Browsing by Author "Tsai, Nu-Man"

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A fusion protein composed of receptor binding domain of vascular endothelial growth factor-A and constant region fragment of antibody: angiogenesis antagonistic activity
(2011-05-01)
Tseng, Feng-Jen
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Liu, Yen-Ku
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Chung, Yo-Shong
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Lin, Yu-Ling
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Chen, Chia-Hung
Vascular endothelial growth factor (VEGF) promotes the growth of solid tumor mainly via VEGF receptor-1 and receptor-2, which are expressed preferentially in proliferating endothelial cells. Therefore, a strategy for simultaneous blockage of both VEGF receptors may have a useful therapeutic effect in tumor growth. In this study, we utilized a fusion protein which is composed of receptor binding domain of VEGF-A (RBDV) and the constant region fragment (Fc) of a human immunoglobulin G1 (IgG1), to interfere with the growth of human umbilical vein endothelial cells (HUVECs) via VEGF receptors. The results showed that RBDV-IgG1 Fc was able to bind with both VEGF receptor-1 and receptor-2. In addition, RBDV-IgG1 Fc could decrease VEGF-induced proliferation and tube formation among HUVECs. Moreover, the cytotoxic test showed RBDV-IgG1 Fc could also enhance the cytotoxic activity of human natural killing cells. The data are suggesting that the fusion protein, RBDV-IgG1 Fc, may have potential as an angiogenesis antagonist for future tumor therapy.
A fusion protein with the receptor binding domain of vascular endothelial growth factor-A (VEGF-A) is an antagonist of angiogenesis in cancer treatment
(2010-11-01)
Tseng, Feng-Jen
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Chen, Yu-Cheng
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Lin, Yu-Ling
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Tsai, Nu-Man
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Lee, Ru-Ping
Vascular endothelial growth factor (VEGF) is an angiogenic factor that signals through VEGFR-1 and VEGFR-2, which are expressed preferentially in proliferating endothelial cells. Thus, simultaneous blockage of both VEGF receptors may provide a more efficient therapeutic response in cancer treatment. We created a recombinant fusion protein (RBDV-IgG1 Fc), which is composed of the receptor binding domain of human VEGF-A (residues 8-109) and the Fc region of human IgG1 immunoglobulin. The recombinant protein can bind to both mouse VEGFR-1 and VEGFR-2 to decrease VEGF-induced proliferation and tube formation of endothelial cells in vitro. In this study, the RBDV-IgG1 Fc fusion protein reduced the effects of proliferation, migration and tube formation induced by VEGF in murine endothelial cells in vitro. In vivo tumor therapy with RBDV-IgG1 Fc resulted in tumor inhibition by reducing angiogenesis. Pathological evidence also shows that RBDV-IgG1 Fc can seriously damage vessels, causing the death of tumor cells. These findings suggest that this chimeric protein has potential as an angiogenesis antagonist in tumor therapy.
A Lipo-PEG-PEI complex for encapsulating curcumin that enhances its antitumor effects on curcumin-sensitive and curcumin-resistance cells
(2012-04-01)
Lin, Yu-Ling
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Liu, Yen-Ku
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Tsai, Nu-Man
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Hsieh, Jui-Hung
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Chen, Chia-Hung
A cationic liposome-PEG-PEI complex (LPPC) was used as a carrier for the encapsulation of hydrophobic curcumin to give curcumin/LPPC. Curcumin/LPPC had an average size less than 270 nm and a zeta potential of approximately 40 mV. The LPPC encapsulation efficiency for curcumin was about 45%. The authors found it surprising that the cytotoxic activity of the curcumin/LPPC was fivefold higher than curcumin when tested on curcumin-sensitive cells and 20-fold more active against curcumin-resistant cells. Curcumin/LPPC treatment caused a cell cycle arrest at G2/M phase, which rapidly resulted in apoptosis. The increased cytotoxic activity of curcumin/LPPC is likely attributable to its rapid accumulation in the cell. In vivo, administration of curcumin/LPPC inhibited about 60 - 90% of tumor growth in mice bearing CT-26 or B16F10 cells. These results demonstrate LPPC encapsulation technology is able to enhance the effects of antitumor drugs. Use of this technology may provide a new tool for cancer therapy, especially for drug-resistant cancer.
