Melanoma biology and treatment: a review of novel regulated cell death-based approaches.

The incidence of melanoma, the most lethal form of skin cancer, has increased due to ultraviolet exposure. The treatment of advanced melanoma, particularly metastatic cases, remains challenging with poor outcomes. Targeted therapies involving BRAF/MEK inhibitors and immunotherapy based on anti-PD1/anti-CTLA4 antibodies have achieved long-term survival rates of approximately 50% for patients with advanced melanoma. However, therapy resistance and inadequate treatment response continue to hinder further breakthroughs in treatments that increase survival rates. This review provides an introduction to the molecular-level pathogenesis of melanoma and offers an overview of current treatment options and their limitations. Cells can die by either accidental or regulated cell death (RCD). RCD is an orderly cell death controlled by a variety of macromolecules to maintain the stability of the internal environment. Since the uncontrolled proliferation of tumor cells requires evasion of RCD programs, inducing the RCD of melanoma cells may be a treatment strategy. This review summarizes studies on various types of nonapoptotic RCDs, such as autophagy-dependent cell death, necroptosis, ferroptosis, pyroptosis, and the recently discovered cuproptosis, in the context of melanoma. The relationships between these RCDs and melanoma are examined, and the interplay between these RCDs and immunotherapy or targeted therapy in patients with melanoma is discussed. Given the findings demonstrating melanoma cell death in response to different stimuli associated with these RCDs, the induction of RCD shows promise as an integral component of treatment strategies for melanoma.

Functional characterization of stap2b in zebrafish vascular development.

The genetic control and signaling pathways of vascular development are not comprehensively understood. Transcription factors Islet2 (Isl2) and nr2f1b are critical for vascular growth in zebrafish, and further transcriptome analysis has revealed potential targets regulated by isl2/nr2f1b. In this study, we focused on the potential activation gene signal-transducing adaptor protein 2b (stap2b) and revealed a novel role of stap2b in vascular development. stap2b mRNA was expressed in developing vessels, suggesting stap2b plays a role in vascularization. Knocking down stap2b expression by morpholino injection or Crispr-Cas9-generated stap2b mutants caused vascular defects, suggesting a role played by stap2b in controlling the patterning of intersegmental vessels (ISVs) and the caudal vein plexus (CVP). The vessel abnormalities associated with stap2b deficiency were found to be due to dysregulated cell migration and proliferation. The decreased expression of vascular-specific markers in stap2b morphants was consistent with the vascular defects observed. In contrast, overexpression of stap2b enhanced the growth of ISVs and reversed the vessel defects in stap2b morphants. These data suggest that stap2b is necessary and sufficient to promote vascular development. Finally, we examined the interaction between stap2b and multiple signaling. We showed that stap2b regulated ISV growth through the JAK-STAT pathway. Moreover, we found that stap2b was regulated by Notch signaling to control ISV growth, and stap2b interacted with bone morphogenetic protein signaling to contribute to CVP formation. Altogether, we demonstrated that stap2b acts downstream of the isl2/nr2f1b pathway to play a pivotal role in vascular development via interaction with multiple signaling pathways.

Effects of quantum size on the thermoelectric properties of bismuth.

First principles and the Boltzmann transport equation have been combined to investigate the effects of quantum size L/λ, the ratio of quantum confinement length L to thermal de Broglie wavelength λ, on the thermoelectric properties of 2D ß-bismuth. It is revealed that the thermoelectric properties of 2D ß-bismuth are highly influenced by quantum size, especially when the L/λ is less than 0.1. Specifically, the Seebeck coefficients of both electrons and holes decrease as the L/λ ratio increases, while the electrical and thermal conductivity show the opposite trend. The results also show that 2D bismuth with three or more layers has semimetal properties, with the first observation of a semiconductor-semimetal transition in 2D bismuth. Moreover, the electron affinity, ionization energy, and work function of 2D ß-bismuth do not exhibit a significant variation or trend with quantum size effects. The detailed electronic structures provide a fundamental understanding of the thermoelectric properties of bismuth, and the obtained results may provide a deep understanding of the relationship between the quantum size and the thermoelectric properties of 2D ß-bismuth.

Diphenyl disulfide potentiates the apoptosis of breast cancer cells through Bax proteolytic activation with accompanying autophagy.

