Blocking elevated p38 MAPK restores efferocytosis and inflammatory resolution in the elderly.

Increasing age alters innate immune-mediated responses; however, the mechanisms underpinning these changes in humans are not fully understood. Using a human dermal model of acute inflammation, we found that, although inflammatory onset is similar between young and elderly individuals, the resolution phase was substantially impaired in elderly individuals. This arose from a reduction in T cell immunoglobulin mucin receptor-4 (TIM-4), a phosphatidylserine receptor expressed on macrophages that enables the engulfment of apoptotic bodies, so-called efferocytosis. Reduced TIM-4 in elderly individuals was caused by an elevation in macrophage p38 mitogen-activated protein kinase (MAPK) activity. Administering an orally active p38 inhibitor to elderly individuals rescued TIM-4 expression, cleared apoptotic bodies and restored a macrophage resolution phenotype. Thus, inhibiting p38 in elderly individuals rejuvenated their resolution response to be more similar to that of younger people. This is the first resolution defect identified in humans that has been successfully reversed, thereby highlighting the tractability of targeting pro-resolution biology to treat diseases driven by chronic inflammation.

Cantharidin (1%), podophyllin (5%), and salicylic acid (30%) formulation in recalcitrant plantar warts: Analysis of 48 patients.

A plantar wart is a benign hyperplasia that appears on the feet due to the human papillomavirus (HPV). One method used for the treatment of recalcitrant plantar warts, those lasting over 2 years or persisting after at least two treatment attempts, is the cantharidin (1%), podophyllin (5%), and salicylic acid (30%) formulation, also known as the CPS formulation. Although this method is in use, there are few studies on it. This study's objective was to ascertain its cure rate. For this retrospective observational study, we reviewed the medical records of patients treated with the CPS formulation at a podiatric clinic specializing in plantar wart treatment. Our sample size was 48 subjects. The CPS formulation had a cure rate of 62.5%. Out of the cured patients, 86.67% (26/30) required one or two applications. There was no observable correlation (p > 0.05) between wart resolution and virus biotype, evolution time, patient's morphological and clinical attributes, location, number of warts, or preceding treatments. The CPS formulation presents a relatively high efficacy rate for treating recalcitrant HPV plantar warts. Still, additional studies are necessary to evaluate its safety and efficiency.

Covalent Binding of Reactive Anhydride of Cantharidin to Biological Amines.

Cantharidin is a terpenoid from coleoptera beetles. Cantharidin has been used to treat molluscum contagiosum and some types of tumors. Cantharidin is highly toxic, and cantharidin poisoning and fatal cases have been reported worldwide. The mechanisms underlying cantharidin-induced toxicity remain unclear. Cantharidin contains anhydride, which may react with biologic amines. This study aimed to examine the chemical reactivity of cantharidin toward nucleophiles and characterize adducts of cantharidin with biologic amines in vitro and in mice. Here two types of conjugates were formed in the incubation of cantharidin under physiologic conditions with free amino acids, a mimic peptide, or amine-containing compounds, respectively. Amide-type conjugates were produced by the binding of cantharidin anhydride with the primary amino group of biologic amines. Imide-type conjugates were generated from the dehydration and cyclization of amide-type conjugates. The structure of the conjugates was characterized by using high-resolution mass spectrometry. We introduced the 14N/15N and 79Br/81Br isotope signatures to confirm the formation of conjugates using L-(ε)15N-lysine, L-lysine-15N2, and bromine-tagged hydrazine, respectively. The structure of imide conjugate was also confirmed by nuclear magnetic resonance experiments. Furthermore, the amide and imide conjugates of cantharidin with amino acids or N-acetyl-lysine were detected in mouse liver and urine. Cantharidin was found to modify lysine residue proteins in mouse liver. Pan-cytochrome P450 inhibitor 1-aminobenzotriazole significantly increased the urine cantharidin-N-acetyl-lysine conjugates, whereas it decreased cantharidin metabolites. In summary, cantharidin anhydride can covalently bind to biologic amines nonenzymatically, which facilitates a better understanding of the role of nonenzymatic reactivity in cantharidin poisoning. SIGNIFICANCE STATEMENT: Anhydride moiety of cantharidin can covalently bind to the primary amino group of biological amines nonenzymatically. Amide and imide conjugates were generated after the covalent binding of cantharidin anhydride with the primary amino groups of amino acids, a mimic peptide, and protein lysine residues. The structure of conjugates was confirmed by 14N/15N and 79Br/81Br isotope signatures using isotope-tagged reagents and nuclear magnetic resonance experiments. This study will facilitate the understanding of the role of nonenzymatic reactivity in cantharidin poisoning.

