Cantharis by photosynthetic bacteria biotransformation: Reduced toxicity and improved antitumor efficacy.[Pubmed: 27041400]

The blister beetle, also known as Mylabris cichorii, is not only widely used in clinical applications in Chinese anticancer medicine, but is also one of the main ingredients in a variety of traditional Chinese medicinal preparations with anticancer activity. However, the strong toxicity exhibited by this beetle species limits its clinical applicability, with the photosynthetic bacteria featuring a strong biological conversion function. Therefore, in this study we explore the use of photosynthetic bacteria for bioconversion of the blister beetle in order to reduce the toxicity and in effort to enhance the overall antitumor effect.

Cloning and functional analysis of farnesyl pyrophosphate synthase (FPPS) gene from Mylabris cichorii.[Pubmed: 26988567]

Cantharidin, a defensive terpene compound synthesized by the meloid beetle (Coleoptera, Meloidae), is an important anticancer agent. However, there has been little study done on how this compound synthesized by the beetle. In this paper, a farnesyl pyrophosphate synthase (FPPS) gene, designated McFPPS, was isolated from Mylabris cichorii by reverse transcription PCR based on conserved domains in other organisms. Multiple alignment analysis showed that the deduced amino acids shared >70% homology with FPPSs from other species and contained typically seven conservative regions. Gene expression profile analysis revealed that McFPPS was expressed throughout the tested growth stages of M. cichorii adults, whereas the transcripts accumulated to the highest level at 20 days in male adults while the highest expression level appeared at 15 days in females. Tissue expression pattern analysis showed that McFPPS was expressed constitutively in all tested tissues and a relatively higher expression level in the alimentary canal of males, but no significant tissue difference in the females. For the first time, a RNA interference strategy was employed to induce a greater suppression of McFPPS mRNA, and thus a sharp decrease in the expression levels of downstream genes and the concentration of product. All these results indicated that McFPPS may be directly involved or play an essential role in the biosynthesis of cantharidin.

De Novo Transcriptome and Expression Profile Analysis to Reveal Genes and Pathways Potentially Involved in Cantharidin Biosynthesis in the Blister Beetle Mylabris cichorii.[Pubmed: 26752526]

The dried body of Mylabris cichorii is well-known Chinese traditional medicine. The sesquiterpenoid cantharidin, which is secreted mostly by adult male beetles, has recently been used as an anti-cancer drug. However, little is known about the mechanisms of cantharidin biosynthesis. Furthermore, there is currently no genomic or transcriptomic information for M. cichorii. In this study, we performed de novo assembly transcriptome of M. cichorii using the Illumina Hiseq2000. A single run produced 9.19 Gb of clean nucleotides comprising 29,247 sequences, including 23,739 annotated sequences (about 81%). We also constructed two expression profile libraries (20-25 day-old adult males and 20-25 day-old adult females) and discovered 2,465 significantly differentially-expressed genes. Putative genes and pathways involved in the biosynthesis of cantharidin were then characterized. We also found that cantharidin biosynthesis in M. cichorii might only occur via the mevalonate (MVA) pathway, not via the methylerythritol 4-phosphate/deoxyxylulose 5-phosphate (MEP/DOXP) pathway or a mixture of these. Besides, we considered that cantharidin biosynthesis might be related to the juvenile hormone (JH) biosynthesis or degradation. The results of transcriptome and expression profiling analysis provide a comprehensive sequence resource for M. cichorii that could facilitate the in-depth study of candidate genes and pathways involved in cantharidin biosynthesis, and may thus help to improve our understanding of the mechanisms of cantharidin biosynthesis in blister beetles.

ISOLATION AND FUNCTIONAL ANALYSIS OF McMenA, A GENE ENCODING A 1,4-DIHYDROXY-2-NAPHTHOATE OCTAPRENYLTRANSFERASE IN Mylabris cichorii.[Pubmed: 25772016]

Cantharidin is a biomolecule with a role in host defense that can also be used as an anticancer drug. The in vivo biosynthetic pathway for cantharidin has been the subject of debate for several decades and the mechanism is not yet completely understood. To study the biosynthetic pathway of cantharidin in blister beetles, Mylabris cichori, a full-length MenA (McMenA) cDNA was cloned based on the partial sequence of the MenA gene from a suppression subtractive hybridization (SSH) library of male and female adult M. cichorii. The cDNA was 1264 base pairs (bp) with an open reading frame of 1026 bp nucleotides encoding a 341 amino acid protein. Analysis of the McMenA amino acid sequence showed that the aspartate rich motif N/DDxxD represented binding sites for prenyl diphosphate via a Mg(2+) ion. Phylogenetic analysis showed that McMenA was most closely related to MenA of Tribolium castaneum, and the amino acid sequence similarity was 86%. The expression pattern of McMenA in adults was analyzed using RT-qPCR, and we found that the highest expression of McMenA occurred during 22-25 days in the sex-separate breeding males, while the lowest expression occurred in females at the same time. Injection with a specific double-strand RNA (dsRNA) of McMenA led to a significant reduction of McMenA mRNA levels after 24 h. Cantharidin and ATP concentrations dropped around the same time. Together, our data showed that the McMenA gene might be involved in cantharidin biosynthesis.

