Publications by authors named "En-Chi Liao"

22 Publications

  • Page 1 of 1

Role of IGFBP-2 in oral cancer metastasis.

Biochim Biophys Acta Mol Basis Dis 2021 Apr 14;1867(7):166143. Epub 2021 Apr 14.

Department of Medical Science, Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Cancer metastasis is one of most main causes of failure in cancer treatment. Nonetheless, more than half of oral cancer patients were diagnosed as advanced oral cancer with dramatically decreased 5-year survival rate to lower than 20%, while the stages become more advanced. In order to improve oral cancer treatment, the identification of cancer metastatic biomarkers and mechanisms is critical. In the current study, two pairs of oral squamous cell carcinoma lines, OC3/C9, and invasive OC3-I5/C9-I5were used as model systems to investigate invasive mechanism as well as to identify potential therapy-associated targets. Based on our previous proteomic analysis, insulin-like growth factor-binding protein 2 (IGFBP-2) was reported participating in oral cancer metastasis. Subsequent studies have applied interference RNA as well as recombinant protein techniques to confirm the roles of IGFBP-2 in oral cancer metastasis and examine their potency in regulating invasion as well as the mechanism IGFBP-2 involved. The results demonstrated that expression of epithelial-mesenchymal transition (EMT) markers including Twist, Snail1, SIP1, profilin, vimentin, uPA and MMP9 were increased in both OC3-I5 and C9-I5 compared to OC3 and C9 cells, while E-cadherin expression was down-regulated in the OC3-I5 and C9-I5 cells. Moreover, IGFBP-2 is shown to affect not only migration and invasion but also wound healing ability and cell proliferation. Our results also revealed that uPA is a downstream target of IGFBP-2 to intermediate oral cancer metastasis. To sum up, the current studies indicated that elevated IGFBP-2 is strongly correlated with oral cancer metastasis and progression, and that it could potentially serve as a prognostic biomarker as well as an innovative target for the treatment of oral cancer invasion.
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http://dx.doi.org/10.1016/j.bbadis.2021.166143DOI Listing
April 2021

Characterization of TGF-β by Induced Oxidative Stress in Human Trabecular Meshwork Cells.

Antioxidants (Basel) 2021 Jan 13;10(1). Epub 2021 Jan 13.

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.

Oxidative stress generated by reactive oxygen species (ROS) plays a critical role in the pathomechanism of glaucoma, which is a multifactorial blinding disease that may cause irreversible damage within human trabecular meshwork cells (HTMCs). It is known that the transforming growth factor-β (TGF-β) signaling pathway is an important component of oxidative stress-induced damage related to extracellular matrix (ECM) fibrosis and activates cell antioxidative mechanisms. To elucidate the dual potential roles and regulatory mechanisms of TGF-β in effects on HTMCs, we established an in vitro oxidative model using hydrogen peroxide (HO) and further focused on TGF-β-related oxidative stress pathways and the related signal transduction. Via a series of cell functional qualitative analyses to detect related protein level alterations and cell fibrosis status, we illustrated the role of TGF-β1 and TGF-β2 in oxidative stress-induced injury by shTGF-β1 and shTGF-β2 knockdown or added recombinant human TGF-β1 protein (rhTGF-β1). The results of protein level showed that p38 MAPK, TGF-β, and its related SMAD family were activated after HO stimulation. Cell functional assays showed that HTMCs with HO exposure duration had a more irregular actin architecture compared to normal TM cells. Data with rhTGF-β1 (1 ng/mL) pretreatment reduced the cell apoptosis rate and amount of reactive oxygen species (ROS), while it also enhanced survival. Furthermore, TGF-β1 and TGF-β2 in terms of antioxidant signaling were related to the activation of collagen I and laminin, which are fibrosis-response proteins. Succinctly, our study demonstrated that low concentrations of TGF-β1 (1 ng/mL) preserves HTMCs from free radical-mediated injury by p-p38 MAPK level and p-AKT signaling balance, presenting a signaling transduction mechanism of TGF-β1 in HTMC oxidative stress-related therapies.
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http://dx.doi.org/10.3390/antiox10010107DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7828702PMC
January 2021

The distribution of cultivable oral anaerobic microbiota identified by MALDI-TOF MS in healthy subjects and in patients with periodontal disease.

J Pharm Biomed Anal 2021 Jan 22;192:113647. Epub 2020 Sep 22.

