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Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, develops rapidly and has a high mortality rate. Relapsed metastasis is the most important factor affecting prognosis and is also the main cause of death for patients with HCC. Cantharidin is a kind of folk medicine for malignant tumors in China. Because of its cytotoxicity, the application of cantharidin is very limited. Magnesium demethylcantharidate (MDC) is a derivative of cantharidin independently developed by our laboratory. Our results show that MDC has anticancer activity and exhibited lower toxicity than cantharidin. However, whether MDC affects the invasion and metastasis of HCC cells and the underlying molecular mechanisms remain obscure. Transwell and Matrigel assays showed that MDC could effectively inhibit the invasion and metastasis of the HCC cell lines SMMC-7721 and SK-Hep1 in a dose-dependent manner. Moreover, MDC significantly inhibited the expression of invasion and metastasis related proteins MMP-2 and MMP-9. In addition, our study found that MDC inhibited the invasion and metastasis of HCC cell lines SMMC-7721 and SK-Hep1 by activating transcription factor FOXO1. Interestingly, the combination of MDC and sorafenib significantly inhibited the invasion and metastasis of HCC cell lines SMMC-7721 and SK-Hep1 compared with the single drug treatment via the activated transcription factor FOXO1. Our work revealed that MDC obviously inhibited the invasion and metastasis of HCC cells, and suggested that MDC could be a potential candidate molecule against the invasion and metastasis of HCC., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Breast cancer is the leading cause of cancer-related death in women worldwide, and despite multiple chemotherapeutic approaches, effective treatment strategies for advanced metastatic breast cancer are still lacking. Metabolic reprogramming is essential for tumor cell growth and propagation, and most cancers, including breast cancer, are accompanied by abnormalities in energy metabolism. Here, we confirmed that sodium cantharidate inhibited cell viability using the Cell Counting Kit-8, clonogenic assay, and Transwell assay. The cell cycle and apoptosis assays indicated that sodium cantharidate induced apoptosis and cell cycle arrest in breast cancer cells. Additionally, proteomic assays, western blots, and metabolic assays revealed that sodium cantharidate converted the metabolic phenotype of breast cancer cells from glycolysis to oxidative phosphorylation. Furthermore, bioinformatics analysis identified possible roles for p53 with respect to the effects of sodium cantharidate on breast cancer cells. Western blot, docking, and phosphatase assays revealed that the regulation of p53 activity by sodium cantharidate was related to its inhibition of protein phosphatase 5 activity. Moreover, sodium cantharidate significantly inhibited tumor growth in tumor-bearing nude mice. In summary, our study provides evidence for the use of sodium cantharidate as an effective and new therapeutic candidate for the treatment of human breast cancer in clinical trials., (Copyright © 2021. Published by Elsevier Inc.)

Nanomedicine can regulate the balance between cytotoxic T lymphocytes (CTLs) and suppressive regulatory T lymphocytes (Tregs), which however has been rarely exploited for cancer immunotherapy. We report a charge-reversal polymer nano-modulator (SP DMC N) activated by tumor microenvironment (TME) for photodynamic immunotherapy of cancer. SP DMC N is constructed by conjugating an immunomodulator (demethylcantharidin, DMC) to the side chains of a photodynamic polymer via an acid-liable linker. The negative charge of SP DMC N ensures its high stability in blood circulation and ideal tumor accumulation; exposure to acidic TME reverses its surface charge to positive, enhancing tumor penetration and locally releasing DMC. Upon near-infrared photoirradiation, SP DMC N generates singlet oxygen to ablate tumors and promote maturation of dendritic cells. Released DMC inhibits protein phosphatase 2 (PP2A) activity and decreases Tregs differentiation. Such combinational action induces a sharp increase in CTL/Treg ratio in TME and effectively inhibits both primary and distant tumors in living mice., (© 2021 Wiley-VCH GmbH.)

Despite the use of many types of chemotherapies for pancreatic cancer, optimal efficacy has not been obtained so far. Pancreatic cancer shows a high incidence of TP53 mutations, inactivating its tumor suppressor activity. In this study, we identified sodium cantharidinate as a novel, potential anti-pancreatic cancer agent that activates p53 function. Sodium cantharidinate reduced the viability of pancreatic cancer cells, including the human primary pancreatic cancer cells, PANC-1, AsPC-1, SW1990 and BXPC-3, in a dose-dependent manner. Sodium cantharidinate induced apoptosis and DNA damage of pancreatic cancer cells. Furthermore, proteome-wide sequencing analysis detected a marked perturbation in p53 signaling pathway on PANC-1 cells upon sodium cantharidinate. Consistent with the previous results, sodium cantharidinate treatment decreased Bcl-2 and mitochondrial cytochrome-c protein expression, as well as phosphorylation of MDM2; meanwhile, it increased the levels of cleaved-caspase-3, cleaved-caspase-9, cleaved-PARP, Bax, and phosphorylated p53, thus inducing the apoptosis of pancreatic cancer cells. The p53-activating effect of sodium cantharidinate was strongly abrogated by treatment with TP53-targeting shRNA. Moreover, sodium cantharidinate inhibited neoplasm growth via the JAK2-STAT3 pathway, which was inhibited by shRNA-TP53 and triggered by combination with gemcitabine. Combination therapy indicated that sodium cantharidinate and gemcitabine synergistically reduced ex vivo and in vivo growth of pancreatic neoplasm. Further docking studies revealed the different binding fates of sodium cantharidinate to activate wild-type p53 function. Thus, sodium cantharidinate could be a potential agent with promising anti-pancreatic cancer efficacy., (© 2020. Science China Press and Springer-Verlag GmbH Germany, part of Springer Nature.)

Background: Sodium cantharidinate (SC) has been broadly applied in lung cancer treatment in China, while its specific function in cervical cancer (CC), a great contributor to death of female reproductive system cancers, remains unclear. Our research evaluated the anti-tumor effects of SC in CC and the mechanism involved., Methods: First, cisplatin (DDP)-resistant Caski-1 and ME180 cell lines were developed and treated with SC. The effects of SC on CC cell growth were then evaluated. Subsequently, the genes targeted by SC were predicted via the bioinformatics website. The correlations between PTPN1 expression and tumor stage, lymph node metastasis and tumor differentiation were examined. We further conducted rescue experiments by overexpressing PTPN1 in CC cells, followed by SC and cisplatin treatments. The activation of the PI3K/AKT pathway in CC cells, and the effect of SC on the growth and drug resistance of Caski-1 cells in vivo were investigated., Results: The sensitivity of Caski-1 and ME180 cells to DDP was increased after SC treatment, which also enhanced the inhibitory effect of DDP on the cell growth. By prediction, we found that SC could target PTPN1. Patients with high expression of PTPN1 had higher clinical stage, lymph node metastasis and lower tumor differentiation. SC inhibited PTPN1 expression. Overexpression of PTPN1 attenuated the effect of SC. Furthermore, PTPN1 activated the PI3K/AKT pathway. Moreover, SC treatment inhibited the growth and drug resistance of Caski-1 cells in vivo., Conclusion: SC promotes drug sensitivity of CC cells to DDP by targeting PTPN1, thereby impairing the PI3K/AKT pathway., Competing Interests: The authors report no conflicts of interest in this work., (© 2021 Chen et al.)

The purpose of this research is to develop a liposomal drug delivery system, which can selectively target hepatocellular carcinoma (HCC) to deliver the antitumor agent N-14NCTDA, a C 14 alkyl chain norcantharimide derivative of norcantharidin. N-14NCTDA-loaded liposomes were successfully prepared by lipid membrane hydration and extrusion methods. SP94, a targeting peptide for HCC cells, was attached to the liposomes loaded with N-14NCTDA by the post-insertion method to obtain SP94 modified liposomes (SP94-LPs). SP94-LPs had a significant cytotoxicity against Hep G2 cells with the IC 50 of 15.395 ± 0.89 μg/mL, which is lower than that of NCTD-S (IC 50  = 20.863 ± 0.56 μg/mL) and GAL-LPs (IC 50  = 24.589 ± 1.02 μg/mL). Compared with conventional liposomes (Con-LPs), SP94-LPs showed greater cellular uptake in Hep G2 cells. Likewise, significant tumor suppression was achieved in H22 tumor-bearing mice which were treated with SP94-LPs. The tumor inhibition rate (IRw) of SP94-LPs was 82 ± 0.98%, obviously higher than that of GAL-LPs (69 ± 1.39%), Con-LPs (60 ± 2.78%), and NCTD-S (51 ± 3.67%). SP94-LPs exhibited a significant hepatocellular carcinoma-targeting activity in vitro and in vivo, which will provide a new alternative for hepatocellular carcinoma treatment in future. Graphical Abstract.

