24 results on '"Junking, Mutita"'
Search Results
2. Therapeutic potential of third-generation chimeric antigen receptor T cells targeting B cell maturation antigen for treating multiple myeloma.
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Rujirachaivej, Punchita, Siriboonpiputtana, Teerapong, Luangwattananun, Piriya, Yuti, Pornpimon, Wutti-in, Yupanun, Choomee, Kornkan, Sujjitjoon, Jatuporn, Chareonsirisuthigul, Takol, Rerkamnuaychoke, Budsaba, Junking, Mutita, and Yenchitsomanus, Pa-thai
- Abstract
Multiple myeloma (MM) is an incurable hematologic malignancy characterized by the rapid proliferation of malignant plasma cells within the bone marrow. Standard therapies often fail due to patient resistance. The US FDA has approved second-generation chimeric antigen receptor (CAR) T cells targeting B-cell maturation antigen (anti-BCMA-CAR2 T cells) for MM treatment. However, achieving enduring clinical responses remains a challenge in CAR T cell therapy. This study developed third-generation T cells with an anti-BCMA CAR (anti-BCMA-CAR3). The CAR incorporated a fully human scFv specific to BCMA, linked to the CD8 hinge region. The design included the CD28 transmembrane domain, two co-stimulatory domains (CD28 and 4-1BB), and the CD3ζ signaling domain (28BBζ). Lentiviral technology generated these modified T cells, which were compared against anti-BCMA-CAR2 T cells for efficacy against cancer. Anti-BCMA-CAR3 T cells exhibited significantly higher cytotoxic activity against BCMA-expressing cells (KMS-12-PE and NCI-H929) compared to anti-BCMA-CAR2 T cells. At an effector-to-target ratio of 10:1, anti-BCMA-CAR3 T cells induced lysis in 75.5 ± 3.8% of NCI-H929 cells, whereas anti-BCMA-CAR2 T cells achieved 56.7 ± 3.4% (p = 0.0023). Notably, after twelve days of cultivation, anti-BCMA-CAR3 T cells nearly eradicated BCMA-positive cells (4.1 ± 2.1%), while anti-BCMA-CAR2 T cells allowed 36.8 ± 20.1% to survive. This study highlights the superior efficacy of anti-BCMA-CAR3 T cells against both low and high BCMA-expressing MM cells, surpassing anti-BCMA-CAR2 T cells. These findings suggest potential for advancing anti-BCMA-CAR3 T cells in chimeric antigen receptor T (CAR-T) therapy for relapsed/refractory MM. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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3. Enhancement of PD-L1-attenuated CAR-T cell function through breast cancer-associated fibroblasts-derived IL-6 signaling via STAT3/AKT pathways.
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Chuangchot, Nisa, Jamjuntra, Pranisa, Yangngam, Supaporn, Luangwattananun, Piriya, Thongchot, Suyanee, Junking, Mutita, Thuwajit, Peti, Yenchitsomanus, Pa-Thai, and Thuwajit, Chanitra
- Subjects
CELL physiology ,INTERLEUKIN-6 ,WESTERN immunoblotting ,T cells ,IMMUNOREGULATION - Abstract
Background: Carcinoma-associated fibroblasts (CAFs) play a critical role in cancer progression and immune cell modulation. In this study, it was aimed to evaluate the roles of CAFs-derived IL-6 in doxorubicin (Dox) resistance and PD-L1-mediated chimeric antigenic receptor (CAR)-T cell resistance in breast cancer (BCA). Methods: CAF conditioned-media (CM) were collected, and the IL-6 level was measured by ELISA. CAF-CM were treated in MDA-MB-231 and HCC70 TNBC cell lines and siIL-6 receptor (IL-6R) knocked down (KD) cells to determine the effect of CAF-derived IL-6 on Dox resistance by flow cytometry and on increased PD-L1 through STAT3, AKT and ERK1/2 pathways by Western blot analysis. After pre-treating with CM, the folate receptor alpha (FRα)-CAR T cell cytotoxicity was evaluated in 2D and 3D spheroid culture assays. Results: The results showed a significant level of IL-6 in CAF-CM compared to that of normal fibroblasts (NFs). The CM with high IL-6 level significantly induced Dox resistance; and PD-L1 expression through STAT3 and AKT pathways in MDA-MB-231 and HCC70 cells. These induction effects were attenuated in siIL-6R KD cells. Moreover, the TNBC cell lines that were CM-treated with STAT3 and an AKT inhibitor had a reduced effect of IL-6 on PD-L1 expression. BCA cells with high IL-6 containing-CM treatment had resistance to cancer cell killing by FRα CAR-T cells compared to untreated cells. Conclusion: These results highlight CAF-derived IL-6 in the resistance of chemotherapy and T cell therapy. Using inhibitors of IL6-STAT3/AKT-PD-L1 axis may provide a potential benefit of Dox and CAR-T cell therapies in BCA patients. [ABSTRACT FROM AUTHOR]
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- 2023
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4. Activation of cytotoxic T lymphocytes by selfdifferentiated myeloid-derived dendritic cells for killing breast cancer cells expressing folate receptor alpha protein.
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Luangwattananun, Piriya, Chiraphapphaiboon, Wannasiri, Thuwajit, Chanitra, Junking, Mutita, and Yenchitsomanus, Pa-Thai
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- 2022
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5. Combination gemcitabine and PD-L1xCD3 bispecific T cell engager (BiTE) enhances T lymphocyte cytotoxicity against cholangiocarcinoma cells.
