Objective:To explore the key molecules regulated by norcantharidin(NCTD)in colon cancer treatment.Methods:We used cell counting kit-8 and 5-ethnyl-20-deoxyuridine/Hoechst staining assays to study the effects of NCTD o...Objective:To explore the key molecules regulated by norcantharidin(NCTD)in colon cancer treatment.Methods:We used cell counting kit-8 and 5-ethnyl-20-deoxyuridine/Hoechst staining assays to study the effects of NCTD on cell proliferation in colon cancer.Annexin V-fluorescein isothiocyanate/propidium iodide staining was used to evaluate apoptosis,whereas Transwell assays were conducted to evaluate migration and invasion.We performed RNA sequencing to analyze the changes in gene expression after treatment.Differential analysis was performed using differential expression sequencing 2(Deseq2)in R.Cytoscape was used to construct a competing endogenous RNA(ceRNA)network and Gene Expression Omnibus(GEO)datasets were used to validate sine oculis homeobox homolog 4(SIX4)expression in colon cancer tissues.Furthermore,the prognostic potential of SIX4 was evaluated using receiver-oper-ating characteristic curves.We conducted an immune infiltration analysis to explore the SIX4 relation-ship with the immune microenvironment in colon cancer.Finally,SIX4 expression,pan-cancer prognosis,tumor mutation burden(TMB)correlations,microsatellite instability(MSI),and mismatch repair(MMR)were analyzed.Results:NCTD inhibited colon cancer cell proliferation(P<0.0001),induced apoptosis(P=0.0007),and suppressed the migration and invasion of colon cancer cells.The H19/miR-193b-3p/SIX4 axis was identified as the key ceRNA network involved in the anticancer activity of NCTD.SIX4 is highly expressed in colon cancer tissues,shortening patient survival and affecting immune infiltration.A pan-cancer analysis showed that SIX4 overexpression affects the survival of various cancers.Finally,we correlated SIX4 expression with TMB,MSI,and MMR expression.Conclusion:NCTD inhibits the malignant behaviour of colon cancer cells.SIX4 is abnormally expressed in multiple tumor types,significantly affecting the overall survival of patients with cancer,and is a core regulatory target of NCTD in the treatment of colon cancer.展开更多
Aim To design and synthesize norcantharidin disodium phosphate derivatives. Methotis Diels-Alder reaction between furan and maleic anhydride afforded dehydronorcantharidin, and subsequent hydrogenation, phosphorylatio...Aim To design and synthesize norcantharidin disodium phosphate derivatives. Methotis Diels-Alder reaction between furan and maleic anhydride afforded dehydronorcantharidin, and subsequent hydrogenation, phosphorylation, and basification gave compounds 1 and 2, separately. Resuits The structures of compounds were confirmed by IR, NMR and FAB-MS. The aqueous solubility of 1 and 2 were improved, compared with the parent compounds, and their activities were more potent than norcantharidin 4 against MGCS03 cell lines. Conclusion The phosphorylation of norcantharidin analogues is an effective way to increase the activity and solubility.展开更多
INTRODUCTIONThe antitumor activity of norcantharidin (NCTD),the demethylated analogue of cantharidin,wasstudied in the early 1980s in China.NCTD has noside effects on urinary organs which cantharidin hasshown and is e...INTRODUCTIONThe antitumor activity of norcantharidin (NCTD),the demethylated analogue of cantharidin,wasstudied in the early 1980s in China.NCTD has noside effects on urinary organs which cantharidin hasshown and is easier to synthesize,and it can inhibitthe proliferation of several tumor cell lines as wellas transplanted tumors.Clinical trials with NCTD asa monotherapeutic agent indicated that NCTD hadbeneficial effects in patients with different kinds展开更多
BACKGROUND: Gallbladder carcinoma is a lethal malignant neoplasm with dismal surgical results. Unfortunately, the adjuvant therapies for gallbladder carcinoma such as chemotherapy and radiotherapy are also disappointi...BACKGROUND: Gallbladder carcinoma is a lethal malignant neoplasm with dismal surgical results. Unfortunately, the adjuvant therapies for gallbladder carcinoma such as chemotherapy and radiotherapy are also disappointing. We reported that norcantharidin (NCTD), a demethylated form of cantharidin, which is an active ingredient of the Chinese medicine Mylabris, was used against human gallbladder carcinoma GBC-SD cells. In the present study, we further studied the mechanism underlying the inhibitory effect of NCTD on growth of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: Human gallbladder carcinoma GBC-SD cells were grown in cell culture and divided into a NCTD group and a control group. The inhibitory effect of NCTD on growth of GBC-SD cells was investigated by evaluation of proliferation, cell cycle, apoptosis and morphological changes of the cells. Cell proliferation was assessed by tetrazolium-based colorimetric assay. The induction of cell cycle arrest and apoptosis was measured by flow cytometry. The morphological changes of the cells were observed by light- and electron-microscopy. To elucidate the anticancer mechanism of NCTD, expression of the proliferation-related gene proteins PCNA, Ki-67, cyclin-D-1 and p27 and the apoptosis-related gene proteins Bcl-2, Bax and Survivin were determined by the streptavidin-biotin complex method and RT-PCR. RESULTS: NCTD inhibited the proliferation of GBCSD cells in a dose- and time-dependent manner, with an IC50 of 56.18 mu g/ml at 48 hours. The flow cytometric profiles revealed that NCTD (at the IC50 for 48 hours) significantly increased the proportion of cells in G(2)/M phase and significantly decreased the proportion of cells in S phase, with a significantly increased rate of cell apoptosis. After treatment with the 48-hour IC50 dose of NCTD, cell shrinkage, vacuolar cytoplasm, membrane budding, karyorrhexis, karyolysis, chromosome condensation and chromatin aggregation in some GBCSD cells were observed by light-microscopy; decreased microvilli, Golgiosome atrophy, mitochondrial swelling, nuclear shrinkage, chromosome condensation and typical apoptosis bodies were seen by electron-microscopy, and the morphological changes of apoptosis occurred in GBCSD cells. The expression of PCNA, Ki-67 and Bcl-2 proteins decreased significantly; the Pix or relative levels of PCNA mRNA, cyclin-D-1 mRNA, Bcl-2 mRNA and Survivin mRNA decreased significantly, whereas the Pix or relative levels of p27 mRNA and Bax mRNA increased significantly. CONCLUSIONS: NCTD inhibits the growth of human gallbladder carcinoma GBC-SD cells in vitro. Its anticancer mechanism may correlate with inhibition of cell proliferation, arrest of the cell cycle, blockage of DNA synthesis, influence on cell metabolism, induction of cell apoptosis and influence on expression of the proliferation-related genes PCNA, Ki-67, cyclin-D-1 and p27, and the apoptosis-related genes Bcl-2, Bax and Survivin in human gallbladder carcinoma GBC-SD cells.展开更多