Institute of Bioinformatics and Structural Biology and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

In this study, we aimed to identify the cultivatable oral anaerobic bacterial distribution in oral cavity by MALDI-TOF Biotyper. The bacterial distribution of three groups, including subjects with/without periodontal disease, two clusters of age (60 years as the cutoff), and before/after treatment, were investigated in this study. There were 38 participants recruited in this study, involving 18 subjects with moderate to severe periodontal-infected patients and 20 healthy controls. Total number of 126 bacterial species were identified by MALDI-TOF MS. The relative abundance of Streptococcus gordonii and Streptococcus intermedius in periodontal patients is higher than healthy controls indicating potential biomarkers for periodontal disease. Participants with periodontal disease were subdivided in to two clusters of age (60 years as the cutoff), 11 and 7 participants were age <60 years and>60 years, respectively. Meanwhile, the incidence of Streptococcus pneumoniae and Streptococcus oralis infection were higher in the subjects above 60 years old than below. Moreover, the bacterial distribution between pre-treatment and post-treatment was similar indicating that basic treatment without the ability to redistribute the microbiota. In summary, the cultivable oral anaerobic bacteria were identified by MALDI-TOF MS and the bacterial distribution shifting was shown to be associated with the progress of periodontal disease to aging and basic treatment. This study provided information for diagnosis and treatment guidelines for oral healthcare.
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http://dx.doi.org/10.1016/j.jpba.2020.113647DOI Listing
January 2021

TGF-β1 signaling protects retinal ganglion cells from oxidative stress via modulation of the HO-1/Nrf2 pathway.

Chem Biol Interact 2020 Nov 24;331:109249. Epub 2020 Sep 24.

Institute of Bioinformatics and Structural Biology, Department of Medical Sciences, National Tsing Hua University, Hsinchu, 300, Taiwan. Electronic address:

Oxidative stress provides a major contribution to the pathogenesis of glaucoma and may induce retinal ganglion cell (RGC) damage. Transforming growth factor β (TGF-β) has appeared as a neuroprotective protein in various indignities. However, the TGF-β mechanism of protective effects against oxidative stress damage in RGCs still undetermined. In our research, we investigated the regulatory mechanisms and potential effects of TGF-β1 & TGF-β2 in hydrogen peroxide (HO)-stimulated oxidative stress of RGCs in vitro. By a series of cell functional qualitative analysis, such as MTT cell viability assay, wound healing ability assay, apoptosis assay, intracellular ROS detection, immunoblot analysis, intracellular GSH content, and high-resolution respirometry, we illustrated the cell state in oxidative stress-induced injury. Results of protein expression showed that TGF-β1 & TGF-β2 was upregulated in RGCs after HO stimulation. Cell functional assays resulted that knockdown of TGF-β1 & TGF-β2 reduced survival rate whereas enhanced apoptosis and accumulation of reactive oxygen species (ROS). Especially TGF-β1 upregulation promoted the protein expression of aldehyde dehydrogenase 3A1 (ALDH3A1) and increased the activity of antioxidant and neuroprotection pathways. Additionally, TGF-β1 & TGF-β2 on antioxidant signaling was related to activation of heme oxygenase-1 (HO-1) and nuclear factor erythroid 2-related factor (Nrf2), which are stress-response proteins. ROS accumulation followed by the accumulation of hypoxia-inducible factor (HIF-1α) caused mitochondrial damage and led to neurodegeneration. In summary, our results demonstrated that TGF-β1 preserves RGCs from free radicals-mediated injury by upregulating the activation of Nrf2 expression and HO-1 signaling balance HIF-1α upregulation, implying a prospective role of TGF-β1 in retinal neuroprotection-related therapies.
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http://dx.doi.org/10.1016/j.cbi.2020.109249DOI Listing
November 2020

The Role of Transforming Growth Factor-Beta in Retinal Ganglion Cells with Hyperglycemia and Oxidative Stress.

Int J Mol Sci 2020 Sep 4;21(18). Epub 2020 Sep 4.

Institute of Bioinformatics and Structural Biology & Department of Medical Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.