Background: Sodium cantharidinate/vitamin B6 (SC/VB6) injection, a famous insect-derived traditional Chinese medicine preparation, has been widely applied as a promising adjunctive drug for hepatocellular carcinoma (HCC). However, its exact clinical efficacy and safety is still not well investigated. In this study, we aimed to summarize the efficacy of SC/VB6 injection on survival, liver function, immune function, and quality of life (QoL) in patients with HCC through the meta-analysis., Methods: All available randomized controlled trials (RCTs) and high-quality prospective cohort studies that investigated the efficacy and safety of SC/VB6 for patients with HCC were searched from ten electronic databases including PubMed, Google Scholar, Cochrane Library, Excerpt Medica Database (Embase), Medline, Web of Science (WOS), Chinese Biomedical Literature Database (CBM), China National Knowledge Infrastructure (CNKI), China Scientific Journal Database (CSJ), and Wanfang Database. Papers in Chinese or English published from January 2000 to July 2020 will be included without any restrictions.Study selection and data extraction will be performed independently by 2 researchers. The clinical outcomes including overall survival (OS), QoL, liver function, immune function, and adverse events, were systematically evaluated. Review Manager 5.3 and Stata 14.0 were used for data analysis, and the quality of the clinical trials was also evaluated., Results: The results of this study will be published in a peer-reviewed journal, and provide a helpful evidence for clinicians to formulate the best postoperative adjuvant treatment strategy for HCC patients., Conclusion: Our study will draw an objective conclusion of the efficacy of SC/VB6 on survival, liver function, immune function, and QoL in patients with HCC., Inplasy Registration Number: INPLASY202070121.

We evaluated the protective effect and toxicity of extracts from Mylabris phalerata Pallas by measuring the activated partial thromboplastin time, prothrombin time, venous thrombosis and acute toxicity in rats. Results showed the petroleum ether and water fractions of M. phalerata inhibited thrombosis but hardly prolonged the activated partial thromboplastin time and prothrombin time in rats. The trichloromethane fraction had obvious toxicity with an LD 50 of 0.2 g/kg in vivo, and contained many cantharidin analogs (CAs) by ultra-performance liquid chromatography-quadrupole ion trap-tandem mass spectrometry (UPLC-QTRAP-MS/MS). CAs are the major potential bioactivity constituent in M. phalerata. An effective and reliable UPLC-QTRAP-MS/MS method was successfully developed to separate and identify CAs. The fragmentation patterns of five purified compounds were applied to elucidate the structure of their analogs. Thirty-four CAs were characterized or tentatively identified, eight of which are proposed to be novel compounds (13-17, 20, 21, 23), and their fragmentation patterns were investigated for the first time. Most importantly, a rapid and reliable UPLC-MS method was developed to identify the CAs of M. phalerata. This method has contributed to the discovery of most of these unknown analogs or their metabolites in M. phalerata effectively and quickly, and does not rely on limited chemical structural diversity libraries., (© 2020 John Wiley & Sons, Ltd.)

Cantharidic acid (CA) is the hydrolysis product of the acid anhydride cantharidin, which is a natural toxin secreted by several species of blister beetles. Several studies have indicated that as an inhibitor of protein phosphatase 2 (PP2A), CA induces apoptosis in various human cancer cells. However, the effect of CA on human nasopharyngeal carcinoma (NPC) cells and the underlying pathways have not been addressed. In our current study, we tested the hypothesis that CA treatment reduces the viability of human NPC cells (HONE-1, NPC-39, and NPC-BM) by inducing apoptosis. Results indicated that CA markedly reduced cell viability, which was revealed by the upregulation of caspase activation in extrinsic and intrinsic apoptosis pathways as well as the upregulation of extracellular-signal-regulated kinase 1/2 (ERK1/2), p38, and c-Jun N-terminal kinase 1/2 (JNK1/2) pathways. Coadministration of a p38 inhibitor (SB203580) with CA abolished the activation of caspase proteins. These findings indicated that CA treatment leads to apoptosis in human NPC cells through the upregulation of caspase activation, mediated particularly by the p38 pathway. Hence, CA is a promising therapeutic agent for human NPC., (© 2020 Wiley Periodicals, Inc.)

Background/aim: To evaluate the anti-cancer mechanism of N-Farnesyl-norcantharimide (NC15)., Materials and Methods: The viability of NC15-treated human leukemic Jurkat T (JKT) cells was assessed using the Kit-8 cell counting method. Flow cytometry analysis, human apoptosis antibody array assay, and whole genome sequencing were adopted to investigate the mechanism underlying the anti-cancer activity of NC15 in JKT cells., Results: The growth inhibition rates of NC15 in JKT cells were about 80% and 95% after treatment with 8 μmol/l NC15 for 24 and 48 h, respectively. The percentages of NC15-treated JKT cells in the sub-G 1 phase at 24 and 48 h were 22.0% and 34.3%, respectively, in contrast to the 1.5% in the control. Next-generation sequencing showed that many tumor suppressor genes (TSG) were up-regulated, while many genes associated with steroid biosynthesis, metabolic pathways, and fatty acid metabolism were down-regulated., Conclusion: NC15 can reduce the cell viability and increase the percentage of JKT cells in the sub-G 1 phase by up-regulating TSG and related genes, and down-regulating the genes for steroid biosynthesis, metabolic pathways and fatty acid metabolism, instead of through apoptosis., (Copyright© 2020, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Osteosarcoma is the most common primary malignant bone tumor in children and adolescents. Overactive EGFR signaling is frequently seen in osteosarcoma cells, and represents a potential therapeutic target. However, feedback activation of STAT3 after EGFR inhibition is linked to treatment resistance, suggesting that combined EGFR/STAT3 inhibition may be needed to overcome this effect. Cantharidin and its analogues have shown strong anticancer effects, including STAT3 inhibition, in several tumor cells. Therefore, we investigated the effects of sodium cantharidate (SC), either as monotherapy and in combination with the EGFR inhibitor erlotinib, on STAT3 activation and osteosarcoma cell growth. Cell viability, migration, and apoptosis assays were performed in human MG63 and U2OS cells, and MG63 xenografts were generated in nude mice to verify the suppression of tumor growth in vivo. Additionally, western blotting and immunohistochemistry were used to verify the STAT3 and EGFR phosphorylation statuses in xenografts. We found that SC repressed cell viability and migration and induced apoptosis in vitro, while combined SC and erlotinib treatment enhanced osteosarcoma growth suppression by preventing feedback activation of STAT3. These data support further development of cantharidin-based combination therapies for metastatic and recurrent/refractory osteosarcoma.

