Publications by authors named "Chang-Yi Wu"

57 Publications

Oxidative Stress-Dependent Synergistic Antiproliferation, Apoptosis, and DNA Damage of Ultraviolet-C and Coral-Derived Sinularin Combined Treatment for Oral Cancer Cells.

Cancers (Basel) 2021 May 18;13(10). Epub 2021 May 18.

PhD Program in Life Science, Department of Biomedical Science and Environmental Biology, College of Life Science, Kaohsiung Medical University, Kaohsiung 80708, Taiwan.

Combined treatment is increasingly used to improve cancer therapy. Non-ionizing radiation ultraviolet-C (UVC) and sinularin, a coral -derived cembranolide, were separately reported to provide an antiproliferation function to some kinds of cancer cells. However, an antiproliferation function using the combined treatment of UVC/sinularin has not been investigated as yet. This study aimed to examine the combined antiproliferation function and explore the combination of UVC/sinularin in oral cancer cells compared to normal oral cells. Regarding cell viability, UVC/sinularin displays the synergistic and selective killing of two oral cancer cell lines, but remains non-effective for normal oral cell lines compared to treatments in terms of MTS and ATP assays. In tests using the flow cytometry, luminescence, and Western blotting methods, UVC/sinularin-treated oral cancer cells exhibited higher reactive oxygen species production, mitochondrial superoxide generation, mitochondrial membrane potential destruction, annexin V, pan-caspase, caspase 3/7, and cleaved-poly (ADP-ribose) polymerase expressions than that in normal oral cells. Accordingly, oxidative stress and apoptosis are highly induced in a combined UVC/sinularin treatment. Moreover, UVC/sinularin treatment provides higher G2/M arrest and γH2AX/8-hydroxyl-2'deoxyguanosine-detected DNA damages in oral cancer cells than in the separate treatments. A pretreatment can revert all of these changes of UVC/sinularin treatment with the antioxidant -acetylcysteine. Taken together, UVC/sinularin acting upon oral cancer cells exhibits a synergistic and selective antiproliferation ability involving oxidative stress-dependent apoptosis and cellular DNA damage with low toxic side effects on normal oral cells.
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http://dx.doi.org/10.3390/cancers13102450DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158103PMC
May 2021

A Marine Terpenoid, Heteronemin, Induces Both the Apoptosis and Ferroptosis of Hepatocellular Carcinoma Cells and Involves the ROS and MAPK Pathways.

Oxid Med Cell Longev 2021 4;2021:7689045. Epub 2021 Jan 4.

Center for Cancer Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan.

Hepatocellular carcinoma (HCC) is a leading cause of death, resulting in over 700 thousand deaths annually worldwide. Chemotherapy is the primary therapeutic strategy for patients with late-stage HCC. Heteronemin is a marine natural product isolated from sp. that has been found to protect against carcinogenesis in cholangiocarcinoma, prostate cancer, and acute myeloid leukemia. In this study, heteronemin was found to inhibit the proliferation of the HCC cell lines HA22T and HA59T and induce apoptosis via the caspase pathway. Heteronemin treatment also induced the formation of reactive oxygen species (ROS), which are associated with heteronemin-induced cell death, and to trigger ROS removal by mitochondrial SOD2 rather than cytosolic SOD1. The mitogen-activated protein kinase (MAPK) signaling pathway was associated with ROS-induced cell death, and heteronemin downregulated the expression of ERK, a MAPK that is associated with cell proliferation. Inhibitors of JNK and p38, which are MAPKs associated with apoptosis, restored heteronemin-induced cell death. In addition, heteronemin treatment reduced the expression of GPX4, a protein that inhibits ferroptosis, which is a novel form of nonapoptotic programmed cell death. Ferroptosis inhibitor treatment also restored heteronemin-induced cell death. Thus, with appropriate structural modification, heteronemin can act as a potent therapeutic against HCC.
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http://dx.doi.org/10.1155/2021/7689045DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7803406PMC
January 2021

In vivo monitoring of carbonic anhydrase expression during the growth of larval zebrafish: a new environment-sensitive fluorophore for responsive turn-on fluorescence.

Chem Commun (Camb) 2020 Sep 25;56(76):11307-11310. Epub 2020 Aug 25.

Department of Chemistry, National Sun Yat-sen University, Kaohsiung 80424, Taiwan.

This study monitors the dynamic progress of a newly developed background-free, target responsive strategy; 2,3-dihydroquinolin-4-imine (DQI) that can instantly respond to environmental changes with fluorescence enhancement, revealing a comprehensive platform for in vivo fluorescence bioimaging of mebrane-bound carbonic anhydrase II in HeLa cells and its expression during the growth of larval zebrafish.
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http://dx.doi.org/10.1039/d0cc03090bDOI Listing
September 2020

Integrated Hypoxia Signaling and Oxidative Stress in Developmental Neurotoxicity of Benzo[a]Pyrene in Zebrafish Embryos.

Antioxidants (Basel) 2020 Aug 11;9(8). Epub 2020 Aug 11.

Department of Bioagricultural Sciences, National Chiayi University, Chiayi City 60004, Taiwan.