A characteristic of diabetes mellitus is hyperglycemia, which is considered with an emphasis on the diabetic retinopathy of progressive neurodegenerative disease. Retinal ganglion cells (RGCs) are believed to be important cells affected in the pathogenesis of diabetic retinopathy. Transforming growth factor-beta (TGF-β) is a neuroprotective protein that helps to withstand various neuronal injuries. To investigate the potential roles and regulatory mechanisms of TGF-β in hyperglycemia-triggered damage of RGCs in vitro, we established RGCs in 5.5, 25, 50, and 100 mM D-glucose supplemented media and focused on the TGF-β-related oxidative stress pathway in combination with hydrogen peroxide (HO). Functional experiments showed that TGF-β1/2 protein expression was upregulated in RGCs with hyperglycemia. The knockdown of TGF-β enhanced the accumulation of reactive oxygen species (ROS), inhibited the cell proliferation rate, and reduced glutathione content in hyperglycemia. Furthermore, the results showed that the TGF-β-mediated enhancement of antioxidant signaling was correlated with the activation of stress response proteins and the antioxidant pathway, such as aldehyde dehydrogenase 3A1 (ALDH3A1), heme oxygenase-1 (HO-1), nuclear factor erythroid 2-related factor (Nrf2), and hypoxia-inducible factor (HIF-1α). Summarizing, our results demonstrated that TGF-β keeps RGCs from hyperglycemia-triggered harm by promoting the activation of the antioxidant pathway, suggesting a potential anti-diabetic therapy for the treatment of diabetic retinopathy.
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http://dx.doi.org/10.3390/ijms21186482DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7554964PMC
September 2020

Targeting UDP-glucose dehydrogenase inhibits ovarian cancer growth and metastasis.

J Cell Mol Med 2020 10 7;24(20):11883-11902. Epub 2020 Sep 7.

Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

More than 70% of patients with ovarian cancer are diagnosed in advanced stages. Therefore, it is urgent to identify a promising prognostic marker and understand the mechanism of ovarian cancer metastasis development. By using proteomics approaches, we found that UDP-glucose dehydrogenase (UGDH) was up-regulated in highly metastatic ovarian cancer TOV21G cells, characterized by high invasiveness (TOV21G ), in comparison to its parental control. Previous reports demonstrated that UGDH is involved in cell migration, but its specific role in cancer metastasis remains unclear. By performing immunohistochemical staining with tissue microarray, we found overexpression of UGDH in ovarian cancer tissue, but not in normal adjacent tissue. Silencing using RNA interference (RNAi) was utilized to knockdown UGDH, which resulted in a significant decrease in metastatic ability in transwell migration, transwell invasion and wound healing assays. The knockdown of UGDH caused cell cycle arrest in the G /G phase and induced a massive decrease of tumour formation rate in vivo. Our data showed that UGDH-depletion led to the down-regulation of epithelial-mesenchymal transition (EMT)-related markers as well as MMP2, and inactivation of the ERK/MAPK pathway. In conclusion, we found that the up-regulation of UGDH is related to ovarian cancer metastasis and the deficiency of UGDH leads to the decrease of cell migration, cell invasion, wound healing and cell proliferation ability. Our findings reveal that UGDH can serve as a prognostic marker and that the inhibition of UGDH is a promising strategy for ovarian cancer treatment.
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http://dx.doi.org/10.1111/jcmm.15808DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7578908PMC
October 2020

Progesterone receptor membrane component 1 is involved in oral cancer cell metastasis.

J Cell Mol Med 2020 09 16;24(17):9737-9751. Epub 2020 Jul 16.

Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

Cancer metastasis is a common cause of failure in cancer therapy. However, over 60% of oral cancer patients present with advanced stage disease, and the five-year survival rates of these patients decrease from 72.6% to 20% as the stage becomes more advanced. In order to manage oral cancer, identification of metastasis biomarker and mechanism is critical. In this study, we use a pair of oral squamous cell carcinoma lines, OC3, and invasive OC3-I5 as a model system to examine invasive mechanism and to identify potential therapeutic targets. We used two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF/TOF MS) to examine the global protein expression changes between OC3 and invasive OC3-I5. A proteomic study reveals that invasive properties alter the expression of 101 proteins in OC3-I5 cells comparing to OC3 cells. Further studies have used RNA interference technique to monitor the influence of progesterone receptor membrane component 1 (PGRMC1) protein in invasion and evaluate their potency in regulating invasion and the mechanism it involved. The results demonstrated that expression of epithelial-mesenchymal transition (EMT) markers including Twist, p-Src, Snail1, SIP1, JAM-A, vimentin and vinculin was increased in OC3-I5 compared to OC3 cells, whereas E-cadherin expression was decreased in the OC3-I5 cells. Moreover, in mouse model, PGRMC1 is shown to affect not only migration and invasion but also metastasis in vivo. Taken together, the proteomic approach allows us to identify numerous proteins, including PGRMC1, involved in invasion mechanism. Our results provide useful diagnostic markers and therapeutic candidates for the treatment of oral cancer invasion.
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http://dx.doi.org/10.1111/jcmm.15535DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7520311PMC
September 2020

Proteomic Analysis of Metastasis-Specific Biomarkers in Pancreatic Cancer: Galectin-1 Plays an Important Metastatic Role in Pancreatic Cancer.

