Norcantharidin Research Articles

RETRACTED ARTICLE: Norcantharidin combined with Coix seed oil synergistically induces apoptosis and inhibits hepatocellular carcinoma growth by downregulating regulatory T cells accumulation

The immune system plays a critical role in exerts effects in the growth and progression of hepatocellular carcinoma (HCC), which needs interacting approaches for effective therapy. In this study, we have found that the Norcantharidin (NCTD) + Coix lacryma-jobi seed oil (CLSO) combination exhibited more potent antitumor effects in an terms of cytotoxicity and apoptotic induction in human HepG2 and HepG2/ADM cells than NCTD or CLSO alone. In vivo, administration of NCTD+CLSO combinations significantly suppressed the formation of tumor in Hepal-1 hepatoma-bearing mice. Furthermore, we found that the in vitro co-cultures of HepG2 or HepG2/ADM cells with PBMCs from healthy donors led to an increase in the number of CD4+CD25+ T cells. This increase was down-regulated by the combination effectively. Down-regulation of FoxP3 mRNA and protein expression occurred during the combination in the co-cultures. The amount of Tregs of Hepal-1 hepatoma-bearing mice was significantly decreased in the combination treated group. The combination down-regulated the expression of FoxP3, CTLA-4 and Tregs related cytokine (TGF-β and IL-10) in the serum of tumor bearing mice. Taken together, these results suggest that the most valuable aspect of the NCTD+CLSO combined use improves the anti-tumor activity and regulates tumor infiltrating Tregs.

Norcantharidin induces mitochondrial-dependent apoptosis through Mcl-1 inhibition in human prostate cancer cells

Norcantharidin (NCTD) is the demethylated form of cantharidin that exhibits anticancer potential in many cancer cell types. Recent reports suggest that NCTD targeting ROS/AMPK and DNA replication signaling pathway could be an effective strategy for the treatment of PCa cells. However, supportive evidence is limited to the effect of NCTD that induction of apoptosis through suppression of the Mcl-1. Here, we show that NCTD induced PCa cell apoptosis and triggered caspase activation, which was associated with mitochondria dysfunction. Mechanistic investigations suggested that NCTD modulated the Akt signaling via increased nuclear translocation and interaction with the myeloid cell leukemia-1 (Mcl-1) promoter by FOXO4, resulting in an apoptotic effect. Moreover, miR-320d, which targets Mcl-1, was significantly upregulated after NCTD treatment. Overexpression of miR-320d by NCTD induced mitochondria dysfunction and apoptosis, which was notably attenuated with a miR-320d inhibitor. In vivo xenograft analysis revealed that NCTD significantly reduced tumor growth in mice with PC3 tumor xenografts. Taken together, our results provide new insights into the critical role of NCTD in suppressing Mcl-1 via epigenetic upregulation of miR-320d, resulting in PCa cell apoptosis.

Abstract 5111: Norcantharidin impairs tumor growth in vivo and inhibits stemness of triple-negative breast cancer cells

Abstract Triple-negative breast cancer (TNBC) is characterized by an abundance of treatment-resistant cancer stem cells (CSC). The absence of a molecular target, coupled with its highly aggressiveness, leads to the lack of an effective therapy for TNBC. Norcantharidin (NCTD) is a synthetic demethylated small-molecule analog of the naturally occurring cantharidin isolated from blister beetles (Mylabris phalerata Pall). Unlike the conventional chemotherapeutics, NCTD toxicity is higher to cancer cells than normal ones, making this small molecule promising for cancer treatment. The aims of this work were: A) To study the effect of NCTD on 4T1 cell line proliferation in vitro. B) To analyze the effect of NCTD on 4T1 derived CSC on self-renewal and clonogenic capacity. C) To evaluate the effect of NCTD on 4T1 tumor growth in vivo. We employed the well-known 4T1 triple-negative breast cancer cell model, which presents a huge proportion of CSC. We observed that NCTD treatment during 96 h significantly reduced 4T1 cell proliferation in vitro. In addition, the IC50 value of NCTD was 27.35 ± 2.83 μM. Related to CSC, NCTD pre-treatment for 96 h impaired CSC self-renewal (Number of secondary mammospheres: Control: 276±39; NCTD: 163±18; p≤0.05) as well as the clonogenic capacity (Number of colonies: Control: 359±38; NCTD: 122±11; p≤0.05). By q-PCR, we observed that NCTD treatment for 48 h significantly induced an increase of Gli-1 and Smooth in CSC, keys member of Sonic Hedgehog pathway. Finally, we performed an in vivo assay, where 4T1 cells were orthotopically inoculated on mammary gland of BALB/c mice, and NCTD was i.p. inoculated twice a week (5mg/kg). We observed that NCTD treatment significantly reduced tumor growth in vivo. Our data suggest that NCTD treatment reduces tumor growth both in vitro and in vivo, possibly through the direct effect on CSC self-renewal and clonogenic capacity, by modulating Sonic Hedgehog pathway. Citation Format: Damian E. Berardi, Guido Cicuttin, Maria A. Taruselli, Stefano M. Cirigliano, Elisa D. Bal de Kier Joffé, Alejandro J. Urtreger, Laura B. Todaro. Norcantharidin impairs tumor growth in vivo and inhibits stemness of triple-negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5111. doi:10.1158/1538-7445.AM2017-5111