Benzo[a]pyrene (B[a]P) is a polycyclic aromatic hydrocarbon formed by the incomplete combustion of organic matter. Environmental B[a]P contamination poses a serious health risk to many organisms because the pollutant may negatively affect many physiological systems. As such, chronic exposure to B[a]P is known to lead to locomotor dysfunction and neurodegeneration in several organisms. In this study, we used the zebrafish model to delineate the acute toxic effects of B[a]P on the developing nervous system. We found that embryonic exposure of B[a]P downregulates and , causing morphological hypoplasia in the telencephalon, ventral thalamus, hypothalamus, epiphysis and posterior commissure. Moreover, hypoxia-inducible factors ( and ) are repressed upon embryonic exposure of B[a]P, leading to reduced expression of the Hif-target genes, and , which are associated with neural differentiation and maintenance. During normal embryogenesis, low-level oxidative stress regulates neuronal development and function. However, our experiments revealed that embryonic oxidative stress is greatly increased in B[a]treated embryos. The expression of was decreased and expression increased in B[a]treated embryos. These transcriptional changes were coincident with increased embryonic levels of HO and malondialdehyde, with the levels in B[a]treated fish similar to those in embryos treated with 120-μM HO. Together, our data suggest that reduced Hif signaling and increased oxidative stress are involved in B[a]induced acute neurotoxicity during embryogenesis.
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http://dx.doi.org/10.3390/antiox9080731DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7464806PMC
August 2020

The Phenoxyphenol Compound 4-HPPP Selectively Induces Antiproliferation Effects and Apoptosis in Human Lung Cancer Cells through Aneupolyploidization and ATR DNA Repair Signaling.

Oxid Med Cell Longev 2020 7;2020:5167292. Epub 2020 Jan 7.

Center for Cancer Research, Kaohsiung Medical University, Kaohsiung 807, Taiwan.

Lung cancer is a leading cause of cancer death worldwide, and non-small-cell lung cancer (NSCLC) accounts for 85% of lung cancer, which is highly metastatic, leading to the poor survival rate of patients. We recently reported that 4-[4-(4-hydroxyphenoxy)phenoxy]phenol (4-HPPP), a phenoxyphenol, exerts antihepatoma effects by inducing apoptosis and autophagy. In this study, we further examined the effect of 4-HPPP and its analogs on NSCLC cells. Colony formation assays showed that 4-HPPP exerts selective cytotoxicity against NSCLC H1299 cells; furthermore, the inhibitory effect of 4-HPPP on the proliferation and migration of NSCLC cells was validated using an zebrafish-based tumor xenograft assay. The flow cytometry-based dichlorofluorescein diacetate (DCF-DA) assays indicated that 4-HPPP caused an increase in reactive oxygen species (ROS) in NSCLC cells, and Western blot assays showed that the major ROS scavenging enzymes superoxide dismutases- (SODs-) 1/2 were upregulated, whereas peroxidase (PRX) was downregulated. Furthermore, 4-HPPP caused both aneuploidization and the accumulation of H2AX, a sensor of DNA damage, as well as the activation of double-strand break (DSB) markers, especially Ataxia-telangiectasia-mutated and Rad3-related (ATR) in NSCLC cells. Our present work suggests that the antiproliferative effects of 4-HPPP on lung cancer cells could be due to its phenoxyphenol structure, and 4-HPPP could be a candidate molecule for treating NSCLC by modulating ROS levels and lowering the threshold of polyploidy-specific cell death in the future.
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http://dx.doi.org/10.1155/2020/5167292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7024103PMC
September 2020

Toxic Effects of Paclobutrazol on Developing Organs at Different Exposure Times in Zebrafish.

Toxics 2019 Dec 6;7(4). Epub 2019 Dec 6.

Department of Bioagricultural Sciences, National Chiayi University, Chiayi City 60004, Taiwan.

To enhance crop productivity and economic profit, farmers often use pesticides that modulate plant growth and prevent disease. However, contamination of ecosystems with agricultural pesticides may impair the health of resident biota. Paclobutrazol (PBZ), an aromatic-containing triazole, is widely applied to many crops in order to promote flowering and fruit setting, while also regulating plant growth and preventing fungus-related diseases. Due to its high mobility, high stability and potential for bioaccumulation, the risks of PBZ to the health of organisms and ecological systems have become a serious concern. In previous studies, we documented the toxicity of PBZ on developing heart, eyes, liver, pancreas and intestine of zebrafish. In this study, we sought to further understand the developmental stage-specific impacts of PBZ on digestive organs and other tissues. Zebrafish were exposed to PBZ beginning at different embryonic stages, and the toxic effects on organs were evaluated at 120 hpf (hours post-fertilization) by in situ hybridization staining with tissue-specific marker genes, such as liver, intestine and pancreas. Unsurprisingly, early-stage embryos exhibited higher sensitivity to PBZ-induced death and developmental hypoplasia of digestive organs. Interestingly, the developing liver and pancreas were more sensitive to PBZ than intestine when embryos were exposed at early stages, but these tissues showed lower sensitivity at later stages. Our delineation of the differential toxic effects of PBZ on developing organs at different exposure timings can serve as a powerful reference for further studies into the mechanisms of PBZ organ toxicity.
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http://dx.doi.org/10.3390/toxics7040062DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6958485PMC
December 2019

C-Ceramide-Induced Rb-Dominant Senescence-Like Phenotype Leads to Human Breast Cancer MCF-7 Escape from 53-Dependent Cell Death.