Breast cancer is a leading cause of cancer-related death worldwide, and chemoresistance often leads to poor patient outcomes. In this study, we investigated the anticancer activity of synthetic diphenyl disulfide (DPDS) in breast cancer cell lines. DPDS inhibited cellular proliferation and viability in a dose-dependent manner and reduced colony formation, an index of clonogenicity. Annexin-V and 7-AAD double staining showed that DPDS could induce the apoptosis of breast cancer cells. Western blotting of the expression of Bax p21 and its cleaved form p18 suggested the activation of p18 Bax-induced apoptosis. Furthermore, the increased expression of the autophagy marker LC3B-II indicated autophagic lysosome accumulation induced by DPDS. Our findings suggest that DPDS has potential as a candidate for treating breast cancer, and further modifications and optimizations are warranted.

Interaction of transcription factors Islet2 and Nr2f1b to control vascular patterning during zebrafish development.

Many regulators controlling arterial identity are well described; however, transcription factors that promote vein identity and vascular patterning have remained largely unknown. We previously identified the transcription factors Islet2 (Isl2) and Nr2f1b required for specification of the vein and tip cell identity mediated by notch pathway in zebrafish. However, the interaction between Isl2 and Nr2f1b is not known. In this study, we report that Nr2f2 plays minor roles on vein and intersegmental vessels (ISV) growth and dissect the genetic interactions among the three transcription factors Isl2, Nr2f1b, and Nr2f2 using a combinatorial knockdown strategy. The double knockdown of isl2/nr2f1b, isl2/nr2f2, and nr2f1b/nr2f2 showed the enhanced defects in vasculature including less completed ISV, reduced veins, and ISV cells. We further tested the genetic relationship among these three transcription factors. We found isl2 can regulate the expression of nr2f1b and nr2f2, suggesting a model where Isl2 functions upstream of Nr2f1b and Nr2f2. We hypothsized that Isl2 and Nr2f1b can function together through cis-regulatory binding motifs. In-vitro luciferase assay results, we showed that Isl2 and Nr2f1b can cooperatively enhance gene expression. Moreover, co-immunoprecipitation results indicated that Isl2 and Nr2f1b interact physically. Together, we showed that the interaction of the Nr2f1b and Nr2f2 transcription factors in combination with the Islet2 play coordinated roles in the vascular development of zebrafish.

Inhibition of visfatin alleviates sepsis-induced intestinal damage by inhibiting Hippo signaling pathway.

BACKGROUND: The aim of this study is to investigate role of Visfatin, one of the pro-inflammatory adipokines, in sepsis-induced intestinal injury and to clarify the potential mechanism. METHODS: C57BL/6 mice underwent cecal ligation and puncture (CLP) surgery to establish sepsis model in vivo. Intestinal epithelial cells were stimulated with LPS to mimic sepsis-induced intestinal injury in vitro. FK866 (the inhibitor of Visfatin) with or without XMU-MP-1 (the inhibitor of Hippo signaling) was applied for treatment. The expression levels of Visfatin, NF-κB and Hippo signaling pathways-related proteins were detected by western blot or immunohistochemistry. The intestinal cell apoptosis and intestinal injury were investigated by TUNEL staining and H&E staining, respectively. ELISA was used to determine the production of inflammatory cytokines. RESULTS: The expression of Visfatin increased in CLP mice. FK866 reduced intestinal pathological injury, inflammatory cytokines production, and intestinal cell apoptosis in sepsis mice. Meanwhile, FK866 affected NF-κB and Hippo signaling pathways. Additionally, the effects of FK866 on inflammatory response, apoptosis, Hippo signaling and NF-κB signaling were partly abolished by XMU-MP-1, the inhibitor of Hippo signaling. In vitro experiments also revealed that FK866 exhibited a protective role against LPS-induced inflammatory response and apoptosis in intestinal cells, as well as regulating NF-κB and Hippo signaling, whereas addition of XMU-MP-1 weakened the protective effects of FK866. CONCLUSION: In short, this study demonstrated that inhibition of Visfatin might alleviate sepsis-induced intestinal injury through Hippo signaling pathway, supporting a further research on Visfatin as a therapeutic target.

Small GTPases and Their Regulators: A Leading Road toward Blood Vessel Development in Zebrafish.

