Yuan-Wu Chen

Triptolide exerts anti-tumor effect on oral cancer and KB cells in vitro and in vivo

Triptolide (TPL), a diterpenoid triepoxide purified from the Chinese herb Tripterygium wilfordii Hook F, has been reported to potentiate the anti-tumor effect in various cancer cells. However, the effect of TPL on oral cancers is not yet evaluated. Herein we first demonstrate that TPL induces prominent growth inhibition and apoptosis in two oral cancer cell lines, SCC25 and OEC-M1 and in KB cells. Our results indicate that TPL induces a dose-dependent apoptosis of these cells at nanomolar concentration. Apoptosis signalings are both activated through time upon TPL treatment detected by elevated caspase-3, 8, 9 activities. In xenograft tumor mouse model, TPL injection successfully inhibits the tumor growth via apoptosis induction which was demonstrated by TUNEL assay. These results demonstrate that TPL exerts anti-tumor effect on oral cancer and KB cells and suggest further the potential of TPL combining with other chemotherapeutic agents or radiotherapy for advanced oral cancer.

Melatonin prolongs islet graft survival in diabetic NOD mice

Abstract:  Islet transplantation has been established as a potential therapy for type 1 diabetes. However, inflammation, allorejection, and on‐going autoimmune damage contribute to early graft loss and failure of islet transplantation. Melatonin is the major secretory product of the pineal gland during the dark period of each day and displays multifunctional properties including the regulation of circadian and seasonal rhythms, antioxidation reactions and immune modulation. Based on the immunosuppressive properties of melatonin, we investigated whether melatonin treatment prolonged the survival of islet grafts in non‐obese diabetic (NOD) mice. The mean islet graft survival time was 7.33 ± 1.51 and 7.75 ± 2.66 days in untreated controls and in the solvent‐treated animals, respectively. Strikingly, the mean survival time of islet grafts in recipients treated with melatonin (200 mg/kg/bw) was 17 ± 7.76 days. Moreover, melatonin treatment reduced the proliferation of splenocytes in NOD mice. Using a T1 and T2 double transgenic mouse model, we found that T helper 1 (Th1) cells in mice treated with melatonin were significantly decreased. The reduction of Th1 cells and T cell proliferation may result from an increase in the immunosuppressive cytokine IL‐10. Our results indicate that melatonin treatment suppresses autoimmune recurrence by inhibiting the proliferation of Th1 cells in NOD mice and thus prolongs the survival of syngeneic islet grafts.

MMP-9 mRNA as a Therapeutic Marker in Acute and Chronic Stages of Arthritis Induced by Type II Collagen Antibody

BACKGROUND/PURPOSE Antibodies against type II collagen (anti-CII) are arthritogenic and central to the initiation of the disease. An animal model of collagen type II-specific monoclonal antibody-induced arthritis (CAIA) has been used for the evaluation of various therapeutic effects in rheumatoid arthritis (RA). We aimed to measure the expression of matrix metalloproteinase (MMP)-9 (gelatinase B), tumor necrosis factor-alpha (TNF-alpha) and interleukin-1beta (IL-1beta) in the acute and chronic stages of the CAIA model for application as a therapeutic marker. METHODS A commercially available antibody cocktail containing four monoclonal anti-type II collagen antibodies were injected into 6 to 8-week-old male BALB/c mice (n=20) and 50 microL lipopolysaccharide was injected 3 days later. The clinical manifestations of RA were recorded and scored at 10 days (acute stage) and 21 days (chronic stage). Then the mice were sacrificed for histologic analysis of the inflamed footpad and gene expression of IL-1beta, TNF-alpha and MMP-9 by ELISA and quantitative polymerase chain reaction amplification. RESULTS Marked inflammation was found in the limb joints of mice at 10 days. Both IL-1beta and MMP-9 expression played a central role in the inflammatory reaction in the acute stage. The expression level of MMP-9 mRNA remained high in the chronic stage of CAIA, but that of IL-1beta mRNA was unexpectedly negligible; the serum level of TNF-alpha in CAIA was undetectable in the acute stage. The expression level of TNF-alpha mRNA was also lower than IL-1beta and MMP-9 in the acute inflammatory stage. CONCLUSION The CAIA model is a fast and highly replicable model of RA. MMP-9 and IL-1beta were highly expressed in the acute stage of CAIA. It is suggested the MMP-9 mRNA level is a suitable marker for both acute and chronic stage, whereas IL-1beta is a marker only for the acute stage of the CAIA murine model.