Int J Mol Sci 2019 Sep 2;20(17). Epub 2019 Sep 2.

Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan.

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 C-ceramide treatment between the two breast cancer cell lines MCF-7 (wild type 53) and MDA-MB-231 (mutant 53) cells. The growth assessment showed that C-ceramide caused significant growth inhibition and apoptosis in MDA-MB-231 cells through down-regulating the expression of mutant 53 whereas up-regulating the expression of pro-apoptotic Bad, and the proteolytic activation of caspase-3. However, senescence-associated (SA)-β-galactosidase (β-gal) was regulated in MCF-7 cells after C-ceramide treatment. The results of proliferation and apoptosis assays showed that MCF-7 cells were more resistant to C-ceramide treatment compared to MDA-MB-231 cells. Furthermore, C-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 C-ceramide-resistant breast cancer cells, which might benefit the strategic development of ceramide-based chemotherapeutics against cancer in the future.
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http://dx.doi.org/10.3390/ijms20174292DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6747432PMC
September 2019

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

FASEB J 2019 09 14;33(9):9959-9973. Epub 2019 Jun 14.

Department of Life Sciences, National Cheng Kung University, Tainan, Taiwan.

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 (), 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.
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http://dx.doi.org/10.1096/fj.201900342RRDOI Listing
September 2019

Combination Therapy of Chloroquine and C₂-Ceramide Enhances Cytotoxicity in Lung Cancer H460 and H1299 Cells.

Cancers (Basel) 2019 Mar 15;11(3). Epub 2019 Mar 15.

Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan.

Non-small cell lung cancer (NSCLC) is a type of malignant cancer, and 85% of metastatic NSCLC patients have a poor prognosis. C₂-ceramide induces G2/M phase arrest and cytotoxicity in NSCLC cells. In this study, the autophagy-inducing effect of C₂-ceramide was demonstrated, and cotreatment with the autophagy inhibitor chloroquine (CQ) was investigated in NSCLC H460 and H1299 cells. The results suggested that C₂-ceramide exhibited dose-dependent anticancer effects in H460 and H1299 cells and autophagy induction. Zebrafish-based acridine orange staining confirmed the combined effects in vivo. Importantly, the combination of a sublethal dose of C₂-ceramide and CQ resulted in additive cytotoxicity and autophagy in both cell lines. Alterations of related signaling factors, including Src and SIRT1 inhibition and activation of the autophagic regulators LAMP2 and LC3-I/II, contributed to the autophagy-dependent apoptosis. We found that C₂-ceramide continuously initiated autophagy; however, CQ inhibited autophagosome maturation and degradation during autophagy progression. Accumulated and non-degraded autophagosomes increased NSCLC cell stress, eventually leading to cell death. This study sheds light on improvements to NSCLC chemotherapy to reduce the chemotherapy dose and NSCLC patient burden.
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http://dx.doi.org/10.3390/cancers11030370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6468447PMC
March 2019

Molecular design of near-infrared fluorescent Pdots for tumor targeting: aggregation-induced emission anti-aggregation-caused quenching.

Chem Sci 2019 Jan 4;10(1):198-207. Epub 2018 Oct 4.

Department of Applied Chemistry , National Chiao Tung University , Hsinchu , Taiwan 30050 . Email:

Semiconducting polymer dots (Pdots) have recently emerged as a new type of ultrabright fluorescent probe that has been proved to be very useful for biomedical imaging. However, Pdots often suffer from serious fluorescence aggregation-caused quenching (ACQ) especially for near-infrared (NIR) fluorescent Pdots. This article compared two strategies to overcome the ACQ effect in near-infrared emissive Pdot systems: aggregation-induced emission (AIE) and anti-aggregation-caused quenching (anti-ACQ). The results show that the anti-ACQ platform outperforms the AIE system. The fluorescence quantum yield of anti-ACQ-based Pdots can be over 50% and the average per-particle brightness of the Pdots is about 5 times higher than that of the commercially available quantum dots. To help understand why the monomer conformations could greatly affect the optical properties of Pdots, molecular dynamics simulations were performed for the first time in such complicated Pdot systems. To demonstrate applications for fluorescence imaging, both microangiography imaging on living zebrafish embryos and specific tumor targeting on mice were performed. We anticipate that these studies will pave the way for the design of new highly fluorescent Pdot systems.
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http://dx.doi.org/10.1039/c8sc03510eDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6333168PMC
January 2019

Phytochemical naphtho[1,2-b] furan-4,5‑dione induced topoisomerase II-mediated DNA damage response in human non-small-cell lung cancer.

Phytomedicine 2019 Feb 28;54:109-119. Epub 2018 Jun 28.

Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan; Center of Excellence for Environmental Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Cancer Center, Department of Medical Research, Kaohsiung Medical University Hospital, Kaohsiung Medical University, Kaohsiung 807, Taiwan; Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan. Electronic address:

Background: Phytochemical naphtho[1,2-b] furan-4,5‑dione (NFD) presenting in Avicennia marina exert anti-cancer effects, but little is known regarding about DNA damage-mediated apoptosis in non-small-cell lung carcinoma (NSCLC).

