Apoptosis induced by norcantharidin in human tumor cells

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

Norcantharidin inhibits growth of human gallbladder carcinoma xenografted tumors in nude mice by inducing apoptosis and blocking the cell cycle in vivo

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.

Inhibitory effect of norcantharidin on the growth of human gallbladder carcinoma GBC-SD cells in vitro

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.

Norcantharidin combined with ABT-737 for hepatocellular carcinoma: Therapeutic effects and molecular mechanisms

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.

Influence of norcantharidin on proliferation,proliferation-related gene proteins prolifera-ting cell nuclear antigen and Ki-67 of human gallbladder carcinoma GBC-SD cells

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.

2D-QSAR Studies on the Norcantharidin Analogues as Protein Phosphatase 1 and 2A Inhibitors

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.

Metal-organic framework IRMOFs coated with a temperaturesensitive gel delivering norcantharidin to treat liver cancer

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.

Preparation and Evaluation of Norcantharidin-encapsulated Liposomes Modified with a Novel CD19 Monoclonal Antibody 2E8

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.

Effect of norcantharidin on proliferation and invasion of human gallbladder carcinoma GBC-SD cells

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 μg/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-67expression. Its anti-invasive activity may be the result of decrease in MMP2 to TIMP2 ratio and reduced cell motility.

Norcantharidin inhibits tumor growth and vasculogenic mimicry of human gallbladder carcinomas by suppression of the PI3-K /MMPs /Ln-5γ2 signaling pathway

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.

去甲斑蝥素抗肿瘤作用机制与联合治疗潜力

目的 对去甲斑蝥素的药理作用机制及临床联用进行系统阐述,并对开发去甲斑蝥素新型给药系统进行展望,为去甲斑蝥素进一步深入研究和临床应用奠定基础。方法 查阅国内外相关文献,对去甲斑蝥素的药理作用机制和临床联合用药情况进行分析和归纳总结。结果 去甲斑蝥素既可以通过单药多靶点机制实现抗肿瘤作用,还具有升高白细胞和调节机体免疫平衡的功能;去甲斑蝥素在临床上可以与化疗药物、免疫药物、靶向药物或放射疗法等联用,实现协同抗癌作用;去甲斑蝥素有望开发出脂肪乳注射液、乳化体和脂质体-乳剂复合体等高靶向、缓控释、低毒性和不良反应少的新型改良系统。结论 去甲斑蝥素临床联用具有广阔的应用前景,有待进一步深入研究。

Induction of Apoptosis in Human Hep3B Hepatoma Cells by Norcantharidin through a p53 Independent Pathway via TRAIL/DR5 Signal Transduction

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,

Protective Effect of Norcantharidin on Collagen-Induced Arthritis Rats

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.

Study on Norcantharidin-induced Apoptosis in SMMC-7721 Cells through Mitochondrial Pathways

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.

Synthesis, Antiproliferative Activity and DNA-Binding Properties of Nitrogen and Sulfur Heterocyclic Norcantharidin Acylamide Acid

Three novel norcantharidin acylamide acids （L1 =N-thiadiazole norcantharidin acylamide acid, CIoHIlN304S; L2=N-thiazole norcantharidin acylamide acid, CjIHI2N204S and L3=N-benzothiazole norcantharidin acylamide acid, C15H14N204S） were synthesized by the reactions of norcantharidin （NCTD=7-oxabicyclo[2,2,1 ]heptane-2,3- dicarboxylic acid anhydride, C8H8O4） with 2-amino-1,3,4-thiadiazole （C2H3N3S）, 2-aminothiazole （C3H4N2S） and 2-aminobenzothiazole （C7H6N2S）, respectively. Their structures were characterized by elemental analysis, IR, and NMR. The inhibition rates of L3 was much higher than those of Lj and L2 against human hepatoma cells SMMC7721 cell lines in vitro. The interaction between the compounds and DNA was studied by means of fluorescence quenching studies and viscosity measurements. The emission intensities decreased obviously with increasing concentration of the compounds in the fluorescence quenching experiments. The linear Stern-Volmer quenching constant Ksq values were 0.62 （Ll）, 0.55 （L2） and 1.08 （L3）, respectively. The binding abilities followed the trend from high to low were L3, L1 and L2, respectively. The results of viscosity measurements showed that L1 and L2 might bind to DNA via partial intercalation, while L3 bound mainly in intercalation.