The targeting effect of Hm2E8b–NCTD–liposomes on B-lineage leukaemia stem cells is associated with the HLF–SLUG axis

To identify an agent with specific activity against B-lineage leukaemia stem cells (B-LSCs), we generated norcantharidin (NCTD)-encapsulated liposomes modified with a novel humanised anti-human CD19 monoclonal antibody, Hm2E8b (Hm2E8b–NCTD–liposomes). These liposomes were specially designed to recognise and kill B-LSCs in vitro, and to decrease non-specific cytotoxicity to untargeted cells. Hm2E8b–NCTD–liposomes selectively ablated B-LSCs through targeting hepatic leukaemia factor (HLF), which is implicated in haematopoietic stem cell regulation and is overexpressed in LSCs. Hm2E8b–NCTD–liposomes decreased HLF protein levels and induced apoptosis in the HAL-01 cell line harbouring the oncoprotein E2A–HLF. This resulted in modulation of the expression of several molecules that govern survival pathways, including HLF, SLUG, NFIL3 and C-Myc, thereby causing the induction of p53 and the mitochondrial caspase cascade. Therefore, the potent in vitro effect of Hm2E8b–NCTD–liposomes on B-LSC activity and survival pathways have the potential to be exploited clinically with appropriate drug combinations.

Preparation and pharmacological evaluation of norcantharidin-conjugated carboxymethyl chitosan in mice bearing hepatocellular carcinoma

In this study, norcantharidin (NCTD), a small-molecule anticancer drug derived from Chinese traditional medicine blister beetle (Mylabris), was conjugated covalently onto carboxymethyl chitosan (CMCS). Then the hepatocellular carcinoma therapeutic properties and liver-protective effects were investigated through orthotopic transplantation tumor model. Results showed that the obtained CMCS-NCTD demonstrated remarkable anti-growth efficacy against hepatocellular 22 in mice. Significant improvement of the liver injury caused by cancer cells was observed in tumor-bearing mice administrated with CMCS-NCTD. Moreover, CMCS-NCTD remarkably increased the serum levels of TNF-α, IFN-γ, TIMP-1 and E-cadherin in mice treated for 12days. Administration of CMCS-NCTD significantly reduced the elevated serum ALT, AST, VEGF and MMP-9 levels of tumor-bearing mice. In addition, activities of SOD and GSH-Px in serum or homogenate of the CMCS-NCTD treated mice were significantly high when compared with model control group. Our data suggested that CMCS-NCTD was a promising candidate as an anti-hepatoma and liver-protection compound.

FAM46C is critical for the anti-proliferation and pro-apoptotic effects of norcantharidin in hepatocellular carcinoma cells