What Is Known and Objective: Sodium cantharidinate has been widely used in lung cancer treatment in China. To investigate whether sodium cantharidinate improves clinical effectiveness in non-small-cell lung cancer, we systematically re-evaluated all related studies., Methods: All studies of cantharidinate for non-small-cell lung cancers (NSCLC) were selected from the MEDLINE, EMBASE, Web of Science (ISI), China National Knowledge Infrastructure Database (CNKI), Chinese Scientific Journals Full-Text Database (VIP), Wanfang, China Biological Medicine Database (CBM), Cochrane Central Register of Controlled Trials (CENTRAL), Chinese clinical trial registry (Chi-CTR), WHO International Clinical Trials Registry Platform (WHO-ICTRP) and US-clinical trials databases (established to September 2017). Their quality was evaluated using the Cochrane evaluation handbook of randomized controlled trials (RCTs) (5.1.0). The data were extracted following PICO principles and synthesized through meta-analysis., Results and Discussion: We included 38 trials involving 2845 patients, but most trials had an unclear risk of bias. Sodium cantharidinate could increase the objective response rate (ORR) (1.52, (1.40-1.66]), disease control rate (DCR) (1.20, [1.16-1.25]) and quality of life (QOL) (1.76, [1.56-1.98]), but not the 1-year overall survival (OS) rate (1.16, [0.91-1.47]) and the 2-year OS rate (1.21, [0.51-2.91]). Subgroup analysis revealed that sodium cantharidinate and vitamin B6 at 0.5, 0.4 or 0.3 mg, and cantharidinate at 0.5 mg could all increase the ORR and DCR. Cantharidinate therapy had a lower risk of neutropenia (0.58, [0.50-0.67]), thrombocytopenia (0.57, [0.45-0.72]), gastrointestinal reaction (0.65, [0.52-0.82]) and nausea/vomiting (0.56, [0.41-0.76]) than that of chemotherapy alone. Sensitivity analysis showed that the results had good robustness., What Is New and Conclusion: Current evidence reveals that sodium cantharidinate can improve tumour responses and QOL with a lower risk of haematotoxicity and gastrointestinal toxicity than chemotherapy alone in NSCLC. However, the evidence does not indicate that it can improve long-term survival rates., (© 2018 John Wiley & Sons Ltd.)

Objective: To systematically evaluate the efficacy and safety of sodium cantharidinate and vitamin B6 (SC/B6) combined with conventional medical treatment (CMT) for the treatment of patients with advanced digestive system neoplasms (DSNs)., Methods: The Cochrane Library, Embase, PubMed, Web of Science, Chinese Scientific Journal Database (VIP), China National Knowledge Infrastructure, and Wanfang databases were searched for clinical trials using SC/B6 for DSNs. Outcome measures, including therapeutic efficacy, quality of life (QoL), and adverse events, were extracted and systematically evaluated., Results: Data from 24 trials including 1,825 advanced DSN patients were included. Compared with CMT alone, its combination with SC/B6 significantly improved the patients' overall response rate (OR =2.25, 95% CI =1.83-2.76, P <0.00001), disease control rate (OR =2.41, 95% CI =1.85-3.15, P <0.00001), and QoL improvement rate (OR =2.75, 95% CI =2.13-3.55, P <0.00001). Moreover, adverse events caused by chemotherapy, including leukopenia, nausea and vomiting, gastrointestinal side effects, hepatotoxicity, diarrhea, transaminase disorder, myelosuppression, anorexia, and anemia, were significantly alleviated ( P <0.05) when SC/B6 was applied to DSN patients. Nephrotoxicity, thrombocytopenia, hand-foot syndrome, and oral mucositis were not significantly alleviated in patients receiving combination therapy ( P >0.05)., Conclusion: The combination of SC/B6 and CMT is more effective in treating DSNs than CMT alone. This combination alleviates the adverse effects associated with chemotherapy and improves the QoL of DSN patients, and its application in the clinic is worth promoting., Competing Interests: Disclosure The authors report no conflicts of interest in this work.

The serine/threonine protein phosphatase type 5 (PP5) is a promising target for designing new antitumor drugs. This enzyme is a member of the PPP phosphatases gene family, which catalyzes a dephosphorylation reaction: a regulatory process in the signal transduction pathway that controls various biological processes. The aim of this work is to study and compare the inhibition of PP5 by ten cantharidin-like inhibitors in order to bring about contributions relevant to the better comprehension of their inhibitory activity. In this theoretical investigation, we used molecular dynamics techniques to understand the role of key interactions that occur in the protein active site; QM calculations were employed to study the interaction mode of these inhibitors in the enzyme. In addition, atoms in molecules (AIM) calculations were carried out to characterize the chemical bonds among the atoms involved and investigate the orbital interactions with their respective energy values. The obtained results suggest that the Arg275, Asn303, His304, His352, Arg400, His427, Glu428, Val429, Tyr451, and Phe446 residues favorably contribute to the interactions between inhibitors and PP5. However, the Asp271 and Asp244 amino acid residues do not favor such interactions for some inhibitors. Through the QM calculations, we can suggest that the reactional energy of the coordination mechanism of these inhibitors in the PP5 active site is quite important and is responsible for the inhibitory activity. The AIM technique employed in this work was essential to get a better comprehension of the transition states acquired from the mechanism simulation. This work offers insights of how cantharidin-like inhibitors interact with human PP5, potentially allowing the design of more specific and even less cytotoxic drugs for cancer treatments. Graphical Abstract Interactions of cantharidin-like inhibitors with human protein phosphatase-5 in a Mg 2+ system.

Cantharidin analogs exhibit anticancer activities, including apoptosis. However, the molecular mechanisms underlying the effects of cantharidic acid (CA), a cantharidin analog, on apoptosis in hepatocellular carcinoma (HCC) cells are unclear. Thus, in this study, we evaluated the anticancer activities of CA by investigating its ability to trigger apoptosis in SK-Hep-1 cells. Our data demonstrated that CA effectively inhibited the proliferation of SK-Hep-1 cells in a dose-dependent manner. Furthermore, CA effectively triggered cell cycle arrest and induced apoptosis, as determined by flow cytometric analysis. Western blotting revealed that CA significantly activated proapoptotic signaling including caspase-3, -8, and -9 in SK-Hep-1 cells. Moreover, treatment of SK-Hep-1 cells with CA induced the activation of ERK, p38, and c-Jun N-terminal kinase. Moreover, the inhibition of p38 by specific inhibitors abolished CA-induced cell apoptosis. In conclusion, our results indicated that CA induces apoptosis in SK-Hep-1 cells through a p38-mediated apoptotic pathway and could be a new HCC therapeutic agent., (© 2017 Wiley Periodicals, Inc.)

Background: Cantharidin has been categorized as highly toxicant in Chinese medicine. But cantharidin can efficiently treat different types of diseases, such as molluscum contagiosum. While cantharidin is quite useful, unfortunately, due to its side effects, increasing regulations have limited access to this useful therapeutic option. Cantharidin's toxic effects have caused it to fall into disuse for most legitimate medical purposes. Although cantharidin generates effects and its advantages must be realized. Recently, cancer affects people's life more and more. Because cantharidin can treat some cancers, so solutions must be used to reduce side effects. This review aims to describe some its analogues, several efficient methods to inhibit the side effects of cantharidin and pharmacogenomics of cantharidin., Methods: We searched for research about cantharidin by entering the database. Then evaluated these papers and analyzed their founding, solution, mechanism, etc., and targeted to screen the papers related to the content of our research, and then sorted them out in accordance with the solution, mechanism research and other content. Finally, these content was unified into a framework., Results: Some cantharidin's analogues were found that they show some similar functions to cantharidin and we found that norcantharidin, acylthiourea derivatives, cantharidinamides, anhydride-modified derivatives and other derivatives have less side effects. The modified cantharidin analogues reduce toxicity in hepatocytes. Cantharidin consists of a six-ring and a five-ring, the moiety of oxygen on the six-ring and the anhydride section exhibit biochemical activity. Protein phosphatases are associated with many cellular processes including apoptosis, cell cycle progression and so on. Cantharidin can cause apoptosis and double-stand breakage of DNA. Cantharidin and norcantharidin can efficiently inhibit the activity of mammalian and plant protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) in vivo. Cantharidin inhibits PP5 at the nanomolar level with an IC50 value of 600 nM. PP5 can manage the cellular survival, death, proliferation and other some intracellular biological activities in mammals. After cantharidin's treatment, the level of EtPP5 mRNA expression was downregulated. Their also can be used to inhibit the Glutathione S-transferases (GSTs), angiogenesis and the expression of A549 human lung cancer cells, trigger eryptosis and induced bladder cancer cell apoptosis. We found that using Vitamin C and ginsenosides and translating cantharidin into nanoparticles can minimize the cantharidin side effects in the patients., Conclusion: Cantharidin can inhibit various tumor cell lines. Cantharidin causes both DNA single- and double- strand breaks and induces apoptosis. Although cantharidin shows some toxicity for human, its anti-cancer effects should be taken seriously. Several viable methods can help solve this problem. The most important pharmacogenomics of cantharidin is that cantharidin can inhibit PPs, because PPs are associated with many cellular processes. This prospect is very broad and needs to continue studying., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