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Wathikthinnakon, Methi, Luangwattananun, Piriya, Sawasdee, Nunghathai, Chiawpanit, Chutipa, Lee, Vannajan Sanghiran, Nimmanpipug, Piyarat, Tragoolpua, Yingmanee, Rotarayanont, Siriphorn, Sangsuwannukul, Thanich, Phanthaphol, Nattaporn, Wutti-in, Yupanun, Somboonpatarakun, Chalermchai, Chieochansin, Thaweesak, Junking, Mutita, Sujjitjoon, Jatuporn, Yenchitsomanus, Pa-thai, and Panya, Aussara
- Subjects
IMMUNE checkpoint proteins ,CYTOTOXIC T cells ,T cells ,CELL death ,GEMCITABINE ,CHOLANGIOCARCINOMA ,ATEZOLIZUMAB ,PROGRAMMED cell death 1 receptors - Abstract
Cholangiocarcinoma (CCA) is a lethal cancer with rapid progression and poor survival. Novel and more effective therapies than those currently available are, therefore, urgently needed. Our research group previously reported the combination of gemcitabine and cytotoxic T lymphocytes to be more effective than single-agent treatment for the elimination of CCA cells. However, gemcitabine treatment of CCA cells upregulates the expression of an immune checkpoint protein (programmed death-ligand 1 [PD-L1]) that consequently inhibits the cytotoxicity of T lymphocytes. To overcome this challenge and take advantage of PD-L1 upregulation upon gemcitabine treatment, we generated recombinant PD-L1xCD3 bispecific T cell engagers (BiTEs) to simultaneously block PD-1/PD-L1 signaling and recruit T lymphocytes to eliminate CCA cells. Two recombinant PD-L1xCD3 BiTEs (mBiTE and sBiTE contain anti-PD-L1 scFv region from atezolizumab and from a published sequence, respectively) were able to specifically bind to both CD3 on T lymphocytes, and to PD-L1 overexpressed after gemcitabine treatment on CCA (KKU213A, KKU055, and KKU100) cells. mBiTE and sBiTE significantly enhanced T lymphocyte cytotoxicity against CCA cells, especially after gemcitabine treatment, and their magnitudes of cytotoxicity were positively associated with the levels of PD-L1 expression. Our findings suggest combination gemcitabine and PD-L1xCD3 BiTE as a potential alternative therapy for CCA. [ABSTRACT FROM AUTHOR]
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- 2022
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6. Antitumor activity of T cells secreting αCD133-αCD3 bispecific T-cell engager against cholangiocarcinoma.
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Sangsuwannukul, Thanich, Supimon, Kamonlapat, Chieochansin, Thaweesak, Choomee, Kornkan, Sujjitjoon, Jatuporn, Junking, Mutita, and Yenchitsomanus, Pa-thai
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T cells ,ANTINEOPLASTIC agents ,CHOLANGIOCARCINOMA ,EPITHELIAL cells - Abstract
Cholangiocarcinoma (CCA) is a lethal cancer of bile duct epithelial cells with a high mortality rate and limited therapeutic options. An effective treatment is, therefore, urgently needed to improve treatment outcomes for these patients. To develop a new therapeutic option, we engineered T cells secreting αCD133-αCD3 bispecific T-cell engager and evaluated their antitumor effects against CD133-expressing CCA cells. The cDNA encoding αCD133-αCD3 bispecific T-cell engager (αCD133-αCD3-ENG) was cloned into pCDH lentiviral construct and its expression was tested in Lenti-X 293T cells. T cells from healthy donors were then transduced with engineered lentiviruses to create T cells secreting αCD133-αCD3 engager to evaluate their antitumor activities. The average transduction efficiency into T cells was approximately 60.03±21.65%. In the co-culture system containing T cells secreting αCD133-αCD3 engager (as effector cells) and mWasabi-luciferase-expressing CCA cells (KKU-100 and KKU-213A; as target cells), the effector T cells exhibited significantly higher cytolytic activities against the target CCA cells (49.0±9.76% and 64.10±13.18%, respectively) than those observed against the untransduced T cells (10.97±10.65%; p = 0.0103 and 9.80±11.05%; p = 0.0054) at an effector-to-target ratio of 5:1. In addition, the secreted αCD133-αCD3 engager significantly redirected both transduced T cells and bystander T cells to kill the target CCA cells (up to 73.20±1.68%; p<0.05). Moreover, the transduced and bystander T cells could kill the target CCA spheroids at a rate approximately 5-fold higher than that of the no treatment control condition (p = 0.0011). Our findings demonstrate proof-of-principle that T cells secreting αCD133-αCD3 engager can be an alternative approach to treating CD133-positive CCA, and they pave the way for future in vivo study and clinical trials. [ABSTRACT FROM AUTHOR]
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- 2022
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7. Development of a Novel Anti-CD19 CAR Containing a Fully Human scFv and Three Costimulatory Domains.