BACKGROUND: Gallbladder carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. We previously reported that norcantharidin (NCTD) is useful against g...BACKGROUND: Gallbladder carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. We previously reported that norcantharidin (NCTD) is useful against growth, proliferation, and invasion of human gallbladder carcinoma GBC-SD cells in vitro. In this study, we further studied the inhibitory effect of NCTD on the growth of xenografted tumors of human gallbladder carcinoma in nude mice in vivo and the underlying mechanisms. METHODS: The tumor xenograft model of human gallbladder carcinoma in nude mice in vivo was established with subcutaneous GBC-SD cells. The experimental mice were randomly divided into control, 5-FU, NCTD, and NCTD+5-FU groups which were given different treatments. Tumor growth in terms of size, growth curve, and inhibitory rate was evaluated. Cell cycle, apoptosis, and morphological changes of the xenografted tumors were assessed by flow cytometry and light/electron microscopy. The expression of the cell cycle-related proteins cyclin-D1 and p27 as well as the apoptosis-related proteins Bcl-2, Box, and survivin were determined by the streptavidin-biotin complex (SABC) method and RT-PCR. RESULTS: NCTD inhibited the growth of the xenografted tumors in a dose- and time-dependent manner. Tumor volume decreased (5.61+/-0.39 vs. 9.78+/-0.61 cm(3), P=0.000) with an increased tumor inhibitory rate (42.63% vs. 0%, P=0.012) in the NTCD group compared with the control group. The apoptosis rate increased (15.08+/-1.49% vs. 5.49+/-0.59%, P=0.0001) along with a decreased percentage of cells in S phase (43.47+/-2.83% vs. 69.85+/-1.96%, P=0.0001) in the NTCD group compared with the control group. The morphological changes of apoptosis such as nuclear shrinkage, chromatin aggregation, chromosome condensation, and typical apoptosis bodies in the xenografted tumor cells induced by NCTD were observed by light and electron microscopy. The expression of cyclin-D1, Bcl-2 and survivin proteins/mRNAs decreased significantly, with increased expression of p27 and Bax proteins/mRNAs in the NCTD group compared with the control group. CONCLUSION: NCTD inhibits the growth of xenografted tumors of human gallbladder carcinoma in nude mice by inducing apoptosis and blocking the cell cycle in vivo.展开更多
AIM: To study the therapeutic effect of norcantharidin(NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. METHODS: Two hepatocellular carcinoma(HCC) cell lines, Hep G2 and SMMC-...AIM: To study the therapeutic effect of norcantharidin(NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. METHODS: Two hepatocellular carcinoma(HCC) cell lines, Hep G2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. Hep G2 and SMMC-7721 cells were cultured in vitro. Liver cancer cells in the logarithmic phase of growth were vaccinated and cultured to the cell wall stage; these cells were treated for 48 h with different concentrations of NCTD, or ABT-737, or NCTD combined with ABT-737. The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium. The expression of Mcl in HCC cells was detected by Western Blotting, and the cells in each group after treatment had apoptosis detected by flow cytometry. The proliferation inhibition rate, the expression of Mcl-1 in cells and the apoptosis inducing effect of treatment were observed in each group, and the effect of NCTD on ABT-737 in the treatment of HCC and its mechanism of action were analyzed.RESULTS: As the concentration of NCTD increased, the cell proliferation inhibition rate gradually decreased; and the treatment effect of ABT-737 1-3 μm combined with NCTD on cell proliferation inhibition was stronger than that of ABT-737 alone. The difference was statistically significant(P < 0.05). In observing the expression of Mcl-1 in cells after the treatment of different concentrations of NCTD, this was partiallyinhibited after treatment with NCTD 15 μm, and the expression of Mcl-1 was almost undetectable after treatment with NCTD 30 μm and 60 μm. The effect on inducing apoptosis with the treatment of ABT-737 or NCTD alone for 48 h was lower than that of the control group. The difference was not statistically significant(P > 0.05). The effect on inducing apoptosis in Hep G2 and SMMC-7721 cells with the treatment of ABT-737 combined with NCTD for 48 h was greater than that of ABT-737 or NCTD alone. The difference was statistically significant(P < 0.05). CONCLUSION: NCTD combined with ABT-737 has a positive role in the treatment of HCC, and it has great value in clinical research.展开更多
AIM: To investigate the effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells in vitro and its anticancer mechanism. METHODS: Human gallbladder carcinoma GBC-SD cells were ...AIM: To investigate the effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells in vitro and its anticancer mechanism. METHODS: Human gallbladder carcinoma GBC-SD cells were cultured by cell culture technique. The growth and the invasiveness of GBC-SD cells in vitro were evaluated by the tetrazolium-based colorimetric assay and by the Matrigel experiment and the crossing-river test. Expression of PCNA, Ki-67, MMP2 and TIMP2 proteins of GBC-SD cells was determined by streptavidin-biotin complex method. RESULTS: In vitro norcantharidin inhibited the growth and proliferation of GBC-SD cells in a dose- and time-dependent manner, with the IC50 value of 56.18 μ/mL at 48 h. Norcantharidin began to inhibit the invasion of GBC-SD cells at the concentration of 5 μg/mL, and the invasive action of GBC-SD cells was inhibited completely and their crossing-river time was prolonged significantly at 40 μg/mL. After treatment with norcantharidin, the expression of PCNA, Ki-67, and MMP2 was significantly decreased. With the increase in TIMP2 expression, the MMP2 to TIMP2 ratio was decreased significantly (P<0.05). CONCLUSION: Norcantharidin inhibits the proliferation and growth of human gallbladder carcinoma cells in vitro at relatively low concentrations by inhibiting PCNA and Ki-67 expression. Its anti-invasive activity may be the result of decrease in MMP2 to TIMP2 ratio and reduced cell motility.展开更多
BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and sh...BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and should be consi- dered for palliative treatment such as chemotherapy and ra- diotherapy. Unfortunately, reports of chemotherapy and radiotherapy for gallbladder carcinoma are disappointing. We investigated the influence of norcantharidin (NCTD) on proliferation, proliferation-related gene proteins PCNA and Ki-67 of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: GBC-SD cell lines of human gallbladder carci- noma were cultured by the cell culture technique. The ex- periment was divided into NCTD group and control group. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The streptavidin-biotin complex method was used to determine the expressions of prolifera- tion-related gene proteins PCNA and Ki-67 of human gall- bladder carcinoma GBC-SD cells. RESULTS: NCTD inhibited the growth and proliferation of GBC-SD cells from 10 mg/L or after 6 hours in a dose- and time-dependent manner, with the IC50 value of 56.18 μg/ ml at 48 hours. After treatment with NCTD, the expression of PCNA (0.932 ±0.031 vs. 0.318 ±0.023, P<0.001) and Ki-67 (0.964 ±0.092 vs. 0.297 ±0.018, P<0.001) proteins were decreased significantly. CONCLUSION: NCTD inhibits the proliferation of human gallbladder carcinoma GBC-SD cells in vitro and the expres- sion of their proliferation-related gene proteins PCNA and Ki-67.展开更多
The Two-dimensional Quantitative Structure-activity Relationship （2D-QSAR） of a series of novel norcantharidin analogues, which exhibit hnhibitory activities of protein phosphatase 1 and 2A （PP1 and PP2A）, has bee...The Two-dimensional Quantitative Structure-activity Relationship （2D-QSAR） of a series of novel norcantharidin analogues, which exhibit hnhibitory activities of protein phosphatase 1 and 2A （PP1 and PP2A）, has been studied with a combined method of ab initio （I/F）, molecular mechanics （MM＋） and statistics. The established 2D-QSAR model （Eq. 1） for PP1 shows a reasonable regressive performance （R2= 0.749）, and the hydrophobic property of this molecule plays a decisive role in determining the inhibitory activity of PP1. In addition, the established 2D-QSAR model （Eq. 2） for PP2A also shows an acceptable regressive performance （R2= 0.701）, and the dipole moment of the molecule determines the inhibitory activity of PP2A.展开更多
BACKGROUND Norcantharidin(NCTD)is suitable for the treatment of primary liver cancer,especially early and middle primary liver cancer.This compound can reduce tumors and improve immune function.However,the side effect...BACKGROUND Norcantharidin(NCTD)is suitable for the treatment of primary liver cancer,especially early and middle primary liver cancer.This compound can reduce tumors and improve immune function.However,the side effects of NCTD have limited its application.There is a marked need to reduce the side effects and increase the efficacy of NCTD.AIM To develop a nanomaterial carrier,NCTD-loaded metal-organic framework IRMOF-3 coated with a temperature-sensitive gel(NCTD-IRMOF-3-Gel),aiming to improve the anticancer activity of NCTD and reduce the drug dose.METHODS NCTD-IRMOF-3-Gel was obtained by a coordination reaction.The apparent characteristics and in vitro release of NCTD-IRMOF-3-Gel were investigated.Cell cytotoxicity assays,flow cytometry,and apoptosis experiments in mouse hepatoma(Hepa1-6)cells were used to determine the anti-liver cancer activity of NCTD-IRMOF-3-Gel in in vitro models.RESULTS The particle size of NCTD-IRMOF-3-Gel was 50-100 nm,and the particle size distribution was uniform.The release curve showed that NCTD-IRMOF-3-Gel had an obvious sustained-release effect.The cytotoxicity assays showed that the free drug NCTD and NCTD-IRMOF-3-Gel treatments markedly inhibited Hepa1-6 cell proliferation,and the inhibition rate increased with increasing drug concentration.By flow cytometry,NCTD-IRMOF-3-Gel was observed to block the Hepa1-6 cell cycle in the S and G2/M phases,and the thermosensitive gel nanoparticles may inhibit cell proliferation by inducing cell cycle arrest.Apoptosis experiments showed that NCTD-IRMOF-3-Gel induced the apoptosis of Hepa1-6 cells.CONCLUSION Our results indicated that the NCTD-IRMOF-3-Gel may be beneficial for liver cancer disease treatment.展开更多
In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2...In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19^＋ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody （mAb）. NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19＋ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19^＋ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG2000-DSPE reached 1：50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19^＋ leukemia cells was significantly higher than that on CD19-1eukemia cells. Similarly, the targeting efficiency of the immtmoliposomes was also higher than that of the NCTDAiposomes on CD 19^＋ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-（4, 5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nahn-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.展开更多