Network toxicology, molecular docking technology, and experimental verification revealed the mechanism of cantharidin-induced testicular injury in mice.

As a protein kinase inhibitor, cantharidin (CTD) exhibits antitumor activities. However, CTD is highly toxic, thereby limiting clinical applications. Moreover, relatively few studies have investigated CTD-induced reproductive toxicity, thus the underlying mechanism remains unclear. In this study, the toxic effects of CTD on mouse testis were confirmed in vivo and the potential mechanism was predicted by network toxicology (NT) and molecular docking technology. Proteins involved in the signaling pathways and core targets were verified. The results showed that different concentrations of CTD induced weight loss increased the testicular coefficient, and caused obvious pathological damage to testicular cells. The NT results showed that the main targets of CTD-induced testicular injury (TI) included AKT1, Caspase 3, Bcl-2, and Bax. The results of pathway enrichment analysis showed that CTD-induced TI was closely related to apoptosis and the PI3K/AKT and HIF-1 signaling pathways. Molecular docking methods confirmed high affinity between CTD and key targets. Western blot analysis showed that CTD inhibited expression of PI3K, AKT, and the anti-apoptotic protein Bcl-2, while promoting expression of the pro-apoptotic proteins Bax and Caspase 3. These results suggest that CTD-induced TI involves multiple targets and pathways, and the underlying mechanism was associated with inhibition of the apoptosis-related PI3K/AKT signaling pathway.

Design and synthesis of cantharidin derivative DCZ5418 as a TRIP13 inhibitor with anti-multiple myeloma activity in vitro and in vivo.

Natural product cantharidin can inhibit multiple myeloma cell growth in vitro, while serious adverse effects limited its clinical application. Therefore, the structural modification of cantharidin is needed. Herein, inspired by the structural similarity of the aliphatic endocyclic moiety in cantharidin and TRIP13 inhibitor DCZ0415, we designed and synthesized DCZ5418 and its nineteen derivatives. The molecular docking study indicated that DCZ5418 had a similar binding mode to TRIP13 protein as DCZ0415 while with a stronger docking score. Moreover, the bioassay studies of the MM-cells viability inhibition, TRIP13 protein binding affinity and enzyme inhibiting activity showed that DCZ5418 had good anti-MM activity in vitro and definite interaction with TRIP13 protein. The acute toxicity test of DCZ5418 showed less toxicity in vivo than cantharidin. Furthermore, DCZ5418 showed good anti-MM effects in vivo with a lower dose administration than DCZ0415 (15 mg/kg vs 25 mg/kg) on the tumor xenograft models. Thus, we obtained a new TRIP13 inhibitor DCZ5418 with improved safety and good activity in vivo, which provides a new example of lead optimization by using the structural fragments of natural products.

In vitro immune-depression and anti-inflammatory activities of cantharidin on gilthead seabream (Sparus aurata) leucocytes activated by λ-carrageenan.