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Wutti-in, Yupanun, Sujjitjoon, Jatuporn, Sawasdee, Nunghathai, Panya, Aussara, Kongkla, Katesara, Yuti, Pornpimon, Yongpitakwattana, Petlada, Thepmalee, Chutamas, Junking, Mutita, Chieochansin, Thaweesak, Poungvarin, Naravat, Yamabhai, Montarop, and Yenchitsomanus, Pa-thai
- Subjects
T cell receptors ,T cells ,ANTIGEN receptors ,CELL receptors ,CANCER cells ,MONOCLONAL antibodies - Abstract
Second-generation anti-CD19-chimeric antigen receptor T cells (anti-CD19-CAR2 T cells) are effective for treating B-cell malignancies; however, anti-CD19-CAR2 T cells can induce human anti-mouse immune responses because anti-CD19 single-chain variable fragment (scFv) in the CAR molecules is derived from a murine FMC63 (mFMC63) monoclonal antibody. Consequently, the persistence of mFMC63-CAR2 T cells and their therapeutic efficiency in patients are decreased, which results in tumor relapse. In an attempt to remedy this shortcoming, we generated a new anti-CD19-CAR T cells containing fully human anti-CD19 scFv (Hu1E7-CAR4 T cells) to pre-clinically evaluate and compare with mFMC63-CAR4 T cells. The human anti-CD19 scFv (Hu1E7) was isolated from a human scFv phage display library and fused to the hinge region of CD8α, the transmembrane domain of CD28, three intracellular costimulatory domains (CD28, 4-1BB, and CD27), and a CD3ζ signaling domain (28BB27ζ). Compared to mFMC63-CAR2 T cells (BBζ) and mFMC63-CAR3 (BB27ζ), the mFMC63-CAR4 T cells (28BB27ζ) exerted superior anti-tumor activity against Raji (CD19
+ ) target cell. The Hu1E7-CAR4 and mFMC63-CAR4 T cells demonstrated comparable cytotoxicity and proliferation. Interestingly, compared to mFMC63-CAR4 T cells, the Hu1E7-CAR4 T cells secreted lower levels of cytokines (IFN-γ and TNF-α), which may be due to the lower binding affinity of Hu1E7-CAR4 T cells. These findings demonstrated the successfulness in creation of a new CAR T cells containing a novel fully human-derived scFv specific to CD19+ cancer cells. In vivo studies are needed to further compare the anti-tumor efficacy and safety of Hu1E7-CAR4 T cells and mFMC63-CAR4 T cells. [ABSTRACT FROM AUTHOR]- Published
- 2022
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8. Piperine suppresses growth and migration of human breast cancer cells through attenuation of Rac1 expression.
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Buranrat, Benjaporn and Junking, Mutita
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- 2022
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9. Anti-mucin 1 chimeric antigen receptor T cells for adoptive T cell therapy of cholangiocarcinoma.
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Supimon, Kamonlapat, Sangsuwannukul, Thanich, Sujjitjoon, Jatuporn, Phanthaphol, Nattaporn, Chieochansin, Thaweesak, Poungvarin, Naravat, Wongkham, Sopit, Junking, Mutita, and Yenchitsomanus, Pa-thai
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T cells ,CELLULAR therapy ,CHOLANGIOCARCINOMA ,MORTALITY ,ANTIGEN receptors - Abstract
Current treatments for cholangiocarcinoma (CCA) are largely unsuccessful due to late diagnosis at advanced stage, leading to high mortality rate. Consequently, improved therapeutic approaches are urgently needed. Chimeric antigen receptor (CAR) T cell therapy is a newly potential therapy that can recognize specific surface antigen without major histocompatibility complex (MHC) restriction. Mucin 1 (MUC1) is an attractive candidate antigen as it is highly expressed and associated with poor prognosis and survival in CCA. We, therefore, set forth to create the fourth-generation CAR (CAR4) construct containing anti-MUC1-single-chain variable fragment (scFv) and three co-stimulatory domains (CD28, CD137, and CD27) linked to CD3ζ and evaluate anti-MUC1-CAR4 T cells in CCA models. Compared to untransduced T cells, anti-MUC1-CAR4 T cells produced increased levels of TNF-α, IFN-γ and granzyme B when exposed to MUC1-expressing KKU-100 and KKU-213A CCA cells (all p < 0.05). Anti-MUC1-CAR4 T cells demonstrated specific killing activity against KKU-100 (45.88 ± 7.45%, p < 0.05) and KKU-213A cells (66.03 ± 3.14%, p < 0.001) at an effector to target ratio of 5:1, but demonstrated negligible cytolytic activity against immortal cholangiocytes. Furthermore, the anti-MUC1-CAR4 T cells could effectively disrupt KKU-213A spheroids. These activities of anti-MUC1-CAR4 T cells supports the development of this approach as an adoptive T cell therapeutic strategy for CCA. [ABSTRACT FROM AUTHOR]
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- 2021
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10. Chimeric Antigen Receptor T Cells Targeting Integrin αvβ6 Expressed on Cholangiocarcinoma Cells.
