Antihepatoma peptide,scolopentide,derived from the centipede scolopendra subspinipes mutilans

BACKGROUND Centipedes have been used to treat tumors for hundreds of years in China.However,current studies focus on antimicrobial and anticoagulation agents rather than tumors.The molecular identities of antihepatoma bioactive components in centipedes have not yet been extensively investigated.It is a challenge to isolate and characterize the effective components of centipedes due to limited peptide purification technologies for animal-derived medicines.AIM To purify,characterize,and synthesize the bioactive components with the strongest antihepatoma activity from centipedes and determine the antihepatoma mechanism.METHODS An antihepatoma peptide(scolopentide)was isolated and identified from the centipede scolopendra subspinipes mutilans using a combination of enzymatic hydrolysis,a Sephadex G-25 column,and two steps of high-performance liquid chromatography(HPLC).Additionally,the CCK8 assay was used to select the extracted fraction with the strongest antihepatoma activity.The molecular weight of the extracted scolopentide was characterized by quadrupole time of flight mass spectrometry(QTOF MS),and the sequence was matched by using the Mascot search engine.Based on the sequence and molecular weight,scolopentide was synthesized using solid-phase peptide synthesis methods.The synthetic scolopentide was confirmed by MS and HPLC.The antineoplastic effect of extracted scolopentide was confirmed by CCK8 assay and morphological changes again in vitro.The antihepatoma effect of synthetic scolopentide was assessed by the CCK8 assay and Hoechst staining in vitro and tumor volume and tumor weight in vivo.In the tumor xenograft experiments,qualified model mice(male 5-week-old BALB/c nude mice)were randomly divided into 2 groups(n=6):The scolopentide group(0.15 mL/d,via intraperitoneal injection of synthetic scolopentide,500 mg/kg/d)and the vehicle group(0.15 mL/d,via intraperitoneal injection of normal saline).The mice were euthanized by cervical dislocation after 14 d of continuous treatment.Mechanistically,flow cytometry was conducted to evaluate the apoptosis rate of HepG2 cells after treatment with extracted scolopentide in vitro.A Hoechst staining assay was also used to observe apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro.CCK8 assays and morphological changes were used to compare the cytotoxicity of synthetic scolopentide to liver cancer cells and normal liver cells in vitro.Molecular docking was performed to clarify whether scolopentide tightly bound to death receptor 4(DR4)and DR5.qRT-PCR was used to measure the mRNA expression of DR4,DR5,fas-associated death domain protein(FADD),Caspase-8,Caspase-3,cytochrome c(Cyto-C),B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax),x-chromosome linked inhibitor-of-apoptosis protein and Cellular fas-associated death domain-like interleukin-1βconverting enzyme inhibitory protein in hepatocarcinoma subcutaneous xenograft tumors from mice.Western blot assays were used to measure the protein expression of DR4,DR5,FADD,Caspase-8,Caspase-3,and Cyto-C in the tumor tissues.The reactive oxygen species(ROS)of tumor tissues were tested.RESULTS In the process of purification,characterization and synthesis of scolopentide,the optimal enzymatic hydrolysis conditions(extract ratio:5.86%,IC_(50):0.310 mg/mL)were as follows:Trypsin at 0.1 g(300 U/g,centipede-trypsin ratio of 20:1),enzymolysis temperature of 46°C,and enzymolysis time of 4 h,which was superior to freeze-thawing with liquid nitrogen(IC_(50):3.07 mg/mL).A peptide with the strongest antihepatoma activity(scolopentide)was further purified through a Sephadex G-25 column(obtained A2)and two steps of HPLC(obtained B5 and C3).The molecular weight of the extracted scolopentide was 1018.997 Da,and the peptide sequence was RAQNHYCK,as characterized by QTOF MS and Mascot.Scolopentide was synthesized in vitro with a qualified molecular weight(1018.8 Da)and purity(98.014%),which was characterized by MS and HPLC.Extracted scolopentide still had an antineoplastic effect in vitro,which inhibited the proliferation of Eca-109(IC_(50):76.27μg/mL),HepG2(IC_(50):22.06μg/mL),and A549(IC_(50):35.13μg/mL)cells,especially HepG2 cells.Synthetic scolopentide inhibited the proliferation of HepG2 cells(treated 6,12,and 24 h)in a concentration-dependent manner in vitro,and the inhibitory effects were the strongest at 12 h(IC_(50):208.11μg/mL).Synthetic scolopentide also inhibited the tumor volume(Vehicle vs Scolopentide,P=0.0003)and weight(Vehicle vs Scolopentide,P=0.0022)in the tumor xenograft experiment.Mechanistically,flow cytometry suggested that the apoptosis ratios of HepG2 cells after treatment with extracted scolopentide were 5.01%(0μg/mL),12.13%(10μg/mL),16.52%(20μg/mL),and 23.20%(40μg/mL).Hoechst staining revealed apoptosis in HepG2 cells after treatment with synthetic scolopentide in vitro.The CCK8 assay and morphological changes indicated that synthetic scolopentide was cytotoxic and was significantly stronger in HepG2 cells than in L02 cells.Molecular docking suggested that scolopentide tightly bound to DR4 and DR5,and the binding free energies were-10.4 kcal/mol and-7.1 kcal/mol,respectively.In subcutaneous xenograft tumors from mice,quantitative real-time polymerase chain reaction and western blotting suggested that scolopentide activated DR4 and DR5 and induced apoptosis in SMMC-7721 Liver cancer cells by promoting the expression of FADD,caspase-8 and caspase-3 through a mitochondria-independent pathway.CONCLUSION Scolopentide,an antihepatoma peptide purified from centipedes,may inspire new antihepatoma agents.Scolopentide activates DR4 and DR5 and induces apoptosis in liver cancer cells through a mitochondria-independent pathway.