Triptolide Transcriptionally Represses HER2 in Ovarian Cancer Cells by Targeting NF-κB

Triptolide (TPL) inhibits the proliferation of a variety of cancer cells and has been proposed as an effective anticancer agent. In this study, we demonstrate that TPL downregulates HER2 protein expression in oral, ovarian, and breast cancer cells. It suppresses HER2 protein expression in a dose- and time-dependent manner. Transrepression of HER2 promoter activity by TPL is also observed. The interacting site of TPL on the HER2 promoter region is located between −207 and −103 bps, which includes a putative binding site for the transcription factor NF-κB. Previous reports demonstrated that TPL suppresses NF-κB expression. We demonstrate that overexpression of NF-κB rescues TPL-mediated suppression of HER2 promoter activity and protein expression in NIH3T3 cells and ovarian cancer cells, respectively. In addition, TPL downregulates the activated (phosphorylated) forms of HER2, phosphoinositide-3 kinase (PI3K), and serine/threonine-specific protein kinase (Akt). TPL also inhibits tumor growth in a mouse model. Furthermore, TPL suppresses HER2 and Ki-67 expression in xenografted tumors based on an immunohistochemistry (IHC) assay. These findings suggest that TPL transrepresses HER2 and suppresses the downstream PI3K/Akt-signaling pathway. Our study reveals that TPL can inhibit tumor growth and thereby may serve as a potential chemotherapeutic agent.

Triptolide ameliorates autoimmune diabetes and prolongs islet graft survival in nonobese diabetic mice.

Objectives Triptolide (TPL) possesses profound immunosuppressive effects and has potential in allograft transplantation. We investigated whether TPL treatment prevents autoimmune diabetes in nonobese diabetic (NOD) mice and prolongs the survival of islet grafts against autoimmune attack or allograft rejection. Methods Diabetic incidence was monitored in TPL-treated NOD mice. Nonobese diabetic or BALB/c islets were transplanted into diabetic recipients treated with TPL. Different T-cell subsets in grafts or spleen were analyzed. The proliferation, apoptosis, cytokines, and activities of AKT, NF&kgr;B, and caspases 3, 8, and 9 of T cells were determined. Results Diabetic incidence was reduced and inflammatory cytokines were decreased in islets and spleen under TPL treatment. T-cell proliferation was reduced and the survival of syngeneic or allogeneic grafts was significantly increased in TPL-treated mice. The populations of CD4, CD8, CD4CD69, CD8CD69, and interferon-&ggr;–producing T cells in islet grafts and spleen were reduced. Triptolide treatment increased the apoptosis of T cells in the spleen of recipients. Levels of phosphorylated protein kinase B and phosphorylated inhibitor of kappa B in splenocytes were reduced and caspases 3, 8, and 9 were increased in TPL-treated mice. Conclusions Triptolide treatment not only reduced the diabetic incidence in NOD mice but also prolonged the survival of syngeneic or allogeneic grafts.

Enhanced Anti-Tumor Activity of Triptolide in Combination with Irradiation for the Treatment of Oral Cancer

Advanced oral cancer has a poor prognosis because of the lack of an effective treatment. We explored the efficiency of combined treatment with triptolide and ionizing radiation for treating oral cancer. Human tongue cancer cells were treated with triptolide, ionizing radiation, or triptolide plus ionizing radiation. Cell proliferation, cell cycle arrest, and apoptotic influences were analyzed by FACS and immunohistochemistry. Tumor potency was examined in an in vivo human tongue cancer cells xenograft mouse model. Our results demonstrated that triptolide caused a marked reduction in colony number that was further enhanced with increasing doses of ionizing radiation. Triptolide increased apoptosis and decreased the expression of anti-apoptotic proteins. In vivo, combination treatment synergistically reduced tumor weight and volume possibly via the induction of apoptosis and reduction in anti-apoptotic protein expression. In conclusion, triptolide plus ionizing radiation treatment had synergistic anti-tumor effects, especially in vivo, and may be a promising combined modality therapy for advanced oral cancer.

IL-1β in Irrigation Fluid and MRNA Expression in Synovial Tissue of the Knee Joint as Therapeutic Markers of Inflammation in Collagen Antibody-Induced Arthritis

Objective: We studied the relationship between the severity of inflammation and IL-1β production and relative expression level of IL-1β mRNA in irrigation fluid and synovial tissue obtained from the knee joint during the acute stage of a murine model of type II collagen antibody-induced arthritis (CAIA). This model is used to identify potential therapeutic markers for treating rheumatoid arthritis. Methods: Irrigation fluid and synovium tissue were harvested from the knee joint of BALB/c mice in acute stage of CAIA induction. The IL-1β protein level was measured by enzyme-linked immunosorbent assay, and the relative expression level of IL-1β mRNA was analyzed by reverse transcription-polymerase chain reaction. Two investigators analyzed expression levels and histopathological changes. Results: IL-1β concentration was higher in irrigation fluid from the knee joint than in the serum in the acute stage of CAIA. The relative expression level of IL-1β mRNA was elevated in synovial tissue. Histopathological changes in the knee joint and foot indicated similar severity. Conclusions: IL-1β concentration in irrigation fluid and relative expression level of IL-1β mRNA in the synovium have potential as therapeutic markers in studying and treating CAIA.