A New Microsphere-Based Immunoassay for Measuring the Activity of Transcription Factors
(2010-12-01)
Lin, Yu-Ling
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Lai, Yun-Ju
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Tsai, Nu-Man
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Peng, Tai-Chu
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Liu, Yen-Ku
There are several traditional and well-developed methods for analyzing the activity of transcription factors, such as EMSA, enzyme-linked immunosorbent assay, and reporter gene activity assays. All of these methods have their own distinct disadvantages, but none can analyze the changes in transcription factors in the few cells that are cultured in the wells of 96-well titer plates. Thus, a new microsphere-based immunoassay to measure the activity of transcription factors (MIA-TF) was developed. In MIA-TF, NeutrAvidin-labeled microspheres were used as the solid phase to capture biotin-labeled double-strand DNA fragments which contain certain transcription factor binding elements. The activity of transcription factors was detected by immunoassay using a transcription factor-specific antibody to monitor the binding with the DNA probe. Next, analysis was performed by flow cytometry. The targets hypoxia-inducible factor-1 alpha (HIF-1 alpha) and nuclear factor-kappa B (NF-kappa B) were applied and detected in this MIA-TF method; the results that we obtained demonstrated that this method could be used to monitor the changes of NF-kappa B or HIF within 50 or 100 ng of nuclear extract. Furthermore, MIA-TF could detect the changes in NF-kappa B or HIF in cells that were cultured in wells of a 96-well plate without purification of the nuclear protein, an important consideration for applying this method to high-throughput assays in the future. The development of MIA-TF would support further progress in clinical analysis and drug screening systems. Overall, MIA-TF is a method with high potential to detect the activity of transcription factors.
A potential role for Helicobacter pylori heat shock protein 60 in gastric tumorigenesis
(2010-02-05)
Lin, Chen-Si
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He, Pei-Juin
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Tsai, Nu-Man
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Li, Chi-Han
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Yang, Shang-Chih
Helicobacter pylon has been found to promote the malignant process leading to gastric cancer. Heat shock protein 60 of H. pylori (HpHSP60) was previously been identified as a potent immunogene. This Study investigates the role of HpHSP60 in gastric cancer carcinogenesis. The effect of HpHSP60 on cell proliferation, anti-death activity, angiogenesis and cell migration were explored. The results showed that HpHSP60 enhanced migration by gastric cancer cells and promoted tube formation by umbilical vein endothelial cells (HUVECs); however, HpHSP60 did not increase cell Proliferation nor was this protein able to rescue gastric cancer cells from death. Moreover, the results also indicated HpHSP60 had different effects on AGS gastric cancer cells or THP-1 monocytic cells in terms of their expression of pro-inflammatory cytokines, which are known to be important to cancer development. We propose that HpHSP60 may trigger the initiation of carcinogenesis by inducing pro-inflammatory cytokine release and by promoting angiogenesis and metastasis. Thus, this extracellular pathogen-derived HSP60 is potentially a vigorous virulence factor that can act as a carcinogen during gastric tumorigenesis. (C) 2010 Elsevier Inc. All rights reserved.
