KEAP1-NRF2 pathway as a novel therapeutic target for EGFR-mutant non-small cell lung cancer.

BACKGROUND: Kelch-like ECH-associated protein 1 (KEAP1)-nuclear factor erythroid-2-related factor 2 (NRF2) pathway is a major regulator protecting cells from oxidative and metabolic stress. Studies have revealed that this pathway is involved in mediating resistance to cytotoxic chemotherapy and immunotherapy, however, its implications in oncogene-addicted tumors are largely unknown. This study aimed to elucidate whether this pathway could be a potential therapeutic target for epidermal growth factor receptor (EGFR)-mutant non-small cell lung cancer. METHODS: We measured the baseline expression of NRF2 using EGFR-mutant parental cells and acquired gefitinib resistant cells. We investigated whether NRF2 inhibition affected cell death in vitro and tumor growth in vivo using a xenograft mouse model, and compared the transcriptional changes before and after NRF2 inhibition. RESULTS: Baseline NRF2 expression was enhanced in PC9 and PC9 with gefitinib resistance (PC9/GR) cells than in other cell lines, with a more prominent expression in PC9/GR. The NRF2 inhibitor induced NRF2 downregulation and cell death in a dose-dependent manner. Co-treatment with an NRF2 inhibitor enhanced osimertinib-induced cell death in vitro, and potentiated tumor growth inhibition in a PC9/GR xenograft model. Finally, RNA sequencing revealed that NRF2 inhibition resulted in the altered expression of multiple genes involved in various signaling pathways. CONCLUSION: We identified that NRF2 inhibition enhanced cell death and inhibited tumor growth in TKI-resistant lung cancer with EGFR-mutation. Thus, NRF2 modulation may be a novel therapeutic strategy to overcome the resistance to EGFR-tyrosine kinase inhibitors.

Mild exposure to fine particulate matter promotes angiogenesis in non-small cell lung carcinoma.

Fine particulate matter (PM2.5) is associated with public health problems worldwide. Especially, PM2.5 induces epigenetic and microenvironmental changes in lung cancer. Angiogenesis is important for the development and growth of cancer and is mediated by angiogenic factors, including vascular endothelial growth factor. However, the effects of mild PM2.5 exposure on angiogenesis in lung cancer remain unclear. In this study, we examined angiogenic effects using relatively lower concentrations of PM2.5 than in other studies and found that PM2.5 increased angiogenic activities in both endothelial cells and non-small cell lung carcinoma cells. PM2.5 also promoted the growth and angiogenesis of lung cancer via the induction of hypoxia-inducible factor-1α (HIF-1α) in a xenograft mouse tumor model. Angiogenic factors, including vascular endothelial growth factor (VEGF), were highly expressed in lung cancer patients in countries with high PM2.5 levels in the atmosphere, and high expression of VEGF in lung cancer patients lowered the survival rate. Collectively, these results provide new insight into the mechanisms by which mild exposure to PM2.5 is involved in HIF-1α-mediated angiogenesis in lung cancer patients.

Angiogenic activities are increased via upregulation of HIF-1α expression in gefitinib-resistant non-small cell lung carcinoma cells.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) have been used to treat patients with non-small cell lung cancer (NSCLC) and activating EGFR mutations; however, the emergence of secondary mutations in EGFR or the acquisition of resistance to EGFR-TKIs can develop and is involved in clinical failure. Since angiogenesis is associated with tumor progression and the blockade of antitumor drugs, inhibition of angiogenesis could be a rational strategy for developing anticancer drugs combined with EGFR-TKIs to treat patients with NSCLC. The signaling pathway mediated by hypoxia-inducible factor-1 (HIF-1) is essential for tumor angiogenesis. The present study aimed to identify the dependence of gefitinib resistance on HIF-1α activity using angiogenesis assays, western blot analysis, colony formation assay, xenograft tumor mouse model and immunohistochemical analysis of tumor tissues. In the NSCLC cell lines, HIF-1α protein expression levels and hypoxia-induced angiogenic activities were found to be increased. In a xenograft mouse tumor model, tumor tissues derived from gefitinib-resistant PC9 cells showed increased protein expression of HIF-1α and angiogenesis within the tumors. Furthermore, inhibition of HIF-1α suppressed resistance to gefitinib, whereas overexpression of HIF-1α increased resistance to gefitinib. The results from the present study provides evidence that HIF-1α was associated with the acquisition of resistance to gefitinib and suggested that inhibiting HIF-1α alleviated gefitinib resistance in NSCLC cell lines.