Identification of suitable reference genes for gene expression studies by qRT-PCR in the blister beetle Mylabris cichorii.[Pubmed: 25368050]

The blister beetle Mylabris cichorii L. (Coleoptera: Meloidae) is a traditional medicinal insect recorded in the Chinese Pharmacopoeia. It synthesizes cantharidin, which kills cancer cells efficiently. Only males produce large amounts of cantharidin. Reference genes are required as endogenous controls for the analysis of differential gene expression in M. cichorii. Our study chose 10 genes as candidate reference genes. The stability of expression of these genes was analyzed by quantitative PCR and determined with two algorithms, geNorm and Normfinder. We recommend UBE3A and RPL22e as suitable reference genes in females and UBE3A, TAF5, and RPL22e in males.

Cantharidin-mediated ultrastructural and biochemical changes in mitochondria lead to apoptosis and necrosis in murine Dalton's lymphoma.[Pubmed: 24029497]

Cantharidin, a type of terpenoid, is the blistering agent of blister beetles frequently used in traditional medicine. The isolation and anticancer activity of cantharidin from blister beetles, Mylabris cichorii has been recently reported by us. This study deals with changes in mitochondrial structure and function and understanding their significance in the underlying mechanism(s) in cantharidin-mediated antitumor effects in Dalton's lymphoma (DL) bearing mice. Cantharidin treatment caused the appearance of abnormal mitochondrial features which included roundish mitochondria with thickened membranes, irregularity in cristae, and appearance of small and large size vacuoles in mitochondria of DL cells. Cantharidin treatment resulted in a decrease in mitochondrial reduced glutathione, succinate dehydrogenase activity, mitochondrial membrane potential, and induced apoptosis and necrosis in DL cells. The decrease/release of mitochondrial cytochrome c were also observed after cantharidin treatment. Flow cytometry-based cell cycle analysis showed a time-dependent accumulation of the sub-G0 population of DL cells, thus, confirming the involvement of apoptosis in tumor cells in cantharidin-mediated antitumor activity. These finding signify that the apoptosis induced by cantharidin in DL cells should involve mitochondrial-dependent pathways. It is suggested that these cantharidin-mediated changes in mitochondria may play a crucial role in its antitumor activity.

Antitumor effect of blister beetles: an ethno-medicinal practice in Karbi community and its experimental evaluation against a murine malignant tumor model.[Pubmed: 23707211]

The blister beetles Epicauta hirticornis and Mylabris cichorii are used as a folk medicine by the Karbi tribe in Karbi Anglong district of Assam, India for the treatment of different human ailments, including cancer cases.

Bioactive component, cantharidin from Mylabris cichorii and its antitumor activity against Ehrlich ascites carcinoma.[Pubmed: 22402807]

The anticancer activity of the extract of blister beetle, Mylabris cichorii has been documented earlier by us. In the present study, the active principle of M. cichorii was isolated and its anticancer efficacy was evaluated against murine Ehrlich ascites carcinoma (EAC). The isolated bioactive compound was characterized to be cantharidin which showed potent antitumor activity and inhibited the proliferation of Ehrlich ascites carcinoma, both in vivo and in vitro. Cantharidin-treated EAC-bearing mice showed about 82% increase in lifespan at the dose of 0.5 mg/kg/day. In vitro cytotoxicity assay with the 3-(4,5 dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide test revealed about 50% cell death at the concentration of 25.8 μg/ml. The fluorescence and transmission electron microscopy revealed that EAC cells treated with cantharidin depicted typical apoptotic morphology with chromatin condensation, nuclear fragmentation into discrete masses, and plasma membrane blebbing which deduce towards the death of these cells. Histological examination of the kidney of cantharidin-treated mice showed glomerular and tubular congestion with abnormal Bowman's capsule, thus, indicating a renal toxicity in the host. Cantharidin-induced renal damage in the host was also manifested by the decreased lactate dehydrogenase isozymes and its possible release from the cells.