Sodium cantharidate induces Apoptosis in breast cancer cells by regulating energy metabolism via the protein phosphatase 5-p53 axis.

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.

Charge-Reversal Polymer Nano-modulators for Photodynamic Immunotherapy of Cancer.

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 (SPDMC N) activated by tumor microenvironment (TME) for photodynamic immunotherapy of cancer. SPDMC 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 SPDMC 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, SPDMC 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.

Combination of Sodium Cantharidinate with Cisplatin Synergistically Hampers Growth of Cervical Cancer.

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.

Sodium cantharidinate, a novel anti-pancreatic cancer agent that activates functional p53.

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.

In Vitro and In Vivo Evaluation of SP94 Modified Liposomes Loaded with N-14NCTDA, a Norcantharimide Derivative for Hepatocellular Carcinoma-Targeting.

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 C14 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 IC50 of 15.395 ± 0.89 µg/mL, which is lower than that of NCTD-S (IC50 = 20.863 ± 0.56 µg/mL) and GAL-LPs (IC50 = 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.

Effect of sodium cantharidinate/vitamin B6 injection on survival, liver function, immune function, and quality of life in patients with hepatocellular carcinoma: Protocol for a meta-analysis.

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.

Inhibition of Jurkat T Cell Growth by N-farnesyl-norcantharimide Through Up-regulation of Tumor Suppressor Genes and Down-regulation of Genes for Steroid Biosynthesis, Metabolic Pathways and Fatty Acid Metabolism.

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-G1 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-G1 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.

Cantharidic acid induces apoptosis in human nasopharyngeal carcinoma cells through p38-mediated upregulation of caspase activation.

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.

Rapid profiling of cantharidin analogs in Mylabris phalerata Pallas by ultra-performance liquid chromatography-quadrupole time-of-flight-tandem mass spectrometry.

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 LD50 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.

Sodium cantharidate targets STAT3 and abrogates EGFR inhibitor resistance in osteosarcoma.

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.

Efficacy and safety of sodium cantharidinate and vitamin B6 injection for the treatment of digestive system neoplasms: a meta-analysis of randomized controlled trials.

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.

Clinical efficacy and safety of sodium cantharidinate plus chemotherapy in non-small-cell lung cancer: A systematic review and meta-analysis of 38 randomized controlled trials.

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.

Interactions of cantharidin-like inhibitors with human protein phosphatase-5 in a Mg2+ system: molecular dynamics and quantum calculations.

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 Mg2+ system.

Overview of Cantharidin and its Analogues.

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.

Cantharidic acid induces apoptosis through the p38 MAPK signaling pathway in human hepatocellular carcinoma.

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.

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.

One-step assembly of polymeric demethylcantharate prodrug/Akt1 shRNA complexes for enhanced cancer therapy.

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.

Comparative assessment of therapeutic safety of norcantharidin, N-farnesyloxy-norcantharimide, and N-farnesyl-norcantharimide against Jurkat T cells relative to human normal lymphoblast: A quantitative pilot study.

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.

Dual-Sensitive Charge-Conversional Polymeric Prodrug for Efficient Codelivery of Demethylcantharidin and Doxorubicin.

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 ß-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.

Synthesis of Canthardin Sulfanilamides and Their Acid Anhydride Analogues via a Ring-Opening Reaction of Activated Aziridines and Their Associated Pharmacological Effects.

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.