ß-arrestin 2 stimulates degradation of HIF-1α and modulates tumor progression of glioblastoma.

The basic function of ß-arrestin 2 (Arrb2) is to negatively regulate the G-protein-coupled receptor signaling pathway through facilitating receptor desensitization and internalization. Arrb2 has also been reported to play various roles in cancer pathology including the proliferation, migration, invasion, metastasis, and apoptosis of solid tumors. However, the molecular mechanisms underlying the tumorigenic capacities of Arrb2 have not been elucidated. Here, we show a novel function of Arrb2: Arrb2 facilitates the degradation of HIF-1α, which is a master regulator of oxygen homeostasis. We also demonstrate that Arrb2 interacts with HIF-1α and stimulates ubiquitin-mediated 26S proteasomal degradation of HIF-1α by recruiting PHD2 and pVHL. Overexpression of Arrb2 in human glioblastoma cells suppresses HIF-1α signaling, tumor growth, and angiogenesis. Consistent with this antitumorigenic effect of Arrb2, low Arrb2 expression levels correlate with high HIF-1α expression and poor glioblastoma patient survival. These results collectively reveal a novel function of Arrb2 in the oxygen-sensing mechanism that directly regulates HIF-1α stability in human cancers and suggest Arrb2 as a new potential therapeutic target for glioblastoma.

Zingerone Suppresses Tumor Development through Decreasing Cyclin D1 Expression and Inducing Mitotic Arrest.

Cancer cells undergo uncontrolled proliferation resulting from aberrant activity of various cell-cycle proteins. Therefore, despite recent advances in intensive chemotherapy, it is difficult to cure cancer completely. Recently, cell-cycle regulators became attractive targets in cancer therapy. Zingerone, a phenolic compound isolated from ginger, is a nontoxic and inexpensive compound with varied pharmacological activities. In this study, the therapeutic effect of zingerone as an anti-mitotic agent in human neuroblastoma cells was investigated. Following treatment of BE(2)-M17 cells with zingerone, we performed a 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay and colony-formation assay to evaluate cellular proliferation, in addition to immunofluorescence cytochemistry and flow cytometry to examine the mitotic cells. The association of gene expression with tumor stage and survival was analyzed. Furthermore, to examine the anti-cancer effect of zingerone, we applied a BALB/c mouse-tumor model using a BALB/c-derived adenocarcinoma cell line. In human neuroblastoma cells, zingerone inhibited cellular viability and survival. Moreover, the number of mitotic cells, particularly those in prometaphase, increased in zingerone-treated neuroblastoma cells. Regarding specific molecular mechanisms, zingerone decreased cyclin D1 expression and induced the cleavage of caspase-3 and poly (ADP-ribose) polymerase 1 (PARP-1). The decrease in cyclin D1 and increase in histone H3 phosphorylated (p)-Ser10 were confirmed by immunohistochemistry in tumor tissues administered with zingerone. These results suggest that zingerone induces mitotic arrest followed by inhibition of growth of neuroblastoma cells. Collectively, zingerone may be a potential therapeutic drug for human cancers, including neuroblastoma.

The Protein Phosphatase PPM1G Destabilizes HIF-1α Expression.