Purpose: To examine whether NFD-induced apoptosis of NSCLC cells is correlated with the induction of DNA damage, and to investigate its underlying mechanism.

Study Design: The anti-proliferative effects of NFD were assessed by MTS Assay Kit FACS assay, and in vivo nude mice xenograft assay. The DNA damage related proteins, the Bcl-2 family and pro-apoptotic factors were examined by immunofluorescence assay, q-PCR, and western blotting. The activity of NF-κB p65 in nuclear extracts was detected using a colorimetric DNA-binding ELISA assay. The inhibitory activity of topoisomerase II (TOPO II) was evaluated by molecular docking and TOPO II catalytic assay.

Results: NFD exerted selective cytotoxicity against NSCLC H1299, H1437 and A549 cells rather than normal lung-embryonated cells MRC-5. Remarkably, we found that NFD activated the hull marker and modulator of DNA damage repairs such as γ-H2AX, ATM, ATR, CHK1, and CHK2 probably caused by the accumulation of intracellular reactive oxygen species (ROS) and inhibition of TOPO II activity. Furthermore, the suppression of transcription factor NF-κB by NFD resulted in significantly decreased levels of pro-survival proteins including Bcl-2 family Bcl-2, Bcl-xL and Mcl-1 and the endogenous inhibitors of apoptosis XIAP and survivin in H1299 cells. Moreover, the nude mice xenograft assay further validated the suppression of H1299 growth by NFD, which is the first report for evaluating the anti-cancer effect of NFD in vivo.

Conclusion: These findings provide a novel mechanism indicating the inhibition of TOPO II activity and NF-κB signaling by NFD, leading to DNA damage and apoptosis of NSCLC tumor cells.
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http://dx.doi.org/10.1016/j.phymed.2018.06.025DOI Listing
February 2019

Protective Effects of Extracts on UVB-Induced Damage in Human Retinal Pigment Epithelial Cells Accompanied by Attenuating ROS and DNA Damage.

Oxid Med Cell Longev 2018 7;2018:4814928. Epub 2018 Nov 7.

Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan.

The medicinal herb fruit has been widely used for improving and maintaining the health of the eyes in the Far East for many centuries. This study is aimed at investigating whether protective effects generated from the aqueous (LBA) and ethanol (LBE) extracts of the fruit existed against oxidative stress-induced apoptosis in human retinal pigment epithelial cells. extracts LBA and LBE exerted the activity of ROS scavenging and rescued UVB irradiation-induced growth inhibition in retinal pigment epithelial ARPE-19 cells. Compared to LBA, the ethanol extract LBE exerted a superior protective activity on UVB-induced growth arrest in ARPE-19 cells. Both extracts significantly reduced cell cycle G-arrest population in ARPE-19 cells. Furthermore, the cytometer-based Annexin V/propidium iodide staining assay further showed that both extracts protected ARPE-19 cells from UVB-induced apoptosis. extracts also reduced the activation of H2AX, a sensor of DNA damage in ARPE-19 cells in a dose-responsive manner. By using Ingenuity Pathway Analysis (IPA), the bioinformatics revealed that the protective effects of both LBA and LBE extracts might be involved in three signaling pathways, especially the Toll-like receptor (TLR) pathway associated with cellular proliferation. Our study suggests that both ethanol and aqueous extracts of exhibit antioxidant activity and rescue UVB-induced apoptosis of ARPE-19 cells. Collectively, the ethanol extract exerts a superior effect on rescuing UVB-induced growth arrest of ARPE-19 compared to the aqueous extract, which might be associated with the activation of TLR signaling. Our present work will benefit the preventive strategy of herbal medicine-based vision protection for treating eye diseases such as age-related macular degeneration in the future.
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http://dx.doi.org/10.1155/2018/4814928DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6247443PMC
January 2019

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

Int J Mol Sci 2018 Nov 30;19(12). Epub 2018 Nov 30.

Center for Stem Cell Research, Kaohsiung Medical University Hospital, Kaohsiung 80708, Taiwan.

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.
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http://dx.doi.org/10.3390/ijms19123823DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6321109PMC
November 2018

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

Int J Mol Sci 2018 Oct 2;19(10). Epub 2018 Oct 2.

Graduate Institute of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.

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 -octanoyl--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.
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http://dx.doi.org/10.3390/ijms19103010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6213533PMC
October 2018

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

Environ Toxicol 2018 Nov 6;33(11):1195-1203. Epub 2018 Sep 6.

Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.

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.
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http://dx.doi.org/10.1002/tox.22625DOI Listing
November 2018

Human non‑small cell lung cancer cells can be sensitized to camptothecin by modulating autophagy.

Int J Oncol 2018 Nov 14;53(5):1967-1979. Epub 2018 Aug 14.

Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan, R.O.C.