去甲斑蝥素通过诱导自噬体聚集促使乳腺癌MDA-MB-231细胞凋亡

探究去甲斑蝥素(norcantharidin,NCTD)对三阴性乳腺癌MDA-MB-231细胞增殖和凋亡的影响。采用Western blot实验检测NCTD对MDA-MB-231细胞中凋亡相关蛋白Bax/Bcl-2、cleaved-PARP/PARP、cleaved-caspase-9、cleaved-caspase-3和MCL-1表达水平的影响;采用Western blot实验检测NCTD对MDA-MB-231细胞中自噬相关蛋白LC3-II/LC3-I,Parkin和PINK1表达水平的影响;采用流式细胞术检测NCTD对MDA-MB-231细胞线粒体膜电位及线粒体活性氧(reactive oxygen species,ROS)变化情况的影响;采用共聚焦显微镜检测NCTD对表达mCherry-EGFP-LC3的MDA-MB-231细胞自噬流的影响;采用流式细胞术检测NCTD联合使用氯喹(chloroquine,CQ)或3-甲基腺嘌呤(3-methyladenine,3-MA)后,对MDA-MB-231细胞凋亡情况的影响。实验结果显示,NCTD可显著上调Bax/Bcl-2、cleaved-PARP/PARP、cleaved-caspase-9、cleavedcaspase-3以及LC3-II/LC3-I蛋白的表达水平,促进Parkin的线粒体易位,阻断自噬流。此外,NCTD联合使用CQ加剧了细胞凋亡,而NCTD联合使用3-MA则减少了细胞凋亡。研究结果表明,NCTD可以诱导自噬体积累并导致MDA-MB-231细胞凋亡。

基于反向分子对接网络药理学探讨去甲斑蝥素治疗肝癌机制

目的:基于反向分子对接网络药理学探讨去甲斑蝥素(NCTD)治疗肝癌(HCC)的关键靶点及作用机制。方法:在ZINC数据库检索“Norcantharidin”获得化合物结构,PharmMapper反向对接预测靶点;Drugbank、OMIM、GeneCards数据库获得肝癌靶点;绘制韦恩图获取交集基因;凭借STRING平台分析NCTD治疗肝癌的靶点,利用Cytoscape 3.7.1可视化构建“成分—靶点—疾病”网络图,经Network Analyzer分析靶点网络拓扑参数提取关键靶点,利用MCODE插件进行模块分析;David做富集分析,并构建“成分疾病—靶点—通路”网络筛出关键基因。结果:获得交集靶点101个,103个分子功能和144条KEGG通路。结论:核心靶点有IGF1、AKT1、HRAS、EGFR、MAPK1,核心通路有癌症通路、PI3K-Akt信号通路、癌症中蛋白聚糖通路、MAPK信号通路、Ras信号通路、前列腺癌通路、化学致癌受体激活通路、肝细胞癌通路。NCTD通过多靶点、通路联合治疗肝癌。

去甲斑蝥素在乳腺癌中的研究进展

乳腺癌是我国最常见的女性恶性肿瘤。传统中药及其提取物因具有单药多靶点、低毒的特点,成为乳腺癌治疗的研究热点。去甲斑蝥素是一种新型人工合成抗肿瘤药物,具有升白细胞、抗肿瘤的作用。其抗肿瘤作用机制包括抗血管生成,促进肿瘤细胞凋亡,抑制肿瘤细胞增殖、侵袭与转移等。以斑蝥为主要来源的中成药在乳腺癌的临床应用中贯穿全程。本文就近年来去甲斑蝥素在乳腺癌中的研究进展进行综述。