Members of the Ras superfamily have been found to perform several functions leading to the development of eukaryotes. These small GTPases are divided into five major subfamilies, and their regulators can "turn on" and "turn off" signals. Recent studies have shown that this superfamily of proteins has various roles in the process of vascular development, such as vasculogenesis and angiogenesis. Here, we discuss the role of these subfamilies in the development of the vascular system in zebrafish.

Nitrobenzoate-Derived Compound X8 Impairs Vascular Development in Zebrafish.

Proper growth and patterning of blood vessels are critical for embryogenesis. Chemicals or environmental hormones may interfere with vascular growth and cause developmental defects. Nitrobenzoate-based compounds have been demonstrated to have a wide range of biological and pharmacological functions, leading to the development of numerous 4-nitrobenzoate derivatives for clinical application. In this study, we tested a novel nitrobenzoate-derived compound, X8, and investigated its effects on vascular development using zebrafish as a model organism. We first determined the survival rate of embryos after the addition of exogenous X8 (0.5, 1, 3, 5, and 10 µM) to the fish medium and determined a sublethal dose of 3 µM for use in further assays. We used transgenic fish to examine the effects of X8 treatment on vascular development. At 25-32 h postfertilization (hpf), X8 treatment impaired the growth of intersegmental vessels (ISVs) and caudal vein plexuses (CVPs). Moreover, X8-treated embryos exhibited pericardial edema and circulatory defects at 60-72 hpf, suggesting the effects of X8 in vasculature. Apoptosis tests showed that the vascular defects were likely caused by the inhibition of proliferation and migration. To investigate the molecular impacts underlying the defects in the vasculature of X8-treated fish, the expression levels of vascular markers, including ephrinb2, mrc1, and stabilin, were assessed, and the decreased expression of those genes was detected, indicating that X8 inhibited the expression of vascular genes. Finally, we showed that X8 treatment disrupted exogenous GS4012-induced angiogenesis in Tg(flk:egfp) zebrafish embryos. In addition, vascular defects were enhanced during cotreatment with X8 and the VEGFR2 inhibitor SU5416, suggesting that X8 treatment causes vascular defects mediated by disruption of VEGF/VEGFR2 signaling. Collectively, our findings indicate that X8 could be developed as a novel antiangiogenic agent.

New Insight into the Effects of Metformin on Diabetic Retinopathy, Aging and Cancer: Nonapoptotic Cell Death, Immunosuppression, and Effects beyond the AMPK Pathway.

Under metabolic stress conditions such as hypoxia and glucose deprivation, an increase in the AMP:ATP ratio activates the AMP-activated protein kinase (AMPK) pathway, resulting in the modulation of cellular metabolism. Metformin, which is widely prescribed for type 2 diabetes mellitus (T2DM) patients, regulates blood sugar by inhibiting hepatic gluconeogenesis and promoting insulin sensitivity to facilitate glucose uptake by cells. At the molecular level, the most well-known mechanism of metformin-mediated cytoprotection is AMPK pathway activation, which modulates metabolism and protects cells from degradation or pathogenic changes, such as those related to aging and diabetic retinopathy (DR). Recently, it has been revealed that metformin acts via AMPK- and non-AMPK-mediated pathways to exert effects beyond those related to diabetes treatment that might prevent aging and ameliorate DR. This review focuses on new insights into the anticancer effects of metformin and its potential modulation of several novel types of nonapoptotic cell death, including ferroptosis, pyroptosis, and necroptosis. In addition, the antimetastatic and immunosuppressive effects of metformin and its hypothesized mechanism are also discussed, highlighting promising cancer prevention strategies for the future.

α2A-adrenoceptor deficiency attenuates lipopolysaccharide-induced lung injury by increasing norepinephrine levels and inhibiting alveolar macrophage activation in acute respiratory distress syndrome.

Acute respiratory distress syndrome (ARDS) is a severe condition with high morbidity and mortality and few interventions. The role of sympathetic stress in the pathogenesis of ARDS has attracted recent research attention. Blockade of α-2 or α2A-adrenoceptor (α2A-AR) has been shown to attenuate lung injury induced by lipopolysaccharide (LPS) in rats. However, the mechanism is unclear. We confirmed the role of α2A-AR in ARDS using knockout mice and alveolar macrophages following LPS stimulation to assess the underlying mechanisms. We found that α2A-AR deficiency decreased the permeability of the alveolar capillary barrier in ARDS mice and suppressed lung inflammation by reducing inflammatory cell infiltration and the production of TNF-α, interleukin (IL)-6, and CXCL2/MIP-2. LPS stimulation decreased NF-κB activation in lung tissues of α2A-AR deficient mice and increased norepinephrine concentrations. In vitro, we found that norepinephrine inhibited the production of TNF-α, IL-6, and CXCL2/MIP-2 and promoted the secretion of IL-10 from LPS-stimulated murine alveolar macrophages. Blockade of α2A-AR by a specific antagonist further inhibited the production of TNF-α, IL-6, and IL-10. Furthermore, norepinephrine down-regulated NF-κB activation in stimulated alveolar macrophages. Altogether, these results suggest that α2A-AR deficiency ameliorates lung injury by increasing norepinephrine concentrations in lung tissues and inhibiting the activation of alveolar macrophages.

Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

VEGF stimulates the formation of new blood vessels by inducing endothelial cell (EC) proliferation and migration. Brefeldin A (BFA)-inhibited guanine nucleotide-exchange protein (BIG)1 and 2 accelerate the replacement of bound GDP with GTP to activate ADP-ribosylation factor (Arf)1, which regulates vesicular transport between the Golgi and plasma membrane. Although it has been reported that treating cells with BFA interferes with Arf1 activation to inhibit VEGF secretion, the role of BIG1 and BIG2 in VEGF trafficking and expression, EC migration and proliferation, and vascular development remains unknown. Here, we found that inactivation of Arf1 reduced VEGF secretion but did not affect the levels of VEGF protein. Interestingly, however, BIG1 and BIG2 knockdown significantly decreased the levels of VEGF mRNA and protein in glioblastoma U251 cells and HUVECs. Furthermore, depletion of BIG1 and BIG2 inhibited HUVEC angiogenesis by diminishing cell migration. Angioblast migration and intersegmental vessel sprouting were also impaired when the BIG2 homolog, Arf guanine nucleotide exchange factor (arfgef)2, was knocked down in zebrafish with endothelial expression of green fluorescent protein (GFP). Depletion of arfgef2 by clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9) also caused defects in vascular development of zebrafish embryos. Taken together, these data reveal that BIG1 and BIG2 participate in endothelial cell angiogenesis.-Lu, F.-I., Wang, Y.-T., Wang, Y.-S., Wu, C.-Y., Li, C.-C. Involvement of BIG1 and BIG2 in regulating VEGF expression and angiogenesis.

C2-Ceramide-Induced Rb-Dominant Senescence-Like Phenotype Leads to Human Breast Cancer MCF-7 Escape from p53-Dependent Cell Death.

Ceramide is a sphingolipid which regulates a variety of signaling pathways in eukaryotic cells. Exogenous ceramide has been shown to induce cellular apoptosis. In this study, we observed that exogenous ceramide induced two distinct morphologies of cell fate following C2-ceramide treatment between the two breast cancer cell lines MCF-7 (wild type p53) and MDA-MB-231 (mutant p53) cells. The growth assessment showed that C2-ceramide caused significant growth inhibition and apoptosis in MDA-MB-231 cells through down-regulating the expression of mutant p53 whereas up-regulating the expression of pro-apoptotic Bad, and the proteolytic activation of caspase-3. However, senescence-associated (SA)-ß-galactosidase (ß-gal) was regulated in MCF-7 cells after C2-ceramide treatment. The results of proliferation and apoptosis assays showed that MCF-7 cells were more resistant to C2-ceramide treatment compared to MDA-MB-231 cells. Furthermore, C2-ceramide treatment induced a time-responsive increase in Rb protein, a key regulator of senescence accompanied with the upregulation of both mRNA level and protein level of SA-genes PAI-1 and TGaseII in MCF-7 but not in MDA-MB-231 cells, suggesting that some cancer cells escape apoptosis through modulating senescence-like phenotype. The results of our present study depicted the mechanism of C2-ceramide-resistant breast cancer cells, which might benefit the strategic development of ceramide-based chemotherapeutics against cancer in the future.

Ketamine exacerbates cortical neuroapoptosis under hyperoxic conditions by upregulating expression of the N-methyl-D-aspartate receptor subunit NR1 in the developing rat brain.