Cantharidin is a natural compound with known therapeutic applications in humans. The aim of this study was to investigate the in vitro effects of cantharidin on gilthead seabream (Sparus aurata) head kidney leucocytes (HKL) stimulated with λ-carrageenan. HKLs were incubated for 24 h with cantharidin (0, 2.5 and 5 µg mL-1) and λ-carrageenan (0 and 1000 µg mL-1). The results showed that HKL viability only decreased by 15.2% after incubated with 5 µg mL-1 of cantharidin and λ-carrageenan. Cantharidin increased the peroxidase activity of HKLs only when incubated in combination with λ-carrageenan. Besides this, cantharidin inhibited the respiratory burst and phagocytic activities. Furthermore, cantharidin induced morphological changes in HKLs (apoptotic and vacuolization signs) that were enhanced when incubated with λ-carrageenan. Considering the analysis of the selected gene expression studied in HKLs [NF-κB subunits (rela, relb, crel, nfkb1, nfkb2), proinflammatory cytokines (il1b, tnfa), anti-inflammatory cytokines (il10, tgfb) and caspases (casp1, casp3, casp8, casp9)], although λ-carrageenan up-regulated the expression of the proinflammatory gene il1b, λ-carrageenan and cantharidin down-regulated its expression in HKLs. In addition, cantharidin up-regulated casp3 and casp9 expression. The casp3 and casp9 gene expression was down-regulated while casp1 gene expression was up-regulated in HKLs incubated with both cantharidin and λ-carrageenan. All the effects of cantharidin are related to its inhibitory effect on protein phosphatases, which induce apoptosis at long exposure times, and minimize the effects of λ-carrageenan. The present results provide detailed insight into the immune-depressive and anti-inflammatory properties of cantharidin on immune cells, which could be of interest to the aquaculture sector.

Application of cantharidin, retinoic acid cream and salicylic acidy in multiple palmoplantar warts.

OBJECTIVE: Multiple palmoplantar warts, caused by human papillomavirus (HPV) infection, were investigated for clinical efficacy using cantharidin, retinoic acid cream, and salicylic acid cream. METHODS: A total of 110 patients with multiple palmoplantar warts were enrolled. The experimental group (54 cases) received a 1:1:1 combination (CRS) of 0.25% cantharidin, 0.1% retinoic acid cream, and 5% salicylic acid, applied with pressurized encapsulation for 8 h every night, three times per week. The control group (56 cases) underwent conventional liquid nitrogen freezing. Monthly follow-ups assessed cure rate, effective rate, dermatological life quality index (DLQI), visual analog scale (VAS), and cost, with evaluations conducted after 3 months. RESULTS: The treatment group exhibited a cure rate of 85.19% and a total effective rate of 96.30%, surpassing the control group with rates of 39.29% and 51.79%, respectively (p < 0.05). The treatment group's DLQI score (1.84 ± 1.06) was significantly lower than the control group's score (6.04 ± 1.78) (p = 0.0005). Additionally, the treatment group's VAS score (1.84 ± 1.06) was notably lower than the control group's score (8.56 ± 1.07) (p < 0.0001). The treatment group's total cost (43.20 ± 2.85) was markedly lower than the control group's cost (206.38 ± 90.81), with a statistically significant difference (p < 0.0001). CONCLUSION: The combination of cantharidin, retinoic acid cream, and salicylic acid with local encapsulation is a safe, effective, economical, and convenient treatment method for multiple palmoplantar warts, exhibiting few side effects and showing promise.

Analysis of cantharidin-induced kidney injury and the protective mechanism of resveratrol in mice determined by liquid chromatography/mass spectrometry-based metabonomics.