Background:Vasculogenic mimicry(VM)is a novel tumor blood supply in some highly aggressive malignant tumors.Recently,we reported VM existed in gallbladder carcinomas(GBCs)and the formation of the special passage throu...Background:Vasculogenic mimicry(VM)is a novel tumor blood supply in some highly aggressive malignant tumors.Recently,we reported VM existed in gallbladder carcinomas(GBCs)and the formation of the special passage through the activation of the PI3K/MMPs/Ln-5γ2 signaling pathway.GBC is a highly aggressive malignant tumor with disappointing treatments and a poor prognosis.Norcantharidin(NCTD)has shown to have multiple antitumor activities against GBCs,etc;however the exact mechanism is not thoroughly elucidated.In this study,we firstly investigated the anti-VM activity of NCTD as a VM inhibitor for GBCs and its underlying mechanisms.Methods:In vitro and in vivo experiments to determine the effects of NCTD on proliferation,invasion,migration,VM formation,hemodynamic and tumor growth of GBC-SD cells and xenografts were respectively done by proliferation,invasion,migration assays,HE staining and CD31-PAS double staining,optic/electron microscopy,tumor assay,and dynamic microMRA.Further,immunohistochemistry,immunofluorescence,Western blotting and RT-PCR were respectively used to examine expression of VM signaling-related markers PI3-K,MMP-2,MT1-MMP and Ln-5γ2 in GBC-SD cells and xenografts in vitro and in vivo.Results:After treatment with NCTD,proliferation,invasion,migration of GBC-SD cells were inhibited;GBC-SD cells and xenografts were unable to form VM-like structures;tumor center-VM region of the xenografts exhibited a decreased signal in intensity;then cell or xenograft growth was inhibited.Whereas all of untreated GBC-SD cells and xenografts formed VM-like structures with the same conditions;the xenograft center-VM region exhibited a gradually increased signal;and facilitated cell or xenograft growth(Figure 1-6).Furthermore,expression of MMP-2 and MT1-MMP products from sections/supernates of 3-D matrices and the xenografts,and expression of PI3-K,MMP-2,MM1-MMP and Ln-5γ2 proteins/mRNAs of the xenografts were all decreased in NCTD or TIMP-2 group(Figure 7-10;all P<0.01,vs.control group);NCTD down-regulated expression of these VM signaling-related markers in vitro and in vivo.Conclusions:NCTD inhibited tumor growth and VM formation of human gallbladder carcinoma GBC-SD cells and xenografts by suppression of the PI3-K/MMPs/Ln-5γ2 signaling pathway.It is firstly concluded that NCTD may be a potential anti-VM agent for human GBCs.展开更多
Objective： To investigate the inhibitory activities of norcantharidin （NCTD）, a demethylated analogue of cantharidin, on Hep3B cells （a human hepatoma cell line） with deficiency of p53. Methods： The survival rat...Objective： To investigate the inhibitory activities of norcantharidin （NCTD）, a demethylated analogue of cantharidin, on Hep3B cells （a human hepatoma cell line） with deficiency of p53. Methods： The survival rate of the Hep3B cells after treating with NCTD was measured by MTT assay. Cell cycle of treated cells was analyzed by flow cytometry, and DNA fragmentation was observed by electrophoresis. The influence of inhibitors for various caspases and anti-death receptors antibodies on the NCTD-induced apoptosis in the cells was determined. Results： NCTD treatment resulted in growth inhibition of Hep3B cells in a dose- and time-dependent manner. Cell cycle analysis of the cells after treatment with NCTD for 48 h shows that NCTD induced G2M phase arrest occursat low concentration （≤25 μ mol/L） but G0G1 phase arrest at high concentration （50 μ mol/L）. The addition of both caspase-3 and caspase-10 inhibitors completely inhibited DNA fragmentation. Addition of anti-TRAIL/DR5 antibody significantly inhibited DNA fragmentation. Conclusion： NCTD may inhibit the proliferation of Hep3B cells by arresting cell cycle at G2M or G0G1 phase, and induce cells apoptosis via TRAIL/DR5 signal transduction through activation of caspase-3 and caspase-10 by a p53-independent pathway,展开更多
Objective： To observe the effect of norcantharidin（NCTD） on collagen-induced arthritis（CIA） rats. Methods： Sixty Sprague-Dawley（SD） rats were randomly divided into 6 groups（n=10）： normal group, CIA model gr...Objective： To observe the effect of norcantharidin（NCTD） on collagen-induced arthritis（CIA） rats. Methods： Sixty Sprague-Dawley（SD） rats were randomly divided into 6 groups（n=10）： normal group, CIA model group（model group）, NCTD low-dose group [1.35 mg/（kg·d）], NCTD middle-dose group [2.7 mg/（kg·d）], NCTD high-dose group [5.4 mg/（kg·d）] and methotrexate（MTX） group [1.8 mg/（kg/w）]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin（H＆E） staining. The serum levels of interleukin（IL） 1β, IL-6, tumor necrosis factor（TNF）-α, vascular endothelial growth factor（VEGF）, IL-17 and transform growth factor（TGF） β were detected by enzyme linked immunosorbent assay（ELISA）. The mRNA expression of retinoid-related orphan nuclear receptor γ t（ROR γ t） and forkhead box P3（Foxp3） in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. Results： MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats＇ ankle joints compared with the model group（P〈0.05 or P〈0.01）. All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats（P〈0.05）. Only middle-and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats（P〈0.05）. However, NCTD has no effect on vascular endothelial growth factor（VEGF） level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group（P〈0.05）. The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group（P〈0.05）. Conclusion： NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.展开更多
Objective： To investigate the mechanism of norcantharidin （NCTD）-induced SMMC-7721 hepatoma cell apoptosis. Methods： SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annex...Objective： To investigate the mechanism of norcantharidin （NCTD）-induced SMMC-7721 hepatoma cell apoptosis. Methods： SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin Ⅴ/propidium iodide staining. The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression. Results： NCTD inhibited SMMC-7721 cell growth in a time-and dose-dependent manner. The cells treated with NCTD showed the loss of mitochondrial membrane potential. The activities of caspase-3, cytochrome c, AIF, and Bax were up-regulated after NCTD treatment at different doses. The expression of Bcl-2 was decreased after treatment with NCTD. Conclusions： NCTD could induce SMMC-7721 cell apoptosis. The activation of the mitochondrial pathway was involved in the process of NCTD-induced SMMC-7721 cell apoptosis.展开更多
基金funded by the National Natural Science Foundation of China(82074284)China National Traditional Chinese Medicine Inheritance and Innovation Team Sub-project(ZYYCXTD-C-202005-10)+1 种基金China Medical Association of Minorities Research Project(2020MZ298-110101)Open Fund Project,Associated Key Laboratory of Traditional Mongolia Medicine Research and Development,National Ethnic Affairs Commission and Ministry of Education of China(MDK2020013).