BACKGROUND: Ketamine and hyperoxia are widely used in obstetric and pediatric settings. Either ketamine or hyperoxia has been reported to cause neuroapoptosis in the developing brain, and ketamine-induced neuronal apoptosis may involve a compensatory upregulation of the N-methyl-D-aspartate (NMDA) receptor NR1 subunit. This study investigated the impact of ketamine administration under hyperoxic conditions on cortical neuroapoptosis and NR1 subunit expression in the infant rat brain. METHODS: Male, 7-day-old rats were randomly allocated to four groups: control, ketamine, hyperoxia, and ketamine + hyperoxia (n = 18 per group). Rats in the control and ketamine groups received subcutaneous injections of either vehicle (saline) or ketamine (50 mg/kg) in room air (21% oxygen). The hyperoxia and ketamine + hyperoxia groups were exposed to 60% oxygen for 2 h after receiving saline or ketamine. Physiological parameters and arterial oxygen saturation were observed. Neuronal apoptosis and the expressions of NR1 mRNA and protein in the frontal cortex were also examined by transferase dUTP nick end labeling (TUNEL) assays, qPCR and Western blot, respectively. RESULTS: Ketamine alone had no effect on paO2 (P > 0.05), but pups exposed to hyperoxia or hyperoxia + ketamine had significantly greater paO2 values compared to control animals (P < 0.01). Animals exposed to ketamine and ketamine + hyperoxia showed higher apoptotic scores, mRNA and protein expression levels of NR1 than control animals (P < 0.01), and ketamine + hyperoxia caused a significantly greater increase than ketamine alone (P < 0.01). CONCLUSIONS: These data suggest that ketamine administration under hyperoxic conditions exacerbates cortical neuroapoptosis in the developing brain, which may be closely associated with an enhancement in NMDA receptor NR1 subunit expression.

Effects of propofol and etomidate anesthesia on cardiovascular miRNA expression: the different profiles?

BACKGROUND: The effects of the intravenous anesthetics propofol and etomidate on circulation are significantly different; however, their differing effects on miRNA expression in the cardiovascular system are not clearly understood. The purpose of this study is to investigate the effects of these two anesthetics on miRNA expression profiles in the heart and blood vessels. METHODS: Rats were randomly divided into a propofol group and an etomidate group. Spontaneous breathing was maintained throughout the anesthesia process and the rats' oxygen supply was ensured. Heart and thoracic aorta tissue was harvested 3 h after induction. The expression profiles of cardiovascular miRNAs were detected by microarray 4.0 analysis. Twelve representative miRNAs were selected for qRT-PCR validation, and their target genes were predicted using bioinformatics methods. RESULTS: Microarray analysis showed 16 differentially expressed miRNAs in heart tissue from the propofol group compared with the etomidate group (10 up-regulated and 6 down-regulated), while in the blood vessels there were 25 altered miRNAs (10 up-regulated, 15 down-regulated). After verifying 12 representative miRNAs via qRT-PCR, the results showed no significant difference in the expression of miRNAs in the heart tissue, but a significant difference in the expression of 5 miRNAs in vessel tissue between the two groups. Bioinformatics analysis predicts that the target genes of the 5 differentially expressed miRNAs are associated with chemical synapse signaling pathways. CONCLUSIONS: Propofol and etomidate have different effects on the expression of cardiovascular miRNAs, and further research is needed to elucidate the functional consequences of these differentially expressed miRNAs.

A novel sulfonyl chromen-4-ones (CHW09) preferentially kills oral cancer cells showing apoptosis, oxidative stress, and DNA damage.

Several functionalized chromones, the key components of naturally occurring oxygenated heterocycles, have anticancer effects but their sulfone compounds are rarely investigated. In this study, we installed a sulfonyl substituent to chromen-4-one skeleton and synthesized CHW09 to evaluate its antioral cancer effect in terms of cell viability, cell cycle, apoptosis, oxidative stress, and DNA damage. In cell viability assay, CHW09 preferentially kills two oral cancer cells (Ca9-22 and CAL 27), less affecting normal oral cells (HGF-1). Although CHW09 does not change the cell cycle distribution significantly, CHW09 induces apoptosis validated by flow cytometry for annexin V and by western blotting for cleaved poly(ADP-ribose) polymerase (PARP), and caspases 3/8/9. These apoptosis signaling expressions are partly decreased by apoptosis inhibitor (Z-VAD-FMK) or free radical scavenger (N-acetylcysteine). Furthermore, CHW09 induces oxidative stress validated by flow cytometry for the generations of reactive oxygen species (ROS) and mitochondrial superoxide (MitoSOX), and the suppression of mitochondrial membrane potential (MMP). CHW09 also induces DNA damage validated by flow cytometry for the increases of DNA double strand break marker γH2AX and oxidative DNA damage marker 8-oxo-2'-deoxyguanosine (8-oxodG). Therefore, our newly synthesized CHW09 induces apoptosis, oxidative stress, and DNA damage, which may lead to preferential killing of oral cancer cells compared with normal oral cells.