Cantharidin (CTD) is the main active component in the traditional Chinese medicine Mylabris and an effective anti-tumor agent. However, it is relatively toxic and exhibits nephrotoxicity, which limits its clinical use. However, its toxic mechanism is not clear. The toxic effects of CTD exposure on the kidney and the protective effect of resveratrol (RES) were studied in a mouse model, by determination of serum biochemical and renal antioxidant indicators, histopathological and ultrastructural observation, and metabonomics. After CTD exposure, serum uric acid, creatinine, and tissue oxidative stress indicators increased, and the renal glomerular and tubular epithelial cells showed clear pathological damage. Ultrastructure observation revealed marked mitochondrial swelling, endoplasmic reticulum dilation, and the presence of autophagy lysosomes in glomerular epithelial cells. RES ameliorated the renal injury induced by CTD. Metabonomics analysis indicated that CTD can induce apoptosis and oxidative damage in kidney cells, mainly by disrupting sphingolipid and glutathione metabolism, increasing sphingosine and sphingomyelin levels, and decreasing glutathione levels. RES counteracts these effects by regulating renal cell proliferation, the inflammatory response, oxidative stress, and apoptosis, by improving the levels of phosphatidylcholine (PC), LysoPC, and lysophosphatidyl glycerol in the glycerophospholipid metabolism pathway, thereby reducing CTD-induced nephrotoxicity. The mechanisms of CTD-induced renal injury and the protective effect of RES were revealed by metabonomics, providing a basis for evaluating clinical treatment regimens to reduce CTD-induced nephrotoxicity.

Functional studies of McSTE24, McCYP305a1, and McJHEH, three essential genes act in cantharidin biosynthesis in the blister beetle (Coleoptera: Meloidae).

Cantharidin is a toxic defensive substance secreted by most blister beetles when attacked. It has been used to treat many complex diseases since ancient times and has recently regained popularity as an anticancer agent. However, the detailed mechanism of the cantharidin biosynthesis has not been completely addressed. In this study, we cloned McSTE24 (encoding STE24 endopeptidase) from terpenoid backbone pathway, McCYP305a1 (encoding cytochrome P450, family 305) and McJHEH [encoding subfamily A, polypeptide 1 and juvenile hormone (JH) epoxide hydrolase] associated to JH synthesis/degradation in the blister beetle Mylabris cichorii (Linnaeus, 1758, Coleoptera: Meloidae). Expression pattern analyses across developmental stages in adult males revealed that the expressions of 3 transcripts were closely linked to cantharidin titer exclusively during the peak period of cantharidin synthesis (20-25 days old). In contrast, at other stages, these genes may primarily regulate different biological processes. When RNA interference with double-stranded RNA suppressed the expressions of the 3 genes individually, significant reductions in cantharidin production were observed in males and also in females following McJHEH knockdown, indicating that these 3 genes might primarily contribute to cantharidin biosynthesis in males, but not in females, while females could self-synthesis a small amount of cantharidin. These findings support the previously hypothesized sexual dimorphism in cantharidin biosynthesis during the adult phase. McCYP305a1 collaborates with its upstream gene McSTE24 in cantharidin biosynthesis, while McJHEH independently regulates cantharidin biosynthesis in males.

Cantharidin.

Cantharidin is the toxic component of blister beetles of the genus Epicauta. Cantharidin is a potent vesicant which causes blisters, erosions, and ulcerations in the gastrointestinal and urinary tracts, and can cause myocardial necrosis. Blister beetles are found over most of North America and specifically contaminate alfalfa at harvest. History of alfalfa feeding, with colic, dysuria, hypocalcemia, and hypomagnesemia are suggestive of blister beetle toxicosis. Myocardial damage causes increased serum cardiac troponin 1. Tentative diagnosis can be made by finding the beetles in feed or ingesta. Definitive diagnosis requires detection of cantharidin in urine or gastric contents. Treatment involves ending exposure, decreasing absorption, controlling pain, using gastroprotectants, and fluids and electrolyte replacement. Prognosis is guarded to poor.

The role of protein phosphatase 2A (PP2A) in the unfolded protein response (UPR) of plants.