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Phanthaphol, Nattaporn, Somboonpatarakun, Chalermchai, Suwanchiwasiri, Kwanpirom, Chieochansin, Thaweesak, Sujjitjoon, Jatuporn, Wongkham, Sopit, Maher, John, Junking, Mutita, and Yenchitsomanus, Pa-thai
- Subjects
CHIMERIC antigen receptors ,T cell receptors ,INTEGRINS ,CHOLANGIOCARCINOMA ,T cells ,FOOT & mouth disease - Abstract
Cholangiocarcinoma (CCA) is a lethal bile duct cancer that responds poorly to current standard treatments. A new therapeutic approach is, therefore, urgently needed. Adoptive T cell transfer using chimeric antigen receptor (CAR) T cells is a new therapeutic modality with demonstrated efficacy in hematologic malignancies. However, its efficacy against solid tumors is modest, and further intensive investigation continues. An important factor that influences the success of CAR T cell therapy is the selection of a target antigen that is highly expressed on cancer cells, but markedly less so in normal cells. Integrin αvβ6 is upregulated in several solid tumors, but is minimally expressed in normal epithelial cells, which suggests integrin αvβ6 as an attractive target antigen for CAR T cell immunotherapy in CCA. We investigated integrin αvβ6 expression in pathological tissue samples from patients with liver fluke-associated CCA. We then created CAR T cells targeting integrin αvβ6 and evaluated their anti-tumor activities against CCA cells. We found overexpression of the integrin αvβ6 protein in 23 of 30 (73.3%) CCA patient tissue samples. Significant association between high integrin αvβ6 expression and short survival time (p = 0.043) was also observed. Lentiviral constructs were engineered to encode CARs containing an integrin αvβ6-binding peptide (A20) derived from foot-and-mouth disease virus fused with a second-generation CD28/CD3ζ signaling domain (A20-2G CAR) or with a fourth-generation CD28/4-1BB/CD27/CD3ζ signaling domain (A20-4G CAR). The A20-2G and A20-4G CARs were highly expressed in primary human T cells transduced with the engineered lentiviruses, and they exhibited high levels of cytotoxicity against integrin αvβ6-positive CCA cells (p < 0.05). Interestingly, the A20-2G and A20-4G CAR T cells displayed anti-tumor function against integrin αvβ6-positive CCA tumor spheroids (p < 0.05). Upon specific antigen recognition, A20-4G CAR T cells produced a slightly lower level of IFN-γ, but exhibited higher proliferation than A20-2G CAR T cells. Thus, the A20-4G CAR T cells with lower level of cytokine production, but with higher proliferation represents a promising potential adoptive T cell therapy for integrin αvβ6-positive CCA. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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11. Fourth-generation chimeric antigen receptor T cells targeting folate receptor alpha antigen expressed on breast cancer cells for adoptive T cell therapy.
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Luangwattananun, Piriya, Junking, Mutita, Sujjitjoon, Jatuporn, Wutti-in, Yupanun, Poungvarin, Naravat, Thuwajit, Chanitra, and Yenchitsomanus, Pa-thai
- Abstract
Purpose: Treatment of breast cancer (BC) by standard methods is effective in the early stage, but ineffective in the advanced stage of disease. To develop an adoptive T cell therapy for advanced and severe BC, we generated fourth-generation chimeric antigen receptor (CAR) T cells targeting folate receptor alpha antigen (FRα) expressed on BC cells, and preclinically evaluated their anti-BC activities. Methods: The fourth-generation FRα-CAR T cells containing extracellular FRα-specific single-chain variable fragment (scFv) and three intracellular costimulatory domains (CD28, 4-1BB, and CD27) linked to CD3ζ were generated using a lentiviral system, and then were evaluated for their anti-BC activities in two-dimensional and three-dimensional (spheroid) cultures. Results: When our fourth-generation FRα-CAR T cells were cocultured with FRα-expressing MDA-MB-231 BC cell line at an effector to target ratio of 20:1, these CAR T cells specifically lysed 88.7 ± 10.6% of the target cells. Interestingly, the cytotoxic lysis of FRα-CAR T cells was more pronounced in target cells with higher surface FRα expression. This specific cytotoxicity of the CAR T cells was not observed when cocultured with FRα-negative MCF10A normal breast-like cell line at the same ratio (34.3 ± 4.7%). When they were cocultured with MDA-MD-231 spheroid, the FRα-CAR T cells exhibited antitumor activity marked with spheroid size reduction and breakage. Conclusion: This proof-of-concept study thus shows the feasibility of using these fourth-generation FRα-CAR T cells for adoptive T cell therapy in BC. [ABSTRACT FROM AUTHOR]
- Published
- 2021
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12. Suppression of TGF-β and IL-10 receptors on self-differentiated dendritic cells by short-hairpin RNAs enhanced activation of effector T-cells against cholangiocarcinoma cells.
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Thepmalee, Chutamas, Panya, Aussara, Sujjitjoon, Jatuporn, Sawasdee, Nunghathai, Poungvarin, Naravat, Junking, Mutita, and Yenchitsomanus, Pa-Thai
- Published
- 2020
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13. Cytolytic Activity of Effector T-lymphocytes Against Hepatocellular Carcinoma is Improved by Dendritic Cells Pulsed with Pooled Tumor Antigens.