Triptolide represses oral cancer cell proliferation, invasion, migration, and angiogenesis in co-inoculation with U937 cells

ObjectivesAdvanced oral cancer is a major public health concern because of a lack of effective prevention and treatment. Triptolide (TPL), a diterpenoid triepoxide derived from the Chinese herb Tripterygium wilfordii, has been demonstrated to possess strong anticancer properties. In this study, we investigated whether TPL exerts anticancer effects on the tumor microenvironment of head and neck squamous cell carcinoma (HNSCC).Materials and methodsHuman macrophage-like U937 cells were co-inoculated with oral cancer SAS cells in a noncontact transwell coculture system. Cytokine expression was detected using ELISA, and cell proliferation was detected using methylene blue. RNA levels were detected using qPCR. Protein levels were detected using Western blot analysis. In vivo experiments involved using xenografted NOD/SCID mice.ResultsOur results demonstrated that TPL inhibited the growth of SAS cells co-inoculated with U937 cells in vitro and in vivo. TPL inhibited the invasion, migration ability, and angiogenesis of SAS cells co-inoculated with U937 cells. Expression of cytokines IL-6, IL-8, and TNF-α was induced by co-inoculation, but TPL repressed their expression.ConclusionTPL suppressed the expression of cytokines IL-6, IL-8, and TNF-α, as well as tumor growth, invasion, migration, and angiogenesis in the co-inoculation of human tongue cancer cells with macrophage-like U937 cells.Clinical relevanceTPL is a potential candidate among novel chemotherapeutic agents or adjuvants for modulating tumor-associated macrophages in a tumor microenvironment of HNSCC.

Daxx and TCF4 interaction links to oral squamous cell carcinoma growth by promoting cell cycle progression via induction of cyclin D1 expression

ObjectivesDeath domain-associated protein (Daxx) has been recently implicated as a positive factor in ovarian cancer and prostate cancer, but the role of Daxx in oral squamous cell carcinoma (OSCC) has never been addressed. Herein, we investigate the expression and function of Daxx in OSCC.Materials and methodsRT-quantitative PCR, Western blotting, and immunohistochemistry were used to evaluation of the expression of Daxx in human OSCC cell lines and clinical surgical specimens. Short hairpin RNA targeting Daxx was transduced by lentivirus infection to knockdown the expression of Daxx in SAS and SCC25 cell lines, and the influence of this knockdown was evaluated by analyzing the growth and the cell cycle in transduced cells. Immunoprecipitation and sequential chromatin immunoprecipitation-quantitative PCR were used to analyze the associations between Daxx, TCF4, and cyclin D1 promoter. Xenograft tumor model was used to evaluate the in vivo tumorigenicity of Daxx in OSCC.ResultsDaxx mRNA and protein expression are elevated in several OSCC cell lines and human OSCC samples in comparison to those in normal tissue. We further find that depletion of Daxx decreases OSCC cell growth activity through G1 cell cycle arrest. Daxx silencing reduces cyclin D1 expression via a Daxx-TCF4 interaction, whereas the Daxx depletion-mediated G1 arrest can be relieved by ectopic expression of cyclin D1. Moreover, we show that in OSCC clinical samples, the expression of Daxx is significantly correlated with that of cyclin D1.ConclusionOur data demonstrate the importance of Daxx in regulation of cyclin D1 expression and provide the first evidence that Daxx exhibits tumor-promoting activity in OSCC.Clinical relevanceDaxx plays an important role in malignant transformation of OSCC and may serves as a target for cancer prevention and treatment.

Dimethyl sulfoxide inhibits spontaneous diabetes and autoimmune recurrence in non-obese diabetic mice by inducing differentiation of regulatory T cells.

Type 1 diabetes mellitus (T1D) is caused by the destruction of insulin-producing β cells in pancreatic islets by autoimmune T cells. Islet transplantation has been established as an effective therapeutic strategy for T1D. However, the survival of islet grafts can be disrupted by recurrent autoimmunity. Dimethyl sulfoxide (DMSO) is a solvent for organic and inorganic substances and an organ-conserving agent used in solid organ transplantations. DMSO also exerts anti-inflammatory, reactive oxygen species scavenger and immunomodulatory effects and therefore exhibits therapeutic potential for the treatment of several human inflammatory diseases. In this study, we investigated the therapeutic potential of DMSO in the inhibition of autoimmunity. We treated an animal model of islet transplantation (NOD mice) with DMSO. The survival of the syngeneic islet grafts was significantly prolonged. The population numbers of CD8, DC and Th1 cells were decreased, and regulatory T (Treg) cell numbers were increased in recipients. The expression levels of IFN-γ and proliferation of T cells were also reduced following DMSO treatment. Furthermore, the differentiation of Treg cells from naive CD4 T cells was significantly increased in the in vitro study. Our results demonstrate for the first time that in vivo DMSO treatment suppresses spontaneous diabetes and autoimmune recurrence in NOD mice by inhibiting the Th1 immune response and inducing the differentiation of Treg cells.