Protein phosphatase 2A (PP2A) is a key regulator of plant growth and development, but its role in the endoplasmic reticulum (ER) stress response remains elusive. In this study, we investigated the function of PP2A under ER stress using loss-of-function mutants of ROOTS CURL of NAPHTHYLPHTHALAMIC ACID1 (RCN1), a regulatory A1 subunit isoform of Arabidopsis PP2A. RCN1 mutants (rcn1-1 and rcn1-2) exhibited reduced sensitivity to tunicamycin (TM), an inhibitor of N-linked glycosylation and inducer of unfolded protein response (UPR) gene expression, resulting in less severe effects compared to wild-type plants (Ws-2 and Col-0). TM negatively impacted PP2A activity in Col-0 plants but did not significantly affect rcn1-2 plants. Additionally, TM treatment did not influence the transcription levels of the PP2AA1(RCN1), 2, and 3 genes in Col-0 plants. Cantharidin, a PP2A inhibitor, exacerbated growth defects in rcn1 plants and alleviated TM-induced growth inhibition in Ws-2 and Col-0 plants. Furthermore, cantharidin treatment mitigated TM hypersensitivity in ire1a&b and bzip28&60 mutants. These findings suggest that PP2A activity is essential for an efficient UPR in Arabidopsis.

Cantharidin induces apoptosis of human triple negative breast cancer cells through mir-607-mediated downregulation of EGFR.

BACKGROUND: Triple negative breast cancer (TNBC) is a major subtype of breast cancer, with limited therapeutic drugs in clinical. Epidermal growth factor receptor (EGFR) is reported to be overexpressed in various TNBC cells. Cantharidin is an effective ingredient in many clinical traditional Chinese medicine preparations, such as Delisheng injection, Aidi injection, Disodium cantharidinate and vitamin B6 injection. Previous studies showed that cantharidin had satisfactory pharmacological activity on a variety of tumors. In this study, we aimed to study the therapeutic potential of cantharidin for TNBC treatment by targeting EGFR, and expound its novel regulator miR-607. METHODS: The effect of cantharidin on breast cancer in vivo was evaluated by 4T1 mice model. Then the effects of cantharidin on TNBC cells was assessed by the MTT, colony formation, and AnnexinV-PE/7AAD staining. Cantharidin acts on EGFR were verified using the cell membrane chromatography, RT-PCR, Western blotting, MTT, and so on. Mechanistic studies were explored by dual-luciferase report assay, RT-PCR, western blotting, and immunofluorescence staining assay. RESULTS: Cantharidin inhibited TNBC cell growth and induce apoptosis by targeting EGFR. miR-607 was a novel EGFR regulator and exhibited suppressive functions on TNBC cell behaviors. Mechanistic study showed that cantharidin blocked the downstream PI3K/AKT/mTOR and ERK/MAPK signaling pathway. CONCLUSION: Our results revealed that cantharidin may be served as a potential candidate for TNBC treatment by miR-607-mediated downregulation of EGFR.

Transcriptomic profiling and differential analysis reveal the renal toxicity mechanisms of mice under cantharidin exposure.

Cantharidin (CTD), extracted from the traditional Chinese medicine mylabris, has shown significant curative effects against a variety of tumors, but its clinical application is limited by its high toxicity. Studies have revealed that CTD can cause toxicity in the kidneys; however, the underlying molecular mechanisms remain unclear. In this study, we investigated the toxic effects in mouse kidneys following CTD treatment by pathological and ultrastructure observations, biochemical index detection, and transcriptomics, and explored the underlying molecular mechanisms by RNA sequencing (RNA-seq). The results showed that after CTD exposure, the kidneys had different degrees of pathological damage, altered uric acid and creatinine levels in serum, and the antioxidant indexes in tissues were significantly increased. These changes were more pronounced at medium and high doses of CTD. RNA-seq analysis revealed 674 differentially expressed genes compared with the control group, of which 131 were upregulated and 543 were downregulated. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses showed that many differentially expressed genes were closely related to the stress response, the CIDE protein family, and the transporter superfamily, as well as the MAPK, AMPK, and HIF-1 pathways. The reliability of the RNA-seq results was verified by qRT-PCR of the six target genes. These findings offer insight into the molecular mechanisms of renal toxicity caused by CTD and provide an important theoretical basis for the clinical treatment of CTD-induced nephrotoxicity.