Lung cancer is a prevalent disease and is one of the leading causes of mortality worldwide. Despite the development of various anticancer drugs, the prognosis of lung cancer is relatively poor. Metastasis of lung cancer, as well as chemoresistance, is associated with a high mortality rate for patients with lung cancer. Camptothecin (CPT) is a well-known anticancer drug, which causes cancer cell apoptosis via the induction of DNA damage; however, the cytotoxicity of CPT easily reaches a plateau at a relatively high dose in lung cancer cells, thus limiting its efficacy. The present study demonstrated that CPT may induce autophagy in two human non‑small cell lung cancer cell lines, H1299 and H460. In addition, the results of a viability assay and Annexin V staining revealed that CPT-induced autophagy could protect lung cancer cells from programmed cell death. Conversely, the cytotoxicity of CPT was increased when autophagy was blocked by 3-methyladenine treatment. Furthermore, inhibition of autophagy enhanced the levels of CPT-induced DNA damage in the lung cancer cell lines. Accordingly, these findings suggested that autophagy exerts a protective role in CPT-treated lung cancer cells, and the combination of CPT with a specific inhibitor of autophagy may be considered a promising strategy for the future treatment of lung cancer.
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http://dx.doi.org/10.3892/ijo.2018.4523DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6192723PMC
November 2018

Coral-Derived Natural Marine Compound GB9 Impairs Vascular Development in Zebrafish.

Int J Mol Sci 2017 Aug 3;18(8). Epub 2017 Aug 3.

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan.

Blood vessels in vertebrates are established and genetically controlled in an evolutionarily-conserved manner during embryogenesis. Disruption of vascular growth by chemical compounds or environmental hormones may cause developmental defects. This study analyzed the vascular impacts of marine compound GB9 in zebrafish. GB9 was isolated from the marine soft coral and had shown anti-neuroinflammatory and anti-nociceptive activities. However, the role of GB9 on vascular development has not been reported. We first tested the survival rate of embryos under exogenous 5, 7.5, 10, and 15 μM GB9 added to the medium and determined a sub-lethal dosage of 10 μM GB9 for further assay. Using transgenic () fish to examine vascular development, we found that GB9 treatment impaired intersegmental vessel (ISV) growth and caudal vein plexus (CVP) patterning at 25 hours post-fertilization (hpf) and 30 hpf. GB9 exposure caused pericardial edema and impaired circulation at 48-52 hpf, which are common secondary effects of vascular defects and suggest the effects of GB9 on vascular development. Apoptic cell death analysis showed that vascular defects were not caused by cell death, but were likely due to the inhibition of migration and/or proliferation by examining ISV cell numbers. To test the molecular mechanisms of vascular defects in GB9-treated embryos, we examined the expression of vascular markers and found the decreased expression of vascular specific markers , , , and . In addition, we examined whether GB9 treatment impairs vascular growth due to an imbalance of redox homeostasis. We found an enhanced effect of vascular defects during GB9 and H₂O₂ co-treatment. Moreover, exogenous -acetyl-cysteine (NAC) treatment rescued the vascular defects in GB9 treated embryos. Our results showed that GB9 exposure causes vascular defects likely mediated by the imbalance of redox homeostasis.
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http://dx.doi.org/10.3390/ijms18081696DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5578086PMC
August 2017

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

Environ Toxicol 2017 Sep 26;32(9):2124-2132. Epub 2017 May 26.

Cancer Center, Kaohsiung Medical University Hospital; Kaohsiung Medical University, Kaohsiung, 80708, Taiwan.

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.
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http://dx.doi.org/10.1002/tox.22425DOI Listing
September 2017

Fine-tune regulation of carboxypeptidase N1 controls vascular patterning during zebrafish development.

Sci Rep 2017 05 12;7(1):1852. Epub 2017 May 12.

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.

Vascular development is regulated by complicated signals and molecules in vertebrates. In this study, we characterized a novel function of carboxypeptidase N1 (Cpn1) in the vasculature. We show that cpn1 mRNA is expressed in developing vessels. The knockdown of cpn1 by morpholino injection impairs the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP), suggesting the role of cpn1 in vascular development. We showed that vascular defects are not caused by cell death but are due to the impairment of migration and proliferation. Consistent with vascular growth defects, loss of cpn1 affects the expression of the vascular markers flt4, mrc1, flk, stabilin, and ephrinb2. Furthermore, the overexpression of cpn1 impaired the growth of ISV and CVP, but the remodeling expression of vascular markers was different from the knockdown of cpn1, indicating the differential regulation mechanisms in cpn1-overexpressing embryos. We examine the interaction between cpn1 and multiple signals and observed that cpn1 is regulated by Notch/VEGF signals for ISV growth and likely regulates BMP signals for CVP patterning. In conclusion, we demonstrate that cpn1 has a critical role in the vascular development of zebrafish. We also reveal a fine-tune regulation of cpn1 that controls vascular patterning mediated by multiple signals.
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http://dx.doi.org/10.1038/s41598-017-01976-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5431830PMC
May 2017

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.

Cancer Cell Int 2017 7;17:37. Epub 2017 Mar 7.

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, 804 Taiwan.

Background: 2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11-indeno[1,2-]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-IC (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.
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http://dx.doi.org/10.1186/s12935-017-0403-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5339964PMC
March 2017

Prdx1-encoded peroxiredoxin is important for vascular development in zebrafish.

FEBS Lett 2017 Mar 8;591(6):889-902. Epub 2017 Mar 8.

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.