J Pharm Biomed Anal 2020 Jul 25;186:113300. Epub 2020 Apr 25.

Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Cancer metastasis is the major cause of death in pancreatic cancer. We have established a pair of pancreatic ductal adenocarcinoma cell line, PANC1 and invasive PANC1-I5, as a model system toinvestigate the metastatic mechanism as well as potential therapeutic targets in pancreatic cancer. We used proteomic analysis based on two-dimensional differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) to examine the global protein expression alterations between PANC1 and PANC1-I5. Proteomic study revealed that 88 proteins are differentially expressed between PANC1-I5 and PANC1 cells, and further functional evaluations through protein expression validation, gene knockout, migration and invasion analysis revealed that galectin-1 is one of the potential players in modulating pancreatic cancer metastasis. To conclude, we have identified numerous proteins might be associated with pancreatic cancer invasiveness in the pancreatic cancer model.
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http://dx.doi.org/10.1016/j.jpba.2020.113300DOI Listing
July 2020

A preliminary study of Parkinson's gene therapy via sono-magnetic sensing gene vector for conquering extra/intracellular barriers in mice.

Brain Stimul 2020 May - Jun;13(3):786-799. Epub 2020 Feb 24.

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan; Institute of Nuclear Engineering and Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Background: Non-virus genetic treatment for Parkinson's disease (PD) via plasmid glial cell-line derived neurotrophic factor (pGDNF) has shown potential for repairing damaged dopaminergic neurons. However, development of this gene therapy is largely hampered by the insufficient transfection efficiency as a result of the cell membrane, lysosome, and cytoskeleton meshwork.

Methods: In this study, we propose the use of polyethylenimine (PEI)-superparamagnetic iron oxide-plasmid DNA (pDNA)-loaded microbubbles (PSp-MBs) in conjunction with focused ultrasound (FUS) and two-step magnetic navigation to provide cavitation, proton sponge effect and magnetic effects to increase the efficiency of gene delivery.

Results: The gene transfection rate in the proposed system was 2.2-fold higher than that of the commercial agent (TransIT®-LT1). The transfection rate could be boosted ∼11%, ∼10%, and 6% by cavitation-magnetic hybrid enhanced cell membrane permeabilization, proton sponge effect, and magnetic-assisted cytoskeleton-reorganization, respectively. In vivo data suggested that effective gene delivery with this system results in a 3.2-fold increase in recovery of dopaminergic neurons and a 3.9-fold improvement in the motor behavior when compared to untreated genetic PD mice.

Conclusions: We proposed that this novel FUS-magnetic hybrid gene delivery platform could be integrated with a variety of therapeutic genes for treating neurodegenerative diseases in the future.
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http://dx.doi.org/10.1016/j.brs.2020.02.024DOI Listing
November 2020

Confers Obesity Resistance and Restores Intestinal Barrier Integrity in Leptin-deficient Obese Mice.

Nutrients 2020 Mar 10;12(3). Epub 2020 Mar 10.

Institute of Bioinformatics and Structural Biology and Department of Medical Sciences, National Tsing Hua University, Hsinchu 30013, Taiwan.

Obesity is associated with metabolic disorders. Thus, obesity prevention and treatment are essential for health. (AC) is a multifunctional medicinal fungus used for the treatment of various diseases and for preventing diet-induced obesity. Leptin deficiency causes over-eating and spontaneous obesity. The concomitant metabolic symptoms are more severe than diet-induced obesity. Here, we used leptin-deficient () mice as an animal model for over-feeding to study the effect of AC on obesity. We fed C57BL/6 mice (WT, ) and mice with AC for four weeks before performing qRT-PCR and immunoblot analysis to elaborate AC-modulated mechanisms. Further, we used Caco-2 cells as a human intestinal epithelial barrier model to examine the effect of AC on intestinal permeability. Our results suggested that AC reduces lipid deposits of the liver and epididymal white adipose tissue (EWAT) by promoting lipid metabolism and inhibiting lipogenesis-associated genes and proteins in mice. Moreover, AC effectively repaired intestinal-barrier injury caused by leptin deficiency and enhanced intestinal barrier integrity in Caco-2 cells. Interestingly, AC significantly reduced body weight and EWAT with no compromise on food intake in mice. Thus, AC effectively reduced obesity caused by leptin-deficiency and can potentially be used as a nutraceutical for treating obesity.
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http://dx.doi.org/10.3390/nu12030726DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7146579PMC
March 2020

Identification of hyperglycemia-associated microbiota alterations in saliva and gingival sulcus.