Norcantharidin (NCTD), a demethylated analog of cantharidin derived from Chinese traditional medicine blister beetle, has been currently used as an anticancer drug for various cancers including hepatocellular carcinoma (HCC). In this study, for a more comprehensive understanding of the targets of NCTD in HCC, next-generation RNA-Seq was utilized. We revealed that the expression of FAM46C, which has been reported as a tumor suppressor for multiple myeloma, was enhanced after NCTD treatment. Re-analysis of TCGA (The Cancer Genome Atlas) LIHC (liver hepatocellular carcinoma) dataset demonstrated that FAM46C expression was significantly lower in HCC tissues than in normal liver tissues. NCTD injection or FAM46C overexpression could mitigate diethylnitrosamine (DEN)-initiated HCC in mice. Ectopic expression of FAM46C in two HCC cell lines, SMCC-7721 and SK-Hep-1, significantly repressed cell proliferation, and increased cells population in G2/M phase and cell apoptotic rate. We also found that FAM46C overexpression caused a notable decrease in Ras expression, MEK1/2 phosphorylation and ERK1/2 phosphorylation. More importantly, FAM46C knockdown significantly weakened the biological effects of NCTD on HCC cells, which suggested NCTD exerted the anticancer functions partially through up-regulating FAM46C. In conclusion, FAM46C, a tumor suppressor for HCC, is important for the anti-proliferation and proapoptotic effects of NCTD.

Asialoglycoprotein receptor-targeted liposomes loaded with a norcantharimide derivative for hepatocyte-selective targeting

In order to overcome the shortcomings associated with the clinical application of norcantharidin (NCTD), including intense irritation and a short half-life, and to obtain a hepatocyte-selective liposome system with high encapsulation efficiency (EE) and low leakage, we synthesized a C14 alkyl chain norcantharimide derivative of NCTD (2-tetradecylhexahydro-1H-4,7-epoxyisoindole-1,3(2H)-dione, N-14NCTDA). Asialoglycoprotein receptor-targeted, galactosylated liposomes loaded with N-14NCTDA (GAL-Lipo) were prepared by the lipid film hydration method. GAL-Lipo with a satisfactory particle size of approximately 120nm has a higher encapsulation efficiency of more than 98.0%, which is markedly increased compared with NCTD loaded liposomes (EE%=47.6%). In addition, GAL-Lipo remained stable for at least 1 month at 4°C. In cytotoxicity assays, GAL-Lipo demonstrated stronger cytotoxicity effects (IC50=24.58μmolL−1) on Hep G2 cells than free N-14NCTDA (100μmol/L) and conventional liposomes (Con-Lipo, 39.49μmol/L) without the GAL modification. GAL-Lipo can continuously accumulate in Hep G2 cells and be internalized into cells via two pathways, namely caveolin-dependent endocytosis and clathrin-dependent asialoglycoprotein receptors (ASGP-R) mediated endocytosis and produces considerably more significant cellular apoptosis. The results of vivo toxicity studies showed that GAL-Lipo dramatically reduced renal toxicity. In addition, GAL-Lipo has a markedly improved pharmacokinetic profile in vivo and a longer circulation time (AUC=6.700±2.964mgL−1h, t1/2z=1.347±0.519h) than Con-Lipo (AUC=2.319±0.121mgL−1h, t1/2z=0.413±0.238h). In conclusion, N-14NCTDA with an ideal logP is a better alternative for the treatment of primary hepatic carcinoma. GAL-Lipo offers an attractive strategy to specifically target hepatocytes via caveolin-dependent and clathrin-dependent asialoglycoprotein receptor-mediated endocytosis resulting in higher anticancer activity and fewer side-effects.

Inhibitory effect of norcantharidin on melanoma tumor growth and vasculogenic mimicry by suppressing MMP-2 expression

A form of microcirculation called vasculogenic mimicry (VM), which constitutes a novel approach for tumor blood supply in certain highly aggressive malignant tumors, was recently reported to contribute to tumor metastasis and poor prognosis in melanoma patients. Development of strategies to target tumor VM may be significant to reduce the recurrence and metastasis of melanoma. Norcantharidin (NCTD) has been shown to inhibit tumor growth and VM of human gallbladder carcinomas. Besides, NCTD could induce melanoma cell apoptosis. However, whether NCTD can inhibit the growth and VM formation of melanoma has not been evaluated. The present study aims to investigate the anti-VM activity of NCTD as a VM inhibitor for melanoma and its potential mechanisms. The anti-VM activity of NCTD was determined in human melanoma A375 cells and xenografts in vitro and in vivo. The findings indicate that NCTD inhibits tumor growth and VM formation of melanoma both in vitro and in vivo by suppressing matrix metalloproteinase-2 expression. The results suggest that NCTD is a potential therapeutic agent targeting VM in melanoma.