Genetic signaling and redox homeostasis are required for proper growth of blood vessels. Here, we report a novel function of peroxiredoxin1 (Prdx1) in vascular development in zebrafish. Knockdown of prdx1 impairs the growth of intersegmental vessel and caudal vein plexus (CVP), and reduces the expression of vascular markers, thus suggesting a role for prdx1 in vasculature and indicating that the antioxidant function of prdx1 is important. We found that H O -treated embryos also have CVP defects and observed synergistic effects when prdx1 knockdown was combined with H O treatment. Moreover, N-acetyl-cysteine treatment rescues the vascular defects in prdx1 morphants. These results suggest that oxidative stress disturbs vascularization. Furthermore, we show that the regulation of prdx1 is mediated by Notch and BMP signals.
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http://dx.doi.org/10.1002/1873-3468.12604DOI Listing
March 2017

Ftr82 Is Critical for Vascular Patterning during Zebrafish Development.

Int J Mol Sci 2017 Jan 13;18(1). Epub 2017 Jan 13.

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung 80424, Taiwan.

Cellular components and signaling pathways are required for the proper growth of blood vessels. Here, we report for the first time that a teleost-specific gene (, member 82) plays a critical role in vasculature during zebrafish development. To date, there has been no description of tripartite motif proteins (TRIM) in vascular development, and the role of is unknown. In this study, we found that mRNA is expressed during the development of vessels, and loss of by morpholino (MO) knockdown impairs the growth of intersegmental vessels (ISV) and caudal vein plexus (CVP), suggesting that plays a critical role in promoting ISV and CVP growth. We showed the specificity of MO by analyzing expression products and expressing mRNA to rescue morphants. We further showed that the knockdown of reduced ISV cell numbers, suggesting that the growth impairment of vessels is likely due to a decrease of cell proliferation and migration, but not cell death. In addition, loss of affects the expression of vascular markers, which is consistent with the defect of vascular growth. Finally, we showed that likely interacts with vascular endothelial growth factor (VEGF) and Notch signaling. Together, we identify teleost-specific as a vascular gene that plays an important role for vascular development in zebrafish.
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http://dx.doi.org/10.3390/ijms18010156DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297789PMC
January 2017

Retinoic Acid Protects and Rescues the Development of Zebrafish Embryonic Retinal Photoreceptor Cells from Exposure to Paclobutrazol.

Int J Mol Sci 2017 Jan 11;18(1). Epub 2017 Jan 11.

Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung City 80424, Taiwan.

Paclobutrazol (PBZ) is a widely used fungicide that shows toxicity to aquatic embryos, probably through rain-wash. Here, we specifically focus on its toxic effect on eye development in zebrafish, as well as the role of retinoic acid (RA), a metabolite of vitamin A that controls proliferation and differentiation of retinal photoreceptor cells, in this toxicity. Embryos were exposed to PBZ with or without RA from 2 to 72 h post-fertilization (hpf), and PBZ-treated embryos (2-72 hpf) were exposed to RA for additional hours until 120 hpf. Eye size and histology were examined. Expression levels of (rod photoreceptor marker), (cone photoreceptor marker), (encoding key enzymes for RA synthesis), and phospho-histone H3 (an M-phase marker) in the eyes of control and treated embryos were examined. PBZ exposure dramatically reduces photoreceptor proliferation, thus resulting in a thinning of the photoreceptor cell layer and leading to a small eye. Co-treatment of PBZ with RA, or post-treatment of PBZ-treated embryos with RA, partially rescues photoreceptor cells, revealed by expression levels of marker proteins and by retinal cell proliferation. PBZ has strong embryonic toxicity to retinal photoreceptors, probably via suppressing the production of RA, with effects including impaired retinal cell division.
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http://dx.doi.org/10.3390/ijms18010130DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5297764PMC
January 2017

An Acetamide Derivative as a Camptothecin Sensitizer for Human Non-Small-Cell Lung Cancer Cells through Increased Oxidative Stress and JNK Activation.

Oxid Med Cell Longev 2016 24;2016:9128102. Epub 2016 Oct 24.

Department of Biotechnology, Kaohsiung Medical University, Kaohsiung 807, Taiwan; Research Center for Environment Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan; Graduate Institute of Biomedical Engineering, National Chung Hsing University, Taichung 402, Taiwan; The Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung 807, Taiwan.

In recent years, combination chemotherapy is a primary strategy for treating lung cancer; however, the issues of antagonism and side effects still limit its applications. The development of chemosensitizer aims to sensitize chemoresistant cancer cells to anticancer drugs and therefore improve the efficacy of chemotherapy. In this study, we examined whether N-[2-(morpholin-4-yl)phenyl]-2-{8-oxatricyclo[7.4.0.0,2,7]trideca-1(9),2(7),3,5,10,12-hexaen-4-yloxy}acetamide (NPOA), an acetamide derivative, sensitizes human non-small-cell lung cancer (NSCLC) H1299 cells towards camptothecin- (CPT-) induced apoptosis effects. Our results demonstrate that the combination of CPT and NPOA enhances anti-lung-cancer effect. The cytometer-based Annexin V/propidium iodide (PI) staining showed that CPT and NPOA cotreatment causes an increased population of apoptotic cells compared to CPT treatment alone. Moreover, Western blotting assay showed an enhancement of Bax expression and caspase cascade leading to cell death of H1299 cells. Besides, CPT and NPOA cotreatment-mediated disruption of mitochondrial membrane potential (MMP) in H1299 cells may function through increasing the activation of the stressed-associated c-Jun N-terminal kinase (JNK). These results showed that NPOA treatment sensitizes H1299 cells towards CPT-induced accumulation of cell cycle S phase and mitochondrial-mediated apoptosis through regulating endogenous ROS and JNK activation. Accordingly, NPOA could be a candidate chemosensitizer of CPT derivative agents such as irinotecan or topotecan in the future.
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http://dx.doi.org/10.1155/2016/9128102DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5098095PMC
March 2017

Inhibitory effect of -ferulic acid on proliferation and migration of human lung cancer cells accompanied with increased endogenous reactive oxygen species and β-catenin instability.

