Urban dust particles disrupt mitotic progression by dysregulating Aurora kinase B-related functions.

Particulate matter (PM), a major component of outdoor air pollution, damages DNA and increases the risk of cancer. Although the harmful effects of PM at the genomic level are known, the detailed mechanism by which PM affects chromosomal stability remains unclear. In this study, we investigated the novel effects of PM on mitotic progression and identified the underlying mechanisms. Gene set enrichment analysis of lung cancer patients residing in countries with high PM concentrations revealed the downregulation of genes associated with mitosis and mitotic structures. We also showed that exposure of lung cancer cells in vitro to urban dust particles (UDPs) inhibits cell proliferation through a prolonged M phase. The mitotic spindles in UDP-treated cells were hyperstabilized, and the number of centrioles increased. The rate of ingression of the cleavage furrow and actin clearance from the polar cortex was reduced significantly. The defects in mitotic progression were attributed to inactivation of Aurora B at kinetochore during early mitosis, and spindle midzone and midbody during late mitosis. While previous studies demonstrated possible links between PM and mitosis, they did not specifically identify the dysregulation of spatiotemporal dynamics of mitotic proteins and structures (e.g., microtubules, centrosomes, cleavage furrow, and equatorial and polar cortex), which results in the accumulation of chromosomal instability, ultimately contributing to carcinogenicity. The data highlight the novel scientific problem of PM-induced mitotic disruption. Additionally, we introduce a practical visual method for assessing the genotoxic outcomes of airborne pollutants, which has implications for future environmental and public health research.

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.

Compound C Inhibits Renca Renal Epithelial Carcinoma Growth in Syngeneic Mouse Models by Blocking Cell Cycle Progression, Adhesion and Invasion.

Compound C (CompC), an inhibitor of AMP-activated protein kinase, reduces the viability of various renal carcinoma cells. The molecular mechanism underlying anti-proliferative effect was investigated by flow cytometry and western blot analysis in Renca cells. Its effect on the growth of Renca xenografts was also examined in a syngeneic BALB/c mouse model. Subsequent results demonstrated that CompC reduced platelet-derived growth factor receptor signaling pathways and increased ERK1/2 activation as well as reactive oxygen species (ROS) production. CompC also increased the level of active Wee1 tyrosine kinase (P-Ser642-Wee1) and the inactive form of Cdk1 (P-Tyr15-Cdk1) while reducing the level of active histone H3 (P-Ser10-H3). ROS-dependent ERK1/2 activation and sequential alterations in Wee1, Cdk1, and histone H3 might be responsible for the CompC-induced G2/M cell cycle arrest and cell viability reduction. In addition, CompC reduced the adhesion, migration, and invasion of Renca cells in the in vitro cell systems, and growth of Renca xenografts in the BALB/c mouse model. Taken together, the inhibition of in vivo tumor growth by CompC may be attributed to the blockage of cell cycle progression, adhesion, migration, and invasion of tumor cells. These findings suggest the therapeutic potential of CompC against tumor development and progression.

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.

Ischemia induces autophagy of endothelial cells and stimulates angiogenic effects in a hindlimb ischemia mouse model.

Although peripheral artery disease (PAD) is a major health problem, there have been limited advances in medical therapies. In PAD patients, angiogenesis is regarded as a promising therapeutic strategy to promote new arterial vessels and improve perfusion of ischemic tissue. Autophagy plays a critical role in catabolic processes for cell survival under normal and stressful conditions and plays fundamental biological roles in various cellular functions. In the present study, we showed that autophagy in endothelial cells is important for the repair and regeneration of damaged tissues. In a hindlimb ischemia mouse model, autophagy was stimulated in endothelial cells of the quadriceps muscle, and adjacent cells proliferated and regenerated. The autophagy pathway was induced under prolonged hypoxia in endothelial cells, and autophagy increased angiogenic activities. Moreover, conditioned media from endothelial cells blocked autophagy and inhibited the proliferation of muscle cells, suggesting that autophagic stimulation in endothelial cells affects the survival of adjacent cells, such as muscle. Collectively, hypoxia/ischemia-induced autophagy angiogenesis, and the damaged tissue surrounded by neo-vessels was regenerated in an ischemia model. Therefore, we strongly suggest that stimulation of autophagy in endothelial cells may be a potent therapeutic strategy in severe vascular diseases, including PAD.

Systematic mutation analysis in rare colorectal cancer presenting ovarian metastases.