Effect of norcantharidin on the proliferation, apoptosis, and cell cycle of human mesangial cells

Aims: Norcantharidin (NCTD) regulates immune system function and reduces proteinuria. We sought to investigate the effect of NCTD on proliferation, apoptosis and cell cycle of cultured human mesangial cells (HMC) in vitro.Methods: HMC cells were divided into a normal control group, and various concentrations of NCTD group (2.5, 5, 10, 20, or 40 μg/mL). Cell proliferation was determined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, apoptosis was detected by Annexin V/propidium iodide (PI) assays, and morphological analysis was performed by Hoechest 33258 staining. Finally, cell cycle was analyzed by flow cytometry.Results: NCTD dose and time dependently inhibits HMC proliferation significantly (p < .05). Apoptosis dose and time dependently increased after NCTD treatment. Cell-cycle analysis revealed that the number of cells in the G2 phase increased significantly, whereas the fraction of cells in the S phase decreased, especially 24 h after 5 μg/ml NCTD treatment.Conclusion: NCTD inhibits HMC cell proliferation, induces apoptosis, and affects the cell cycle.

Repression of YAP by NCTD disrupts NSCLC progression.

The efficacy of available lung cancer therapeutic interference is significantly limited by various resistance mechanisms to those drugs. Activation of the oncogene YAP underlying the initiation, progression, and metastasis of lung cancer associates with poor prognosis and confers drug resistance against targeted therapy. In this study, we evaluated the specificity of norcantharidin (NCTD) in repressing YAP to inhibit non-small cell lung carcinoma (NSCLC) progression. Our study revealed that YAP signal pathways were aberrantly activated in lung cancer tissues and cells which rendered more proliferative and invasive phenotypes to human lung cancer cells. We confirmed that NCTD specifically repressed YAP signaling pathway to interfere the YAP-mediated non-small cell lung carcinoma progression and metastasis via arresting cell cycle, enhancing apoptosis and inducing senescence. We also found NCTD-mediated repression of YAP decreased epithelial-to-mesenchymal transition (EMT) and reduced the motile and invasive cellular phenotype in vitro via enhancing E-cadherin and decreasing fibronectin/vimentin. Mechanistic investigations revealed that NCTD transcriptionally downregulated YAP and post-translationally modulated the subcellular redistribution of YAP between nucleus and cytoplasm. Collectively, our results indicated that NCTD is a novel therapeutic drug candidate for NSCLC which specifically and sensitively target YAP signal pathway.

Norcantharidin-induced Apoptosis of AGS Human Gastric Cancer Cells Through Reactive Oxygen Species Production, and Caspase- and Mitochondria-dependent Signaling Pathways.

Norcantharidin (NCTD) was purified from mylabris, the dried body of the Chinese blister beetle. NCTD has been shown to exhibit anticancer activities in many human cancer cell lines, but there are no reports to show whether it induces apoptosis of human gastric cancer cells. Therefore, in the present study, we investigated NCTD-induced cell death and associated protein expression in human gastric cancer AGS cells in vitro. Cell morphological changes, viability and cell-cycle distribution were examined and analyzed by phase-contrast microscopy and flow cytometric assays. Flow cytometry was also used to measure the levels of reactive oxygen species (ROS), Ca2+, mitochondrial membrane potential (Ψm) and activity of caspases. The results indicated that NCTD induced cell morphological changes, reduced total viable cell number and induced G0/G1 phase arrest. NCTD also increased ROS production and reduced the Ψm and increased caspase-9 activity in AGS cells. Western blotting also found that NCTD increased the pro-apoptotic proteins such as BCL2-associated X protein (BAX) and BH3 interacting-domain death agonist (BID) and increased the release of cytochrome c, apoptosis inducing factor (AIF) and endonuclease G (Endo G) release from mitochondria in AGS cells. NCTD also significantly increased the expression of active forms of caspase-3 and -8 and -9 and reduced the expression of caspase-4 and -12 in AGS cells. Based on these observations, we suggest that NCTD-induced apoptotic cell death may be through mitochondria- and caspase-dependent pathways.