文摘Objective:To explore the key molecules regulated by norcantharidin(NCTD)in colon cancer treatment.Methods:We used cell counting kit-8 and 5-ethnyl-20-deoxyuridine/Hoechst staining assays to study the effects of NCTD on cell proliferation in colon cancer.Annexin V-fluorescein isothiocyanate/propidium iodide staining was used to evaluate apoptosis,whereas Transwell assays were conducted to evaluate migration and invasion.We performed RNA sequencing to analyze the changes in gene expression after treatment.Differential analysis was performed using differential expression sequencing 2(Deseq2)in R.Cytoscape was used to construct a competing endogenous RNA(ceRNA)network and Gene Expression Omnibus(GEO)datasets were used to validate sine oculis homeobox homolog 4(SIX4)expression in colon cancer tissues.Furthermore,the prognostic potential of SIX4 was evaluated using receiver-oper-ating characteristic curves.We conducted an immune infiltration analysis to explore the SIX4 relation-ship with the immune microenvironment in colon cancer.Finally,SIX4 expression,pan-cancer prognosis,tumor mutation burden(TMB)correlations,microsatellite instability(MSI),and mismatch repair(MMR)were analyzed.Results:NCTD inhibited colon cancer cell proliferation(P<0.0001),induced apoptosis(P=0.0007),and suppressed the migration and invasion of colon cancer cells.The H19/miR-193b-3p/SIX4 axis was identified as the key ceRNA network involved in the anticancer activity of NCTD.SIX4 is highly expressed in colon cancer tissues,shortening patient survival and affecting immune infiltration.A pan-cancer analysis showed that SIX4 overexpression affects the survival of various cancers.Finally,we correlated SIX4 expression with TMB,MSI,and MMR expression.Conclusion:NCTD inhibits the malignant behaviour of colon cancer cells.SIX4 is abnormally expressed in multiple tumor types,significantly affecting the overall survival of patients with cancer,and is a core regulatory target of NCTD in the treatment of colon cancer.
文摘Aim To design and synthesize norcantharidin disodium phosphate derivatives. Methotis Diels-Alder reaction between furan and maleic anhydride afforded dehydronorcantharidin, and subsequent hydrogenation, phosphorylation, and basification gave compounds 1 and 2, separately. Resuits The structures of compounds were confirmed by IR, NMR and FAB-MS. The aqueous solubility of 1 and 2 were improved, compared with the parent compounds, and their activities were more potent than norcantharidin 4 against MGCS03 cell lines. Conclusion The phosphorylation of norcantharidin analogues is an effective way to increase the activity and solubility.
文摘INTRODUCTIONThe antitumor activity of norcantharidin (NCTD),the demethylated analogue of cantharidin,wasstudied in the early 1980s in China.NCTD has noside effects on urinary organs which cantharidin hasshown and is easier to synthesize,and it can inhibitthe proliferation of several tumor cell lines as wellas transplanted tumors.Clinical trials with NCTD asa monotherapeutic agent indicated that NCTD hadbeneficial effects in patients with different kinds
文摘BACKGROUND: Gallbladder carcinoma is a lethal malignant neoplasm with dismal surgical results. Unfortunately, the adjuvant therapies for gallbladder carcinoma such as chemotherapy and radiotherapy are also disappointing. We reported that norcantharidin (NCTD), a demethylated form of cantharidin, which is an active ingredient of the Chinese medicine Mylabris, was used against human gallbladder carcinoma GBC-SD cells. In the present study, we further studied the mechanism underlying the inhibitory effect of NCTD on growth of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: Human gallbladder carcinoma GBC-SD cells were grown in cell culture and divided into a NCTD group and a control group. The inhibitory effect of NCTD on growth of GBC-SD cells was investigated by evaluation of proliferation, cell cycle, apoptosis and morphological changes of the cells. Cell proliferation was assessed by tetrazolium-based colorimetric assay. The induction of cell cycle arrest and apoptosis was measured by flow cytometry. The morphological changes of the cells were observed by light- and electron-microscopy. To elucidate the anticancer mechanism of NCTD, expression of the proliferation-related gene proteins PCNA, Ki-67, cyclin-D-1 and p27 and the apoptosis-related gene proteins Bcl-2, Bax and Survivin were determined by the streptavidin-biotin complex method and RT-PCR. RESULTS: NCTD inhibited the proliferation of GBCSD cells in a dose- and time-dependent manner, with an IC50 of 56.18 mu g/ml at 48 hours. The flow cytometric profiles revealed that NCTD (at the IC50 for 48 hours) significantly increased the proportion of cells in G(2)/M phase and significantly decreased the proportion of cells in S phase, with a significantly increased rate of cell apoptosis. After treatment with the 48-hour IC50 dose of NCTD, cell shrinkage, vacuolar cytoplasm, membrane budding, karyorrhexis, karyolysis, chromosome condensation and chromatin aggregation in some GBCSD cells were observed by light-microscopy; decreased microvilli, Golgiosome atrophy, mitochondrial swelling, nuclear shrinkage, chromosome condensation and typical apoptosis bodies were seen by electron-microscopy, and the morphological changes of apoptosis occurred in GBCSD cells. The expression of PCNA, Ki-67 and Bcl-2 proteins decreased significantly; the Pix or relative levels of PCNA mRNA, cyclin-D-1 mRNA, Bcl-2 mRNA and Survivin mRNA decreased significantly, whereas the Pix or relative levels of p27 mRNA and Bax mRNA increased significantly. CONCLUSIONS: NCTD inhibits the growth of human gallbladder carcinoma GBC-SD cells in vitro. Its anticancer mechanism may correlate with inhibition of cell proliferation, arrest of the cell cycle, blockage of DNA synthesis, influence on cell metabolism, induction of cell apoptosis and influence on expression of the proliferation-related genes PCNA, Ki-67, cyclin-D-1 and p27, and the apoptosis-related genes Bcl-2, Bax and Survivin in human gallbladder carcinoma GBC-SD cells.