Integrative analysis of the molecular signature of target genes involved in the antitumor effects of cantharidin on hepatocellular carcinoma.

BACKGROUND: Cantharidin (CTD) is the active ingredient of Chinese medicine, which has been traditionally used in multiple cancers treatment, especially in hepatocellular carcinoma (HCC). However, a comprehensive analysis of the CTD-related molecular mechanism is still necessary to understand its functions in HCC treatment. This study aimed to reveal the novel molecular targets and regulatory networks of CTD in HCC. METHODS: A model of H22 tumour-bearing mice was constructed, and the function of CTD in tumour growth was evaluated. An integrated approach of CTD associated transcriptional profiling and biological systems analysis was used to identify key regulators involved in antitumour pathways. The identified differential expression patterns were supported by the results of Gene Ontology (GO) term and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyse, and by protein-protein interaction (PPI) network construction. The relationships between gene expression and tumour immunity were evaluated using Tumour Immune Estimation Resource (TIMER). Prognostic value was analyzed with Kaplan-Meier plotter. RESULTS: In the present study, the therapeutic effect of CTD on HCC was evaluated in vivo. We obtained the CTD-related transcriptional profiles, systematically and intuitively illustrated its possible pharmacological mechanisms in HCC through multiple targets and signalling pathways. These results revealed that the CTD-related differentially expressed genes were involved in autophagy, transcription factors (TFs) related transcriptional regulation, fatty acid metabolism and immune response in HCC. We found that MAPT, TOP2A, CENPF and MEFV were hub genes of CTD targets involved in autophagy regulation. Totally, 14 TFs have been confirmed to be critical for transcriptional regulation, and 33 TF targets were identified as the hub genes in transcriptional mis-regulation pathway in cancer. These TFs were associated with the immune response and immune cell infiltration. In addition, the downregulated genes were significantly enriched in metabolic regulation pathways, especially fatty acid metabolism after CTD treatment. Furthermore, the network of CTD associated miRNAs with these fatty acid metabolism-related targets was constructed in HCC. CONCLUSIONS: Taken together, our results comprehensively elucidated that CTD could act on multiple targets in HCC therapy, affecting autophagy, transcriptional regulation, the immune response and fatty acid metabolism. Our results provide a foundation for the study of the molecular mechanistic of CTD and its clinical application in the treatment of HCC.

Molluscum Contagiosum Virus Evasion of Immune Surveillance: A Review.

BACKGROUND: Molluscum contagiosum (MC) is an acute infection caused by the molluscum contagiosum virus (MCV) with a worldwide incidence of approximately 8,000 cases per 100,000 individuals annually. Greater than 90% of MC cases occur in the pediatric population, and affected adults are more likely to be younger or immunocompromised. MC has minimal inflammation initially; however, a strong inflammatory response can occur during resolution of the infection, termed the beginning of the end (BOTE). MC infections may last months to years, and it is hypothesized that persistent infections may be due to suppression of immunity by MCV proteins, thus affecting MC’s clinical progression. OBJECTIVE: We reviewed the current proposed mechanisms of MCV immune evasion and discuss potential therapeutic options for MC treatment. METHODS: A literature search was conducted using electronic databases (Pubmed, Google Scholar, Medline). RESULTS: We compiled 18 original research articles and identified 11 proteins produced by MCV that are postulated to participate in evasion of host immunity through various molecular pathways. These proteins and/or their downstream pathways may be influenced by MC treatments in phase 3 development, including berdazimer gel 10.3% and VP-102 cantharidin, 0.7%. CONCLUSION: MCV is distinctive in evading immune surveillance by inhibiting or dampening several immune pathways via the production of viral proteins. The result is decreasing local inflammatory response which contributes to the prolonged survival of MCV in the epidermis. Persistent MC can be a nuisance for some patients and treatment may be desired. Currently, no treatment has been approved by the US Food and Drug Administration (FDA). Two approaches in the pipeline may affect the immune avoidance mechanisms; nevertheless, their exact mechanisms between the potential therapeutics and viral proteins remain enigmatic. J Drugs Dermatol. 2023;22(2):182-189. doi:10.36849/JDD.7230.