Arch Biochem Biophys 2020 03 23;682:108278. Epub 2020 Jan 23.

Institute of Bioinformatics and Structural Biology and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Oral microbes are a contributing factor to hyperglycemia by inducing an increase in insulin resistance resulting in uncontrolled blood glucose levels. However, the relationship between the distribution of oral flora and hyperglycemia is still controversial. Combining the power of MALDI-Biotyper with anaerobic bacterial culture, this study explores the correlation between anaerobic bacteria in the oral cavity and blood glucose levels. The results demonstrated that altered blood glucose levels contributed to a varied bacterial distribution in the oral cavity. Specifically, Veillonella spp. and Prevotella spp. were identified in a higher proportion in people with elevated blood glucose levels. Six bacterial species identified in this study (Prevotella melaninogenica, Campylobacter rectus, Streptococcus gordonii, Streptococcus mitis, Streptococcus salivarius, and Veillonella parvula) not only demonstrated a positive association with higher blood glucose levels, but also likely contribute to the development of the condition. The data demonstrated MALDI-TOF MS to be a simpler, faster, and more economical clinical identification tool that provides clarity and depth to the research on blood glucose and oral microbiota.
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http://dx.doi.org/10.1016/j.abb.2020.108278DOI Listing
March 2020

Normalization of Tumor Vasculature by Oxygen Microbubbles with Ultrasound.

Theranostics 2019 25;9(24):7370-7383. Epub 2019 Sep 25.

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, Hsinchu, Taiwan.

Tumor microenvironment influences the efficacy of anti-cancer therapies. The dysfunctional tumor vasculature limits the efficiency of oxygenation and drug delivery to reduce treatment outcome. A concept of tumor vascular normalization (VN), which inhibits angiogenesis to improve vessel maturity, blood perfusion, and oxygenation, has been demonstrated under the anti-angiogenic therapy. The efficiency of drug delivery and penetration is increased by enhancing perfusion and reducing interstitial fluid pressure during the time window of VN. However, anti-angiogenic agents only induce transient VN and then prune vessels to aggravate tumor hypoxia. To repair tumor vessels without altering vessel density, we proposed to induce tumor VN by local oxygen release via oxygen microbubbles with ultrasound. With tumor perfusion enhancement under ultrasound contrast imaging tracing, the time window of VN was defined as 2-8 days after a single oxygen microbubble treatment. The enhanced tumor oxygenation after oxygen microbubble treatment inhibited hypoxia inducible factor-1 alpha (HIF-1α)/vascular endothelial growth factor (VEGF) pathway to improve the morphology and function of tumor vasculature. The pericyte coverage and Hoechst penetration of tumor vessels increased without any changes to the vessel density. Finally, the intratumoral accumulation of anti-cancer drug doxorubicin could be increased 3-4 folds during tumor VN. These findings demonstrate that regulating tumor oxygenation by oxygen microbubbles could normalize dysfunctional vessels to enhance vascular maturity, blood perfusion, and drug penetration. Furthermore, ultrasound perfusion imaging provides a simple and non-invasive way to detect the VN time window, which increases the feasibility of VN in clinical cancer applications.
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http://dx.doi.org/10.7150/thno.37750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6831304PMC
September 2020

Role of PGRMC1 in cell physiology of cervical cancer.

Life Sci 2019 Aug 16;231:116541. Epub 2019 Jun 16.

Dept. of Medical Sciences & Inst. of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Aims: The most frequent cancers among women worldwide. The mortality of cervical cancer has declined significantly primarily due to the widespread use of Pap smear tests as a screening test and therapeutic vaccination. However, cervical cancer still remains a severe disease among the female population, as the prognosis of metastatic cervical cancer is very poor.

Key Methods: In this study, we performed 2D-DIGE and MALDI-TOF/TOF MS to analyze differentially expressed proteins between HeLa and invasive HeLa-I5 cells..

Key Findings: According to our proteomics data, 68 differentially expressed proteins between the HeLa and HeLa-I5 cells were identified. One of these differentially expressed proteins, Progesterone receptor membrane component 1 (PGRMC1), was selected as a candidate for further studies. To correlate the role of PGRMC1 with cellular migration and cancer progression, small interfering RNA (siRNA) was used to knockdown the expression of PGRMC1. Similar function of PGRMC1 was also observed in two other cervical cancer lines, CaSki and ME-180.