文摘BACKGROUND: Gallbladder carcinoma, a lethal malignant neoplasm with poor prognosis, has dismal results of surgical resection and chemoradiotherapy. We previously reported that norcantharidin (NCTD) is useful against growth, proliferation, and invasion of human gallbladder carcinoma GBC-SD cells in vitro. In this study, we further studied the inhibitory effect of NCTD on the growth of xenografted tumors of human gallbladder carcinoma in nude mice in vivo and the underlying mechanisms. METHODS: The tumor xenograft model of human gallbladder carcinoma in nude mice in vivo was established with subcutaneous GBC-SD cells. The experimental mice were randomly divided into control, 5-FU, NCTD, and NCTD+5-FU groups which were given different treatments. Tumor growth in terms of size, growth curve, and inhibitory rate was evaluated. Cell cycle, apoptosis, and morphological changes of the xenografted tumors were assessed by flow cytometry and light/electron microscopy. The expression of the cell cycle-related proteins cyclin-D1 and p27 as well as the apoptosis-related proteins Bcl-2, Box, and survivin were determined by the streptavidin-biotin complex (SABC) method and RT-PCR. RESULTS: NCTD inhibited the growth of the xenografted tumors in a dose- and time-dependent manner. Tumor volume decreased (5.61+/-0.39 vs. 9.78+/-0.61 cm(3), P=0.000) with an increased tumor inhibitory rate (42.63% vs. 0%, P=0.012) in the NTCD group compared with the control group. The apoptosis rate increased (15.08+/-1.49% vs. 5.49+/-0.59%, P=0.0001) along with a decreased percentage of cells in S phase (43.47+/-2.83% vs. 69.85+/-1.96%, P=0.0001) in the NTCD group compared with the control group. The morphological changes of apoptosis such as nuclear shrinkage, chromatin aggregation, chromosome condensation, and typical apoptosis bodies in the xenografted tumor cells induced by NCTD were observed by light and electron microscopy. The expression of cyclin-D1, Bcl-2 and survivin proteins/mRNAs decreased significantly, with increased expression of p27 and Bax proteins/mRNAs in the NCTD group compared with the control group. CONCLUSION: NCTD inhibits the growth of xenografted tumors of human gallbladder carcinoma in nude mice by inducing apoptosis and blocking the cell cycle in vivo.
文摘AIM: To study the therapeutic effect of norcantharidin(NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. METHODS: Two hepatocellular carcinoma(HCC) cell lines, Hep G2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. Hep G2 and SMMC-7721 cells were cultured in vitro. Liver cancer cells in the logarithmic phase of growth were vaccinated and cultured to the cell wall stage; these cells were treated for 48 h with different concentrations of NCTD, or ABT-737, or NCTD combined with ABT-737. The cell proliferation inhibition rate was detected by methyl thiazolyl tetrazolium. The expression of Mcl in HCC cells was detected by Western Blotting, and the cells in each group after treatment had apoptosis detected by flow cytometry. The proliferation inhibition rate, the expression of Mcl-1 in cells and the apoptosis inducing effect of treatment were observed in each group, and the effect of NCTD on ABT-737 in the treatment of HCC and its mechanism of action were analyzed.RESULTS: As the concentration of NCTD increased, the cell proliferation inhibition rate gradually decreased; and the treatment effect of ABT-737 1-3 μm combined with NCTD on cell proliferation inhibition was stronger than that of ABT-737 alone. The difference was statistically significant(P < 0.05). In observing the expression of Mcl-1 in cells after the treatment of different concentrations of NCTD, this was partiallyinhibited after treatment with NCTD 15 μm, and the expression of Mcl-1 was almost undetectable after treatment with NCTD 30 μm and 60 μm. The effect on inducing apoptosis with the treatment of ABT-737 or NCTD alone for 48 h was lower than that of the control group. The difference was not statistically significant(P > 0.05). The effect on inducing apoptosis in Hep G2 and SMMC-7721 cells with the treatment of ABT-737 combined with NCTD for 48 h was greater than that of ABT-737 or NCTD alone. The difference was statistically significant(P < 0.05). CONCLUSION: NCTD combined with ABT-737 has a positive role in the treatment of HCC, and it has great value in clinical research.
基金Supported by the Scientific Foundation of Tongji University, China, No. KPB027
文摘AIM: To investigate the effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells in vitro and its anticancer mechanism. METHODS: Human gallbladder carcinoma GBC-SD cells were cultured by cell culture technique. The growth and the invasiveness of GBC-SD cells in vitro were evaluated by the tetrazolium-based colorimetric assay and by the Matrigel experiment and the crossing-river test. Expression of PCNA, Ki-67, MMP2 and TIMP2 proteins of GBC-SD cells was determined by streptavidin-biotin complex method. RESULTS: In vitro norcantharidin inhibited the growth and proliferation of GBC-SD cells in a dose- and time-dependent manner, with the IC50 value of 56.18 μ/mL at 48 h. Norcantharidin began to inhibit the invasion of GBC-SD cells at the concentration of 5 μg/mL, and the invasive action of GBC-SD cells was inhibited completely and their crossing-river time was prolonged significantly at 40 μg/mL. After treatment with norcantharidin, the expression of PCNA, Ki-67, and MMP2 was significantly decreased. With the increase in TIMP2 expression, the MMP2 to TIMP2 ratio was decreased significantly (P<0.05). CONCLUSION: Norcantharidin inhibits the proliferation and growth of human gallbladder carcinoma cells in vitro at relatively low concentrations by inhibiting PCNA and Ki-67 expression. Its anti-invasive activity may be the result of decrease in MMP2 to TIMP2 ratio and reduced cell motility.
文摘BACKGROUND: Gallbladder carcinoma is a highly lethal and aggressive disease with early metastasis, strong invasion and poor prognosis. Most patients with this disease are at the advanced and un-resectable stage and should be consi- dered for palliative treatment such as chemotherapy and ra- diotherapy. Unfortunately, reports of chemotherapy and radiotherapy for gallbladder carcinoma are disappointing. We investigated the influence of norcantharidin (NCTD) on proliferation, proliferation-related gene proteins PCNA and Ki-67 of human gallbladder carcinoma GBC-SD cells in vitro. METHODS: GBC-SD cell lines of human gallbladder carci- noma were cultured by the cell culture technique. The ex- periment was divided into NCTD group and control group. The tetrazolium-based colorimetric assay was used to evaluate cell growth. The streptavidin-biotin complex method was used to determine the expressions of prolifera- tion-related gene proteins PCNA and Ki-67 of human gall- bladder carcinoma GBC-SD cells. RESULTS: NCTD inhibited the growth and proliferation of GBC-SD cells from 10 mg/L or after 6 hours in a dose- and time-dependent manner, with the IC50 value of 56.18 μg/ ml at 48 hours. After treatment with NCTD, the expression of PCNA (0.932 ±0.031 vs. 0.318 ±0.023, P<0.001) and Ki-67 (0.964 ±0.092 vs. 0.297 ±0.018, P<0.001) proteins were decreased significantly. CONCLUSION: NCTD inhibits the proliferation of human gallbladder carcinoma GBC-SD cells in vitro and the expres- sion of their proliferation-related gene proteins PCNA and Ki-67.