Antibodies against Helicobacter pylori heat shock protein 60 aggravate HSP60-mediated proinflammatory responses
(2011-08-01)
Liao, Kuang-Wen
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Lin, Chen-Si
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Chen, Wen-Liang
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Yang, Chu-Ting
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Lin, Ching-Min
Anti-Helicobacter pylori heat shock protein 60 (HpHSP60) antibodies are Usually found in H. pylori-infected patients and are known to be associated with the progression of gastric diseases. However, the effects of these antibodies on the functions of HpHSP60 have not been identified. This study aims to investigate the effects of the interaction between anti-HSP60 antibodies and HpHSP60 on inflammatory responses. Anti-HpHSP60 polyclonal sera and monoclonal antibodies (mAbs) were produced to evaluate their effects on HpHSP60-induced IL-8 and TNF-alpha activity. The results indicated that anti-HpHSP60 polyclonal sera collected from patients infected with H. pylori or from rabbit and mice immunized with HpHSP60 could significantly enhance HpHSP60-mediated IL-8 and TNF-alpha secretion from monocytic THP-1 cells. Similar effects were also found with anti-HpHSP60 mAbs. Further analysis revealed that this phenomenon was only carried out by anti-HpHSP60 antibody but not by other non-specific mAbs. Moreover, the non-specific mAbs decreased the synergism of HpHSP60 and anti-HpHSP60 mAbs in proinflammatory cytokine induction. Herein, we have examined the role of anti-HpHSP60 antibody in host immune responses for the first time. This study demonstrated that H. pylori HSP60/mAbs could modulate helico-bacterial pathogenesis by increasing IL-8 and TNF-alpha production. The pathogen-specific antibodies may execute potential immune functions rather than recognize or neutralize microbes. (C) 2011 Elsevier Ltd. All rights reserved.
Antitumor Effect of n-Butylidenephthalide Encapsulated on B16/F10 Melanoma Cells In Vitro with a Polycationic Liposome Containing PEI and Polyethylene Glycol Complex
(2018-12-01)
Gao, Hong-Wei
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Chang, Kai-Fu
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Huang, Xiao-Fan
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Lin, Yu-Ling
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Weng, Jun-Cheng
Advanced melanoma can metastasize to distal organs from the skin and yield an aggressive disease and poor prognosis even after treatment with chemotherapeutic agents. The compound n-Butylidenephthalide (BP) is isolated from Angelica sinensis, which is used to treat anemia and gynecological dysfunction in traditional Chinese medicine. Studies have indicated that BP can inhibit cancers, including brain, lung, prostate, liver, and colon cancers. However, because BP is a natural hydrophobic compound, it is quickly metabolized by the liver within 24 h, and thus has limited potential for development in cancer therapy. This study investigated the anticancer mechanisms of BP through encapsulation with a novel polycationic liposome containing polyethylenimine (PEI) and polyethylene glycol complex (LPPC) in melanoma cells. The results demonstrated that BP/LPPC had higher cytotoxicity than BP alone and induced cell cycle arrest at the G(0)/G(1) phase in B16/F10 melanoma cells. The BP/LPPC-treated cell indicated an increase in subG(1) percentage and TUNEL positive apoptotic morphology through induction of extrinsic and intrinsic apoptosis pathways. The combination of BP and LPPC and clinical drug 5-Fluorouracil had a greater synergistic inhibition effect than did a single drug. Moreover, LPPC encapsulation improved the uptake of BP values through enhancement of cell endocytosis and maintained BP cytotoxicity activity within 24 h. In conclusion, BP/LPPC can inhibit growth of melanoma cells and induce cell arrest and apoptosis, indicating that BP/LPPC has great potential for development of melanoma therapy agents.
Antitumor Effects of N-Butylidenephthalide Encapsulated in Lipopolyplexs in Colorectal Cancer Cells
(2020-05-01)
Chang, Kai-Fu
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Chang, Jinghua Tsai
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Huang, Xiao-Fan
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Lin, Yu-Ling
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Liao, Kuang-Wen
Colorectal cancer (CRC) is the third most common type of cancer and the second most common cause of cancer-related death in the world. N-Butylidenephthalide (BP), a natural compound, inhibits several cancers, such as hepatoma, brain tumor and colon cancer. However, due to the unstable structure, the activity of BP is quickly lost after dissolution in an aqueous solution. A polycationic liposomal polyethylenimine and polyethylene glycol complex (LPPC), a new drug carrier, encapsulates both hydrophobic and hydrophilic compounds, maintains the activity of the compound, and increases uptake of cancer cells. The purpose of this study is to investigate the antitumor effects and protection of BP encapsulated in LPPC in CRC cells. The LPPC encapsulation protected BP activity, increased the cytotoxicity of BP and enhanced cell uptake through clathrin-mediated endocytosis. Moreover, the BP/LPPC-regulated the expression of the p21 protein and cell cycle-related proteins (CDK4, Cyclin B1 and Cyclin D1), resulting in an increase in the population of cells in the G(0)/G(1) and subG(1) phases. BP/LPPC induced cell apoptosis by activating the extrinsic (Fas, Fas-L and Caspase-8) and intrinsic (Bax and Caspase-9) apoptosis pathways. Additionally, BP/LPPC combined with 5-FU synergistically inhibited the growth of HT-29 cells. In conclusion, LPPC enhanced the antitumor activity and cellular uptake of BP, and the BP/LPPC complex induced cell cycle arrest and apoptosis, thereby causing death. These findings suggest the putative use of BP/LPPC as an adjuvant cytotoxic agent for colorectal cancer.
