLa cells. We further demonstrated in vivo that mollugin suppressed the growth of tumor xenografts derived from HeLa cells. Taken together, mollugin may be a valuable candidate for cancer treatment by targeting NF-κB.

Shikonin suppresses proliferation and induces cell cycle arrest through the inhibition of hypoxia-inducible factor-1α signaling.

Hypoxia enhances the development of solid tumors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that is dominantly expressed under hypoxia in solid tumor cells and is a key factor of tumor regulation. HIF-1α regulates several target genes involved in many aspects of cancer progression, including angiogenesis, metastasis, and cell proliferation, as well as imparting resistance to cancer treatment. In this study, we assessed shikonin, which derives from the traditional medical herb Lithospermum erythrorhizon, for its anti-cancer effects in hypoxia-induced human colon cancer cell lines. Shikonin showed potent inhibitory activity against hypoxia-induced HIF-1α activation in various human cancer cell lines and efficient scavenging activity of hypoxia-induced reactive oxygen species in tumor cells. Further analysis revealed that shikonin inhibited HIF-1α protein synthesis without affecting the expression of HIF-1α mRNA or degrading HIF-1α protein. It was subsequently shown to attenuate the activation of downstream mTOR/p70S6K/4E-BP1/eIF4E kinase. Shikonin also dose-dependently caused the cell cycle arrest of activated HCT116 cells and inhibited the proliferation of HCT116 and SW620 cells. Moreover, it significantly inhibited tumor growth in a xenograft modal. These findings suggest that shikonin could be considered for use as a potential drug in human colon cancer therapy.

Imperatorin efficiently blocks TNF-α-mediated activation of ROS/PI3K/Akt/NF-κB pathway.

Inflammation contributes to development and progression in a variety of cancers, including cervical cancer, which is the second leading cause of cancer deaths in women worldwide. In this study, we examined the anti-inflammatory effects of imperatorin, a psoralen-type furanocoumarin from the fruits of Angelica dahurica, in tumor necrosis factor-α (TNF-α)-stimulated HeLa cells by investigating its impact on the production and expression of cytokines and the major signal-transduction pathways. We found this compound significantly inhibited the TNF-α-induced expression of NF-κB target genes, such as COX-2, cyclin  D1, MMP-9, VEGF, IL-6 and Bcl-xL in a concentration-dependent manner. Further analysis revealed that imperatorin was a potent inhibitor of NF-κB activation by the suppression of TNF-α-induced IKKα/ß phosphorylation, IκB phosphorylation and degradation, and NF-κB p65 nuclear translocation. We also demonstrated that imperatorin downregulated TNF-α-induced activation of PI3K/Akt. Furthermore, our findings show that imperatorin inhibits TNF-α-induced ROS generation. Taken together, imperatorin can blunt inflammation by inhibiting the ROS-mediated activation of the PI3K/Akt/NF-κB pathway.

Imperatorin suppresses proliferation and angiogenesis of human colon cancer cell by targeting HIF-1α via the mTOR/p70S6K/4E-BP1 and MAPK pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Angelica dahurica is a commonly used traditional Chinese medicine to treat migraine headache, toothache and cancer. Imperatorin is an active natural furocoumarin component originating from Angelica dahurica and has been shown to exhibit multiple bioeffector functions, including anti-cancer activity. However, the mechanism by which imperatorin inhibits tumor growth is not fully understood. AIM OF THE STUDY: The aim of this study was to investigate the effectiveness of imperatorin as a treatment of cancer and to identify the underlying mechanisms of its anticancer activity. MATERIALS AND METHODS: HCT116, HeLa, and Hep3B cells were used in this study. Major assays were promoter-reporter gene assay, MTT, western blot analysis, immunofluorescence assay, reverse transcription-PCR (RT-PCR), flow cytometric analysis, clonogenic assay, EdU labeling and immunofluorescence, xenografted assay, and VEGF ELISA. RESULTS: We here demonstrated the effect of imperatorin on hypoxia-inducible factor-1 (HIF-1) activation. Imperatorin showed a potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines. This compound markedly decreased the hypoxia-induced accumulation of HIF-1α protein dose-dependently, whereas it did not affect the expressions of HIF-1ß and topoisomerase-I (Topo-I). Further analysis revealed that imperatorin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, the phosphorylation levels of mammalian target of rapamycin (mTOR), ribosomal protein S6 kinase (p70S6K), eIF4E binding protein-1 (4E-BP1), eukaryotic initiation factor 4E (eIF4E), extracellular signal-regulated kinase-1/2 (ERK1/2), SAPK/JNK and p38 were significantly suppressed by imperatorin. Furthermore, imperatorin prevented hypoxia-induced expression of HIF-1 target genes and flow cytometric analysis indicated that imperatorin induced G1 phase arrest in human colon cancer cell (HCT116). We found that imperatorin administration inhibits tumor growth and blocks tumor angiogenesis in a xenograft tumor model. CONCLUSIONS: These results show that imperatorin inhibited HIF-1α protein synthesis by downregulating the mTOR/p70S6K/4E-BP1 and MAPK pathways. These conclusions suggest that imperatorin is an effective inhibitor of HIF-1 and provide new perspectives into the mechanism of its anticancer activity.