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 C 14 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 (IC 50 =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 -1 h, t 1/2z =1.347±0.519h) than Con-Lipo (AUC=2.319±0.121mgL -1 h, 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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

This report demonstrated a one-step assembly for co-delivering chemotherapeutics and therapeutic nucleic acids, constructed by integrating drug molecules into a nucleic acid condensing polymeric prodrug through degradable linkages. Demethylcantharate was selected as the model drug and pre-modified by esterifying its two carboxylic groups with 2-hydroxyethyl acrylate. The synthesized demethylcantharate diacrylate was then used to polymerize with linear polyethyleneimine (PEI 423) through a one-step Michael-addition reaction. The obtained cationic polymeric demethylcantharate prodrug was used to pack Akt1 shRNA into complexes through a one-step assembly. The formed complexes could release the parent drug demethylcantharate and Akt1 shRNA through the hydrolysis of ester bonds. Cellular assays involving cell uptake, cytotoxicity, and cell migration indicated that demethylcantharate and Akt1 shRNA co-delivered in the present form significantly and synergistically suppress the growth and metastasis of three human cancer cells. This work suggests that incorporating drug molecules into a nucleic acid-packing cationic polymer as a polymeric prodrug in a degradable form is a highly convenient and efficient way to co-deliver drugs and nucleic acids for cancer therapy., (Copyright © 2016 Elsevier B.V. All rights reserved.)

A tumor is a complicated system, and tumor cells are typically heterogeneous in many aspects. Polymeric drug delivery nanocarriers sensitive to a single type of biosignals may not release cargos effectively in all tumor cells, leading to low therapeutic efficacy. To address the challenges, here, we demonstrated a pH/reduction dual-sensitive charge-conversional polymeric prodrug strategy for efficient codelivery. Reduction-sensitive disulfide group and acid-labile anticancer drug (demethylcantharidin, DMC)-conjugated β-carboxylic amide group were repeatedly and regularly introduced into copolymer chain simultaneously via facile CuAAC click polymerization. The obtained multifunctional polymeric prodrug P(DMC), mPEG-b-poly(disulfide-alt-demethylcantharidin)-b-mPEG was further utilized for DOX encapsulation. Under tumor tissue/cell microenvironments (pH 6.5 and 10 mM GSH), the DOX-loaded polymeric prodrug nanoparticles (P(DMC)@DOX NPs) performed surface negative-to-positive charge conversion and accelerated/sufficient release of DMC and DOX. The remarkably enhanced cellular internalization and cytotoxicity in vitro, especially against DOX-resistant SMMC-7721 cells, were demonstrated. P(DMC)@DOX NPs in vivo also exhibited higher tumor accumulation and improved antitumor efficiency compared to P(SA)@DOX NPs with one drug and without charge-conversion ability. The desired multifunctional polymeric prodrug strategy brings a new opportunity for cancer chemotherapy.

The therapeutic safety of an anticancer drug is one of the most important concerns of the physician treating the cancer patient. Half maximal inhibitory concentration (IC50) and hillslope are usually used to represent the strength and sensitivity of an anticancer drug on cancer cells. The therapeutic safety of the anticancer drug can be assessed by comparing the IC50 and hillslope of anticancer drugs on cancer cells relative to normal cells. Since there are situations where "more anticancer activity" implies "more toxicity," the safety of an anticancer drug in these situations is hard to evaluate by using IC50 and hillslope alone. In a previous study, the "net effect" index was devised to represent the net therapeutic effects of one anticancer drug relative to the other. However, the therapeutic safety of one specific anticancer drug alone was not defined in the "net effect" index. This study introduced the "safety index (SI)" to quantify the degree of safety of an anticancer drug by using 4-parameter logistic model on cancer cells relative to normal cells. The therapeutic safety of norcantharidin (NCTD), N-farnesyloxy-norcantharimide (NOC15), and N-farnesyl-norcantharimide (NC15) in the treatment of Jurkat T cells relative to human normal lymphoblast was compared using the newly defined SI. We found that the SI of NOC15 and NC15 was significantly higher than that of NCTD, suggesting that both NOC15 and NC15 can damage more cancer cells and less normal cells than NCTD. We conclude that both NOC15 and NC15 are safer anticancer drugs than NCTD in the treatment of Jurkat T cells relative to human normal lymphoblast. The SI can be further applied to the screening, developments, and applications of anticancer drugs in the future.

To investigate the stability and the conversion rules of cantharidin and cantharidic acid contained in the Mylabris aqueous solution under different conditions. The contents of cantharidin and cantharidic acid under different conditions (pH, temperature and light) were determined by HPLC-TQ-MS. The results showed that the content of cantharidin was gradually decreased with the rising of pH value, while on the contrary, the content of cantharidic acid was increased gradually; after Mylabris aqueous solution with different pH values were placed at 25, 40 ℃ and 25 ℃ respectively for lighting for 90 days, the samples were collected for analysis. The results showed the contents of cantharidin and cantharidic acid were changed greatly in the first 10 days, mainly including the decrease of cantharidic acid and increase of cantharidin when the solution was acidic, and the increase of cantharidic acid and decrease of cantharidin when the solution was alkaline. After that, the contents of the above two components basically remained stable. This study revealed that pH was the main factor to affect the contents of cantharidin and cantharidic acid, and they could be converted into each other with the changes of pH value. High temperature and light can accelerate the speed of achieving such balance. The study can provide certain reference for the quality control of the medicines using the Mylabris as raw material., Competing Interests: The authors of this article and the planning committee members and staff have no relevant financial relationships with commercial interests to disclose., (Copyright© by the Chinese Pharmaceutical Association.)

The cantharidinimide derivatives, 5a-h, including sulfanilamides containing pyrimidyl, pyrazinyl, hydrogen, thiazolyl, and oxazolyl groups were synthesized. Modification of cantharidinimide by means of the reaction of activated aziridine ring opening led to the discovery of a novel class of antitumor compounds. The analogues 10i-k, 11l-n, 12o-p, and 16q-s were obtained from treating cantharidinimide 6 and analogues (7, 8, and 13) with activated aziridines, which produced a series of ring-opened products including normal and abnormal types. Some of these compounds showed cytotoxic effects in vitro against HL-60, Hep3B, MCF7, and MDA-MB-231 cancer cells. The most potent cytostatic compound, N-cantharidinimido-sulfamethazine (5a), exhibited anti-HL-60 and anti-Hep3B cell activities. Two compounds 5g and 5h displayed slight effects on the Hep3B cell line, while the other compounds produced no response in these four cell lines.

This study investigated the anticancer effects of N-farnesyloxy-norcantharimide (NOC15), a newly synthesized norcantharidin (NCTD) analogue, on human leukemic Jurkat T cells and the signaling pathway underlying its effects. We found that the half maximal inhibitory concentration (IC50) of NOC15 on Jurkat T cells is 1.4 μmol/l, which is 11.14-fold (=15.6÷1.4) smaller than the 15.6 μmol/l of NCTD on Jurkat T cells, whereas the IC50 of NOC15 on human normal lymphoblast (HNL) is 207.9 μmol/l, which is 8.17-fold (=1698.0÷207.8) smaller than the 1698.0 μmol/l of NCTD on HNL cells. These results indicated that NOC15 exerts a higher anticancer effect on Jurkat T cells and has higher toxicity toward HNL cells than NCTD. Thus, NOC15 is 1.36-fold (=11.14÷8.17) beneficial as an anticancer agent toward Jurkat T cells compared with NCTD. Moreover, NOC15 can increase the percentage of cells in the sub-G1 phase and reduce the cell viability of Jurkat T cells, stimulate p38 and extracellular signal-regulated protein kinase 1/2 (ERK1/2) of mitogen-activated protein kinases (MAPKs) signaling pathway, and inhibit calcineurin expression and interleukin-2 (IL-2) production. However, NOC15 exerted no effects on the Jun-N-terminal kinase 1/2 (JNK1/2) signaling pathway, the production of IL-8, and tumor necrosis factor-α. We conclude that the anticancer activity of the newly synthesized NOC15 is 1.36-fold beneficial than NCTD as an anticancer agent and that NOC15 can increase the percentage of cells in the sub-G1 phase through the stimulation of p38 and ERK1/2 of the MAPK signaling pathway and the inhibition of calcineurin expression and IL-2 production. The NOC15 may have the potential of being developed into an anticancer agent in the future.