Characterizing the polymeric status of Helicobacter pylori heat shock protein 60
(2009-10-16)
Lin, Ching-Yi
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Huang, Yu-Shan
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Li, Chi-Han
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Hsieh, Yuan-Ting
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Tsai, Nu-Man
Helicobacter pylori heat shock protein 60 (HpHsp60) was first identified as an adhesion molecule associated with H. pylori infection. Here we have analyzed the structure of HpHsp60 via amino acid BLAST, circular dichroism, and electrophoresis and the results indicate that most recombinant HpHsp60 molecules exist as dimers or tetramers, which is quite different from Escherichia coli Hsp60. Treatment of human monocytic cells THP-1 with HpHsp60 was found to up-regulate a panel of cytokines including IL-1 alpha, IL-8, IL-10, IFN-gamma, TNF-alpha, TGF-beta, GRO, and RANTES. Carboxymethylated HpHsp60 molecules with a switched oligomeric status were able to further enhance NF-kappa B-mediated IL-8 and TNF-alpha secretion in THP-1 cells compared to unmodified HpHsp60 molecules. These results indicated that the oligomeric status of HpHsp60s might have an important role in regulating host inflammation and thus help facilitate H. pylori persistent infection. (C) 2009 Elsevier Inc. All rights reserved.
Development of a computational promoter with highly efficient expression in tumors
(2018-04-27)
Ho, Shu-Yi
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Chang, Bo-Hau
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Chung, Chen-Han
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Lin, Yu-Ling
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Chuang, Cheng-Hsun
Background: Gene therapy is a potent method to increase the therapeutic efficacy against cancer. However, a gene that is specifically expressed in the tumor area has not been identified. In addition, nonspecific expression of therapeutic genes in normal tissues may cause side effects that can harm the patients' health. Certain promoters have been reported to drive therapeutic gene expression specifically in cancer cells; however, low expression levels of the target gene are a problem for providing good therapeutic efficacy. Therefore, a specific and highly expressive promoter is needed for cancer gene therapy. Methods: Bioinformatics approaches were utilized to analyze transcription factors (TFs) from high-throughput data. Reverse transcription polymerase chain reaction, western blotting and cell transfection were applied for the measurement of mRNA, protein expression and activity. C57BL/6JNarl mice were injected with pD5-hrGFP to evaluate the expression of TFs. Results: We analyzed bioinformatics data and identified three TFs, nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kappa B), cyclic AMP response element binding protein (CREB), and hypoxia-inducible factor-1 alpha (HIF-1 alpha), that are highly active in tumor cells. Here, we constructed a novel mini-promoter, D5, that is composed of the binding sites of the three TFs. The results show that the D5 promoter specifically drives therapeutic gene expression in tumor tissues and that the strength of the D5 promoter is directly proportional to tumor size. Conclusions: Our results show that bioinformatics may be a good tool for the selection of appropriate TFs and for the design of specific mini-promoters to improve cancer gene therapy.