Exogenous C8-Ceramide Induces Apoptosis by Overproduction of ROS and the Switch of Superoxide Dismutases SOD1 to SOD2 in Human Lung Cancer Cells.

Ceramides, abundant sphingolipids on the cell membrane, can act as signaling molecules to regulate cellular functions including cell viability. Exogenous ceramide has been shown to exert potent anti-proliferative effects against cancer cells, but little is known about how it affects reactive oxygen species (ROS) in lung cancer cells. In this study, we investigated the effect of N-octanoyl-D-erythro-sphingosine (C8-ceramide) on human non-small-cell lung cancer H1299 cells. Flow cytometry-based assays indicated that C8-ceramide increased the level of endogenous ROS in H1299 cells. Interestingly, the ratio of superoxide dismutases (SODs) SOD1 and SOD2 seem to be regulated by C8-ceramide treatment. Furthermore, the accumulation of cell cycle G1 phase and apoptotic populations in C8-ceramide-treated H1299 cells was observed. The results of the Western blot showed that C8-ceramide causes a dramatically increased protein level of cyclin D1, a critical regulator of cell cycle G1/S transition. These results suggest that C8-ceramide acts as a potent chemotherapeutic agent and may increase the endogenous ROS level by regulating the switch of SOD1 and SOD2, causing the anti-proliferation, and consequently triggering the apoptosis of NSCLC H1299 cells. Accordingly, our works may give a promising strategy for lung cancer treatment in the future.

α-Melanocyte-Stimulating Hormone Attenuates Neovascularization by Inducing Nitric Oxide Deficiency via MC-Rs/PKA/NF-κB Signaling.

α-melanocyte-stimulating hormone (α-MSH) has been characterized as a novel angiogenesis inhibitor. The homeostasis of nitric oxide (NO) plays an important role in neovascularization. However, it remains unclear whether α-MSH mitigates angiogenesis through modulation of NO and its signaling pathway. The present study elucidated the function and mechanism of NO signaling in α-MSH-induced angiogenesis inhibition using cultured human umbilical vein endothelial cells (HUVECs), rat aorta rings, and transgenic zebrafish. By Griess reagent assay, it was found α-MSH dose-dependently reduced the NO release in HUVECs. Immunoblotting and immunofluorescence analysis revealed α-MSH potently suppressed endothelial and inducible nitric oxide synthase (eNOS/iNOS) expression, which was accompanied with inhibition of nuclear factor kappa B (NF-κB) activities. Excessive supply of NO donor l-arginine reversed the α-MSH-induced angiogenesis inhibition in vitro and in vivo. By using antibody neutralization and RNA interference, it was delineated that melanocortin-1 receptor (MC1-R) and melanocortin-2 receptor (MC2-R) participated in α-MSH-induced inhibition of NO production and NF-κB/eNOS/iNOS signaling. This was supported by pharmaceutical inhibition of protein kinase A (PKA), the downstream effector of MC-Rs signaling, using H89 abolished the α-MSH-mediated suppression of NO release and eNOS/iNOS protein level. Therefore, α-MSH exerts anti-angiogenic function by perturbing NO bioavailability and eNOS/iNOS expression in endothelial cells.

The LIM-homeodomain transcription factor Islet2a promotes angioblast migration.

Angioblasts of the developing vascular system require many signaling inputs to initiate their migration, proliferation and differentiation into endothelial cells. What is less studied is which intrinsic cell factors interpret these extrinsic signals. Here, we show the Lim homeodomain transcription factor islet2a (isl2a) is expressed in the lateral posterior mesoderm prior to angioblast migration. isl2a deficient angioblasts show disorganized migration to the midline to form axial vessels and fail to spread around the tailbud of the embryo. Isl2a morphants have fewer vein cells and decreased vein marker expression. We demonstrate that isl2a is required cell autonomously in angioblasts to promote their incorporation into the vein, and is permissive for vein identity. Knockout of isl2a results in decreased migration and proliferation of angioblasts during intersegmental artery growth. Since Notch signaling controls both artery-vein identity and tip-stalk cell formation, we explored the interaction of isl2a and Notch. We find that isl2a expression is negatively regulated by Notch activity, and that isl2a positively regulates flt4, a VEGF-C receptor repressed by Notch during angiogenesis. Thus Isl2a may act as an intermediate between Notch signaling and genetic programs controlling angioblast number and migration, placing it as a novel transcriptional regulator of early angiogenesis.