Development, optimization and in vitro evaluation of norcantharidin loadedself-nanoemulsifying drug delivery systems (NCTD-SNEDDS)

This study focused on developing a self-nanoemulsifying drug delivery system (SNEDDS) containing bioactive surfactants under an efficient screening approach for overcoming problems associated with the delivery of norcantharidin (NCTD), a high dose chemotherapy agent having pH dependent solubility. Preliminary screening was implemented to select proper components combination. Besides the solubility of NCTD in the oil phase, emulsifying efficiency, droplet size and size distribution were also employed to select components of the SNEDDS. Moreover, the influence of surfactant and co-surfactant on the interfacial tension and droplets of nanoemulsions were investigated to further understand the mechanism of spontaneous emulsification. Co-surfactant addition promoted the emulsification via reducing the water/oil interfacial tension and viscosity. Ternary phase diagrams were constructed to investigate the phase behavior and designate the optimum systems. The alternative formulations were characterized for cloud point, dilution robustness, droplet size, polydispersity index (PDI) and transmission electron microscopy (TEM). In vitro dissolution study showed that the dissolution rate of optimized formulation (NCTD 10 mg/g, EO 50 wt.%, Cremophor EL 35 wt.%, ethylene glycol 15 wt.%) was slower than drug suspension under the same conditions, confirming that the developed SNEDDS formulation would exhibit sustained release potential.

Atg5 siRNA inhibits autophagy and enhances norcantharidin-induced apoptosis in hepatocellular carcinoma

Background: Cantharidin is a terpenoid isolated from Chinese blister beetles, and norcantharidin (NCTD) is a demethylated analog of cantharidin. It has been reported that cantharidin and norcantharidin have anticancer activities. Growing evidence suggests that inhibiting autophagy can induce apoptosis in the human hepatoma cell line HepG2. The objective of the present study was to determine whether inhibition of autophagy enhances NCTD-induced apoptosis in HepG2 cells. Methods: HepG2 cells were cultured in DMEM containing NCTD. Autophagy was upregulated in the presence of HBSS media supplemented with Ca2+ and Mg2+ and 10 mM HEPES and downregulated in the presence of 3-methyladenine (3-MA) and Atg5 siRNA. Autophagy, cell viability, and the expression of apoptotic proteins were assessed in HepG2 cells. Results: Our data showed that cell apoptosis generally increased after norcantharidin treatment in HepG2 cells. Expression of LC3-II, an autophagosomemarker, increased when cells were treated with HBSS media. It also increased cell viability. However, in the presence of 3-MA and Atg5 siRNA, autophagy was inhibited, LC3-II expression decreased and cell apoptosis increased. There was increased expression of Bax, cytochrome c, cleaved caspase-3, caspase-9 and PARP and the mitochondrial membrane potential was disrupted. Additionally, increased apoptosis was accompanied by increased reactive oxygen species (ROS) production. Conclusion: NCTD has anticancer activity, and Atg5 siRNA-mediated downregulation of autophagy enhanced its anticancer actions due to ROS generation and activation of the mitochondrial apoptosis pathway.

Norcantharidin blocks Wnt/β-catenin signaling via promoter demethylation of WIF-1 in glioma.

Glioma is one of the most common primary intracranial tumors, and the prognosis is poor even though much treatment management is employed. Wnt/β-catenin signaling has been reported to be associated with glioma. Norcantharidin (NCTD) is the demethylated analog of cantharidin isolated from blister beetles, and it is reported to possess anticancer activity but less nephrotoxicity than cantharidin. Accordingly, we aimed to investigate NCTD as an anti-neoplastic drug that inhibits the Wnt/β‑catenin pathway via promoter demethylation of Wnt inhibitory factor-1 (WIF-1) in glioma growth in vitro. In the present study, we report that NCTD inhibited cell proliferation, induced apoptosis and cell cycle arrest, and suppressed cell migration and invasion in vitro. Moreover, we observed that the expression levels of WIF-1 mRNA and protein in the NCTD-treated cells were increased significantly compared with these levels in the negative control (NC) cells. Promoter demethylation was observed in the NCTD‑treated cells. In contrast, aberrant methylation was observed in the NC cells. Additionally, more investigation revealed that NCTD suppressed activity of Wnt/β-catenin signaling and transcription of β-catenin/TCF-4. Furthermore, the expression of apoptosis-related proteins Bcl-2 and cleaved caspase-3 indicated significant cell apoptosis. We provide initial evidence that NCTD reactivates WIF-1 from a methylation state, and downregulates canonical Wnt/β-catenin signaling. Our findings revealed that NCTD is effective for glioma in vitro and may be a new therapeutic option in vivo.

