The Effects Of Scorpion Analgesic Peptide,BmK AGAP Added To Lidocaine On Nerve Blocks And The Antitumor Effect BmK AGAP On Breast Cancer In Vitro And In Vivo

SECTION ONE: The effect of Bm K AGAP added to lidocaine on nerve blocks.BackgroundThe incidences of systemic toxicity and other complications associated with the existing local anesthetics can occur at the clinical concentration level and vary with the anesthetic techniques,type of surgery and patient factor.This evidence suggests the need for therapeutic interventions in regional and peripheral anesthesia.Many analgesics have been tested and demonstrated to be clinically beneficial when added to local anesthetics for regional or peripheral anesthesia.Despite the existence and the benefits of these analgesic adjuvants,study reports indicate that there is a need for adjuncts that will provide faster recovery without compromising anesthetic reliability.Some neurotoxins have high potency for nerve block.Tetrodotoxin and saxitoxins have high affinity and great specificity for voltage-gated sodium channels(VGSCs),especially Nav 1.7 but lower affinity for Nav 1.8 and Nav 1.5.Emerging evidence shows that scorpion venom peptide Bm K AGAP mediate analgesic activity by binding to voltage-independently at site-3 of sodium channels.Animal studies have shown that scorpion venom peptide,Bm K AGAP exhibit potent analgesic activity with minimum advert effects compared with morphine.The current study was designed to test the hypothesis that scorpion venom peptide,Bm K AGAP added to lidocaine in intrathecal injection or sciatic nerve block causes a dose-dependent increase intensity of sensory block and prolong the duration of analgesia without significant adverse effects.MethodsIntrathecal injectionPartial sciatic nerve ligation was performed on 84 rats to induce a rapid onset and longlasting mechanical allodynia.Preservative-free lidocaine(0.5%)and Bm K AGAP(0.5,1,1.5,or 2 mg/kg)were prepared.The rats were randomly assigned to one of seven groups.Group A(n=12)received intrathecal injection of saline as the negative control;Group B(n=12)received intrathecal injection of lidocaine;Group C(n=12)received intrathecal injection of lidocaine + Bm K AGAP(0.5 mg/kg);Group D(n=12)received intrathecal injection of lidocaine + Bm K AGAP(1 mg/kg);Group E(n=12)received intrathecal injection of lidocaine + Bm K AGAP(1.5 mg/kg);Group F(n=12)received intrathecal injection of lidocaine + Bm K AGAP(2 mg/kg);Group G(n = 12)received intrathecal injection of Bm K AGAP(2 mg/kg)alone and Group H(n=12)sham.We operated on the rats representing the sham group and the sciatic nerve exposed but not ligated.The von Frey filaments were used to assessed mechanical allodynia in rats.Sciatic nerve block.We performed partial sciatic nerve ligation on 60 rats to induce a rapid onset and longlasting mechanical allodynia.Equal volume(600?l)of preservative-free lidocaine(0.5%)and Bm K AGAP(2 or 1 mg/kg)were prepared with saline(0.9%).The rats were randomly allocated into five groups.Group A(n=12)received saline as the negative control;Group B(n=12)received equal volume of lidocaine and saline;Group C(n=12)received lidocaine and Bm K AGAP(1 mg/kg);Group D(n=12)received lidocaine along with Bm K AGAP(2 mg/kg)and Group E(n=12)sham.We operated on the rats representing the sham group(n=12)and the sciatic nerve exposed but not ligated.The von Frey filaments were used to assessed mechanical allodynia in rats.ResultsIntrathecal injectionThe data showed that rats from the lidocaine group exhibited 9.45 g of increased PWT compared with 1.40 g of baseline PWT.Rats from the Bm K AGAP(2 mg/kg)alone groups exhibited increased 4.93 PWT compared with 3.00 g baseline PWT.The rats from the lidocaine + Bm K AGAP(0.5 mg/kg)group exhibited 11.40 g of increased PWT compared with 1.54 g of baseline PWT.Rats from the lidocaine + Bm K AGAP(1 mg/kg)group showed 12.31 g increased PWT compared with baseline PWT of 1.20 g.Similarly,rats from lidocaine + Bm K AGAP(1.5 mg/kg)group exhibited increased PWT of 13.37 g compared with baseline PWT of 1.35 g,whereas the rats from the lidocaine + Bm K AGAP(2 mg/kg)group presented 13.99 g increased PWT compared with 1.29 g baseline PWT.It was observed that rats supplemented with Bm K AGAP exhibited dose-dependent increased PWT compared with rats that were injected with lidocaine alone.The data showed that rats supplemented with Bm K AGAP(0.5,1,1.5,2 mg/kg)to lidocaine showed significantly prolonged