Mayor Anna Beatriz R, Guevarra Leonardo A, Santiago-Bautista Myla R, Santiago Librado A
The Graduate School, University of Santo Tomas, Manila, Philippines.
Research Center for Natural and Applied Sciences, University of Santo Tomas, Manila, Philippines.
J Venom Anim Toxins Incl Trop Dis. 2020 Aug 3;26:e20190104. doi: 10.1590/1678-9199-JVATITD-2019-0104.
Spider venom is a potential source of pharmacologically important compounds. Previous studies on spider venoms reported the presence of bioactive molecules that possess cell-modulating activities. Despite these claims, sparse scientific evidence is available on the cytotoxic mechanisms in relation to the components of the spider venom. In this study, we aimed to determine the cytotoxic fractions of the spider venom extracted from and to ascertain the possible mechanism of toxicity towards human lung adenocarcinoma (A549) cells.
Spider venom was extracted by electrostimulation. Components of the extracted venom were separated by reversed-phase high performance liquid chromatography (RP-HPLC) using a linear gradient of 0.1% trifluoroacetic acid (TFA) in water and 0.1% TFA in 95% acetonitrile (ACN). Cytotoxic activity was evaluated by the MTT assay. Apoptotic or necrotic cell death was assessed by microscopic evaluation in the presence of Hoechst 33342 and Annexin V, Alexa Fluor 488 conjugate fluorescent stains, and caspase activation assay. Phospholipase A (PLA) activity of the cytotoxic fractions were also measured.
We observed and isolated six fractions from the venom of collected from Aurora, Zamboanga del Sur. Four of these fractions displayed cytotoxic activities. Fractions AT5-1, AT5-3, and AT5-4 were found to be apoptotic while AT5-6, the least polar among the cytotoxic components, was observed to induce necrosis. PLA activity also showed cytotoxicity in all fractions but presented no relationship between specific activity of PLA and cytotoxicity.
The venom of spider, an endemic tarantula species in the Philippines, contains components that were able to induce either apoptosis or necrosis in A549 cells.
蜘蛛毒液是具有重要药理活性化合物的潜在来源。先前对蜘蛛毒液的研究报道了具有细胞调节活性的生物活性分子的存在。尽管有这些说法，但关于蜘蛛毒液成分的细胞毒性机制的科学证据却很少。在本研究中，我们旨在确定从[蜘蛛名称未给出]提取的蜘蛛毒液的细胞毒性组分，并确定其对人肺腺癌（A549）细胞的可能毒性机制。
通过电刺激提取蜘蛛毒液。使用0.1%三氟乙酸（TFA）的水溶液和0.1% TFA的95%乙腈（ACN）线性梯度，通过反相高效液相色谱（RP-HPLC）分离提取毒液的成分。通过MTT法评估细胞毒性活性。在存在Hoechst 33342和膜联蛋白V、Alexa Fluor 488共轭荧光染料的情况下，通过显微镜评估和半胱天冬酶激活试验评估凋亡或坏死性细胞死亡。还测量了细胞毒性组分的磷脂酶A（PLA）活性。
我们从南三宝颜省奥罗拉采集的[蜘蛛名称未给出]毒液中观察并分离出六个组分。其中四个组分具有细胞毒性活性。发现组分AT5-1、AT5-3和AT5-4具有凋亡作用，而细胞毒性成分中极性最小的AT5-6则诱导坏死。PLA活性在所有组分中也显示出细胞毒性，但PLA的比活性与细胞毒性之间没有关系。
菲律宾特有的狼蛛物种[蜘蛛名称未给出]的毒液含有能够在A549细胞中诱导凋亡或坏死的成分。