大黄素-8-O-β-D-葡萄糖苷联合去甲斑蝥素体内外抗肿瘤作用及其机制

目的探究大黄素-8-O-β-D-葡萄糖苷(EG)与去甲斑蝥素(NCTD)联用的抗肿瘤作用及机制。方法①EG和NCTD与人肝癌HepG2细胞和人肺癌A549细胞作用48 h。给药方案:EG和NCTD分别单用(终浓度均为60~960μmol·L-1)、同时联用(EG+NCTD,1∶1同时给药,终浓度分别为30~480μmol·L-1)、序贯联用方案Ⅰ(EG→NCTD,EG作用24 h后除去EG,加入NCTD继续作用24 h,终浓度均为60~960μmol·L-1)和序贯联用方案Ⅱ(NCTD→EG,NCTD作用24 h后除去NCTD,加入EG继续作用24 h,终浓度均为60~960μmol·L-1)。MTT法检测细胞存活率,并计算相应的联合指数(CI)值,判断药物联合作用效应。随后利用反向找靶技术并构建蛋白-蛋白互作(PPI)网络筛选两药联用潜在的关键靶点。流式细胞术分析EG和NCTD单用及NCTD→EG序贯联用对HepG2和A549细胞凋亡的影响,Western印迹法检测胱天蛋白酶3(CASP3)和活化CASP3蛋白表达。②建立肝癌H22移植瘤小鼠模型,设模型对照(给予PBS)、5-氟脲嘧啶(5-Fu)阳性对照(30 mg·kg^(-1))、EG单用(4 mg·kg^(-1))、NCTD单用(4 mg·kg^(-1))、两药同时联用(EG+NCTD,各2 mg·kg^(-1))及序贯联用(NCTD→EG,NCTD 4 mg·kg^(-1)6 d,EG 4 mg·kg^(-1)6 d)组,均ip给药,每天1次,共12 d。末次给药后24 h小鼠称重后处死,剥离移植瘤组织称重,计算肿瘤抑制率。结果①EG和NCTD单用、同时联用及2种方案序贯联用均呈浓度依赖性抑制HepG2和A549细胞存活(P<0.01),在一定浓度范围内具有协同作用。EG和NCTD单用及同时联用三者比较,NCTD单用IC50最低,同时联用未表现出更好的作用。与两者单用比较,NCTD→EG序贯联用IC50最低。基于反向找靶及PPI分析筛选出关键靶点CASP3,表明两药联用可能涉及细胞凋亡通路。流式细胞术和Western印迹实验结果表明,与EG和NCTD单用组相比,NCTD→EG序贯联用明显增加HepG2和A549细胞凋亡率(P<0.01),且活化CASP3蛋白表达增加(P<0.01),而CASP3蛋白表达无明显变化。②肝癌H22移植瘤模型小鼠体内实验结果表明,与模型对照组相比,NCTD单用、同时联用、NCTD→EG序贯联用和5-Fu阳性对照组瘤重均显著降低(P<0.05),抑瘤率分别为42.4%,47.8%,50.4%和69.0%;EG单用抑瘤作用不明显。同时联用组小鼠体重较模型对照组升高(P<0.05),5-Fu组显著降低(P<0.01)。结论EG与NCTD联用具有协同抗肿瘤作用,其机制与诱导肿瘤细胞凋亡有关。

去甲斑蝥素片辅助ERCP治疗食管癌的应用研究

目的观察去甲斑蝥素片辅助内镜逆行胰胆管造影术(retrograde cholangiopancreatography,ERCP)治疗食管癌对术后胆道感染的防治效果。方法本文为前瞻性研究。选取2021年5月至2022年12月南阳医学高等专科学校第一附属医院收治的115例食管癌患者为研究对象,采用电脑随机分组法将入组患者分为对照组(57例)和观察组(58例)。对照组男34例、女23例,年龄(61.22±5.32)岁,采用ERCP治疗;观察组男35例、女23例,年龄(60.45±5.17)岁,采用去甲斑蝥素片辅助ERCP治疗。对比两组患者炎症因子、免疫功能指标、血清肿瘤标志物水平及术后不良事件发生情况。采用t检验、χ^(2)检验。结果治疗前,两组患者炎症因子、免疫功能指标及血清肿瘤标志物水平比较,差异均无统计学意义(均P>0.05)。治疗后,观察组肿瘤坏死因子-α(TNF-α)[(3.52±0.46)ng/L]、白细胞介素-6(IL-6)[(135.45±10.69)ng/L]、IL-8[(15.22±5.24)μg/L]均低于对照组[(4.79±1.24)ng/L、(140.77±10.88)ng/L、(18.44±5.27)μg/L],差异均有统计学意义(t=7.306、2.645、3.286,均P<0.05)。治疗后,观察组总T细胞(CD3)[(65.29±10.76)%]、干扰素-γ(IFN-γ)[(10.21±3.44)ng/L]、IL-12[(61.25±10.71)μg/L]均高于对照组[(60.77±10.31)%、(8.24±2.39)ng/L、(56.25±10.15)μg/L],差异均有统计学意义(t=2.300、3.561、2.569,均P<0.05)。治疗后,观察组癌胚抗原(CEA)[(4.24±1.36)μg/L]、糖类抗原19-9(CA19-9)[(7.49±2.11)U/ml]、糖类抗原50(CA50)[(5.11±1.27)μg/L]均低于对照组[(5.82±1.25)μg/L、(10.61±4.24)U/ml、(7.25±2.14)μg/L],差异均有统计学意义(t=6.483、5.009、6.535,均P<0.05)。治疗后,观察组不良事件发生率为6.90%(4/58),低于对照组[17.54%(10/57)],差异有统计学意义(χ^(2)=5.277,P<0.05)。结论去甲斑蝥素片辅助ERCP能有效缓解食管癌患者炎症反应并增强机体免疫功能,同时还可下调肿瘤标志物水平并降低胆道感染等不良事件发生风险。