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Chieochansin, Thaweesak, Thepmalee, Chutamas, Grainok, Janya, Junking, Mutita, and Yenchitsomanus, Pa-thai
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Cellular immunotherapy is a promising new therapeutic approach for hepatocellular carcinoma (HCC), which has a high recurrence rate, irrespective of the treatment administered. In this study, we attempted to improve the cytolytic activity of effector T-lymphocytes against HCC. T-lymphocytes were activated by monocyte-derived dendritic cells (DCs) pulsed with cell lysate or RNA prepared from HCC cell lines. Monocytes were activated for differentiation into DCs by treatment with the IL4 and GM-CSF. DCs were pulsed with cell lysate or RNA prepared from a single cell line or combinations of two or three HCC cell lines, and then co-cultured with autologous T-lymphocytes with the intent of creating specific cytotoxicity. We discovered that DCs pulsed with total RNA effectuated greater T-lymphocyte function than DCs pulsed with total cell lysate, as evidenced by greater cytolytic activities against HCC target cells. The percentage of Huh7, HepG2, and SNU449 cell apoptosis at effector:target ratio of 10:1 was 42.6 ± 4.5% (p = 0.01), 33.6 ± 3.1% (p = 0.007), and 21.4 ± 1.4% (p < 0.001), respectively. DCs pulsed with pools of antigens prepared from three cell lines improved the cytolytic function of effector T-lymphocytes by approximately two-fold (p < 0.001), which suggests that this approach be further developed and applied for adoptive transfer treatment of HCC. [ABSTRACT FROM AUTHOR]
- Published
- 2019
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14. Cytotoxic activity of effector T cells against cholangiocarcinoma is enhanced by self-differentiated monocyte-derived dendritic cells.
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Panya, Aussara, Thepmalee, Chutamas, Sawasdee, Nunghathai, Sujjitjoon, Jatuporn, Phanthaphol, Nattaporn, Junking, Mutita, Wongkham, Sopit, and Yenchitsomanus, Pa-thai
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CANCER cells ,DENDRITIC cells ,ANTINEOPLASTIC agents ,CELLULAR therapy ,CANCER treatment - Abstract
Cholangiocarcinoma (CCA) is a cancer of the bile ducts that is associated with poor prognosis and poor treatment outcome. Approximately one-third of CCA patients can undergo surgery, but the recurrence rate is high and chemotherapy often cannot satisfactorily prolong survival. Cellular immunotherapy based on adoptive T-cell transfer is a potential treatment for CCA; however, the development of this technology and the search for an appropriate tumor-associated antigen are still ongoing. To enhance the cytotoxic activity of effector T cells against CCA, we developed self-differentiated monocyte-derived dendritic cells (SD-DC) presenting cAMP-dependent protein kinase type I-alpha regulatory subunit (PRKAR1A), which is an overexpressed protein that plays a role in the regulation of tumor growth to activate T cells for CCA cell killing. Dendritic cells (DCs) transduced with lentivirus harboring tri-cistronic cDNA sequences (SD-DC-PR) could produce granulocyte-macrophage colony-stimulating factor, interleukin-4, and PRKAR1A. SD-DC showed similar phenotypes to those of DCs derived by conventional method. Autologous effector T cells (CD3+, CD8+) activated by SD-DC-PR exhibited greater cytotoxic activity against CCA than those activated by conventionally-derived DCs. Effector T cells activated by SD-DC-PR killed 60% of CCA cells at an effector-to-target ratio of 15:1, which is approximately twofold greater than the cell killing performance of those stimulated with control DC. The cytotoxic activities of effector T cells activated by SD-DC-PR against CCA cells were significantly associated with the expression levels of PRKR1A in CCA cells. This finding that SD-DC-PR effectively stimulated autologous effector T cells to kill CCA cells may help to accelerate the development of novel therapies for treating CCA. [ABSTRACT FROM AUTHOR]
- Published
- 2018
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15. Inhibition of IL-10 and TGF-β receptors on dendritic cells enhances activation of effector T-cells to kill cholangiocarcinoma cells.
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Thepmalee, Chutamas, Panya, Aussara, Junking, Mutita, Chieochansin, Thaweesak, and Yenchitsomanus, Pa-thai
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- 2018
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16. Vivo-morpholino oligomers strongly inhibit dengue virus replication and production.
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Phumesin, Patta, Junking, Mutita, Panya, Aussara, Yongpitakwattana, Petlada, Noisakran, Sansanee, Limjindaporn, Thawornchai, and Yenchitsomanus, Pa-thai
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DENGUE viruses ,DENGUE hemorrhagic fever ,VIRAL genomes ,OLIGOMERS ,SEROTYPES ,RNA sequencing ,DISEASE risk factors - Abstract
Dengue virus (DENV) infection is a worldwide public health problem, which can cause severe dengue hemorrhagic fever (DHF) and life-threatening dengue shock syndrome (DSS). There are currently no anti-DENV drugs available, and there has been an intensive search for effective anti-DENV agents that can inhibit all four DENV serotypes. In this study, we tested whether vivo-morpholino oligomers (vivo-MOs), whose effect on DENV infection has not previously been studied, can inhibit DENV infection. Vivo-MOs were designed to target the top of 3’ stem-loop (3’ SL) in the 3’ UTR of the DENV genome and tested for inhibition of DENV infection in monkey kidney epithelial (Vero) cells and human lung epithelial carcinoma (A549) cells. The results showed that vivo-MOs could bind to a DENV RNA sequence and markedly reduce DENV-RNA, protein, and virus production in infected Vero and A549 cells. Vivo-MOs at a concentration of 4 µM could inhibit DENV production by more than 10
4 -fold when compared to that of an untreated control. In addition, vivo-MOs also inhibited DENV production in U937 cells and primary human monocytes. Therefore, vivo-MOs targeting to the 3’ SL in the 3’ UTR of DENV genomes are effective and have the potential to be developed as anti-DENV agents. [ABSTRACT FROM AUTHOR]- Published
- 2018
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17. γ-COPI mediates the retention of kAE1 G701D protein in Golgi apparatus – a mechanistic explanation of distal renal tubular acidosis associated with the G701D mutation.