Encapsulated n-Butylidenephthalide Efficiently Crosses the Blood-Brain Barrier and Suppresses Growth of Glioblastoma
(2020-01-01)
Lin, Yu-Ling
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Huang, Xiao-Fan
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Chang, Kai-Fu
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Liao, Kuang-Wen
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Tsai, Nu-Man
Background: n-Butylidenephthalide (BP) has anti-tumor effects on glioblastoma. However, the limitation of BP for clinical application is its unstable structure. A polycationic liposomal polyethylenimine (PEI) and polyethylene glycol (PEG) complex (LPPC) has been developed to encapsulate BP for drug structure protection. The purpose of this study was to investigate the anti-cancer effects of the BP/LPPC complex on glioblastoma in vitro and in vivo. Methods: DBTRG-05MG tumor bearing xenograft mice were treated with BP and BP/LPPC and then their tumor sizes, survival, drug biodistribution were measured. RG2 tumor bearing F344 rats also treated with BP and BP/LPPC and then their tumor sizes by magnetic resonance imaging for evaluation blood-brain barrier (BBB) across and drug therapeutic effects. After treated with BP/LPPC in vitro, cell uptake, cell cycle and apoptotic regulators were analyzed for evaluation the therapeutic mechanism. Results: In athymic mice, BP/LPPC could efficiently suppress tumor growth and prolong survival. In F334 rats, BP/LPPC crossed the BBB and led to tumor shrinkage. BP/LPPC promoted cell cycle arrest at the G(0)/G(1) phase and triggered the extrinsic and intrinsic cell apoptosis pathways resulting cell death. BP/LPPC also efficiently suppressed VEGF, VEGFR1, VEGFR2, MMP2 and MMP9 expression. Conclusion: BP/LPPC was rapidly and efficiently transported to the tumor area across the BBB and induced cell apoptosis, anti-angiogenetic and anti-metastatic effects in vitro and in vivo.
Helicobacter pylori-derived Heat shock protein 60 enhances angiogenesis via a CXCR2-mediated signaling pathway
(2010-06-25)
Lin, Chen-Si
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He, Pei-Juin
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Hsu, Wei-Tung
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Wu, Ming-Shiang
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Wu, Chang-Jer
Helicobacter pylori is a potent carcinogen associated with gastric cancer malignancy. Recently. H. pylori Heat shock protein 60 (HpHSP60) has been reported to promote cancer development by inducing chronic inflammation and promoting tumor cell migration. This study demonstrates a role for HpHSP60 in angiogenesis, a necessary precursor to tumor growth. We showed that HpHSP60 enhanced cell migration and tube formation, but not cell proliferation, in human umbilical vein endothelial cells (HUVECs). HpHSP60 also indirectly promoted HUVEC proliferation when HUVECs were co-cultured with supernatants collected from HpHSP60-treated AGS or THP-1 cells. The angiogenic array showed that HpHSP60 dramatically induced THP-1 cells and HUVECs to produce the chemotactic factors IL-8 and GRO. Inhibition of CXCR2, the receptor for IL-8 and GRO, or downstream PLC beta 2/Ca2+-mediated signaling, significantly abolished HpHSP60-induced tube formation. In contrast, suppression of MAP K or PI3 K signaling did not affect HpHSP60-mediated tubulogenesis. These data suggest that HpHSP60 enhances angiogenesis via CXCR2/PLC beta 2/Ca2+ signal transduction in endothelial cells. (C) 2010 Elsevier Inc. All rights reserved.
In Situ Activation of Pituitary-Infiltrating T Lymphocytes in Autoimmune Hypophysitis
(2017-03-06)
Lin, Han-Huei
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Gutenberg, Angelika
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Chen, Tzu-Yu
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Tsai, Nu-Man
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Lee, Chia-Jung
Autoimmune hypophysitis (AH) is a chronic inflammatory disease characterized by infiltration of T and B lymphocytes in the pituitary gland. The mechanisms through which infiltrating lymphocytes cause disease remain unknown. Using a mouse model of AH we assessed whether T lymphocytes undergo activation in the pituitary gland. Infiltrating T cells co-localized with dendritic cells in the pituitary and produced increased levels of interferon-gamma and interleukin-17 upon stimulation in vitro. Assessing proliferation of CD3- and B220-postive lymphocytes by double immunohistochemistry (PCNA-staining) and flow cytometry (BrdU incorporation) revealed that a discrete proportion of infiltrating T cells and B cells underwent proliferation within the pituitary parenchyma. This proliferation persisted into the late disease stage (day 56 post-immunization), indicating the presence of a continuous generation of autoreactive T and B cells within the pituitary gland. T cell proliferation in the pituitary was confirmed in patients affected by autoimmune hypophysitis. In conclusion, we show that pituitary-infiltrating lymphocytes proliferate in situ during AH, providing a previously unknown pathogenic mechanism and new avenues for treatment.