This study investigated the anticancer effects of two newly synthesized norcantharidin analogs, N-farnesyloxy-norcantharimide (NOC15) and N-farnesyl-norcantharimide (NC15), in L1210 cells and in a syngeneic mouse leukemia model (L1210 cell line plus DBA/2 mice). We found that the half-maximal inhibitory concentration (IC50) of NOC15 and NC15 on L1210 cells is 1.56 and 2.62 μmol/l, respectively, and that the IC50 of NOC15 and NC15 on human normal lymphoblast is 207.9 and 2569 μmol/l, respectively. In cell cycle analysis, NOC15 could increase the sub-G1 phase, whereas NC15 could induce G2/M arrest. Annexin-V apoptosis assay indicated that both NOC15 and NC15 could induce cell apoptosis. In the syngeneic mouse leukemia model, both NOC15 and NC15 could increase the survival days of mice and decrease the tumor weight. Moreover, both NOC15 and NC15 could retard the increase in peripheral blood leukocyte count due to L1210 cells. In the subcutaneous (s.c.) group, the treatment with NOC15 could retard the decrease in the weight of the liver and the spleen caused by L1210 cells, whereas the treatment with NC15 could retard the decrease in the weight of the spleen caused by L1210 cells. We conclude that the new compounds NOC15 and NC15 have strong anticancer activity and low toxicity both in vitro and in vivo. NOC15 and NC15 may have the potential to be developed into anticancer agents in the future.

Glutathione S-transferases (GSTs) play a key role in detoxification of xenobiotics in organisms. However, their other functions, especially response to the natural toxin cantharidin produced by beetles in the Meloidae and Oedemeridae families, are less known. We obtained GST cDNAs from three sources: Cydia pomonella (CpGSTd1), Sus scrofa (SsGSTα1), and Triticum aestivum (TaGSTf3). The predicted molecular mass is 24.19, 25.28 and 24.49 kDa, respectively. These proteins contain typical N-terminal and C-terminal domains. Recombinant GSTs were heterologously expressed in Escherichia coli as soluble fusion proteins. Their optimal activities are exhibited at pH 7.0-7.5 at 30 °C. Activity of CpGSTd1 is strongly inhibited by cantharidin and cantharidic acid, but is only slightly suppressed by the demethylated analog of cantharidin and cantharidic acid. Enzymatic assays revealed that cantharidin has no effect on SsGSTα1 activity, while it significantly stimulates TaGSTf3 activity, with an EC50 value of 0.3852 mM. Activities of these proteins are potently inhibited by the known GST competitive inhibitor: S-hexylglutathione (GTX). Our results suggest that these GSTs from different sources share similar structural and biochemical characteristics. Our results also suggest that CpGSTd1 might act as a binding protein with cantharidin and its analogs., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: To evaluate the efficacy of mannatide combined with sodium cantharidate vitamin B6 in the treatment of malignant pleural effusions., Materials and Methods: Data for 69 patients with malignant pleural effusions who did not receive systemic chemotherapy were collected. Injection into the thorax using mannatide combined with sodium cantharidate vitamin B6 was performed for 37 patients in the experimental group and mannatide combined with cisplatin for 32 patients in the control group. Objective responses, KPS (Karnofsky Scoring) and incidences of side effects between the two groups were compared., Results: 13 patients reached CR (complete response) and 11 PR (partial response) in the experimental group, while 12 patients reached CR and 9 PR in the control group, the difference in overall objective responses between the two groups not being significant (66.7% vs 63.6%, p=0.806). However, improvement of KPS in the experimental group was greater than in the control group; total side-effect incidences during the period of treatment were 22.2% (8/36) and 54.5% (18/33), respectively (p=0.006)., Conclusions: Regimen of mannatide combined with sodium cantharidate vitamin B6 had better improvement in quality-of-life and symptom relief, with a lower side-effect incidence in treatment of malignant pleural effusions.

Silver complexes have been shown to possess antimicrobial and anticancer properties. Ag-SP-DNC, a novel silver and singly protonated dehydronorcantharidin complex, was synthesized in our previous study. In this study, we offer evidence that Ag-SP-DNC elicits a reactive oxygen species (ROS)-mediated mitochondrial apoptosis in lung cancer cells. Ag-SP-DNC inhibited the growth of A549 cells by inducing G2/M phase cell cycle arrest and apoptosis. Ag-SP-DNC induced apoptosis was associated with the levels of intracellular ROS. The further study revealed that Ag-SP-DNC disrupted the mitochondrial membrane potential, induced the caspase-3 activation and led to the translocation of apoptosis inducing factor and endonucleaseG to the nucleus. These findings have important implications for the development of silver complexes for anticancer applications., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

Objective: The present study observed the clinical outcomes of the sodium cantharidinate/vitamin B6/GP regimen in the treatment of middle-late stage Non-small-cell lung carcinoma (NSCLC)., Materials and Methods: Eighty-six cases of NSCLC were selected and randomized into two groups. Forty-five cases in the treatment group were subject to 30 ml cantharidinate/vitamin B6+ GP regimen, for four 21-day cycles. Forty-one cases in the control group were subject to regular GP regimen, for four 21-day cycles., Results: The effectiveness rate was 57.8% in the treatment group and 36.6% in the control group, suggesting statistical difference (P < 0.05); life quality in the treatment group was significantly better than that in the control group (P < 0.05); the occurrence rate of the toxic/adverse effects were significantly lower in the treatment group compared with that in the control group (P < 0.05)., Conclusion: Sodium cantharidinate/vitamin B6/GP regimen had fair effectiveness and synergistically improved the clinical outcomes. It lowered the toxic/adverse effects and its application is worth further investigation and promotion.

Objective: The present study evaluated the treatment effectiveness of sodium cantharidinate/vitamin B6 in patient with middle/late stage primary liver cancer., Materials and Methods: A 3-month follow-up study on 104 patients with primary liver cancer was carried out. Regular medication treatment was applied to 41 patients and sodium cantharidinate/vitamin B6 combined with the regular medication was applied to 63 patients. The treatment effectiveness and prognosis were evaluated using the statistical methods., Results: At the end of the treatment, no significant difference was detected between the two groups; 1-month follow-up survey showed that in the treatment group, the death rate was lower, the treatment gain was maintained longer and the tumor morphology was maintained better, compared with the control group; 3-month follow-up study showed that there was not significance difference between the two groups., Conclusion: Sodium cantharidinate/vitamin B6 might be used as auxiliary drug in patients with primary liver cancer and could improve the treatment outcomes for a short-term period.