Although colorectal cancer is one of the most lethal cancer types in the world, its metastasis to the ovary is rare, compared to metastasis to other organs. Consequently, the genomic basis for colon-to-ovary metastasis remains unstudied, due to limited available patients, and thus there have been no attempts to construct individual-specific networks. Due to its rarity, the small sample size makes common mutations difficult to find. To overcome this problem, we herein attempted to apply a biological connectivity map called a sample-specific network (SSN), to reveal common biological functions in three samples. Our three samples were compared to a clinical dataset contained in The Cancer Genome Atlas (TCGA) Colorectal Adenocarcinoma (COAD), showing different mutational spectra, compared to matched samples based on age, gender, microsatellite instability (MSI) status, and tumor, node, metastasis (TNM) stage. The SSNs for the three samples revealed significant correlations of the mutation statuses of several apoptosis genes, in contrast to the TCGA-matched samples. Further analysis of a targeted-gene panel sequencing dataset for colon-to-ovary metastasis of primary tumor samples also confirmed significant correlations of the mutational statuses among apoptosis genes. In summary, using SSN, we successfully identified a common function (apoptosis) among our three patients having colon-to-ovary metastasis, despite no common mutations in the three patients. Such computational analyses could facilitate productive study of rare cancers and other diseases.

Comparison of Microbiota Variation in Korean Healthy Adolescents with Adults Suggests Notable Maturity Differences.

Comparative studies of microbiome variation in world populations and different developmental stages of organisms are essential to decipher the linkages among microbiome, health, and disease. Notably, the gut microbiota are believed to mature in early life. In this context, we compared the gut microbiota diversity in Korean adolescent healthy samples (KAHSs) to healthy Korean adults (HKAs) as well as the Human Microbiome Project healthy samples (HMPHSs), the latter being one of the largest adult cohorts, based on organismal composition, alpha- and beta-diversities, function/pathway prediction analysis, and co-occurrence networks. We found that the gut microbiota compositions, including the ratios of firmicutes to bacteroidetes, between KAHSs and HMPHSs were different, and the diversities of KAHSs were less than those of HMPHSs. The predicted functions, for example, secondary bile acid synthesis and insulin signaling of KAHSs and HMPHSs, were also significantly different. Genus-level networks showed that co-occurrences among different taxa more frequently happened in HMPHSs than in KAHSs. Even though both KAHSs and HMPHSs represent healthy microbiomes, comparisons showed substantial differences, likely implicating different diets, environments, and demographics. Interestingly, we observed lower microbial diversities and less frequent co-occurrences among different taxa in KAHSs than adult HMPHSs and HKAs. These new findings collectively suggest that the adolescent gut microbiota in the present Korean sample did not reach the extent of maturity of adult microbiota diversity. In all, further population studies of microbiome variation across geographies and developmental stages are warranted, and should usefully inform future diagnostics and therapeutics innovation targeting the microbiome.

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.

CCAR2 negatively regulates IL-8 production in cervical cancer cells.

Cell cycle and apoptosis regulator 2 (CCAR2) is a multifaceted protein that controls diverse cellular functions; however, its function in cancer is unclear. To better understand its potential role in cancer, we examined gene expression patterns regulated by CCAR2 in cervical cancer cells. Cytokine and chemokine production by CCAR2-deficient cells increased under oxidative conditions. In particular, H2O2-treated CCAR2-depleted cells showed a significant increase in interleukin-8 (IL-8) production, indicating a negative regulation of IL-8 by CCAR2. Upregulation of IL-8 expression in CCAR2-deficient cells occurred via activation of transcription factor AP-1. The negative correlation between CCAR2 and IL-8 expression was confirmed by examining mRNA and protein levels in tissues from cervical cancer patients. Furthermore, CCAR2-regulated IL-8 expression is associated with a shorter survival of cervical cancer patients. Overall, the data suggest that CCAR2 plays a critical role in controlling both the cancer secretome and cancer progression.

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.

Neuromedin B receptor antagonism inhibits migration, invasion, and epithelial-mesenchymal transition of breast cancer cells.

Neuromedin B (NMB) acts as an autocrine growth factor and a pro-angiogenic factor. Its receptor, NMB receptor (NMB-R), is overexpressed in solid tumors. In the present study, we showed that an NMB-R antagonist, PD168368, suppresses migration and invasion of the human breast cancer cell line MDA-MB-231. In addition, PD168368 reduced epithelial-mesenchymal transition (EMT) of breast cancer cells by E-cadherin upregulation and vimentin downregulation. Moreover, we found that PD168368 potently inhibits in vivo metastasis of breast cancer. Taken together, these findings suggest that NMB-R antagonism may be an alternative approach to prevent breast cancer metastasis, and targeting NMB-R may provide a novel therapeutic strategy for breast cancer treatment.