duration of increased PWT compared with rats injected with lidocaine only.Sciatic nerve blockThe data showed that rats from the lidocaine group exhibited increased 9.61 g of PWT.Rats from the Bm K AGAP(2 mg/kg)alone demonstrated increased 4.48 g PWT compared with 1.70 g baseline PWT.The rats from the lidocaine + Bm K AGAP(1 mg/kg)group exhibited increased 12.17 g of PWT,whereas the rats from the lidocaine + Bm K AGAP(2mg/kg)group exhibited increased 14.40 g of PWT compared with the baseline PWT.There was a prolonged duration of increased paw withdrawal threshold in rats that were supplemented with Bm K AGAP compared with rats that received lidocaine injection alone.We realized a significant(P< 0.0001)prolonged duration of increased paw withdrawal threshold in rats that were supplemented with Bm K AGAP(2 mg/kg)compared with rats that received Bm K AGAP(1 mg/kg).ConclusionIn conclusion,the findings of this study suggest that Bm K AGAP added to lidocaine in intrathecal injection or sciatic nerve block causes dose-dependent increase intensity of sensory block and prolong the duration of analgesia without significant adverse effect.This emerging evidence is an essential first step in encouraging future possible clinical use of Bm K AGAP as an analgesic adjuvant in regional or peripheral anesthesia to improve perioperative analgesia.SECTION TWO: The antitumor effect of the scorpion venom analgesic peptide,Bm K AGAP inhibits stemness and epithelial-mesenchymal transition in breast cancer by down-regulating PTX3 in vitro and in vivoBackgroundEven though the current conventional cancer drugs have demonstrated to be making progress,pharmacotherapies are still challenged with long-term solutions to pain treatment in cancer.Pain is the major and distressing symptom of cancer.Opioids analgesics are the most frequently used analgesic for perioperative anesthetic and cancerrelated pain.Morphine and fentanyl are used for many decades with anti-tumor to treat patients with cancer metastasis.However,previous study reports showed that morphine and fentanyl promote cancer cell Stemness,EMT,and drug resistance.Scorpion venom analgesic peptide,Bm K AGAP reported exhibiting potent analgesic and anti-tumor activity in mice may present as an alternative therapeutic agent for cancer and pain.The peptide is reported to induce apoptosis and inhibits proliferation of human colon cancer cells.The roles of the scorpion venom peptide Bm K AGAP in regulating breast cancer cell Stemness,EMT,migration,and invasion are yet to be determined.ObjectiveTo investigate the roles of the scorpion venom analgesic peptide,Bm K AGAP in regulating cancer cell stemness,Epithelial-Mesenchymal Transition,migration,and invasion of breast cancer in vitro and in vivo.MethodsWe treated MDA-MB-231 and MCF-7 human breast cancer cells with different concentrations of Bm K AGAP and performed q PCR,ELISA,Western blot,immunofluorescence staining,Sphere formation,colony assay,transwell migration,and invasion assay to determine the effects of Bm K AGAP on breast cancer cells Stemness,Epithelial-Mesenchymal Transition,migration,and invasion.ResultsThe data of the analysis of q PCR,ELISA,Western blot,immunofluorescence staining,Sphere formation,colony assay,transwell migration and invasion assay showed that the r Bm K AGAP decreased the expression of Oct4,Sox2,N-cadherin,Snail,and increased the expression of E-cadherin at the m RNA and protein levels in breast cancer cells.The xenograft tumor mouse model showed inhibition of xenograft tumor growth,stem-like features and EMT by r Bm K AGAP.The data showed that r Bm K AGAP inhibits breast cancer cell Stemness,EMT,migration,and invasion by down-regulating PTX3 through NF-k B and Wnt/?-Catenin Signaling Pathway in vitro and in vivo.ConclusionsThe data on the antitumor effects suggest Bm K AGAP may find use in inhibiting cancer cell stemness,epithelial-mesenchymal transition,migration and invasion,and for treating cancer-related pain.High expression of PTX3 increased stem-like features(Oct4,Sox2,and Nanog),decreased the expression of E-cadherin and increased other epithelialmesenchymal transition markers.Thus,PTX3 may be a potential target for regulating cancer cell stemness and epithelial-mesenchymal transition,and a plausible therapeutic target or strategy.Bm K AGAP inhibition of breast cancer cell stemness,epithelialmesenchymal transition,migration,and invasion may be a useful therapeutic approach for breast cancer and related pain.