Comparative analysis of diverse toxins from a new pharmaceutical centipede,Scolopendra mojiangica

As the oldest venomous animals,centipedes use their venom as a weapon to attack prey and for protection.Centipede venom,which contains many bioactive and pharmacologically active compounds,has been used for centuries in Chinese medicine,as shown by ancient records.Based on comparative analysis,we revealed the diversity of and differences in centipede toxin-like molecules between Scolopendra mojiangica,a substitute pharmaceutical material used in China,and S.subspinipes mutilans.More than 6000 peptides isolated from the venom were identified by electrospray ionization-tandem mass spectrometry(ESI-MS/MS)and inferred from the transcriptome.As a result,in the proteome of S.mojiangica,246 unique proteins were identified:one in five were toxin-like proteins or putative toxins with unknown function,accounting for a lower percentage of total proteins than that in S.mutilans.Transcriptome mining identified approximately 10 times more toxin-like proteins,which can characterize the precursor structures of mature toxinlike peptides.However,the constitution and quantity of the toxin transcripts in these two centipedes were similar.In toxicity assays,the crude venom showed strong insecticidal and hemolytic activity.These findings highlight the extensive diversity of toxin-like proteins in S.mojiangica and provide a new foundation for the medical-pharmaceutical use of centipede toxin-like proteins.

Clinical consequences of centipede bite:Is it neurotoxic?

The primary purpose of this article was to review the current literature regarding the clinical consequences of centipede envenomation in humans,in order to determine whether the bite of these arthropods is neurotoxic to humans or not. A thorough search of the literature regarding the clinical consequences of centipede bites in humans was applied,with great respect to neurological symptoms potentially caused by such bites. Centipede bite commonly causes only local reactions,which usually resolve within a few days without sequelae. The patients in the majority of centipede envenomations describe a painful but benign syndrome. However,mild constitutional symptoms are relatively frequent. Remarkably,centipedes can rarely cause severe systematic reactions such as anaphylaxis or even hypotension and myocardial ischemia. Factors such as patient age,comorbidity,anatomic site of envenomation,and size/species of centipede should be considered when evaluating a centipede envenomation victim. According to the current literature,the centipede bite does not seem to be neurotoxic to humans. However,it commonly causes symptoms mediated by the nervous system. These include local and generalized symptoms,with the first dominated by sensory disturbances and the second by non-specific symptoms such as headache,anxiety and presyncope. Based on our results,the answer to our study's question is negative. The centipede bite is not neurotoxic to humans. However,it commonly causes symptoms mediated by the nervous system,which include primarily local pain and sensory disturbances,as well as generalized non-specific symptoms such as headache,anxiety and vagotonia.

Design, Analysis and Experimental Performance of a Bionic Piezoelectric Rotary Actuator

This study presents a piezoelectric rotary actuator which is equipped with a bionic driving mechanism imitating the cen- tipede foot. The configuration and the operational principle are introduced in detail. The movement model is established to analyze the motion of the actuator. We establish a set of experimental system and corresponding experiments are conducted to evaluate the characteristics of the prototype. The results indicate that the prototype can be operated stably step by step and all steps have high reproducibility. The driving resolutions in forward and backward motions are 2.31 grad and 1.83 μrad, respectively. The prototype can also output a relatively accurate circular motion and the maximum output torques in forward and backward directions are 76.4 Nmm and 70.6 Nmm, respectively. Under driving frequency of 1 Hz, the maximum angular velocities in forward and backward directions are 1029.3 grad·s^-1 and 1165 μrad·s^-1 when the driving voltage is 120 V. Under driving voltage of 60 V, the angular velocities in forward and backward motions can be up to 235100 grad·s^-1 and 153650 μrad·s^-1 when the driving frequency is 1024 Hz. We can obtain the satisfactory angular velocity by choosing a proper driving voltage and frequency for the actuator.