Hypoxia-inducible factors (HIFs) are key regulators of hypoxic responses, and their stability and transcriptional activity are controlled by several kinases. However, the regulation of HIF by protein phosphatases has not been thoroughly investigated. Here, we found that overexpression of Mg2+/Mn2+-dependent protein phosphatase 1 gamma (PPM1G), one of Ser/Thr protein phosphatases, downregulated protein expression of ectopic HIF-1α under normoxic or acute hypoxic conditions. In addition, the deficiency of PPM1G upregulated protein expression of endogenous HIF-1α under normoxic or acute oxidative stress conditions. PPM1G decreased expression of HIF-1α via the proteasomal pathway. PPM1G-mediated HIF-1α degradation was dependent on prolyl hydroxylase (PHD), but independent of von Hippel-Lindau (VHL). These data suggest that PPM1G is critical for the control of HIF-1α-dependent responses.

New Targets for Parkinson's Disease: Adhesion G Protein-Coupled Receptor B1 is Downregulated by AMP-Activated Protein Kinase Activation.

While progressive dopaminergic neurodegeneration is responsible for the cardinal motor defects in Parkinson's disease (PD), new diagnostics and therapeutic targets are necessary to effectively address this major global health burden. We evaluated whether the adhesion G protein-coupled receptor B1 (ADGRB1, formerly BAI1, brain-specific angiogenesis inhibitor 1) might contribute to dopaminergic neuronal loss. We used bioinformatic analyses, as well as in vitro and in vivo PD models. We report in this study that ADGRB1 is decreased in PD and that the ADGRB1 level is specifically decreased in dopaminergic neurons in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-intoxicated mice. In primary mouse mesencephalic neurons and human neuroblastoma cell lines, 1-methyl-4-phenylpyridinium (MPP+), a toxic metabolite of MPTP, suppressed the expression of ADGRB1. Moreover, we applied a network generation tool, Ingenuity Pathway Analysis®, with the transcriptomics dataset to extend the upstream regulatory pathway of ADGRB1 expression. AMP-activated protein kinase (AMPK) was predicted as a regulator, and consequently, 5-aminoimidazole-4-carboxamide ribonucleotide, a specific activator of AMPK, reduced the ADGRB1 protein level. Finally, ADGRB1 overexpression decreased nuclear condensation induced by MPP+ treatment. Taken together, we observed that decreased ADGRB1 by activation of AMPK induced neuronal cell death in MPTP/MPP+-mediated PD models, suggesting that ADGRB1 might potentially play a survival role in the neurodegenerative pathway of PD. These data offer new insights into dopaminergic cell death with therapeutic implications for neurodegenerative disorders.

The Neuroprotective Effects of Cinnamic Aldehyde in an MPTP Mouse Model of Parkinson's Disease.

Cinnamic aldehyde (CA), a key flavor compound in cinnamon essential oil, has been identified as an anti-oxidant, anti-angiogenic, and anti-inflammatory material. Recently, the neuroprotective effects of CA have been reported in various neurodegenerative disorders, including Parkinson's disease (PD). In neurons, autophagy is tightly regulated, and consequently, the dysregulation of autophagy may induce neurodegenerative disorders. In the present study, we found that the selective dopaminergic neuronal death in the substantia nigra of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse models was prevented by CA. Stimulation of microtubule-associated protein light chain 3 (LC3) puncta mediated by MPTP treatment was decreased by CA. Moreover, down-regulated p62 in the substantia nigra of MPTP mice was increased by administration of CA. Finally, we showed that blockage of autophagy using autophagy inhibitors protected the 1-methyl-4-phenylpyridinium (MPP⁺)-mediated death of BE(2)-M17 cells. Together these results suggest that CA has a neuroprotective effect in a PD model and that inhibition of autophagy might be a promising therapeutic target for PD.

Evaluation of non-thermal plasma-induced anticancer effects on human colon cancer cells.