基金supported by the China Scholarship Council (CSC [2006] No. 3085)
文摘The Two-dimensional Quantitative Structure-activity Relationship （2D-QSAR） of a series of novel norcantharidin analogues, which exhibit hnhibitory activities of protein phosphatase 1 and 2A （PP1 and PP2A）, has been studied with a combined method of ab initio （I/F）, molecular mechanics （MM＋） and statistics. The established 2D-QSAR model （Eq. 1） for PP1 shows a reasonable regressive performance （R2= 0.749）, and the hydrophobic property of this molecule plays a decisive role in determining the inhibitory activity of PP1. In addition, the established 2D-QSAR model （Eq. 2） for PP2A also shows an acceptable regressive performance （R2= 0.701）, and the dipole moment of the molecule determines the inhibitory activity of PP2A.
基金Supported by National Natural Science Foundation of China,No.82074025 and No.82074271the Heilongjiang Traditional Chinese Medicine Research Project,No.ZHY18-047and Scientific Research Project of Heilongjiang Health Committee,No.2020-293.
文摘BACKGROUND Norcantharidin(NCTD)is suitable for the treatment of primary liver cancer,especially early and middle primary liver cancer.This compound can reduce tumors and improve immune function.However,the side effects of NCTD have limited its application.There is a marked need to reduce the side effects and increase the efficacy of NCTD.AIM To develop a nanomaterial carrier,NCTD-loaded metal-organic framework IRMOF-3 coated with a temperature-sensitive gel(NCTD-IRMOF-3-Gel),aiming to improve the anticancer activity of NCTD and reduce the drug dose.METHODS NCTD-IRMOF-3-Gel was obtained by a coordination reaction.The apparent characteristics and in vitro release of NCTD-IRMOF-3-Gel were investigated.Cell cytotoxicity assays,flow cytometry,and apoptosis experiments in mouse hepatoma(Hepa1-6)cells were used to determine the anti-liver cancer activity of NCTD-IRMOF-3-Gel in in vitro models.RESULTS The particle size of NCTD-IRMOF-3-Gel was 50-100 nm,and the particle size distribution was uniform.The release curve showed that NCTD-IRMOF-3-Gel had an obvious sustained-release effect.The cytotoxicity assays showed that the free drug NCTD and NCTD-IRMOF-3-Gel treatments markedly inhibited Hepa1-6 cell proliferation,and the inhibition rate increased with increasing drug concentration.By flow cytometry,NCTD-IRMOF-3-Gel was observed to block the Hepa1-6 cell cycle in the S and G2/M phases,and the thermosensitive gel nanoparticles may inhibit cell proliferation by inducing cell cycle arrest.Apoptosis experiments showed that NCTD-IRMOF-3-Gel induced the apoptosis of Hepa1-6 cells.CONCLUSION Our results indicated that the NCTD-IRMOF-3-Gel may be beneficial for liver cancer disease treatment.
基金supported by a grant from the National Natural Sciences Foundation of China (No. 30170391)the Zhejiang Provincial Fund of Natural Sciences (No:Z205166)the Zhejiang Provincial Fund of Science and Technology Bureau (No:2007C23007)
文摘In this study, norcanthridin （NCTD）-encapsulated liposomes were modified with a novel murine anti-human CD19 monoclonal antibody 2E8 （2E8-NCTD-liposomes） and the targeting efficiency and specific cytotoxicity of 2E8-NCTD-liposomes to CD19^＋ leukemia cells were evaluated. BALB/c mice were injected with 2E8 hybridoma cells to obtain 2E8 monoclonal antibody （mAb）. NCTD-liposomes were prepared by using film dispersion method. 2E8 mAbs were linked to NCTD-liposomes using post-incorporation technology. Flow cytometry showed that the targeting efficiency of purified 2E8 mAbs on CD19＋ Nalm-6 cells was 99.93%. The purified 2E8 mAbs were conjugated with NCTD-liposomes to prepare 2E8-NCTD-liposomes whose targeting efficiency on CD19^＋ Nalm-6 was also 95.82%. The average size of 2E8-NCTD-liposomes was 118.32 nm in diameter. HPLC showed that the encapsulation efficiency of NCTD was 46.51%. When the molar ratio of 2E8/Mal-PEG2000-DSPE reached 1：50, we obtained the liposomes with 9 2E8 molecules per liposome. The targeting efficiency of 2E8-NCTD-liposomes on CD19^＋ leukemia cells was significantly higher than that on CD19-1eukemia cells. Similarly, the targeting efficiency of the immtmoliposomes was also higher than that of the NCTDAiposomes on CD 19^＋ leukemia cells. Those results were consistent with those observed by laser scanning confocal microscopy. 3-（4, 5-Dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） assay demonstrated that 2E8-NCTD-liposomes specifically killed Nalm-6 cells in a dose- and time-dependent manner. The viability of Nalm-6 cells treated by 2E8-NCTD-liposomes was significantly lower than that of Molt-3 cells and it was also significantly lower than that of Nahn-6 cells treated with the same concentration of NCTD-liposomes or free NCTD. We are led to concluded that 2E8 antigen can serve as a specific targeting molecule of B lineage hematopoietic malignancies for liposome targeting, and 2E8-NCTD-liposomes can be used as a new and effective means for the treatment of B lineage hematopoietic malignancies.