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

To study the therapeutic effect of norcantharidin (NCTD) combined with ABT-737 on hepatocellular carcinoma cells and the molecular mechanism. Two hepatocellular carcinoma (HCC) cell lines, HepG2 and SMMC-7721, were selected. ABT-737 and NCTD were allocated into groups to be used alone or in combination. HepG2 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. 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 partially inhibited 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 HepG2 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). NCTD combined with ABT-737 has a positive role in the treatment of HCC, and it has great value in clinical research.

Norcantharidin induces autophagy-related prostate cancer cell death through Beclin-1 upregulation by miR-129-5p suppression.

Norcantharidin (NCTD) has an anticancer potential to allow it to be used in the treatment of some malignant cancers. However, whether NCTD may have similar anticancer effects on prostate cancer (PC) is unknown. Here, we aimed to examine the effects of NCTD on PC cells and the underlying mechanisms. We found that NCTD dose-dependently inhibited the PC cell growth, in either a cell counting kit-8 (CCK-8) assay or a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Moreover, NCTD dose-dependently increased the PC cell autophagy, through upregulation of Beclin-1. Furthermore, the Beclin-1 protein, but not mRNA, was regulated by NCTD in PC cells, suggesting post-transcriptional control of Beclin-1 by NCTD. Finally, microRNA (miR)-129-5p was found to be regulated by NCTD, and bioinformatics analyses showed that miR-129-5p targeted the 3'-UTR of Beclin-1 mRNA to inhibit its translation, which was confirmed by luciferase reporter assay. Together, these data suggest that NCTD may upregulate Beclin-1 through suppression of miR-129-5p to induce autophagic cell death and cell proliferation arrest in PC cells. Our study sheds light on using NCTD as a novel treatment for PC.

Norcantharidin modulates miR-655-regulated SENP6 protein translation to suppresses invasion of glioblastoma cells.

Norcantharidin (NCTD) is currently used as an anticancer drug for the treatment of some malignant cancers. However, whether it may have therapeutic effects on glioblastoma multiforme (GBM) remains unknown. Moreover, the underlying mechanisms have not been completely elucidated. Recently, SUMO-specific protease 6 (SENP6) has been shown as a tumor suppressor in some cancers. Nevertheless, whether it is involved in the pathogenesis of GBM has not been examined. Here, we studied the effects of NCTD on GBM cells. We found that NCTD dose-dependently increased SENP6 protein, but not messenger RNA (mRNA), in GBM cells, resulting in the suppression of cell invasion. Depletion of SENP6 in GBM cells significantly attenuated the NCTD-induced suppression of GBM cell invasion, while overexpression of SENP6 in GBM cells mimicked the effects of NCTD on cell invasion. Moreover, NCTD dose-dependently decreased the levels of microRNA-655 (miR-655), which bound to 3'-UTR of SENP6 mRNA to inhibit its translation. Overexpression of miR-655 decreased SENP6 in GBM cells, while depletion of miR-655 increased SENP6 protein in GBM cells. Taken together, our data demonstrates a previously unappreciated control of NCTD to suppress GBM cell invasion through modulation of miR-655-regulated SENP6 protein translation.

Evaluation of DNA damage induced by norcantharidin in human cultured lymphocytes

Norcantharidin (NCTD) is currently used in the treatment of several cancers such as leukemia, melanoma and hepatoma. The mechanism of action of NCTD is suggested to involve induction of apoptosis of cancer cells via production of reactive oxygen species. In this study, the genotoxic effect of different concentrations of NCTD (1, 10 and 20 μm) in human lymphocytes was investigated using sister chromatid exchanges (SCEs) and chromosomal aberrations (CAs) assays. The results revealed that NCTD significantly increased the rate of SCEs (p < 0.05) in a dose-dependent manner. In addition, NCTD significantly increased the number of high-frequency cells (SCEs ≥ 8, p < 0.05). However, NCTD did not have any significant effect on the rate of CAs (p > 0.05). In addition, no significant differences were detected in the mitotic index or proliferative index at examined doses (up to 20 μm). In conclusion, NCTD is genotoxic to human cultured lymphocytes as measured by SCE assay.