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Duangtum, Natapol, Junking, Mutita, Phadngam, Suratchanee, Sawasdee, Nunghathai, Castiglioni, Andrea, Charngkaew, Komgrid, Limjindaporn, Thawornchai, Isidoro, Ciro, and Yenchitsomanus, Pa-thai
- Abstract
Mutations of the solute carrier family 4 member 1 (SLC4A1) gene encoding kidney anion (chloride/bicarbonate ion) exchanger 1 (kAE1) can cause genetic distal renal tubular acidosis (dRTA). Different SLC4A1 mutations give rise to mutant kAE1 proteins with distinct defects in protein trafficking. The mutant kAE1 protein may be retained in endoplasmic reticulum (ER) or Golgi apparatus, or mis-targeted to the apical membrane, failing to display its function at the baso-lateral membrane. The ER-retained mutant kAE1 interacts with calnexin chaperone protein; disruption of this interaction permits the mutant kAE1 to reach the cell surface and display anion exchange activity. However, the mechanism of Golgi retention of mutant kAE1 G701D protein, which is otherwise functional, is still unclear. In the present study, we show that Golgi retention of kAE1 G701D is due to a stable interaction with the Golgi-resident protein, coat protein complex I (COPI), that plays a role in retrograde vesicular trafficking and Golgi-based quality control. The interaction and co-localization of kAE1 G701D with the γ-COPI subunit were demonstrated in human embryonic kidney (HEK-293T) cells by co-immunoprecipitation and immunofluorescence staining. Small interference RNA (siRNA) silencing of COPI expression in the transfected HEK-293T cells increased the cell surface expression of transgenic kAE1 G701D, as shown by immunofluorescence staining. Our data unveil the molecular mechanism of Golgi retention of kAE1 G701D and suggest that disruption of the COPI-kAE1 G701D interaction could be a therapeutic strategy to treat dRTA caused by this mutant. [ABSTRACT FROM AUTHOR]
- Published
- 2017
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18. A Peptide Inhibitor Derived from the Conserved Ectodomain Region of DENV Membrane (M) Protein with Activity Against Dengue Virus Infection.
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Panya, Aussara, Sawasdee, Nunghathai, Junking, Mutita, Srisawat, Chatchawan, Choowongkomon, Kiattawee, and Yenchitsomanus, Pa ‐ thai
- Subjects
DENGUE ,THERAPEUTICS ,PEPTIDES ,VIRAL vaccines ,ANTIVIRAL agents ,MEMBRANE proteins ,VIREMIA ,ENZYME inhibitors - Abstract
Dengue virus ( DENV) infection is a public health problem worldwide; thus, the development of a vaccine and anti- DENV drugs is urgently needed. It has been observed that low levels of viremia in DENV-infected individuals are associated with mild disease outcomes; therefore, reduction of DENV load should offer therapeutic benefits. Disruption of protein-protein interactions on the surface of DENV by a peptide that mimics part of its structural protein may affect stability of the virion structure and inhibit viral entry into host cells. To test this hypothesis, we generated a novel peptide inhibitor that mimics the conserved ectodomain region of DENV membrane (M) protein, MLH40 peptide, for DENV inhibition assays. MLH40 inhibited all four serotypes of the virus ( DENV1-4) at half maximal inhibition concentration of 24-31 μ m. MLH40 at 100 μ m blocked DENV2 attachment to cells by 80%. The inhibitory activity of MLH40 against DENV was consistently observed with different cell types, including Vero, A549, and Huh7 cells. Prediction of MLH40 binding by a molecular docking program indicated that its N-terminal loop may interact with DENV envelope (E) proteins and alter their dimer conformation. Thus, MLH40 may serve as a lead-peptide inhibitor for the development of an anti- DENV drug. [ABSTRACT FROM AUTHOR]
- Published
- 2015
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19. Adaptor Protein 1A Facilitates Dengue Virus Replication.
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Yasamut, Umpa, Tongmuang, Nopprarat, Yenchitsomanus, Pa-thai, Junking, Mutita, Noisakran, Sansanee, Puttikhunt, Chunya, Chu, Justin Jang Hann, and Limjindaporn, Thawornchai
- Subjects
ADAPTOR proteins ,DENGUE viruses ,VIRAL replication ,VIRAL envelopes ,PROTEIN expression ,VIRION - Abstract
Rearrangement of membrane structure induced by dengue virus (DENV) is essential for replication, and requires host cellular machinery. Adaptor protein complex (AP)-1 is a host component, which can be recruited to components required for membrane rearrangement. Therefore, dysfunction of AP-1 may affect membrane organization, thereby decreasing replication of virus in infected cells. In the present study, AP-1-dependent traffic inhibitor inhibited DENV protein expression and virion production. We further clarified the role of AP-1A in the life cycle of DENV by RNA interference. AP-1A was not involved in DENV entry into cells. However, it facilitated DENV RNA replication. Viral RNA level was reduced significantly in Huh7 cells transfected with AP-1A small interfering RNA (siRNA) compared with control siRNA. Transfection of naked DENV viral RNA into Huh7 cells transfected with AP-1A siRNA resulted in less viral RNA and virion production than transfection into Huh7 cells transfected with control siRNA. Huh7 cells transfected with AP-1A siRNA showed greater modification of membrane structures and fewer vesicular packets compared with cells transfected with control siRNA. Therefore, AP-1A may partly control DENV-induced rearrangement of membrane structures required for viral replication. [ABSTRACT FROM AUTHOR]