The different types of cell death occurring in HCT116 colorectal cancer cell after the treatment of cisplatin, carboplatin, oxaliplatin, DMC, Pt(NH3)2(demethylcantharidin:DMC) and Pt(R,R-1,2-diaminocyclohexane: DACH)(DMC) were examined. Pt(NH3)2(DMC) and Pt(R,R-DACH)(DMC) are the two DMC-integrated platinum complexes:Pt(R,R-DACH)(DMC) with the same Pt moiety as oxaliplatin and Pt(NH3)2(DMC) akin to carboplatin. Using the light scattering properties of cells combined with propidium iodide (PI) red fluorescence to distinguish between early apoptotic and necrotic cells, the results confirmed that apoptosis, which triggered by cisplatin, carboplatin and oxaliplatin, was the major type of cell death, while the major DMC-induced cell death type was necrosis. The increase in the necrotic cell population was observed after Pt((NH3)2(DMC) treatment when compared with carboplatin; therefore, the DMC ligand in Pt(NH3)2(DMC) contributing to cell death was demonstrated. However, the DMC ligand in Pt(R,RDACH)( DMC) failed to elevate the necrotic cell population significantly in contrast to oxaliplatin, thus Pt(R,R-DACH) in Pt(R,RDACH)( DMC) dominantly contributed to cell death. The IC50 value (antiproliferative activity) reflects the net effect of drugs on cell proliferation resulting from inhibition of cell growth and division, and induction of cell death. The sub-G1 populations representing the sum of the amounts of late apoptotic cells and necrotic cells after the treatment of cispatin, carboplatin, oxaliplatin, DMC, Pt(NH3)2(DMC) and Pt(R,R-DACH)(DMC) were found to be not correlated with the corresponding IC50 values;therefore, the rate of cell growth and division inhibition rather than the rate of induction of cell death dictated to the IC50 values. This combined analysis of IC50 values and the sub-G1 population data also reveals that the platinum compounds containing R,R-DACH are most efficient in inhibiting cell growth and division, while carboplatin induces cell death most rapidly. When the Pt-DNA adducts are believed to be major cytotoxic species, the combined analysis of the IC50 values and the sub-G1 population data infers that the R,R-DACH-Pt-1,2-d(GpG) intrastrand cross-links caused by oxaliplatin treatment are more effective in inducing cell growth and division inhibition, while the (NH3)2Pt-1,3- d(GpXpG) intrastrand cross-links caused by carboplatin treatment can trigger cell death more rapidly. The rate of cell growth and division inhibition and the cell death rate induced by the main cisplatin-DNA adducts:(NH3)2Pt-1,2-d(GpG) intrastrand cross-links lie in between.

Invasion and metastasis are major causes of malignant tumor-associated mortality. The present study aimed to investigate the molecular events underlying inhibitory effect of N-benzylcantharidinamide, a novel synthetic analog of cantharidin, on matrix metalloproteinase-9 (MMP-9)-mediated invasion in highly metastatic hepatocellular carcinoma Hep3B cells. In this investigation, among six analogs of cantharidin, only N-benzylcantharidinamide has the inhibitory action on MMP-9 expression at non-toxic dose. The MMP-9 expression and invasion of Hep3B cells were significantly suppressed by treatment of N-benzylcantharidinamide in a dose-dependent manner. On the other hand, the transcriptional activity of MMP-9 promoter and nuclear levels of NF-κB and AP-1 as the main transcriptional factors inducing MMP-9 expression were not affected by it although the level of MMP-9 mRNA was reduced by treatment of N-benzylcantharidinamide. Interestingly, the stability of MMP-9 mRNA was significantly reduced by N-benzylcantharidinamide-treatment. In addition, the cytosolic translocation of human antigen R (HuR), which results in the increase of MMP-9 mRNA stability through interaction of HuR with 3'-untranslated region of MMP-9 mRNA, was suppressed by treatment of N-benzylcantharidinamide, in a dose-dependent manner. Taken together, it was demonstrated, for the first time, that N-benzylcantharidinamide suppresses MMP-9 expression by reducing HuR-mediated MMP-9 mRNA stability for the inhibition of invasive potential in highly metastatic Hep3B cells., (Copyright © 2014 Elsevier Inc. All rights reserved.)

A Pt (IV) complex which combined the bioactivities of both oxaliplatin and demethylcantharidin (DMC) is synthesized and delivered by a polymer-drug conjugate for combination chemotherapy. Oxaliplatin is released from the polymer-drug conjugate within cancer cell by reduction to attack nuclear DNA, while a dose of DMC is also hydrolyzed subsequently to block DNA damage-induced defense mechanisms by serine/threonine phosphatase 2A (PP2A) inhibition. In vitro evaluation shows that the polymer-drug conjugate with dual modes of action upon cancer cells displays higher cytotoxicity against SKOV-3 cells than that of free drugs. This enhanced cytotoxicity is attributed to the synergistic effect between oxaliplatin and DMC, as well as the effective intracellular internalization of the micelles observed by confocal laser scanning microscopy (CLSM) imaging., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cantharidin, a natural toxin produced by the blister beetle, was reported to be toxic to some pests, but the mechanism of its toxicity in insects remains undefined. We found that cantharidin exerted in vivo and in vitro inhibitory effects on protein serine/threonine phosphatases (PSPs) of Plutella xylostella. Five PSP genes, PP1, PP2A, PP4, PP5, and PP6, were cloned from P. xylostella. Phosphatase domain alignment showed a high similarity. Recombinant PxPP5 (rPxPP5) was expressed in Escherichia coli and purified. Cantharidin and its 11 analogs were used to perform the rPxPP5 activity inhibition assay in vitro. Cantharidin strongly inhibited rPxPP5 activity competitively, with an IC50 of 0.38 μM. All analogs also showed inhibitory activity, with an IC50 of 7.42-538.38 μM. The rank of IC50 values was found to be consistent with their toxicities in P. xylostella larvae with a correlation coefficient (R(2)) of 0.87. 3D models of the phosphatase domains of these five PxPSPs were constructed which superimposed well indicating a high structural similarity demonstrating that the chemicals used may be inhibitors of the other four PxPSPs. Binding model analysis of cantharidin and its analogs which interacted with PxPP5 showed that the cantharidin-derived moiety was anchored to the active site, explaining their inhibitory effect on rPxPP5 in vitro. Results of binding free energy calculations are also well in line with their inhibition effects on rPxPP5, with a correlation coefficient (R(2)) of 0.72. In light of the above results we argue that protein serine/threonine phosphatases are the targets of cantharidin and its analogs acting on P. xylostella.

Background: [corrected] Cantharidin, and its derivatives can not only inhibit the proliferation of tumor cells, but can also induce tumor cell apoptosis. It shows cantharidin exhibits a wide range of reactivity in anticancer. The objective of this paper was to study the inhibitory effect of sodium cantharidinate on human hepatoma HepG2 cells., Materials and Methods: MTT assay was used to detect the proliferation of HepG2 cells, and immunohisto-chemical method was used to detect the change in VEGF, protein level, and to determine the inhibitory effect of sodium cantharidinate on human hepatoma HepG2 cells., Results: As results, sodium cantharidinate significantly inhibited the growth of HepG2 cells in a time-and dose-dependent manner., Conclusion: We conclude that sodium cantharidinate has an inhibitory effect on human hepatoma HepG2 cells.

Chemical and pharmacological information on cantharidin-based small molecules was analyzed. The review summarizes new facts about blister beetles' metabolites for the period 2006-2012. General synthetic approaches to cantharidin-based small molecules as well as their chemical transformations and biological activities related to cantharidin, norcantharidin, cantharidimide, and norcantharimide analogs, especially their inhibitory activity of phosphoprotein phosphatases in cancer treatment, were discussed in this mini review, which could help to design new small molecule modulators for other biological models., (© 2013 John Wiley & Sons A/S.)

Three new transition metal complexes [Mn2(DCA)2(bipy)2]·5H2O (1), [M2(DCA)2(bipy)2(H2O)]·10H2O(M=Ni(II)(2);Zn(II)(3)), (DCA=demethylcantharate, 7-oxabicyclo[2,2,1]heptane-2,3-dicarboxylate, C8H8O5) were synthesized and characterized by elemental analysis, molar conductance, infrared spectra and X-ray diffraction techniques. Each metal ion was six-coordinated in complexes. Complex 1 has a Mn2O2 center. Complexes 2 and 3 have asymmetric binuclear structure. Great amount of intermolecular hydrogen-bonding and π-π(*) stacking interactions were formed in these complex structures. The DNA-binding properties of complexes were investigated by electronic absorption spectra and viscosity measurements. The DNA binding constants Kb/(Lmol(-1)) were 1.71×10(4) (1), 2.62×10(4) (2) and 1.59×10(4) (3) at 298 K. The complexes could quench the intrinsic fluorescence of bovine serum albumin (BSA) strongly through static quenching. The protein binding constants Ka/(L mol(-1)) were 7.27×10(4) (1), 4.55×10(4) (2) and 7.87×10(4) L mol(-1) (3) and binding site was one. The complexes bind more tightly with DNA and BSA than with ligands. Complexes 1 and 3 had stronger inhibition ratios than Na2(DCA) against human hepatoma cells (SMMC-7721) lines and human gastric cancer cells (MGC80-3) lines in vitro. Complex 3 showed the strongest antiproliferative activity against SMMC-7721 (IC50=29.46±2.12 μmol L(-1)) and MGC80-3 (IC50=27.02±2.38 μmol L(-1)), which shows potential in anti-cancer drug development., (Copyright © 2013 Elsevier B.V. All rights reserved.)