Significance: PGRMC1 plays an essential role in regulating cancer progression and metastasis of cervical cancer cells, thus serving as a potential therapeutic target for cervical cancer.
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http://dx.doi.org/10.1016/j.lfs.2019.06.016DOI Listing
August 2019

Proteomic analysis of evodiamine-induced cytotoxicity in thyroid cancer cells.

J Pharm Biomed Anal 2018 Oct 9;160:344-350. Epub 2018 Aug 9.

Institute of Bioinformatics and Structural Biology & Department of Medical Sciences, National Tsing Hua University, Hsinchu 300, Taiwan. Electronic address:

Evodiamine is a natural product extracted from herbal plants such as Tetradium which has shown to have anti-fat uptake and anti-proliferation properties. However, the effects of evodiamine on the behavior of thyroid cancers are largely unknown. To determine if evodiamine might be useful in the treatment of thyroid cancer and its cytotoxic mechanism, we analyzed the impact of evodiamine treatment on differential protein expression in human thyroid cancer cell line ARO using lysine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS). This study demonstrated 77 protein features that were significantly changed in protein expression and revealed evodiamine-induced cytotoxicity in thyroid cancer cells involves dysregulation of protein folding, cytoskeleton, cytoskeleton regulation and transcription control. Our work shows that this combined proteomic strategy provides a rapid method to study the molecular mechanisms of evodiamine-induced cytotoxicity in thyroid cancer cells. The identified targets may be useful for further evaluation as potential targets in thyroid cancer therapy.
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http://dx.doi.org/10.1016/j.jpba.2018.08.008DOI Listing
October 2018

Proteomic analysis of honokiol-induced cytotoxicity in thyroid cancer cells.

Life Sci 2018 Aug 5;207:184-204. Epub 2018 Jun 5.

Institute of Bioinformatics and Structural Biology, Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

Aims: Honokiol is a natural product extracted from herbal plants such as the Magnolia species which have been shown to exhibit anti-tumor and anti-metastatic properties. However, the effects of honokiol on thyroid cancers are largely unknown.

Materials And Methods: To determine whether honokiol might be useful for the treatment of thyroid cancer and to elucidate the mechanism of toxicity of honokiol, we analyzed the impact of honokiol treatment on differential protein expression in human thyroid cancer cell line ARO using lysine-labeling two-dimensional difference gel electrophoresis (2D-DIGE) combined with mass spectrometry (MS).

Key Findings: This study revealed 178 proteins that showed a significant change in expression levels and also revealed that honokiol-induced cytotoxicity in thyroid cancer cells involves dysregulation of cytoskeleton, protein folding, transcription control and glycolysis.

Significance: Our work shows that combined proteomic strategy provides a rapid method to study the molecular mechanisms of honokiol-induced cytotoxicity in thyroid cancer cells. The identified targets may be useful for further evaluation as potential targets in thyroid cancer therapy.
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http://dx.doi.org/10.1016/j.lfs.2018.06.002DOI Listing
August 2018

Proteomic investigating the cooperative lethal effect of EGFR and MDM2 inhibitors on ovarian carcinoma.

Arch Biochem Biophys 2018 Jun 12;647:10-32. Epub 2018 Apr 12.

Institute of Bioinformatics and Structural Biology and Department of Medical Science, National Tsing Hua University, Hsinchu, Taiwan. Electronic address:

With the concept of precision medicine, combining multiple molecular-targeting therapies has brought new approaches to current cancer treatments. Malfunction of the tumor suppressor protein, p53 is a universal hallmark in human cancers. Under normal conditions, p53 is degraded through an ubiquitin-proteosome pathway regulated by its negative regulator, MDM2. In contrast, cellular stress such as DNA damage will activate p53 to carry out DNA repair, cell cycle arrest, and apoptosis. In this study, we focused on ovarian carcinoma with high EGFR and MDM2 overexpression rate. We assessed the effects of combined inhibition by MDM2 (JNJ-26854165) and EGFR (gefitinib) inhibitors on various ovarian cell lines to determine the importance of these two molecular targets on cell proliferation. We then used a proteomic strategy to investigate the relationship between MDM2 and EGFR inhibition to explore the underlying mechanisms of how their combined signaling blockades work together to exert cooperative inhibition. Our results demonstrated that all four cell lines were sensitive to both individual and combined, MDM2 and EGFR inhibition. The proteomic analysis also showed that gefitinib/JNJ-treated CAOV3 cells exhibited downregulation of proteins involved in nucleotide biosynthesis such as nucleoside diphosphate kinase B (NME2). In conclusion, our study showed that the combined treatment with JNJ and gefitinib exerted synergistic inhibition on cell proliferation, thereby suggesting the potential application of combining MDM2 inhibitors with EGFR inhibitors for enhancing efficacy in ovarian cancer treatment.
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http://dx.doi.org/10.1016/j.abb.2018.04.004DOI Listing
June 2018

Angiogenesis-targeting microbubbles combined with ultrasound-mediated gene therapy in brain tumors.