文摘Background:Vasculogenic mimicry(VM)is a novel tumor blood supply in some highly aggressive malignant tumors.Recently,we reported VM existed in gallbladder carcinomas(GBCs)and the formation of the special passage through the activation of the PI3K/MMPs/Ln-5γ2 signaling pathway.GBC is a highly aggressive malignant tumor with disappointing treatments and a poor prognosis.Norcantharidin(NCTD)has shown to have multiple antitumor activities against GBCs,etc;however the exact mechanism is not thoroughly elucidated.In this study,we firstly investigated the anti-VM activity of NCTD as a VM inhibitor for GBCs and its underlying mechanisms.Methods:In vitro and in vivo experiments to determine the effects of NCTD on proliferation,invasion,migration,VM formation,hemodynamic and tumor growth of GBC-SD cells and xenografts were respectively done by proliferation,invasion,migration assays,HE staining and CD31-PAS double staining,optic/electron microscopy,tumor assay,and dynamic microMRA.Further,immunohistochemistry,immunofluorescence,Western blotting and RT-PCR were respectively used to examine expression of VM signaling-related markers PI3-K,MMP-2,MT1-MMP and Ln-5γ2 in GBC-SD cells and xenografts in vitro and in vivo.Results:After treatment with NCTD,proliferation,invasion,migration of GBC-SD cells were inhibited;GBC-SD cells and xenografts were unable to form VM-like structures;tumor center-VM region of the xenografts exhibited a decreased signal in intensity;then cell or xenograft growth was inhibited.Whereas all of untreated GBC-SD cells and xenografts formed VM-like structures with the same conditions;the xenograft center-VM region exhibited a gradually increased signal;and facilitated cell or xenograft growth(Figure 1-6).Furthermore,expression of MMP-2 and MT1-MMP products from sections/supernates of 3-D matrices and the xenografts,and expression of PI3-K,MMP-2,MM1-MMP and Ln-5γ2 proteins/mRNAs of the xenografts were all decreased in NCTD or TIMP-2 group(Figure 7-10;all P<0.01,vs.control group);NCTD down-regulated expression of these VM signaling-related markers in vitro and in vivo.Conclusions:NCTD inhibited tumor growth and VM formation of human gallbladder carcinoma GBC-SD cells and xenografts by suppression of the PI3-K/MMPs/Ln-5γ2 signaling pathway.It is firstly concluded that NCTD may be a potential anti-VM agent for human GBCs.
文摘Objective： To investigate the inhibitory activities of norcantharidin （NCTD）, a demethylated analogue of cantharidin, on Hep3B cells （a human hepatoma cell line） with deficiency of p53. Methods： The survival rate of the Hep3B cells after treating with NCTD was measured by MTT assay. Cell cycle of treated cells was analyzed by flow cytometry, and DNA fragmentation was observed by electrophoresis. The influence of inhibitors for various caspases and anti-death receptors antibodies on the NCTD-induced apoptosis in the cells was determined. Results： NCTD treatment resulted in growth inhibition of Hep3B cells in a dose- and time-dependent manner. Cell cycle analysis of the cells after treatment with NCTD for 48 h shows that NCTD induced G2M phase arrest occursat low concentration （≤25 μ mol/L） but G0G1 phase arrest at high concentration （50 μ mol/L）. The addition of both caspase-3 and caspase-10 inhibitors completely inhibited DNA fragmentation. Addition of anti-TRAIL/DR5 antibody significantly inhibited DNA fragmentation. Conclusion： NCTD may inhibit the proliferation of Hep3B cells by arresting cell cycle at G2M or G0G1 phase, and induce cells apoptosis via TRAIL/DR5 signal transduction through activation of caspase-3 and caspase-10 by a p53-independent pathway,
基金Supported by the National Natural Science Foundation of China(No.81273837)
文摘Objective： To observe the effect of norcantharidin（NCTD） on collagen-induced arthritis（CIA） rats. Methods： Sixty Sprague-Dawley（SD） rats were randomly divided into 6 groups（n=10）： normal group, CIA model group（model group）, NCTD low-dose group [1.35 mg/（kg·d）], NCTD middle-dose group [2.7 mg/（kg·d）], NCTD high-dose group [5.4 mg/（kg·d）] and methotrexate（MTX） group [1.8 mg/（kg/w）]. Anesthetized rats were sacrificed by luxation of cervical vertebra after 4 weeks of administration. The arthritis scores were evaluated twice a week. The pathological changes in the ankle joints of rats were observed by hematoxylin-eosin（H＆E） staining. The serum levels of interleukin（IL） 1β, IL-6, tumor necrosis factor（TNF）-α, vascular endothelial growth factor（VEGF）, IL-17 and transform growth factor（TGF） β were detected by enzyme linked immunosorbent assay（ELISA）. The mRNA expression of retinoid-related orphan nuclear receptor γ t（ROR γ t） and forkhead box P3（Foxp3） in peripheral blood lymphocytes were confirmed by real-time polymerase chain reaction. Results： MTX and high-dose NCTD not only decreased the arthritis scores but also alleviated the pathological changes in CIA rats＇ ankle joints compared with the model group（P〈0.05 or P〈0.01）. All doses of NCTD significantly inhibited the serum levels of IL-6, IL-17 and TNF-α in CIA rats（P〈0.05）. Only middle-and high-dose of NCTD prominently decreased serum IL-1β and TGF-β levels of CIA rats（P〈0.05）. However, NCTD has no effect on vascular endothelial growth factor（VEGF） level in CIA rats. The Foxp3 mRNA expression in all NCTD groups were increased significantly than in the model group（P〈0.05）. The mRNA expression of RORγt in NCTD high-dose group was decreased apparently in comparison with the model group（P〈0.05）. Conclusion： NCTD showed therapeutic effect on CIA rats by inhibition of cytokines and regulation of Th17/Treg cells.
基金Supported by National Natural Science Foundation of China(No. 2007Z002A)Grants of Putuo Hospital Affiliated to Shanghai University of Traditional Chinese MedicineGrants from the Department of Biochemistry and Molecular Biology,Department of Hematology and Oncology, University of MiamiMiller School of Medicine
文摘Objective： To investigate the mechanism of norcantharidin （NCTD）-induced SMMC-7721 hepatoma cell apoptosis. Methods： SMMC-7721 cell growth inhibition was measured by the MTT method. Apoptosis was detected by Annexin Ⅴ/propidium iodide staining. The mitochondrial membrane potential was measured by flow cytometry. Western blot analysis was used to evaluate the level of cytochrome c, caspase-3, AIF, Bcl-2 and Bax expression. Results： NCTD inhibited SMMC-7721 cell growth in a time-and dose-dependent manner. The cells treated with NCTD showed the loss of mitochondrial membrane potential. The activities of caspase-3, cytochrome c, AIF, and Bax were up-regulated after NCTD treatment at different doses. The expression of Bcl-2 was decreased after treatment with NCTD. Conclusions： NCTD could induce SMMC-7721 cell apoptosis. The activation of the mitochondrial pathway was involved in the process of NCTD-induced SMMC-7721 cell apoptosis.