In vivo amelioration of endogenous antitumor autoantibodies via low-dose P4N through the LTA4H/activin A/BAFF pathway
(2016-11-29)
Lin, Yu-Ling
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Tsai, Nu-Man
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Hsieh, Cheng-Hao
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Ho, Shu-Yi
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Chang, Jung
Cancer progression is associated with the development of antitumor autoantibodies in patients\' sera. Although passive treatment with antitumor antibodies has exhibited remarkable therapeutic efficacy, inhibitory effects on tumor progression by endogenous antitumor autoantibodies (EAAs) have been limited. In this study, we show that P4N, a derivative of the plant lignan nordihydroguaiaretic acid (NDGA), enhanced the production of EAAs and inhibited tumor growth at low noncytotoxic concentrations via its immunoregulatory activity. Intratumoral injection of P4N improved the quantity and quality of EAAs, and passive transfer of P4N-induced EAAs dramatically suppressed lung metastasis formation and prolonged the survival of mice inoculated with metastatic CT26 tumor cells. P4N-induced EAAs specifically recognized two surface antigens, 78-kDa glucose-regulated protein (GRP78) and F1F0 ATP synthase, on the plasma membrane of cancer cells. Additionally, P4N treatment led to B-cell proliferation, differentiation to plasma cells, and high titers of autoantibody production. By serial induction of autocrine and paracrine signals in monocytes, P4N increased B-cell proliferation and antibody production via the leukotriene A4 hydrolase (LTA4H)/activin A/B-cell activating factor (BAFF) pathway. This mechanism provides a useful platform for studying and seeking a novel immunomodulator that can be applied in targeting therapy by improving the quantity and quality of the EAAs.
Inhibition of breast cancer with transdermal tamoxifen-encapsulated lipoplex
(2016-02-19)
Lin, Yu-Ling
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Chen, Chia-Hung
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Wu, Hsin-Yi
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Tsai, Nu-Man
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Jian, Ting-Yan
Background: Tamoxifen is currently used for the treatment of both early and advanced estrogen receptor (ER) positive breast cancer in pre- and post-menopausal women. However, using tamoxifen routinely to inhibit endogenous or exogenous estrogen effects is occasionally difficult because of its potential side effects. Objectives: The aim of this study is to design a local drug delivery system to encapsulate tamoxifen for observing their efficacy of skin penetration, drug accumulation and cancer therapy. Methods: A cationic liposome-PEG-PEI complex (LPPC) was used as a carrier for the encapsulation of tamoxifen and forming 'LPPC/TAM' for transdermal release. The cytotoxicity of LPPC/TAM was analyzed by MTT. The skin penetration, tumor growth inhibition and organ damages were measured in xenograft mice following transdermal treatment. Results: LPPC/TAM had an average size less than 270 nm and a zeta-potential of approximately 40 mV. LPPC/TAM displayed dramatically increased the cytotoxic activity in all breast cancer cells, especially in ER-positive breast cancer cells. In vivo, LPPC drug delivery helped the fluorescent dye penetrating across the skim and accumulating rapidly in tumor area. Administration of LPPC/TAM by transdermal route inhibited about 86 % of tumor growth in mice bearing BT474 tumors. This local treatment of LPPC/TAM did not injury skin and any organs. Conclusion: LPPC-delivery system provided a better skin penetration and drug accumulation and therapeutic efficacy. Therefore, LPPC/TAM drug delivery maybe a useful transdermal tool of drugs utilization for breast cancer therapy.