Non-thermal atmospheric-pressure plasma has been introduced in various applications such as sterilization, wound healing, blood coagulation, and other biomedical applications. The most attractive application of non-thermal atmospheric-pressure plasma is in cancer treatment, where the plasma is used to produce reactive oxygen species (ROS) to facilitate cell apoptosis. We investigate the effects of different durations of exposure to dielectric-barrier discharge (DBD) plasma on colon cancer cells using measurement of cell viability and ROS levels, western blot, immunocytochemistry, and Raman spectroscopy. Our results suggest that different kinds of plasma-treated cells can be differentiated from control cells using the Raman data.

Effect of ginsenoside Rh-2 via activation of caspase-3 and Bcl-2-insensitive pathway in ovarian cancer cells.

Ginsenoside has been reported to have therapeutic effects for some types of cancer, but its effect on ovarian cancer cells has not been evaluated. In this study, we monitored the effects of ginsenoside-Rh2 (Rh2) on the inhibition of cell proliferation and the apoptotic process in the ovarian cancer cell line SKOV3 using an MTT assay and TUNEL assay. We found that Rh2 inhibited cell proliferation and significantly induced apoptosis. We confirmed the apoptotic effects of Rh2 using western blot analysis of apoptosis-related proteins. Specifically, the levels of cleaved poly ADP ribose polymerase (PARP) and cleaved caspase-3 significantly increased in SKOV3 cells treated with Rh2. Therefore, Rh2 clearly suppressed the growth of SKOV3 cells in vitro, which was associated with induction of the apoptosis pathway. Moreover, the migration assay showed that Rh2 inhibited the invasive ability of SKOV3 cells. Taken together, our results suggest that Rh2 has anticancer effects in SKOV3 cells through inhibition of cell proliferation and induction of apoptosis. Considering the therapeutic potential of Rh2, more studies should be carried out to facilitate the future application of this natural product as a potential anti-cancer agent.

Zingerone suppresses angiogenesis via inhibition of matrix metalloproteinases during tumor development.

Angiogenesis is an essential step for tumor survival and progression, and the inhibition of angiogenesis is a good strategy for tumor therapeutics. In this study, we investigated the therapeutic effect of zingerone in a mouse tumor model. Zingerone suppressed tumor progression and tumor angiogenesis. Moreover, we found that zingerone inhibited the angiogenic activities of endothelial cells by both direct and indirect means. A mechanistic study showed that the activities of MMP-2 and MMP-9 in tumor cells were decreased by treatment with zingerone. Interestingly, zingerone-mediated inhibition of MMP-2 and MMP-9 was involved in the JNK pathway. In conclusion, zingerone showed strong anti-angiogenic activity via the inhibition of MMP-2 and MMP-9 during tumor progression, suggesting that zingerone may be a potential therapeutic drug for human cancers.

Cinnamic aldehyde suppresses hypoxia-induced angiogenesis via inhibition of hypoxia-inducible factor-1α expression during tumor progression.

During tumor progression, hypoxia-inducible factor 1 (HIF-1) plays a critical role in tumor angiogenesis and tumor growth by regulating the transcription of several genes in response to a hypoxic environment and changes in growth factors. This study was designed to investigate the effects of cinnamic aldehyde (CA) on tumor growth and angiogenesis and the mechanisms underlying CA's anti-angiogenic activities. We found that CA administration inhibits tumor growth and blocks tumor angiogenesis in BALB/c mice. In addition, CA treatment decreased HIF-1α protein expression and vascular endothelial growth factor (VEGF) expression in mouse tumors and Renca cells exposed to hypoxia in vitro. Interestingly, CA treatment did not affect the stability of von Hippel-Lindau protein (pVHL)-associated HIF-1α and CA attenuated the activation of mammalian target of rapamycin (mTOR) pathway. Collectively, these findings strongly indicate that the anti-angiogenic activity of CA is, at least in part, regulated by the mTOR pathway-mediated suppression of HIF-1α protein expression and these findings suggest that CA may be a potential drug for human cancer therapy.

Salicin, an extract from white willow bark, inhibits angiogenesis by blocking the ROS-ERK pathways.

