Spider venom components decrease glioblastoma cell migration and invasion through RhoA-ROCK and Na+/K+-ATPase β2: potential molecular entities to treat invasive brain cancer

Abstract

Background Glioblastoma (GB) cells have the ability to migrate and infiltrate the normal parenchyma, leading to the formation of recurrent tumors often adjacent to the surgical extraction site. We recently showed that Phoneutria nigriventer spider venom (PnV) has anticancer effects mainly on the migration of human GB cell lines (NG97 and U-251). The present work aimed to investigate the effects of isolated components from the venom on migration, invasiveness, morphology and adhesion of GB cells, also evaluating RhoA-ROCK signaling and Na+/K+-ATPase β2 (AMOG) involvement. Methods Human (NG97) GB cells were treated with twelve subfractions (SFs—obtained by HPLC from PnV). Migration and invasion were evaluated by scratch wound healing and transwell assays, respectively. Cell morphology and actin cytoskeleton were shown by GFAP and phalloidin labeling. The assay with fibronectin coated well plate was made to evaluate cell adhesion. Western blotting demonstrated ROCK and AMOG levels and a ROCK inhibitor was used to verify the involvement of this pathway. Values were analyzed by the GraphPad Prism software package and the level of significance was determinate using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparisons test. Results Two (SF1 and SF11) of twelve SFs, decreased migration and invasion compared to untreated control cells. Both SFs also altered actin cytoskeleton, changed cell morphology and reduced adhesion. SF1 and SF11 increased ROCK expression and the inhibition of this protein abolished the effects of both subfractions on migration, morphology and adhesion (but not on invasion). SF11 also increased Na+/K+-ATPase β2. Conclusion All components of the venom were evaluated and two SFs were able to impair human glioblastoma cells. The RhoA effector, ROCK, was shown to be involved in the mechanisms of both PnV components. It is possible that AMOG mediates the effect of SF11 on the invasion. Further investigations to isolate and biochemically characterize the molecules are underway.

Spider venom-derived peptide JZTX-14 prevents migration and invasion of breast cancer cells via inhibition of sodium channels

“…Spider venom is a rich natural product with significant antitumour effects in human lung adenocarcinoma, leukaemia, liver cancer, glioblastoma, and breast cancer ( Gao et al, 2007 ; Liu et al, 2012 ; Lian et al, 2018 ; Barreto Dos Santos et al, 2019 ; Barreto et al, 2020 ; Lopez et al, 2020 ; de Avelar Júnior et al, 2022 ). We isolated and purified natural JZTX-14 from spider venom, which consists of 31 amino acids with three pairs of disulfide bonds.…”

The β2-Subunit (AMOG) of Human Na+, K+-ATPase Is a Homophilic Adhesion Molecule

“…Based on the new findings reported here, we infer that β 2 /AMOG is a heterophilic astrocyte-neuron adhesion molecule that developed a self-avoidance mechanism in a similar way to how neurons avoid forming synapses with themselves [ 38 ]. Interestingly, recent studies have related the β 2 /AMOG with glioblastoma (GB), the most devastating type of glioma [ 21 , 30 , 39 , 40 , 41 ]. Those studies depict the β 2 /AMOG as a tumor suppressor cell adhesion molecule in astrocytes.…”

Isolated Components From Spider Venom Targeting Human Glioblastoma Cells and Its Potential Combined Therapy With Rapamycin

“…The cell culture was maintained in a humidified atmosphere at 37°C and 5% CO 2 until semi-confluence (about 90% of the total surface area). For the assays, cells were transferred after careful scraping of 48- or 96-well plates (Corning, Inc.), at an initial density of 1 × 10 4 cells per well, and incubated at 37°C for 72 h. The cells were then treated with 14 μg/ml and 280 μg/ml of PnV ( Rapôso, 2017 ), F1, F2, and F3 (0.1, 1, and 10 μg/ml) ( Barreto dos Santos et al, 2019 ), or SF 1–11 (0.1 and 1 μg/ml), for 1, 5, 24, and/or 72 h, depending on the method ( Barreto dos Santos et al, 2019 ; Barreto et al, 2020 ), according to the following assays, while the control cells were maintained in the medium for the same times. For the mTOR inhibition assay, cells were incubated with 50 nM of rapamycin (#051357-13 Cayman Chemical) ( Foster and Toschi, 2009 ).…”

“…It has been shown that biomolecules from scorpion and spider venoms have a chemotherapeutic effect on glioma, neuroblastoma, leukemia, lymphoma, breast cancer, lung cancer, hepatoma, and pancreatic and prostate cancer, among others ( Rapôso, 2017 ). Our group demonstrated that Phoneutria nigriventer spider venom (PnV) decreased cell viability and proliferation, impaired cell cycle and induced apoptosis of human GB lineages ( Barreto dos Santos et al, 2019 ), and reduced or eradicated the development of GB in a preclinical trial developed in mice ( Bonfanti et al, 2020 ), and PnV-isolated components impaired cell migration and adhesion through RhoA-ROCK and Na + /K + -ATPase ( Barreto et al, 2020 ).…”