Chin Med 2016 1;11:45. Epub 2016 Oct 1.

Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, 807 Taiwan.

Background: -ferulic (FA) acid exhibits antioxidant effects in vitro. However, the underlying mechanism of -FA activity in cellular physiology, especially cancer physiology, remains largely unknown. This study investigated the cellular physiological effects of -FA on the H1299 human lung cancer cell line.

Methods: The 2,2-diphenyl-1-picrylhydrazyl assay was used to determine free radical scavenging capability. Assessment of intracellular reactive oxygen species (ROS) was evaluated using oxidized 2',7'-dichlorofluorescin diacetate and dihydroethidium staining. Trypan blue exclusion, colony formation, and anchorage-independent growth assays were used to determine cellular proliferation. Annexin V staining assay was used to assess cellular apoptosis by flow cytometry. Wound healing and Boyden's well assays were used to detect the migration and invasion of cells. Gelatin zymography was used to detect matrix metalloproteinase (MMP-2 and MMP-9) activity. Western blotting was used to detect expression levels of various signaling pathway proteins.

Results: DPPH assay results indicated that -FA exerted potent antioxidant effects. However, -FA increased intracellular ROS levels, including hydrogen peroxide and superoxide anion, in H1299 cells. -FA treatment inhibited cellular proliferation and induced moderate apoptotic cell death at the highest concentration used (0.6 mM). Furthermore, -FA moderately inhibited the migration of H1299 cells at the concentrations of 0.3 and 0.6 mM and attenuated MMP-2 and MMP-9 activity. -FA caused the phosphorylation of β-catenin, resulting in proteasomal degradation of β-catenin. Conversely, -FA treatment increased the expression of pro-apoptotic factor Bax and decreased the expression of pro-survival factor survivin.

Conclusion: Various concentrations (0.06-0.6 mM) of -FA exert both anti-proliferation and anti-migration effects in the human lung cancer cell line H1299.
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http://dx.doi.org/10.1186/s13020-016-0116-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5045596PMC
October 2016

A Quinone-Containing Compound Enhances Camptothecin-Induced Apoptosis of Lung Cancer Through Modulating Endogenous ROS and ERK Signaling.

Arch Immunol Ther Exp (Warsz) 2017 Jun 27;65(3):241-252. Epub 2016 Sep 27.

Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.

The natural compound camptothecin (CPT) derivatives have widely been used for anti-cancer treatments, including lung cancer. However, many chemoresistant cancer cells often develop a relatively higher threshold for inducing apoptosis, causing a limited efficacy of anti-cancer drugs. Likewise, lung cancer cells acquire chemoresistance against CPT analogs, such as irinotecan and topotecan, finally resulting in an unsatisfied outcome and poor prognosis of lung cancer patients. TFPP is a quinone-containing compound as a candidate for CPT-based combination chemotherapy. In this study, we examined the effect of TFPP and CPT cotreatment on non-small cell lung cancer (NSCLC) cells. Cell proliferation and flow cytometry-based Annexin-V/PI staining assays demonstrated the synergistic effect of TFPP on CPT-induced apoptosis in both NSCLC A549 and H1299 cells. The results of CPT and TFPP cotreatment cause the regulation of the ERK-Bim axis and the activation of mitochondrial-mediated caspase cascade, including caspase-9 and caspase-3. Besides, TFPP significantly enhanced CPT-induced endogenous reactive oxygen species (ROS) in the two NSCLC cells. In contrast, the treatment of N-acetyl-L-cysteine (NAC), an ROS scavenger, rescues the apoptosis of NSCLC cells induced by TFPP and CPT cotreatment, suggesting that the synergistic effect of TFPP on CPT-induced anti-NSCLC cells is through upregulating ROS production. Consequently, our results suggest that TFPP sensitizes NSCLC towards CPT-based chemotherapy may act through decreasing the apoptosis-initiating threshold. Therefore, TFPP may be a promising chemosensitizer for lung cancer treatment, and the underlying mechanism warrants further.
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http://dx.doi.org/10.1007/s00005-016-0424-8DOI Listing
June 2017

The LIM-homeodomain transcription factor Islet2a promotes angioblast migration.

Dev Biol 2016 06 25;414(2):181-92. Epub 2016 Apr 25.

Biochemistry and Molecular Biology, Cumming School of Medicine, University of Calgary, 3330 Hospital Drive NW, Calgary AB, Canada, T2N 4N1. Electronic address:

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.
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http://dx.doi.org/10.1016/j.ydbio.2016.04.019DOI Listing
June 2016

Antiproliferation of Cryptocarya concinna-derived cryptocaryone against oral cancer cells involving apoptosis, oxidative stress, and DNA damage.