Artemisinin inhibits inflammatory response via regulating NF-κB and MAPK signaling pathways.

Artemisinin, isolated from the Chinese plant Artemisia annua, has been used for many years to treat different forms of malarial parasites. In this study, we explored the anti-inflammatory activity of artemisinin and the underlying mechanism of this action. We demonstrated that the anti-inflammatory effects of artemisinin in TPA-induced skin inflammation in mice. Then the artemisinin significantly inhibited the expression of NF-κB reporter gene induced by TNF-α in a dose-dependent manner. Artemisinin also inhibited TNF-α induced phosphorylation and degradation of IκBα, p65 nuclear translocation. Artemisinin also has an impact on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2) and receptor interacting protein 1 (RIP1). Furthermore, pretreatment of cells with artemisinin prevented the TNF-α-induced expression of NF-κB target genes, such as anti-apoptosis (c-IAP1, Bcl-2, and FLIP), proliferation (COX-2, cyclinD1), invasion (MMP-9), angiogenesis (VEGF), and major inflammatory cytokines (TNF-α, iNOS, and MCP1). We also proved that artemisinin potentiated TNF-α-induced apoptosis. Moreover, artemisinin significantly impaired the ROS production and phosphorylation of p38 and ERK, but did not affect the phosphorylation of JNK. Taken together, artemisinin may be a potentially useful therapeutic agent for inflammatory-related diseases.

Dihydromyricetin suppresses TNF-α-induced NF-κB activation and target gene expression.

Nuclear factor-kappa B (NF-κB) has been reported to play a pivotal role in many physiological processes including inflammation, apoptosis, and angiogenesis. We discovered a potent natural NF-κB inhibitor, dihydromyricetin, from the traditional herb Ampelopsis grossedentata, which has a long history of use in food and medicine. In this study, we demonstrated the effect of dihydromyricetin on NF-κB activation in TNF-α-induced HeLa cells. Dihydromyricetin was found to markedly inhibit the phosphorylation and degradation of the inhibitor of NF-κB alpha (IκBα), and subsequent nuclear translocation of p65. Dihydromyricetin also has an impact on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2), and receptor-interacting protein 1 (RIP1). Furthermore, the current results reveal that dihydromyricetin led to the downregulation of target genes involved in inflammation, proliferation, as well as potentiation of TNF-α-induced apoptosis through suppressing the activation of NF-κB. In conclusion, our data indicate that dihydromyricetin may be a potentially useful therapeutic agent for inflammatory diseases.

Baicalein inhibits TNF-α-induced NF-κB activation and expression of NF-κB-regulated target gene products.

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, immunity, apoptosis and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified baicalein from Scutellaria baicalensis as an inhibitor of NF-κB activation. As examined by the NF-κB luciferase reporter assay, we found that baicalein suppressed TNF-α-induced NF-κB activation in a dose-dependent manner. It also inhibited TNF-α-induced nuclear translocation of p65 through inhibition of phosphorylation and degradation of IκBα. Furthermore, baicalein blocked the TNF-α-induced expression of NF-κB target genes involved in anti-apoptosis (cIAP-1, cIAP-2, FLIP and BCL-2), proliferation (COX-2, cyclin D1 and c-Myc), invasion (MMP­9), angiogenesis (VEGF) and major inflammatory cytokines (IL-8 and MCP1). The flow cytometric analysis indicated that baicalein potentiated TNF-α-induced apoptosis and induced G1 phase arrest in HeLa cells. Moreover, baicalein significantly blocked activation of p38, extracellular signal-regulated kinase 1/2 (ERK1/2). Our results imply that baicalein could be a lead compound for the modulation of inflammatory diseases as well as certain cancers in which inhibition of NF-κB activity may be desirable.

Perillyl alcohol efficiently scavenges activity of cellular ROS and inhibits the translational expression of hypoxia-inducible factor-1α via mTOR/4E-BP1 signaling pathways.

Perillyl alcohol (POH) is a dietary monoterpene present in a variety of plants with a pure or mixed form, and it is one of the very few natural substances with anticancer activity. However, the mechanism by which POH unleashes its anticancer activity in tumor cells remains unclear. We here demonstrated the effect of POH on hypoxia-inducible factor-1α (HIF-1α) activation. POH showed the potent inhibitory activity against HIF-1 activation induced by hypoxia in various human cancer cell lines and efficient scavenging activity of cellular Reactive oxygen species (ROS) by hypoxia in tumor cells. Further analysis revealed that POH inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Moreover, we found that suppression of HIF-1α accumulation by POH correlated with strong de-phosphorylation of mammalian target of rapamycin (mTOR) and eIF4E binding protein-1 (4E-BP1), and eukaryotic initiation factor 4E (eIF4E). These results showed that POH inhibited HIF-1α protein synthesis through the inhibition of mTOR/4E-BP1 signaling pathways. Furthermore, POH increased the expression of p53, p21, induced cell cycle arrest in the G1 phase as well as decreased cyclin D1, c-Myc, and Skp2 expression. In vivo studies further confirmed the inhibitory effect of POH on the expression of HIF-1α proteins, leading to a decrease growth of HCT116 cells in a xenograft tumor model. There results show that POH is an effective inhibitor of HIF-1 and provide new perspectives in to the mechanism of its anticancer activity.