- Published
- 2015
- Full Text
- View/download PDF
20. Testosterone reduces AGTR1 expression to prevent β-cell and islet apoptosis from glucotoxicity.
- Author
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Kooptiwut, Suwattanee, Hanchang, Wanthanee, Semprasert, Namoiy, Junking, Mutita, Limjindaporn, Thawornchai, and Yenchitsomanus, Pa-thai
- Subjects
TESTOSTERONE ,APOPTOSIS ,HYPOGONADISM ,TREATMENT of diabetes ,OXIDATIVE stress - Abstract
Hypogonadism in men is associated with an increased incidence of type 2 diabetes. Supplementation with testosterone has been shown to protect pancreatic ß-cell against apoptosis due to toxic substances including streptozotocin and high glucose. One of the pathological mechanisms of glucose-induced pancreatic ß-cell apoptosis is the induction of the local rennin-angiotensin-aldosterone system (RAAS). The role of testosterone in regulation of the pancreatic RAAS is still unknown. This study aims to investigate the protective action of testosterone against glucotoxicity-induced pancreatic ß-cell apoptosis via alteration of the pancreatic RAAS pathway. Rat insulinoma cell line (INS-1) cells or isolated male mouse islets were cultured in basal and high-glucose media in the presence or absence of testosterone, losartan, and angiotensin II (Ang II), then cell apoptosis, cleaved caspase 3 expression, oxidative stress, and expression of angiotensin II type 1 receptor (AGTR1) and p47
phox mRNA and protein were measured. Testosterone and losartan showed similar effects in reducing pancreatic ß-cell apoptosis. Testosterone significantly reduced expression of AGTR1 protein in INS-1 cells cultured in high-glucose medium or high-glucose medium with Ang II. Testosterone decreased the expression of AGTR1 and p47phox mRNA and protein in comparison with levels in cells cultured in high-glucose medium alone. Furthermore, testosterone attenuated superoxide production when co-cultured with high-glucose medium. In contrast, when cultured in basal glucose, supplementation of testosterone did not have any effect on cell apoptosis, oxidative stress, and expression of AGT1R and p47phox . In addition, high-glucose medium did not increase cleaved caspase 3 in AGTR1 knockdown experiments. Thus, our results indicated thattestosterone prevents pancreatic ß-cell apoptosis dueto glucotoxicity through reduction of the expression of ATGR1 and its signaling pathway. [ABSTRACT FROM AUTHOR]- Published
- 2015
- Full Text
- View/download PDF
21. Role of Adaptor Proteins and Clathrin in the Trafficking of Human Kidney Anion Exchanger 1 ( kAE1) to the Cell Surface.
- Author
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Junking, Mutita, Sawasdee, Nunghathai, Duangtum, Natapol, Cheunsuchon, Boonyarit, Limjindaporn, Thawornchai, and Yenchitsomanus, Pa‐thai
- Subjects
ADAPTOR proteins ,CLATHRIN ,ION exchange (Chemistry) ,CELL membranes ,ACID-base equilibrium ,KIDNEY tubules ,PROTEIN-protein interactions - Abstract
Kidney anion exchanger 1 ( kAE1) plays an important role in acid-base homeostasis by mediating chloride/bicarbornate (Cl
− / HCO3 − ) exchange at the basolateral membrane of α-intercalated cells in the distal nephron. Impaired intracellular trafficking of kAE1 caused by mutations of S LC4A1 encoding kAE1 results in kidney disease - distal renal tubular acidosis ( dRTA). However, it is not known how the intracellular sorting and trafficking of kAE1 from trans-Golgi network ( TGN) to the basolateral membrane occurs. Here, we studied the role of basolateral-related sorting proteins, including the mu1 subunit of adaptor protein ( AP) complexes, clathrin and protein kinase D, on kAE1 trafficking in polarized and non-polarized kidney cells. By using RNA interference, co-immunoprecipitation, yellow fluorescent protein-based protein fragment complementation assays and immunofluorescence staining, we demonstrated that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin (but not AP-1 mu1B, PKD1 or PKD2) play crucial roles in intracellular sorting and trafficking of kAE1. We also demonstrated colocalization of kAE1 and basolateral-related sorting proteins in human kidney tissues by double immunofluorescence staining. These findings indicate that AP-1 mu1A, AP-3 mu1, AP-4 mu1 and clathrin are required for kAE1 sorting and trafficking from TGN to the basolateral membrane of acid-secreting α-intercalated cells. [ABSTRACT FROM AUTHOR]- Published
- 2014
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22. Suppression of galectin-3 expression enhances apoptosis and chemosensitivity in liver fluke-associated cholangiocarcinoma.