BMC Complement Altern Med 2016 Mar 8;16:94. Epub 2016 Mar 8.

Department of Biomedical Science and Environmental Biology, Kaohsiung Medical University, Kaohsiung, Taiwan.

Background: Cryptocarya-derived crude extracts and their compounds have been reported to have an antiproliferation effect on several types of cancers but their impact on oral cancer is less well understood.

Methods: We examined the cell proliferation effect and mechanism of C. concinna-derived cryptocaryone (CPC) on oral cancer cells in terms of cell viability, apoptosis, reactive oxygen species (ROS), mitochondrial depolarization, and DNA damage.

Results: We found that CPC dose-responsively reduced cell viability of two types of oral cancer cells (Ca9-22 and CAL 27) in MTS assay. The CPC-induced dose-responsive apoptosis effects on Ca9-22 cells were confirmed by flow cytometry-based sub-G1 accumulation, annexin V staining, and pancaspase analyses. For oral cancer Ca9-22 cells, CPC also induced oxidative stress responses in terms of ROS generation and mitochondrial depolarization. Moreover, γH2AX flow cytometry showed DNA damage in CPC-treated Ca9-22 cells. CPC-induced cell responses in terms of cell viability, apoptosis, oxidative stress, and DNA damage were rescued by N-acetylcysteine pretreatment, suggesting that oxidative stress plays an important role in CPC-induced death of oral cancer cells.

Conclusions: CPC is a potential ROS-mediated natural product for anti-oral cancer therapy.
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http://dx.doi.org/10.1186/s12906-016-1073-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4784356PMC
March 2016

BPIQ, a novel synthetic quinoline derivative, inhibits growth and induces mitochondrial apoptosis of lung cancer cells in vitro and in zebrafish xenograft model.

BMC Cancer 2015 Dec 16;15:962. Epub 2015 Dec 16.

Department of Medicinal and Applied Chemistry, Kaohsiung Medical University, Kaohsiung, 807, Taiwan.

Background: 2,9-Bis[2-(pyrrolidin-1-yl)ethoxy]-6-{4-[2-(pyrrolidin-1-yl)ethoxy] phenyl}-11H-indeno[1,2-c]quinolin-11-one (BPIQ) is a derivative from 6-arylindeno[1,2-c]quinoline. Our previous study showed the anti-cancer potential of BPIQ compared to its two analogues topotecan and irinotecan. In the study, the aim is to investigate the potency and the mechanism of BPIQ against lung cancer cells.

Methods: Both in vitro and zebrafish xenograft model were performed to examine the anti-lung cancer effect of BPIQ. Flow cytometer-based assays were performed for detecting apoptosis and cell cycle distribution. Western blot assay was used for detecting the changes of apoptotic and cell cycle-associated proteins. siRNA knockdown assay was performed for confirming the apoptotic role of Bim.

Results: Both in vitro and zebrafish xenograft model demonstrated the anti-lung cancer effect of BPIQ. BPIQ-induced proliferative inhibition of H1299 cells was achieved through the induction of G2/M-phase arrest and apoptosis. The results of Western blot showed that BPIQ-induced G2/M-phase arrest was associated with a marked decrease in the protein levels of cyclin B and cyclin-dependent kinase 1 (CDK1). The up-regulation of pro-apoptotic Bad, Bim and down-regulation of pro-survival XIAP and survivin was observed following BPIQ treatment.

Conclusions: BPIQ-induced anti-lung cancer is involved in mitochondrial apoptosis. BPIQ could be a promising anti-lung cancer drug for further applications.
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http://dx.doi.org/10.1186/s12885-015-1970-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4682281PMC
December 2015

Nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development.

J Biomed Sci 2015 Nov 17;22:104. Epub 2015 Nov 17.

Department of Biological Sciences, National Sun Yat-sen University, Kaohsiung, Taiwan.

Background: The specification of vein and the patterning of intersegmental vessels (ISV) controlled by transcription factor is not fully characterized. The orphan nuclear receptor Chicken ovalbumin upstream promoter transcription factor II (CoupTFII, a.k.a NR2F2) positively regulates vein identity in mice. In this study, we show that nr2f1b is important for vein and tip cell identity during zebrafish development.

Results: Nr2f1b mRNA is expressed in ventral lateral mesoderm at 15S stage and in vessels at 24 hpf consistent with a role in early vascular specification. Morpholino knockdown of nr2f1b results in a decrease in both vein cell number and expression of the vein specific marker flt4 and mrc1, suggested its role in venous specification. We also show loss of nr2f1b reduced ISV cell number and impairs ISV growth, which is likely due to the impairment of angiogenic cells migration and/or proliferation by time-lapse imaging. Consequently, nr2f1b morphants showed pericardial edema and circulation defects. Overexpression of nr2f1b under the fli promoter increases the number of venous cells and ISV endothelial cells indicated the function of nr2f1b is required and necessary for vascular development. We further showed that nr2f1b likely interact with Notch signalling. nr2f1b expression is increased in rbpsuh morphants and DAPT-treatment embryos suggested nr2f1b is negatively regulated by Notch activity.

Conclusions: We show nr2f1b control venous specification and angiogenic patterning during zebrafish vascular development, which is mediated by Notch signalings.
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http://dx.doi.org/10.1186/s12929-015-0209-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647328PMC
November 2015