Zinc finger protein 91 (ZFP91) activates HIF-1α via NF-κB/p65 to promote proliferation and tumorigenesis of colon cancer.

Zinc finger protein 91 (ZFP91) has been reported to be involved in various biological processes. However, the clinical significance and biological role of ZFP91 in colon cancer remains unknown. Here, we show that ZFP91 expression is upregulated in patients with colon cancer. We found that ZFP91 upregulated HIF-1α at the levels of promoter and protein in colon cancer cells. Using chromatin immunoprecipitation, electrophoretic mobility shift assay and luciferase reporter gene assay, we found that NF-κB/p65 is required for the binding of ZFP91 to the HIF-1α promoter at -197/-188 base pairs and for the transcriptional activation of HIF-1α gene mediated by ZFP91. Flow cytometry, 5-ethynyl-2'-deoxyuridine (EdU) incorporation and tumor xenograft assay demonstrated that ZFP91 enhanced cell proliferation of colon cancer through upregulating HIF-1α in vitro and in vivo. Furthermore, ZFP91 is positively associated with HIF-1α in human colon cancer. Thus, we concluded that ZFP91 activates transcriptional coregulatory protein HIF-1α through transcription factor NF-κB/p65 in the promotion of proliferation and tumorigenesis in colon cancer cell. ZFP91 may serve as a driver gene to activate HIF-1α transcription in the development of cancer.

Kamebakaurin inhibits the expression of hypoxia-inducible factor-1α and its target genes to confer antitumor activity.

Hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor that mediates the adaptation of tumor cells and tissues to the hypoxic microenvironment, has attracted considerable interest as a potential therapeutic target. Kamebakaurin is a diterpenoid compound isolated from Isodon excia (Maxin.) Hara, which has been used for anti-inflammatory activities. However, its antitumor activity along with molecular mechanism has not been reported. Kamebakaurin showed potent inhibitory activity against HIF-1 activation induced by hypoxia or CoCl2 in various human cancer cell lines. This compound significantly decreased the hypoxia-induced accumulation of HIF-1α protein, whereas it did not affect the expression of topoisomerase-I (Topo-I). Further analysis revealed that kamebakaurin inhibited HIF-1α protein synthesis, without affecting the expression level of HIF-1α mRNA or degradation of HIF-1α protein. Furthermore, kamebakaurin prevented hypoxia-induced expression of HIF-1 target genes for vascular endothelial growth factor (VEGF) and erythropoietin (EPO). However, kamebakaurin caused cell growth inhibition via cell cycle arrest at G1 phase in tumor cells. In vivo studies, we further confirmed the inhibitory effect of kamebakaurin on the expression of HIF-1α proteins, leading to growth inhibition of HCT116 cells in a xenograft tumor model. These results show that kamebakaurin is an effective inhibitor of HIF-1 and provide new perspectives into its anticancer activity.

4',6-Dihydroxy-4-methoxyisoaurone inhibits TNF-α-induced NF-κB activation and expressions of NF-κB-regulated target gene products.

The nuclear factor-κB (NF-κB) transcription factors control many physiological processes including inflammation, apoptosis, and angiogenesis. In our search for NF-κB inhibitors from natural resources, we identified 4',6-dihydroxy-4-methoxyisoaurone (ISOA) as an inhibitor of NF-κB activation from the seeds of Trichosanthes kirilowii. However, the mechanism by which ISOA inhibits NF-κB activation is not fully understood. In the present study, we demonstrated the effect of ISOA on NF-κB activation in TNF-α-stimulated HeLa cells. This compound suppressed NF-κB activation through the inhibition of IκB kinase (IKK) activation. ISOA also has an influence on upstream signaling of IKK through the inhibition of expression of adaptor proteins, TNF receptor-associated factor 2 (TRAF2) and receptor interacting protein 1 (RIP1). Consequently, ISOA blocked the phosphorylation and degradation of the inhibitor of NF-κB alpha (IκBα), and subsequent phosphorylation and nuclear translocation of p65. The suppression of NF-κB activation by ISOA led to the down-regulation of target genes involved in inflammation, proliferation, as well as potentiation of TNF-α-induced apoptosis. Taken together, this study extends our understanding on the mechanisms underlying the anti-inflammatory and anti-cancer activities of ISOA. Our findings provide new insight into the molecular mechanisms and a potential application of ISOA for inflammatory diseases as well as certain cancers.