A novel strategy for combination chemotherapy (platinum and demethylcantharidin) via a polymer-(tandem drugs) conjugate for enhanced cancer treatment is demonstrated. Cisplatin can be released inside cell by reduction to attack DNA, while DMC will be hydrolyzed subsequently to block DNA-damage-induced defense mechanisms by serine/threonine phosphatase PP2A inhibition. Synergistic effect of the polymer-(tandem drugs) conjugate causes complete suppression of H22 liver tumor xenografts without recurrence., (Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

This review summarizes the progress that has been made recently in the medicinal chemistry of cantharidin, a potent antitumor agent from traditional Chinese medicine. Thousands of analogs have been synthesized on the basis of cantharidin, a part of which shows excellent properties, in particular, norcantharidin and norcantharimide. Despite the enormous efforts made, the intriguing bioactivities, mechanism, indications, and their interplay are still ill-defined. This review provides our up-to-date understanding in connection with the therapeutic use, mechanism, structure-activity relationship (SAR) and interesting properties of cantharidin analogs. Considerable development in the design of cantharidin analogs, in combination with mechanistic studies, has laid a foundation for transforming novel antitumor drugs into the clinic.

Norcantharidin (NCTD) is currently used for anticancer therapy but the exact mechanism of action remains unknown. Pre-replicative complexes (pre-RCs) are essential for cell DNA replication and highly related to malignant proliferation. Here, we examined the inhibitory effect of NCTD on pre-RC components in HepG2 cells. We showed that NCTD induced degradation of Cdc6 and Mcm2 in a dose-dependent manner. Under 100 μM NCTD concentration, about 70% of Cdc6 and 50% of Mcm2 were degraded. In addition, the nuclear translocation of Mcm6 was inhibited by NCTD. Further studies aiming at G1 synchronous cells showed that, NCTD reduced the chromatin-bound Cdc6, Mcm2 and Mcm6. Moreover, the cells were blocked from entering the S phase and accumulated at the G1 phase when released synchronously into the cell cycle. Consistently, the DNA replication was inhibited by NCTD. Finally, the combination NCTD with Cdc6 depletion lead to more severe cytotoxicity (88%) than NCTD (52%) and Cdc6 depletion (39%) alone. A synergic cytotoxicity was observed between Cdc6 depletion and NCTD. In conclusion, our results demonstrate that NCTD inhibits pre-RC assembly; subsequently blocks the G1 to S transition; and inhibits DNA replication in HepG2 cells. Pre-RCs are an intriguing target for cancer therapy, which merits further investigations for anticancer development.

Four new transition metal complexes (Habtz)(2)[M(DCA)(2)]·6H(2)O (M=Co(II) (1), Ni(II) (2), Cu(II) (3), Zn(II) (4); DCA=demethylcantharate, 7-oxabicyclo [2.2.1]heptane-2,3-dicarboxylate, C(8)H(8)O(5); Habtz=2-aminobenzothiazole acid, C(7)H(7)N(2)S) were synthesized and characterized by elemental analysis, molar conductance, infrared spectra and thermogravimetric analysis. The coordination number of complex was six. The X-ray diffraction analysis indicated that complex 3 crystallized in the triclinic crystal system with P1¯ space group. The DNA-binding properties of the complexes were investigated by electronic absorption spectra, fluorescence spectra, viscosity measurements. Title complexes could bind to DNA via partial intercalative mode. The K(b) of the complexes were 5.33×10(4) (1), 7.04×10(4) (2), 9.91×10(4) (3) and 5.03×10(4) L mol(-1) (4). The results of agarose gel electrophoresis showed that Cu(II) complex could cleave pBR322 plasmid DNA via radical-based mechanism. The complexes could quench the intrinsic fluorescence of bovine serum albumin (BSA) through a static quenching with the binding constants K(a) of 1.11×10(4) (1), 1.24×10(6) (2), 8.42×10(5) (3) and 1.75×10(4) L mol(-1) (4). The complexes had intense antiproliferative activities against human hepatoma cell lines (SMMC7721) and human gastric cancer cells (MGC80-3) lines in vitro. Cu(II) complex had the strongest activity against human gastric cancer cells., (Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.)

Three novel transition metal complexes (Hapy)(2)[M(DCA)(2)]·6H(2)O (M = Mn(II) (1), Ni(II) (2), Cu(II) (3); DCA = demethylcantharate, 7-oxabicyclo[2.2.1]heptane-2,3-dicarboxylate, C(8)H(8)O(5); Hapy = 2-aminopyridine acid, C(5)H(7)N(2)) were synthesized and characterized by elemental analysis, infrared spectra, thermogravimetric analysis and X-ray diffraction. DNA binding properties of the complexes were investigated by electronic absorption spectra, fluorescence spectra and viscosity measurements. Results indicated the complexes could bind to DNA through partial intercalation mode with binding constants K ( b )/(L·mol(-1)) of 1.91 × 10(4) (1), 5.13 × 10(4) (2) and 1.12 × 10(5) (3) at 298 K. Meanwhile, the interactions of the complexes with BSA were also studied by fluorescence spectra. The results suggested that the complexes could quench the fluorescence of BSA through static quenching with the binding constants K ( A )/(L·mol(-1)) of 1.44 × 10(6) (1), 1.14 × 10(7) (2) and 2.98 × 10(4) (3). And the main contribution was tryptophan residues of BSA. The antiproliferative activity test revealed that complexes showed more intense inhibition ratios against human hepatoma cells lines and human gastric cancer cells lines in vitro. Copper(II) complex (3) possesses the strongest inhibition ratio against human hepatoma cells.

The lab made an effort to prepare some biological active cantharidinimines by heating the reactant 1 and 2a-g, 5h-i and 7j-r amines to suitable temperature with ethanol to provide 18 N-thiazolyl-, sulfanyl-, aminopyridyl-, bromopyridyl-, alkylpyridyl- and hydroxypyridylcantharidinimines 3a-g, 4a-c, 6h-i and 8j-r in yield of 4-77% (Chart 1). These cantharidinimine derivatives were tested for their capabilities to suppress growth of the human carcinoma cell lines, HL-60, MCF7, Neuro-2a and A549, because the incidence rate is more prominent in Asian countries than western countries. Compounds 3c-d and 6h-i were found to have some antitumor activity in HL-60 but less activity in MCF cell and compounds 8j-l displayed some inhibition effects to A549 cell line, but less effect to Neuro-2a cell line. Compounds 8m-r had no cytotoxic effect against both cell lines. The cytotoxic effects of these cantharidinimine compounds seemed to be better than the cantharidinimide compounds which we had mentioned several years ago.

Using small molecule probes to understand gene function is an attractive approach that allows functional characterization of genes that are dispensable in standard laboratory conditions and provides insight into the mode of action of these compounds. Using chemogenomic assays we previously identified yeast Crg1, an uncharacterized SAM-dependent methyltransferase, as a novel interactor of the protein phosphatase inhibitor cantharidin. In this study we used a combinatorial approach that exploits contemporary high-throughput techniques available in Saccharomyces cerevisiae combined with rigorous biological follow-up to characterize the interaction of Crg1 with cantharidin. Biochemical analysis of this enzyme followed by a systematic analysis of the interactome and lipidome of CRG1 mutants revealed that Crg1, a stress-responsive SAM-dependent methyltransferase, methylates cantharidin in vitro. Chemogenomic assays uncovered that lipid-related processes are essential for cantharidin resistance in cells sensitized by deletion of the CRG1 gene. Lipidome-wide analysis of mutants further showed that cantharidin induces alterations in glycerophospholipid and sphingolipid abundance in a Crg1-dependent manner. We propose that Crg1 is a small molecule methyltransferase important for maintaining lipid homeostasis in response to drug perturbation. This approach demonstrates the value of combining chemical genomics with other systems-based methods for characterizing proteins and elucidating previously unknown mechanisms of action of small molecule inhibitors., Competing Interests: The authors have declared that no competing interests exist.