J Control Release 2017 06 11;255:164-175. Epub 2017 Apr 11.

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan. Electronic address:

The major challenges in gene therapy for brain cancer are poor transgene expression due to the blood-brain barrier (BBB) and neurologic damage caused by conventional intracerebral injection. Non-viral gene delivery using ultrasound-targeted microbubbles (MBs) oscillation via the systematic transvascular route is attractive, but there is currently no high-yielding and targeted gene expression method. In this study, we developed a non-viral and angiogenesis-targeting gene delivery approach for efficient brain tumor gene therapy without brain damage. We developed a VEGFR2-targeted and cationic microbubbles (VCMBs) gene vector for use with transcranial focused ultrasound (FUS) exposure to allow transient gene delivery. The system was tested in a brain tumor model using the firefly luciferase gene and herpes simplex virus type 1 thymidine kinase/ganciclovir (pHSV-TK/GCV) with VCMBs under FUS exposure for transgene expression and anti-tumor effect. In vitro data showed that VCMBs have a high DNA-loading efficiency and high affinity for cancer cells. In vivo data confirmed that this technique enhanced gene delivery into tumor tissues without affecting normal brain tissues. The VCMBs group resulted in higher luciferase expression (3.8 fold) relative to the CMBs group (1.9 fold), and the direct injection group. The tumor volume on day 25 was significantly smaller in rats treated with the pHSV-TK/GCV system using VCMBs under FUS (9.7±5.2mm) than in the direct injection group (40.1±4.3mm). We demonstrated the successful use of DNA-loaded VCMBs and FUS for non-viral, non-invasive and targeted gene delivery to brain tumors.
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http://dx.doi.org/10.1016/j.jconrel.2017.04.010DOI Listing
June 2017

Proteomic Analysis of Various Rat Ocular Tissues after Ischemia-Reperfusion Injury and Possible Relevance to Acute Glaucoma.

Int J Mol Sci 2017 Feb 5;18(2). Epub 2017 Feb 5.

Institute of Bioinformatics and Structural Biology & Department of Medical Sciences, National Tsing Hua University, Hsinchu 300, Taiwan.

Glaucoma is a group of eye diseases that can cause vision loss and optical nerve damage. To investigate the protein expression alterations in various intraocular tissues (i.e., the cornea, conjunctiva, uvea, retina, and sclera) during ischemia-reperfusion (IR) injury, this study performed a proteomic analysis to qualitatively investigate such alterations resulting from acute glaucoma. The IR injury model combined with the proteomic analysis approach of two-dimensional difference gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) was used to monitor the protein expression alterations in two groups of specimens (an IR injury group and a control group). The analysis results revealed 221 unique differentially expressed proteins of a total of 1481 proteins in the cornea between the two groups. In addition, 97 of 1206 conjunctival proteins, 90 of 1354 uveal proteins, 61 of 1180 scleral proteins, and 37 of 1204 retinal proteins were differentially expressed. These findings imply that different ocular tissues have different tolerances against IR injury. To sum up, this study utilized the acute glaucoma model combined with 2D-DIGE and MALDI-TOF MS to investigate the IR injury affected protein expression on various ocular tissues, and based on the ratio of protein expression alterations, the alterations in the ocular tissues were in the following order: the cornea, conjunctiva, uvea, sclera, and retina.
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http://dx.doi.org/10.3390/ijms18020334DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5343869PMC
February 2017

Folate-conjugated gene-carrying microbubbles with focused ultrasound for concurrent blood-brain barrier opening and local gene delivery.

Biomaterials 2016 11 12;106:46-57. Epub 2016 Aug 12.