- Author
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Wongkham, Sopit, Junking, Mutita, Wongkham, Chaisiri, Sripa, Banchob, Chur-in, Siri, and Araki, Norie
- Abstract
Cholangiocarcinoma (CCA) is a fatal disease with high resistance to anticancer drugs. This is probably in part due to enhanced resistance to apoptosis. We have previously shown that galectin-3 (Gal-3), a β-galactoside-binding lectin, is highly expressed in CCA tissues. In this study, we demonstrated further that Gal-3 plays a direct role in anti-apoptosis regardless of the apoptotic insults. The anti-apoptotic activity and chemoresistance of CCA cells were related to Gal-3 expression level. Suppression of Gal-3 expression with siRNA stimulated apoptosis. siGal-3-K626 transiently depleted Gal-3 expression to the baseline and dramatically induced apoptosis, while siGal-3-K402 suppressed Gal-3 expression by 50% and provoked cell apoptosis, but only under apoptotic insults (hypoxic conditions or short UV radiation). These actions were reversed in Gal-3 overexpressing CCA cells. The correlation between the degree of anti-apoptotic activity and the level of endogenous Gal-3 was demonstrated. Suppression of Gal-3 expression in CCA cells with siGal-3-K402 significantly enhanced apoptosis induced by cisplatin or 5-fluorouracil by approximately 10 times, whereas overexpression of Gal-3 led to an increased resistance to drugs. In summary, the present study showed that the cellular level of Gal-3 might contribute to the anti-apoptotic activity and chemoresistance of CCA cells. Hence, Gal-3 expression level in cancer cells or tissues may be a marker for predicting chemotherapeutic response, and Gal-3 may be a specific gene-targeting therapy option for treating CCA. ( Cancer Sci 2009; 00: 000–000) [ABSTRACT FROM AUTHOR]
- Published
- 2009
- Full Text
- View/download PDF
23. Estradiol Prevents High Glucose-Induced β-cell Apoptosis by Decreased BTG2 Expression.
- Author
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Kooptiwut, Suwattanee, Kaewin, Suchada, Semprasert, Namoiy, Sujjitjoon, Jatuporn, Junking, Mutita, Suksri, Kanchana, and Yenchitsomanus, Pa-thai
- Abstract
Hyperglycemia stimulates several pathways to induce pancreatic β-cell apoptosis. In our previous study by mRNA analysis, we demonstrated that B-cell translocation gene 2 (BTG
2 ) expression was up-regulated in INS-1 cells cultured under high glucose conditions, but this effect was reversed by estrogen. In the present study, we demonstrated that BTG2 mRNA and protein expressions in both INS-1 cells and mouse pancreatic islets increased under high glucose conditions compared to those cultured under basal glucose conditions, while in the presence of estrogen, the BTG2 mRNA and protein expressions decreased. SiRNA-BTG2 significantly reduced cell apoptosis, cleaved-caspase 3, and Bax, compared to the siRNA-control in INS-1 cultured under high glucose conditions. We further demonstrated that BTG2 promoter activity was activated under high glucose conditions whereas estrogen significantly reduced it. The effects of estrogen on BTG2 expression were inhibited by estrogen receptor inhibitors. Also, under high glucose conditions, p53 and Bax mRNA and protein expressions increased, but they decreased in the presence of estrogen. Again, the effect of estrogen on p53 and Bax expression was inhibited by estrogen receptor inhibitors. Taken together, this study demonstrates that estrogen reduces pancreatic β-cell apoptosis under high glucose conditions via suppression of BTG2, p53, and Bax expressions. [ABSTRACT FROM AUTHOR]- Published
- 2018
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24. Enhanced cytotoxic activity of effector T-cells against cholangiocarcinoma by dendritic cells pulsed with pooled mRNA.
- Author
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Junking, Mutita, Grainok, Janya, Thepmalee, Chutamas, Wongkham, Sopit, and Yenchitsomanus, Pa-Thai
- Subjects
IMMUNOTHERAPY ,CHOLANGIOCARCINOMA ,T cells ,DENDRITIC cells ,TUMOR antigens ,THERAPEUTICS - Abstract
Cholangiocarcinoma is a malignancy of bile duct epithelia with an increasing in incidence rate worldwide. Surgery is the only curative treatment, while adjuvant chemotherapy and radiotherapy render poor responses. Cell-based immunotherapy is a potential strategy for cholangiocarcinoma treatment. However, variation of tumor antigens in cholangiocarcinoma leads to the ineffectiveness of cell-based immunotherapy. In this study, we examined the activation of effector T-cells by dendritic cells pulsed with protein lysate or total RNA from cholangiocarcinoma cell lines for their cytolytic activity against cholangiocarcinoma. Broad-spectrum antigen types with respect to RNA antigen sources were obtained from combination of three cholangiocarcinoma cell lines (KKU-213, KKU-100, and KKU-055). Compared with protein lysate–pulsed dendritic cells, total RNA–pulsed dendritic cells induced anti-tumor effector T-cell response with higher killing ability to KKU-100 and KKU-213 cells compared with protein lysate–pulsed dendritic cells. Moreover, pooled messenger RNA from three cholangiocarcinoma cell lines significantly increased the specific killing capacity of activated lymphocytes against KKU-213 cells. These results suggest that activation of anti-tumor effector T-cells against cholangiocarcinoma by RNA-pulsed dendritic cells is more effective than that by protein lysate–pulsed dendritic cells. In addition, pulsing dendritic cells with pooled messenger RNA from multiple cell lines enhanced the efficacy of a cellular immune response against cholangiocarcinoma. [ABSTRACT FROM AUTHOR]
- Published
- 2017
- Full Text
- View/download PDF
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