Inhibitory Effects of Chloroform Extracts Derived from Corbicula fluminea on the Release of Pro-inflammatory Cytokines
(2012-04-25)
Lin, Ching-Min
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Lin, Yu-Ling
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Tsai, Nu-Man
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Wu, Hsin-Yi
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Ho, Shu-Yi
Corbicula fluminea, the primary freshwater bivalve cultivated in Taiwan, was formerly used as a remedy for hepatitis. Recent reports indicate that C. fluminea has many bioactivities, but it remains unknown whether C. fluminea affects inflammation. This study explored the anti-inflammatory activity of C.fluminea. C. fluminea was first treated with chloroform to obtain clam chloroform extracts (CCEs). On the basis of the assay for the release of pro-inflammatory cytokines in vitro and in vivo, the results show that the CCEs significantly lowered the release of lipopolysaccharide (LPS)-induced pro-inflammatory cytokines. Additionally, the CCEs reduced LPS-induced organ damage. Real-time polymerase chain reaction analysis suggested that CCEs inhibit the LPS-induced mRNA expression of interleukin-1 beta and tumor necrosis factor-alpha. Western blot analysis indicated that the CCEs increased expression of I kappa B and attenuated the phosphorylation of I kappa B. Gas chromatography mass spectrometry suggests that phytosterols and fatty adds are responsible for the anti-inflammatory properties of CCEs. Taken together, CCEs have the potential to be developed as an anti-inflammatory functional food.
Lipo-PEG-PEI complex as an intracellular transporter for protein therapeutics
(2019-01-01)
Lin, Yu-Ling
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Chen, Chia-Hung
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Liu, Yen-Ku
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Huang, Tse-Hung
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Tsai, Nu-Man
Background: Protein or peptide drugs are emerging therapeutics for treating human diseases. However, current protein drugs are typically limited to acting on extracellular/cell membrane components associated with the diseases, while intracellular delivery of recombinant proteins replaces or replenishes faulty/missing proteins and remains inadequate. In this study, we developed a convenient and efficient intracellular protein delivery vehicle. Materials and methods: A cationic liposomal polyethylenimine and polyethylene glycol complex (LPPC) was developed to noncovalently capture proteins for protein transfer into cells via endocytosis. beta-glucuronidase (beta G) was used in vitro and in vivo as a model enzyme to demonstrate the enzymatic activity of the intracellular transport of a protein. Results: The endocytosed protein/LPPC complexes escaped from lysosomes, and the bound protein dissociated from LPPC in the cytosol. The enzymatic activity of beta G was well preserved after intracellular delivery in vitro and in vivo. Conclusion: Using LPPC as an intracellular protein transporter for protein therapeutics, we illustrated that LPPC may be an effective and convenient tool for studying diseases and developing therapeutics.
Lipoplex-based targeted gene therapy for the suppression of tumours with VEGFR expression by producing anti-angiogenic molecules
(2020-04-09)
Ho, Shu-Yi
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Chen, Pin-Rong
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Chen, Chia-Hung
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Tsai, Nu-Man
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Lin, Yu-Hsin
Background The anti-angiogenic fusion protein RBDV-IgG1 Fc (RBDV), which comprises the receptor-binding domain of vascular endothelial growth factor-A (VEGF-A), has shown antitumour effects by reducing angiogenesis in vivo. This study used the cationic lipoplex lipo-PEG-PEI-complex (LPPC) to simultaneously encapsulate both the RBDV targeting protein and the RBDV plasmid (pRBDV) without covalent bonds to assess VEGFR targeting gene therapy in mice with melanoma in vivo. Results LPPC protected the therapeutic transgene from degradation by DNase, and the LPPC/RBDV complexes could specifically target VEGFR-positive B16-F10 cells both in vitro and in vivo. With or without RBDV protein-targeting direction, the pRBDV-expressing RBDV proteins were expressed and reached a maximal concentration on the 7th day in the sera after transfection in vivo and significantly elicited growth suppression against B16-F10 melanoma but not IgG1 control proteins. In particular, LPPC/pRBDV/RBDV treatment with the targeting molecules dramatically inhibited B16-F10 tumour growth in vivo to provide better therapeutic efficacy than the treatments with gene therapy with IgG1 protein targeting or administration of a protein drug with RBDV. Conclusions The simultaneous combination of the LPPC complex with pRBDV gene therapy and RBDV protein targeting might be a potential tool to conveniently administer targeted gene therapy for cancer therapy.