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.

AK-1, a SIRT2 inhibitor, destabilizes HIF-1α and diminishes its transcriptional activity during hypoxia.

Sirtuin family proteins are involved in the regulation of hypoxic responses which are primarily dependent on a hypoxia-inducible factor (HIF). However, few studies have examined the use of sirtuin inhibitors to regulate HIF. The present study examined the effect of a SIRT2-specific inhibitor, AK-1, on hypoxic responses. Under hypoxic conditions, AK-1 increased the ubiquitination of HIF-1α in a VHL-dependent manner, leading to the degradation of HIF-1α via a proteasomal pathway. Downregulation of HIF-1α expression reduced its transcriptional activity and, eventually, reduced the expression of BNIP3, one of HIF-1 target genes, in AK-1-treated cells. These data demonstrate that SIRT2 inhibition attenuates hypoxic responses, and that SIRT2 inhibitors may have potential as treatments for hypoxia-associated pathological conditions.

Age-related changes in pial arterial structure and blood flow in mice.

Age-related cerebral blood flow decreases are thought to deteriorate cognition and cause senescence, although the related mechanism is unclear. To investigate the relationships between aging and changes in cerebral blood flow and vasculature, we obtained fluorescence images of young (2-month-old) and old (12-month-old) mice using indocyanine green (ICG). First, we found that the blood flow in old mice's brains is lower than that in young mice and that old mice had more curved pial arteries and fewer pial artery junctions than young mice. Second, using Western blotting, we determined that the ratio of collagen to elastin (related to cerebral vascular wall distensibility) increased with age. Finally, we found that the peak ICG intensity and blood flow index decreased, whereas the mean transit time increased, with age in the middle cerebral artery and superior sagittal sinus. Age-related changes in pial arterial structure and composition, concurrent with the observed changes in the blood flow parameters, suggest that age-related changes in the cerebral vasculature structure and distensibility may induce altered brain blood flow.

Exendin-4 protects hindlimb ischemic injury by inducing angiogenesis.

Exendin-4, an analog of glucagon-like peptide-1, has shown to have beneficial effects on endothelial function, and was recently approved for the treatment of diabetes. In previous studies, we showed that exendin-4 induces angiogenesis in in vitro and ex vivo assays; in this study, we assessed the proangiogenic effects of exendin-4 in vivo using a mouse hindlimb ischemia model. Treatment with exendin-4 for three days mitigated hindlimb and gastrocnemius muscle fiber necrosis. Hindlimb perfusion was determined using indocyanine green fluorescence dynamics that showed, significantly higher blood flow rate to the ischemic hindlimbs in an exendin-4-treated group. Immunohistochemistry assay showed that exendin-4 increased CD31-positive areas in the gastrocnemius muscle of ischemic limbs. Furthermore, treatment of the hindlimbs of ischemic mice with exendin-4 increased vascular endothelial growth factor (VEGF) and phospho-extracellular signal-related kinase (ERK) on western blot analysis. Our data demonstrate that exendin-4 prevents hindlimb ischemic injury by inducing vessels via VEGF angiogenic-related pathways. These findings suggest that exendin-4 has potential as a therapeutic agent for vascular diseases that stimulate angiogenesis.

Zingerone activates VMAT2 during MPP(+) -induced Cell Death.

Parkinson's disease (PD) is characterized by a progressive and selective loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) and striatum. 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is used to produce an animal model for PD, and it is converted to 1-methyl-4-phenylpyridine (MPP(+)) in animals. MPP(+) accumulation leads to neuronal cell death. Vesicular monoamine transporter 2 (VMAT2) regulates the accumulation of monoamine neurotransmitters into synaptic vesicles and is involved in neuroprotection against neurotoxin-induced cell death. Recently, zingerone has been reported to reduce oxidative stress and inhibit inflammation. Therefore, we examined the effect of zingerone on neuronal cell death in a PD model. In an MPP(+) and MPTP-mediated PD model, neuronal cell survival was increased by zingerone without modifying neuroinflammation or reactive oxygen species generation. Zingerone also induced ERK activation and VMAT2 expression, leading to the attenuation of MPP(+)-induced neuronal cell death. Our current results suggest that zingerone has a neuroprotective effect in a PD model.