Salicin has been studied as a potent antiinflammatory agent. Angiogenesis is an essential process for tumor progression, and negative regulation of angiogenesis provides a good strategy for antitumor therapy. However, the potential medicinal value of salicin on antitumorigenic and antiangiogenic effects remain unexplored. In this study, we examined the antitumorigenic and antiangiogenic activity of salicin and its underlying mechanism of action. Salicin suppressed the angiogenic activity of endothelial cells, such as migration, tube formation, and sprouting from an aorta. Moreover, salicin reduced reactive oxygen species production and activation of the extracellular signal-regulated kinase pathway. The expression of vascular endothelial growth factor was also decreased by salicin in endothelial cells. When the salicin was administered to mice, salicin inhibited tumor growth and angiogenesis in a mouse tumor model. Taken together, salicin targets the signaling pathways mediated by reactive oxygen species and extracellular signal-regulated kinase, providing new perspectives into a potent therapeutic agent for hypervascularized tumors.

Antiangiogenic effects of p-coumaric acid in human endothelial cells.

p-Coumaric acid, a hydroxy derivative of cinnamic acid, has been known to possess antioxidant and anticancer activities. Despite its potential contribution to chemopreventive effects, the mechanism by which p-coumaric acid exerts its antiangiogenic actions remains elusive. In this study, we revealed that p-coumaric acid inhibited the sprouting of endothelial cells in rat aortic rings and inhibited the tube formation and migration of endothelial cells. We observed that p-coumaric acid could downregulate mRNA expression levels of the key angiogenic factors vascular endothelial growth factor and basic fibroblast growth factor. Also, we demonstrated that p-coumaric acid inhibited both the AKT and ERK signaling pathways, which are known to be crucial for angiogenesis. Using a mouse model, we also showed that p-coumaric acid effectively suppressed tumor growth in vivo by lowering hemoglobin contents. Collectively, these findings indicate that p-coumaric acid possesses potent anticancer properties due to the inhibition of angiogenesis in vivo.

Melatonin suppresses tumor progression by reducing angiogenesis stimulated by HIF-1 in a mouse tumor model.

The sustained expansion of a tumor mass requires new blood vessel formation to provide rapidly proliferating tumor cells with an adequate supply of oxygen and nutrients. Hypoxia-inducible factor-1 (HIF-1) plays an essential role in tumor angiogenesis and growth by regulating the transcription of genes in response to hypoxic stress. This study was designed to investigate the effects of melatonin on tumor growth and angiogenesis, as well as the mechanism underlying the antitumor activities of melatonin. In this study, we show that the administration of melatonin inhibits tumor growth and blocks tumor angiogenesis in mice. Moreover, melatonin diminished the expression of the HIF-1α protein within the tumor mass during tumorigenesis. Our findings suggest that melatonin is a promising anti-angiogenic therapeutic agent targeting HIF-1α in cancer. Considering that HIF-1α is overexpressed in a majority of human cancers, melatonin could offer a potent therapeutic agent for cancer.

The anti-angiogenic activities of glycyrrhizic acid in tumor progression.

Glycyrrhizic acid (GA) is the bioactive compound of licorice and has been used as a herbal medicine because of its anti-viral, anti-cancer, and anti-inflammatory properties. This study was designed to investigate the effects of GA on tumor growth, angiogenesis, and the mechanisms underlying the anti-angiogenic activities of GA. We observed that GA inhibited tumor growth and angiogenesis in mice. GA decreased angiogenic activities, such as the migration, invasion, and tube formation of endothelial cells. We also demonstrated that GA reduced the production of reactive oxygen species and activation of ERK in endothelial cells. Our findings suggest that GA is a promising anti-angiogenic therapeutic agent that targets the ERK pathway. Considering that angiogenesis is highly stimulated in the majority of cancers, GA could offer a potent therapeutic agent for cancer.

Reduced expression of FASN through SREBP-1 down-regulation is responsible for hypoxic cell death in HepG2 cells.