Quality Evaluation of Traditional Chinese Medicine Mylabris Based on Heavy Metal Risk Assessment and Analysis of Pharmacological Component Contents.

As a highly representative traditional Chinese anti-tumor medicinal material, the biomass of Mylabris is collected from the wild. However, the living environments of Mylabris is differ, so Mylabris may be contaminated by heavy metal pollution depending on the environment. These environments may also affect the amount of biosynthesis of its medicinal ingredient, cantharidin, there by affecting the quality of Mylabris. In this study, we determined the heavy metal content in Mylabris from different origins by using ICP-MS, evaluated the risk posed by these heavy metals, and recommended theoretical maximum limits of heavy metals in medicinal Mylabris. The results show that the Cu content in Mylabris is substantially higher than that in Cr, As, Pb, Cd, and Hg. A quantitative risk assessment showed that Mylabris poses no noncarcinogenic risks. The results of the total carcinogenic risk value showed that origins S12 and S13 pose carcinogenic risk by Cr and As, and the rest of the origins were in the human-tolerable carcinogenic risk range. We found large differences in the cantharidin content in Mylabris from different origins. In general, the Mylabris from origins S2, S3 and S4 had a higher in vivo cantharidin content, which proved that the quality of the medicinal materials was higher here than in other production areas. Finally, we providing a reference for the quality evaluation of medicinal Mylabris materials.

Use of different ligand modification liposomes to evaluate the anti-liver tumor activity of cantharidin.

In this study, cantharidin(CTD), a bioactive terpenoid in traditional Chinese medicine cantharidin, was selected as a model component to construct novel nano liposome delivery systems for hepatocellular carcinoma therapy. Previous studies have shown that although cantharidin has definite curative effects on primary liver cancer, it is associated with numerous toxic and side effects. Therefore, based on the glycyrrhetinic acid (GA) binding site and the asialoglycoprotein receptor (ASGPR) on the hepatocyte membrane, the surface of CTD liposomes was modified with stearyl alcohol galactoside (SA-Gal) or/and the newly synthesized 3-succinic-30-stearyl deoxyglycyrrhetinic acid (11-DGA-Suc) ligands, and the physicochemical properties, pharmacokinetics, in vivo and in vitro anti-liver tumor activity and its mechanism of modified liposomes were investigated. Compared to CTD-lip, SA-Gal-CTD-lip, and 11-DGA-Suc + SA-Gal-CTD-lip, 11-DGA-Suc-CTD-lip showed stronger cytotoxicity and increased inhibition of HepG2 cell migration had the highest apoptosis rate. The cell cycle results indicated that HepG2 cells was arrested mainly at G0/G1phase and G2/M phase. The results of in vivo pharmacokinetic experiments revealed that the distribution of modified liposomes in the liver was significantly increased compared with that of unmodified liposome. In vivo tumor inhibition experiment showed that 11-DGA-Suc-CTD-lip had excellent tumor inhibition, and the tumor inhibition rates was 80.96%. The 11-DGA-Suc-CTD-lip group also displayed the strongest proliferation inhibition with the lowest proliferation index of 7% in PCNA assay and the highest apoptotic index of 49% in TUNEL assay. Taken together, our findings provide a promising solution for improving the targeting of nano liposomes and further demonstrates the encouraging potential of poor solubility and high toxicity drugs applicable to tumor therapy.