Department of Biomedical Engineering and Environmental Sciences, National Tsing Hua University, No. 101, Section 2, Kuang-Fu Road, Hsinchu 30013, Taiwan. Electronic address:

Previous studies have demonstrated that circulating DNA-encapsulated microbubbles (MBs) combined with focused ultrasound (FUS) can be used for local blood-brain barrier (BBB) opening and gene delivery. However, few studies focused on how to increase the efficiency of gene delivery to brain tumors after the released gene penetrating the BBB. Here, we proposed the use of folate-conjugated DNA-loaded cationic MBs (FCMBs). When combined with FUS as a trigger for BBB opening, FCMBs were converted into nanometer-sized vesicles that were transported to the brain parenchyma. The FCMBs can selectively aggregate around tumor cells that overexpressed the folate receptor, thus enhancing gene delivery via folate-stimulated endocytosis. Our results confirmed that FCMBs can carry DNA on the surface of the MB shell and have good targeting ability on C6 glioma cells. In addition, the optimized FUS parameters for FCMBs-enhanced gene delivery were confirmed by cell experiments (center frequency = 1 MHz; acoustic pressure = 700 kPa; pulse repetition frequency = 5 Hz; cycle number = 10000; exposure time = 1 min; FCMBs concentration = 4 × 10(7) MB/mL). In vivo data also indicated that FCMBs show better gene transfection efficiency than MBs without folate conjugation and the traditional approach of directly injecting the gene. This study described our novel development of multifunctional MBs for FUS-triggered gene delivery/therapy.
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http://dx.doi.org/10.1016/j.biomaterials.2016.08.017DOI Listing
November 2016

MMP-13 is involved in oral cancer cell metastasis.

Oncotarget 2016 03;7(13):17144-61

Department of Medical Sciences and Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu, Taiwan.

The oral cancer cell line OC3-I5 with a highly invasive ability was selected and derived from an established OSCC line OC3. In this study, we demonstrated that matrix metalloproteinases protein MMP-13 was up-regulated in OC3-I5 than in OC3 cells. We also observed that expression of epithelial-mesenchymal transition (EMT) markers including Twist, p-Src, Snail1, SIP1, JAM-A, and vinculin were increased in OC3-I5 compared to OC3 cells, whereas E-cadherin expression was decreased in the OC3-I5 cells. Using siMMP-13 knockdown techniques, we showed that siMMP-13 not only reduced the invasion and migration, but also the adhesion abilities of oral cancer cells. In support of the role of MMP-13 in metastasis, we used MMP-13 expressing plasmid-transfected 293T cells to enhance MMP-13 expression in the OC3 cells, transplanting the MMP-13 over expressing OC3 cells into nude mice led to enhanced lung metastasis. In summary, our findings show that MMP-13 promotes invasion and metastasis in oral cancer cells, suggesting altered expression of MMP-13 may be utilized to impede the process of metastasis.
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http://dx.doi.org/10.18632/oncotarget.7942DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4941377PMC
March 2016

Identification of Up- and Down-Regulated Proteins in Pemetrexed-Resistant Human Lung Adenocarcinoma: Flavin Reductase and Calreticulin Play Key Roles in the Development of Pemetrexed-Associated Resistance.

J Proteome Res 2015 Nov 22;14(11):4907-20. Epub 2015 Oct 22.

Department of Medical Science and Institute of Bioinformatics and Structural Biology, National Tsing Hua University , No. 101, Kuang-Fu Road Sec. 2, Hsinchu 30013, Taiwan.

Drug resistance is one of the major causes of cancer chemotherapy failure. In the current study, we used a pair of lung adenocarcinoma cell lines, A549 and the pemetrexed-resistant A549/PEM cells, as a model to monitor resistance-dependent cellular responses and identify potential therapeutic targets. By means of 2D differential gel electrophoresis (2D-DIGE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), we investigated the global protein expression alterations induced by pemetrexed treatment and resistance. The proteomic result revealed that pemetrexed exposure obviously altered the expression of 81 proteins in the A549 cells, whereas no significant response was observed in the similarly treated A549/PEM cells, hence implying an association between these proteins and the drug-specific response. Moreover, 72 proteins including flavin reductase and calreticulin demonstrated differential expression between the A549 and A549/PEM cells, indicating baseline resistance. Additional tests employed siRNA silencing, protein overexpression, cell viability analysis, and analysis of apoptosis to examine and confirm the potency of flavin reductase and calreticulin proteins in the development of pemetrexed resistance. In summary, by using a proteomic approach, we identified numerous proteins, including flavin reductase and calreticulin, involved in pemetrexed drug resistance-developing mechanisms. Our results provide useful diagnostic markers and therapeutic candidates for pemetrexed-resistant lung cancer treatment.
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http://dx.doi.org/10.1021/acs.jproteome.5b00794DOI Listing
November 2015