Cells under hypoxic stress either activate an adaptive response or undergo cell death. Although some mechanisms have been reported, the exact mechanism behind hypoxic cell death remains unclear. Recently, increased expression of fatty acid synthase (FASN) has been observed in various human cancers. In highly proliferating cells, tumor-associated FASN is considered necessary for both membrane lipids production and post-translational protein modification, but the exact mechanisms are not fully understood. Further, FASN overexpression is associated with aggressive and malignant cancer diseases and FASN inhibition induces apoptosis in cancer cells. For this reason, FASN is emerging as a key target for the potential diagnosis and treatment of various cancers. Here, we observed decreased FASN expression under hypoxic cell death conditions in HepG2 cells. Thus, we examined the effect of decreased FASN expression on hypoxia-induced cell death in HepG2 cells and also investigated the mechanism responsible for reduction of FASN expression under hypoxic cell death conditions. As a result, reduction of FASN expression resulted in hypoxic cell death via malonyl-CoA accumulation. In addition, SREBP-1 restored FASN reduction and hypoxia-induced apoptosis. Taken together, we suggest that hypoxic cell death is promoted by the reduced expression of FASN through SREBP-1 down-regulation.

Coenzyme Q10 decreases basic fibroblast growth factor (bFGF)-induced angiogenesis by blocking ERK activation.

Coenzyme Q10 (CoQ10) is an essential factor of the mitochondrial respiratory chain and has effective antioxidant properties. Therefore, CoQ10 has been used in a variety of clinical applications and used as a nutritional supplement to treat several human diseases. Here, we tested the effects of CoQ10 on angiogenesis stimulated by basic fibroblast growth factor (bFGF). CoQ10 significantly inhibited bFGF-induced angiogenesis in a mouse Matrigel plug and the sprouting of endothelial cells in rat aortic rings. In addition, CoQ10 decreased the ability of tube formation, migration, and invasion in endothelial cells. When CoQ10 was used to inhibit angiogenesis in endothelial cells, the expression of vascular endothelial growth factor (VEGF) and the phosphorylation of ERK were decreased. Taken together, these results indicate that CoQ10 is able to act as an antiangiogenic regulator, and its inhibitory activity is mediated by blocking an ERK-dependent pathway. This study suggests that CoQ10 may be used a therapeutic agent to decrease neovascularization in several diseases, including solid tumors.

Ethyl pyruvate stabilizes hypoxia-inducible factor 1 alpha via stimulation of the TCA cycle.

Ethyl pyruvate is a simple derivative of the endogenous metabolite pyruvate. Pyruvate is the starting substrate for the tricarboxylic acid (TCA) cycle and plays a central role in intermediary metabolism. The present study was to determine whether ethyl pyruvate affects the expression of hypoxia-inducible factor 1 alpha (HIF-1alpha) and to explore the mechanism of HIF-1alpha regulation. We found that ethyl pyruvate increased HIF-1alpha stability via inhibition of pVHL-mediated degradation. Furthermore, ethyl pyruvate enhanced the reactive oxygen species (ROS) generated through the TCA cycle in mitochondria. Taken together, our results support a novel role for ethyl pyruvate in HIF-1alpha stabilization by which high rates of the TCA cycle can promote ROS production.

AMP-activated protein kinase is activated in Parkinson's disease models mediated by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

The selective loss of dopaminergic neurons in the substantia nigra pars compacta is a feature of Parkinson's disease (PD). 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced neurotoxicity is the most common experimental model used to investigate the pathogenesis of PD. Administration of MPTP in mice produces neuropathological defects as observed in PD and 1-methyl-4-pyridinium (MPP(+)) induces cell death when neuronal cell cultures are used. AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis. In the present study, we demonstrated that AMPK is activated by MPTP in mice and MPP(+) in SH-SY5Y cells. The inhibition of AMPK by compound C resulted in an increase in MPP(+)-induced cell death. We further showed that overexpression of AMPK increased cell viability after exposure to MPP(+) in SH-SY5Y cells. Based on these results, we suggest that activation of AMPK might prevent neuronal cell death and play a role as a survival factor in PD.