Liquid chromatography–tandem mass spectrometry evaluation of the pharmacokinetics of a diacid metabolite of norcantharidin loaded in folic acid-targeted liposomes in mice

A previous study has reported diacid metabolite (DM) as the stable form of norcantharidin (NCTD), which is almost 100% metabolized to DM-NCTD. However, the unreliable pharmacokinetic characteristics of DM-NCTD could result in low bioavailability, hindering the clinical use of DM-NCTD in the treatment of diseases. A liposomal drug delivery system could overcome the shortcomings of DM-NCTD by improving the relative bioavailability (Fr), reducing drug toxicity, and increasing the therapeutic efficacy. However, there are no data concerning the pharmacokinetics of a DM-NCTD-loaded liposomal drug delivery system in animals, which is required for assessing its safety profile. Therefore, a rapid and sensitive liquid chromatography–tandem mass spectrometry (LC–MS/MS) method was developed and validated for the determination of DM-NCTD in mouse plasma. Standard curves were linear (r=0.9966) over the range 10.0–1.00×104ng/ml in mouse plasma with a lower limit of quantification (LLOQ) of 10ng/ml. This study successfully investigated the pharmacokinetics of DM-NCTD and DM-NCTD encapsulated in polyethylene glycol (PEG)-Liposomes (DM-NCTD/PEG-Liposome) or folic acid (FA)-PEG-Liposomes (DM-NCTD/FA-PEG-Liposome) in Kunming mice after a single intravenous dose of 2mg/kg. The plasma profile data of the three groups adhered to a two-compartment model. Compared with the DM-NCTD group, the Liposome groups had longer circulation times following intravenous administration in mice, and the Fr of DM-NCTD increased significantly (P<0.05). Furthermore, the area under the concentration–time curve (AUC) declined with an increase in the volume of distribution (Vd) from the PEG-Liposome to the FA-PEG-Liposome groups, which indicates a more efficient removal of the drug from the plasma of the FA-PEG-Liposome group. This result suggests a possible increased risk of DM-NCTD intoxication in normal tissues with FA-PEG-Liposomes. Based on this study, further investigation of the biodistribution of DM-NCTD/FA-PEG-Liposomes in healthy animals is warranted. In addition, the plausibility of formulating a safe DM-NCTD-loaded system without increasing toxicity against normal tissues needs to be determined.

Intraarterial administration of norcantharidin attenuates ischemic stroke damage in rodents when given at the time of reperfusion: novel uses of endovascular capabilities.

OBJECT Matrix metalloprotease-9 (MMP-9) plays a critical role in infarct progression, blood-brain barrier (BBB) disruption, and vasogenic edema. While systemic administration of MMP-9 inhibitors has shown neuroprotective promise in ischemic stroke, there has been little effort to incorporate these drugs into endovascular modalities. By modifying the rodent middle cerebral artery occlusion (MCAO) model to allow local intraarterial delivery of drugs, one has the ability to mimic endovascular delivery of therapeutics. Using this model, the authors sought to maximize the protective potential of MMP-9 inhibition by intraarterial administration of an MMP-9 inhibitor, norcantharidin (NCTD). METHODS Spontaneously hypertensive rats were subjected to 90-minute MCAO followed immediately by local intraarterial administration of NCTD. The rats' neurobehavioral performances were scored according to the ladder rung walking test results and the Garcia neurological test for as long as 7 days after stroke. MRI was also conducted 24 hours after the stroke to assess infarct volume and BBB disruption. At the end of the experimental protocol, rat brains were used for active MMP-9 immunohistochemical analysis to assess the degree of MMP-9 inhibition. RESULTS NCTD-treated rats showed significantly better neurobehavioral scores for all days tested. MR images also depicted significantly decreased infarct volumes and BBB disruption 24 hours after stroke. Inhibition of MMP-9 expression in the ischemic region was depicted on immunohistochemical analysis, wherein treated rats showed decreased active MMP-9 staining compared with controls. CONCLUSIONS Intraarterial NCTD significantly improved outcome when administered at the time of reperfusion in a spontaneously hypertensive rat stroke model. This study suggests that supplementing endovascular revascularization with local neuroprotective drug therapy may be a viable therapeutic strategy.