Objective: To study the effect of sodium cantharidinate on the angiogenesis of nude mice with human gastric cancer., Methods: Nude mice xenograft models of human gastric cancer were established by injecting gastric carcinoma cell BGC823 into peritoneal. Expression of VEGF and MVD labeling by CD34 in human gastric cancer cells were measured by immunohistochemistry., Results: Expression scores of VEGF in medium dose and high dose group with sodium cantharidinate treatment were lower than those in low dose and control group (P < 0.01). There was no significant difference between medium dose and high dose group or low dose and control group (P > 0.05). MVD values in medium and high dose group with sodium cantharidinate treatment were lower than those in low dose and control group (P < 0.01), but there was no significant difference between medium dose and high dose group (P > 0.05)., Conclusions: sodium cantharidinate can inhibit the growth of the tumor by down-regulating VEGF expression of the tumour cell and the angiogenesis of the tumour.

Failure to cure many cancers once they are disseminated has been attributed to the presence of resistant cancer stem cells. Cantharidin, a natural compound isolated from the beetles and other insects has been traditionally used as anticancer agent, but limited by its significant toxicity. It has shown that cantharidin can force cancer cells prematurely into cell cycle and subsequently induce apoptotic cell death through the inhibition of protein phosphatase 2A (PP2A). In this study, we showed that a synthesized analog of cantharidin, LB1, with significant PP2A inhibition activity but without apparent toxicity, greatly enhanced the effectiveness of the standard anti-sarcoma chemotherapeutic agent, doxorubicin (DOX), in the xenograft growth inhibition and lung metastases prevention of an aggressive sarcoma derived from transformed mesenchymal stem cells in syngeneic rats. We report here on the possibility of, pharmacologic inhibition of PP2A with low toxicity cantharidin derivatives may be a useful strategy to enhance the effectiveness of DNA-damaged chemotherapeutic drugs against stem cell-derived cancer., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Objectives: To establish a HPLC method for determination of N-methylcantharidimide in dogs' plasma and to study the pharmacokinetics of N-methylcantharidimide in dogs'., Method: The plasma samples were extracted by methanol. The acetonitrile and the purified water composed mobile phase. The flow rate was 0. 7 mL x min(-1), ultraviolet detection wavelength was at 212 nm., Result: The calibration curve was linear over the range from 0.01-10.0 mg x L(-1) with a correlation coefficiency of 0.996 3. The lower limit of quantitation was 0.01 mg x L(-1). The mean recovery was 92.3%. the relative standard deviation (RSD) of intra-day and inter-day were all less than 10%. After intravenous administration of N-methylcantharidimide with 3 dosages of 10, 15, 20 mg x kg(-1) to dogs, the corresponding distribution half-livers (t1/2alpha) were 1.8, 2.1, 1.7 min, and the elimination half-lives (t1/2beta) were 144,139, 146 min, respectively., Conclusion: This method is convenient, accurate and reliable. It can be used for determination of N-methylcantharidimide in dogs' plasma and pharmacokinetic studies.

Previous research indicates that cantharidin, norcantharidin and their analogues exhibit anticancer activity due to their inhibition of cancer cell lines such as HL60, HT29 and L1210. The anticancer activities of cantharidin, norcantharidin and their analogues involve the suppression of serine/threonine protein phosphatases (PPs) activity. However, cantharidin is not suitable for cancer therapy because of its high cytotoxicity in vitro (IC(50) = 21 microM in primary cultured rat hepatocytes). In this study, synthetic cantharidin analogues with a structure of aminothiazole compounds 3-9 and a structure of anhydride compounds 10-12 were screened for anticancer activities and cytotoxic effects on human hepatocellular carcinoma cell (HCC) lines HepG2, Sk-Hep1, and primary cultured rat hepatocytes. Experimental results indicated that compounds 3-9 did not perform as expected with regard to anticancer activity and exhibited lower cytotoxicity. Compound 10 promoted apoptosis in HepG2 (IC(50) = 62 microM) and SK-Hep1(IC(50) = 151 microM) cell lines. Compounds 11 and 12 had anticancer potential similar to that of compound 10. After treatment with compounds 3-12, primary cultured rat hepatocytes exhibited no cytotoxicity (IC(50) > 200 microM). By investigating the structure-activity relationship (SAR) of these analogues as a whole, this study suggests that the anhydride ether oxygen such as in cantharidin, norcantharidin and compounds 10-12 may be correlated with HCC survival suppression. The results further suggest that the elimination of bridging ether oxygen on the ring, such as in compounds 10-12, can decrease cytotoxicity., (2010 Elsevier Masson SAS. All rights reserved.)

Cantharidin (1) and norcantharidin (2) are potent protein phosphatase 1 and 2A inhibitors that also display high levels of anticancer activity against a broad range of tumor cells lines. Surprisingly, Delta-5,6-ethyl norcantharidin (3, cis-tetrahydrofurano[3,4-c]furan-1,3-dione) displays neither phosphatase inhibition nor anticancer activity. This suggests that the 5,6-ethyl bridge is pivotal to both anti-cancer and protein phosphatase activity. Additionally bioisosteric replacement of the ethereal oxygen has no effect on biological activity nor does modification of the anhydride moiety. Unlike the parent norcantharidin, anhydride ring opening has no effect on either protein phosphatase inhibition or anti-cancer activity. Additionally, this work highlights the discovery of the octyl substituted, cis-5-benzyl-2-hexyltetrahydro-2H,3aH-pyrrolo[3,4-c]pyrrole-1,3-dione, 9p, and the octyl substituted, cis-octyltetrahydro-5H-furo[3,4-c]pyrrole-4,6-dione, 8p, as two new cytotoxic agents which are equipotent (9p) with, and more potent (8p) than norcantharidin., (Crown Copyright (c) 2010. Published by Elsevier Masson SAS. All rights reserved.)

Natural product extracts are a rich source of small molecules that display antitumor activity. Cantharidin, in the form of the dried body of the Chinese blister beetles: Mylabris phalerata or M. cichorii, displays antitumor activity and induces apoptosis in many types of tumor cells. Cantharidin has been used as an anticancer agent by the Chinese for the treatment of hepatoma and oesophageal carcinoma for a long time. Although cantharidin is a natural toxin that possesses potent anti-tumor properties, its clinical application is limited due to severe side-effects and highly toxic nature. Therefore, some modified cantharidin analogues are synthesized chemically in order to achieve a comparable antitumour property to the mother compound but simultaneously produce a less toxic effect on non-cancer cells. In recent years, based on the structure of cantharidin, there has been intense interest in developing potent and selective inhibitors of PP1 and PP2A on tumour cells. Though numerous analogues of cantharidin have been synthesized and researched with tumour cell lines, there is little success on clinical application because of the potential toxicity of cantharidin derivates. The focus of this review is to describe how cantharidin and cantharidin derivates participate in antitumour processes in tumour cells, and discuss the molecular mechanisms of cantharidin and cantharidin derivates on tumour cells.

Cantharidin (1) and its derivatives are of significant interest as serine/threonine protein phosphatase 1 and 2A inhibitors. Additionally, compounds of this type have displayed growth inhibition of various tumour cell lines. To further explore both of these inhibition pathways, a number of amide-acid norcantharidin analogues (15-26) were prepared. Compounds 23 and 24, containing two carboxylic acid residues, showed good PP1 and PP2A activity, with IC(50) values of approximately 15 and approximately 3 mum, respectively. Substituted aromatic amide analogues 45, 48, 49, 52, 53, and 54 also displayed good PP1 and PP2A inhibition, with IC(50) values in the range of 15-10 microM (PP1) and 11-5 microM (PP2A). However, bulky ortho substituents on the aromatic ring caused the aromatic ring to be skewed from the NCO planarity, leading to a decrease in PP1 and PP2A inhibition. A number of analogues, 20, 22, 25 and 46, showed excellent tumour growth inhibition, with 46 in particular being more potent than the lead, norcantharidin 2.