Liposomal n-butylidenephthalide protects the drug from oxidation and enhances its antitumor effects in glioblastoma multiforme
(2015-01-01)
Lin, Yu-Ling
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Chang, Kai-Fu
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Huang, Xiao-Fan
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Hung, Che-Lun
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Chen, Shyh-Chang
Background: The natural compound n-butylidenephthalide (BP) can pass through the blood-brain barrier to inhibit the growth of glioblastoma multiforme tumors. However, BP has an unstable structure that reduces its antitumor activity and half-life in vivo. Objective: The aim of this study is to design a drug delivery system to encapsulate BP to enhance its efficacy by improving its protection and delivery. Methods: To protect its structural stability against protein-rich and peroxide solutions, BP was encapsulated into a lipo-PEG-PEI complex (LPPC). Then, the cytotoxicity of BP/LPPC following preincubation in protein-rich, acid/alkaline, and peroxide solutions was analyzed by MTT. Cell uptake of BP/LPPC was also measured by confocal microscopy. The therapeutic effects of BP/LPPC were analyzed in xenograft mice following intratumoral and intravenous injections. Results: When BP was encapsulated in LPPC, its cytotoxicity was maintained following preincubation in protein-rich, acid/alkaline, and peroxide solutions. The cytotoxic activity of encapsulated BP was higher than that of free BP (similar to 4.5-to 8.5-fold). This increased cytotoxic activity of BP/LPPC is attributable to its rapid transport across the cell membrane. In an animal study, a subcutaneously xenografted glioblastoma multiforme mouse that was treated with BP by intratumoral and intravenous administration showed inhibited tumor growth. The same dose of BP/LPPC was significantly more effective in terms of tumor inhibition. Conclusion: LPPC encapsulation technology is able to protect BP's structural stability and enhance its antitumor effects, thus providing a better tool for use in cancer therapy.
Sodium Dodecyl Sulfate-Modified Doxorubicin-Loaded Chitosan-Lipid Nanocarrier with Multi Polysaccharide-Lecithin Nanoarchitecture for Augmented Bioavailability and Stability of Oral Administration In Vitro and In Vivo
(2016-05)
Su, Chia-Wei
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Chiang, Min-Yu
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Lin, Yu-Ling
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Tsai, Nu-Man
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Chen, Yen-Po
For oral anti-cancer drug delivery, a new chitosan-lipid nanoparticle with sodium dodecyl sulfate modification was designed and synthesized using a double emulsification. TEM examination showed that the DOX-loaded nanoparticles, termed D-PL/TG NPs, exhibited a unique core shell configuration composed of multiple amphiphilic chitosan-lecithin reverse micelles as the core and a triglyceride shell as a physical barrier to improve the encapsulation efficiency and reduce the drug leakage. In addition, the D-PL/TG NPs with sodium dodecyl sulfate modification on the surface have enhanced stability in the GI tract and increased oral bioavailability of doxorubicin. In vitro transport studies performed on Caco-2 monolayers indicated that the D-PL/TG NPs enhanced the permeability of DOX in the Caco-2 monolayers by altering the transport pathway from passive diffusion to transcytosis. The in vivo intestinal absorption assay suggested that the D-PL/TG NPs were preferentially absorbed through the specialized membranous epithelial cells (M cells) of the Peyer\'s patches, resulting in a significant improvement (8-fold) in oral bioavailability compared to that of free DOX. The experimental outcomes in this work demonstrate that the D-PL/TG NPs provide an exciting opportunity for advances in the oral administration of drugs with poor bioavailability that are usually used in treating tough and chronic diseases.