Cantharidin and Its Analogue Norcantharidin Inhibit Metastasis-Inducing Genes S100A4 and MACC1.

Colorectal cancer (CRC) is the third most prevalent and second deadliest cancer worldwide. In addition, metastasis directly causes up to 90% of all CRC deaths, highlighting the metastatic burden of the disease. Biomarkers such as S100A4 and MACC1 aid in identifying patients with a high risk of metastasis formation. High expression of S100A4 or MACC1 and to a greater extent the combination of both biomarkers is a predictor for metastasis and poor patient survival in CRC. MACC1 is a tumor-initiating and metastasis-promoting oncogene, whereas S100A4 has not been shown to initiate tumor formation but can, nevertheless, promote malignant tumor growth and metastasis formation. Cantharidin is a natural drug extracted from various blister beetle species, and its demethylated analogue norcantharidin has been shown in several studies to have an anti-cancer and anti-metastatic effect in different cancer entities such as CRC, breast cancer, and lung cancer. The impact of the natural compound cantharidin and norcantharidin on S100A4 and MACC1 gene expression, cancer cell migration, motility, and colony formation in vitro was tested. Here, for the first time, we have demonstrated that cantharidin and norcantharidin are transcriptional inhibitors of S100A4 and MACC1 mRNA expression, protein expression, and motility in CRC cells. Our results clearly indicate that cantharidin and, to a lesser extent, its analogue norcantharidin are promising compounds for efficient anti-metastatic therapy targeting the metastasis-inducing genes S100A4 and MACC1 for personalized medicine for cancer patients.

Five New Cantharidin Derivatives from the Insect Mylabris cichorii L. and Their Potential against Kidney Fibrosis In Vitro.

Five new monoterpenoids including three 1-hydroxymethyl-2-methyl cantharimide-type derivatives (1, 2, and 5) and two 1,2-dimethyl cantharimide-type derivatives (3 and 4), together with three known compounds (6-8) were isolated from the insect Mylabris cichorii Linnaeus. The structures of these new compounds, including their absolute configurations, were characterized by detailed analysis of NMR, chemical derivatization, and quantum chemical ECD calculations. All of the compounds were tested for their biological activity against kidney fibrosis. The results revealed that compounds 2, 4, and 7 could inhibit kidney fibrosis in vitro at 40 µM by inhibiting the expression of fibronectin and collagen I in TGF-ß1-induced NRK-52e cells.

In vitro effects of cantharidin on gilthead seabream (Sparus aurata) head-kidney leucocytes.

Cantharidin is a toxic vesicant terpene used in folk and traditional medicine due to its various therapeutic effects. Since there are no previous data on the effect of cantharidin in fish, this study aimed to investigate the in vitro related-inflammatory effects of cantharidin in gilthead seabream (Sparus aurata L.) head-kidney leucocytes (HKLs). In the first experiment, the HKLs were incubated with 0, 5 and 10 µg mL-1 of cantharidin for 24 h to delimit its possible toxic effects. In a second experiment, leucocytes were incubated with ranging concentrations from 0 to 10 µg mL-1 for 3, 6, or 12 h. Cell viability was higher in acidophilic granulocytes than in monocytes/macrophages and lymphocytes. Cantharidin caused apoptosis as was evidenced by transmission electron microscopy. In addition, cantharidin produced a time- and dose-dependent decrease of respiratory burst and phagocytic activities in HKLs, while their peroxidase activity was increased at 24 h of incubation with 5 and 10 µg mL-1 of cantharidin. Different changes in the gene expression were observed after incubation with cantharidin. While the gene expression of tnfa, il1b and crel was up-regulated in HKLs, the nfkb1 and igmh genes were down-regulated in comparison to the expression found in control HKLs. Present results offer a first view of the possible effects and action mechanisms of cantharidin in HKLs, as well as its implication in the inflammatory process, which could be of interest not only for basic research but also in the aquaculture sector.