Dual roles of extracellular signal-regulated kinase (ERK) in quinoline compound BPIQ-induced apoptosis and anti-migration of human non-small cell lung cancer cells.

BACKGROUND: 2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11H-indeno[1,2-c]quinoline-11-one (BPIQ), is a synthetic quinoline analog. A previous study showed the anti-cancer potential of BPIQ through modulating mitochondrial-mediated apoptosis. However, the effect of BPIQ on cell migration, an index of cancer metastasis, has not yet been examined. Furthermore, among signal pathways involved in stresses, the members of the mitogen-activated protein kinase (MAPK) family are crucial for regulating the survival and migration of cells. In this study, the aim was to explore further the role of MAPK members, including JNK, p38 and extracellular signal-regulated kinase (ERK) in BPIQ-induced apoptosis and anti-migration of human non-small cell lung cancer (NSCLC) cells. METHODS: Western Blot assay was performed for detecting the activation of MAPK members in NSCLC H1299 cells following BPIQ administration. Cellular proliferation was determined using a trypan blue exclusion assay. Cellular apoptosis was detected using flow cytometer-based Annexin V/propidium iodide dual staining. Cellular migration was determined using wound-healing assay and Boyden's chamber assay. Zymography assay was performed for examining MMP-2 and -9 activities. The assessment of MAPK inhibition was performed for further validating the role of JNK, p38, and ERK in BPIQ-induced growth inhibition, apoptosis, and migration of NSCLC cells. RESULTS: Western Blot assay showed that BPIQ treatment upregulates the phosphorylated levels of both MAPK proteins JNK and ERK. However, only ERK inhibitor rescues BPIQ-induced growth inhibition of NSCLC H1299 cells. The results of Annexin V assay further confirmed the pro-apoptotic role of ERK in BPIQ-induced cell death of H1299 cells. The results of wound healing and Boyden chamber assays showed that sub-IC50 (sub-lethal) concentrations of BPIQ cause a significant inhibition of migration in H1299 cells accompanied with downregulating the activity of MMP-2 and -9, the motility index of cancer cells. Inhibition of ERK significantly enhances BPIQ-induced anti-migration of H1299 cells. CONCLUSIONS: Our results suggest ERK may play dual roles in BPIQ-induced apoptosis and anti-migration, and it would be worthwhile further developing strategies for treating chemoresistant lung cancers through modulating ERK activity.

Sinularin induces oxidative stress-mediated G2/M arrest and apoptosis in oral cancer cells.

Soft corals-derived natural product, sinularin, was antiproliferative against some cancers but its effect and detailed mechanism on oral cancer cells remain unclear. The subject of this study is to examine the antioral cancer effects and underlying detailed mechanisms in terms of cell viability, oxidative stress, cell cycle analysis, and apoptosis analyses. In MTS assay, sinularin dose-responsively decreased cell viability of three oral cancer cells (Ca9-22, HSC-3, and CAL 27) but only little damage to oral normal cells (HGF-1). This cell killing effect was rescued by the antioxidant N-acetylcysteine (NAC) pretreatment. Abnormal cell morphology and induction of reactive oxygen species (ROS) were found in sinularin-treated oral cancer Ca9-22 cells, however, NAC pretreatment also recovered these changes. Sinularin arrested the Ca9-22 cells at G2/M phase and dysregulated the G2/M regulatory proteins such as cdc2 and cyclin B1. Sinularin dose-responsively induced apoptosis on Ca9-22 cells in terms of flow cytometry (annexin V and pancaspase analyses) and western blotting (caspases 3, 8, 9) and poly (ADP-ribose) polymerase (PARP). These apoptotic changes of sinularin-treated Ca9-22 cells were rescued by NAC pretreatment. Taken together, sinularin induces oxidative stress-mediated antiproliferation, G2/M arrest, and apoptosis against oral cancer cells and may be a potential marine drug for antioral cancer therapy.