The requirement for polyamines for intestinal epithelial cell migration is mediated through Rac1

The rapid migration of intestinal epithelial cells is important to the healing of mucosal ulcers and wounds. This cell migration requires the presence of polyamines and the activation of RhoA. RhoA activity, however, is not sufficient for migration because polyamine depletion inhibited the migration of IEC-6 cells expressing constitutively active RhoA. The current study examines the role of Rac1 and Cdc42 in cell migration and whether their activities are polyamine-dependent. Polyamine depletion with alpha-difluoromethylornithine inhibited the activities of RhoA, Rac1, and Cdc42. This inhibition was prevented by supplying exogenous putrescine in the presence of alpha-difluoromethylornithine. IEC-6 cells transfected with constitutively active Rac1 and Cdc42 migrated more rapidly than vector-transfected cells, whereas cells expressing dominant negative Rac1 and Cdc42 migrated more slowly. Polyamine depletion had no effect on the migration of cells expressing Rac1 and only partially inhibited the migration of those expressing Cdc42. Although polyamine depletion caused the disappearance of actin stress fibers in cells transfected with empty vector, it had no effect on cells expressing Rac1. Constitutively active Rac1 increased RhoA and Cdc42 activity in both normal and polyamine-depleted cells. These results demonstrate that Rac1, RhoA, and Cdc42 are required for optimal epithelial cell migration and that Rac1 activity is sufficient for cell migration in the absence of polyamines due to its ability to activate RhoA and Cdc42 as well as its own effects on the process of cell migration. These data imply that the involvement of polyamines in cell migration occurs either at Rac1 itself or upstream from Rac1.     

Regulation by GDI of RhoA/Rho-kinase-induced Ca2+ sensitization of smooth muscle myosin II

We characterized the role of guanine nucleotide dissociation inhibitor (GDI) in RhoA/Rho-kinase-mediated Ca2+ sensitization of smooth muscle. Endogenous contents (approximately 2-4 microM) of RhoA and RhoGDI were near stoichiometric, whereas a supraphysiological GDI concentration was required to relax Ca2+ sensitization of force by GTP and guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). GDI also inhibited Ca2+ sensitization by GTP. G14V RhoA, by alpha-adrenergic and muscarinic agonists, and extracted RhoA from membranes. GTPgammaS translocated Rho-kinase to a Triton X-114-extractable membrane fraction. GTP. G14V RhoA complexed with GDI also induced Ca2+ sensitization, probably through in vivo dissociation of GTP. RhoA from the complex, because it was reversed by addition of excess GDI. GDI did not inhibit Ca2+ sensitization by phorbol ester. Constitutively active Cdc42 and Rac1 inhibited Ca2+ sensitization by GTP. G14V RhoA. We conclude that 1) the most likely in vivo function of GDI is to prevent perpetual "recycling" of GDP. RhoA to GTP. RhoA; 2) nucleotide exchange (GTP for GDP) on complexed GDP. RhoA/GDI can precede translocation of RhoA to the membrane; 3) activation of Rho-kinase exposes a hydrophobic domain; and 4) Cdc42 and Rac1 can inhibit Ca2+ sensitization by activated GTP. RhoA.     

Invasion of T-lymphoma cells: cooperation between Rho family GTPases and lysophospholipid receptor signaling.

Rho-like GTPases orchestrate distinct cytoskeletal changes in response to receptor stimulation. Invasion of T-lymphoma cells into a fibroblast monolayer is induced by Tiam1, an activator of the Rho-like GTPase Rac, and by constitutively active V12Rac1. Here we show that activated V12Cdc42 can also induce invasion of T-lymphoma cells. Activated RhoA potentiates invasion, but fails by itself to mimic Rac and Cdc42. However, invasion is inhibited by the Rho-inactivating C3 transferase. Thus, RhoA is required but not sufficient for invasion. Invasion of T-lymphoma cells is critically dependent on the presence of serum. Serum can be replaced by the serum-borne lipids lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P) (10(-7)-10(-6) M), which act on distinct G protein-linked receptors to activate RhoA and phospholipase C (PLC)-Ca2+ signaling. LPA- and S1P-induced invasion is preceded by Rho-dependent F-actin redistribution and pseudopodia formation. However, expression of both V14RhoA and V12Rac1 does not bypass the LPA/S1P requirement for invasion, indicating involvement of an additional signaling pathway independent of RhoA. The PLC inhibitor U-73122, but not the inactive analog U-73343, abolishes invasion. Our results indicate that T-lymphoma invasion is driven by Tiam1/Rac or Cdc42 activation, and is dependent on LPA/S1P receptor-mediated RhoA and PLC signaling pathways which lead to pseudopod formation and enhanced infiltration.     

Localized suppression of RhoA activity by Tyr31/118-phosphorylated paxillin in cell adhesion and migration

RhoA activity is transiently inhibited at the initial phase of integrin engagement, when Cdc42- and/or Rac1-mediated membrane spreading and ruffling predominantly occur. Paxillin, an integrin-assembly protein, has four major tyrosine phosphorylation sites, and the phosphorylation of Tyr31 and Tyr118 correlates with cell adhesion and migration. We found that mutation of Tyr31/118 caused enhanced activation of RhoA and premature formation of stress fibers with substantial loss of efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells. These phenotypes were similar to those induced by RhoA(G14V) in parental cells, and could be abolished by expression of RhoA(T19N), Rac1(G12V), or p190RhoGAP in the mutant-expressing cells. Phosphorylated Tyr31/118 was found to bind to two src homology (SH)2 domains of p120RasGAP, with coprecipitation of endogenous paxillin with p120RasGAP. p190RhoGAP is known to be a major intracellular binding partner for the p120RasGAP SH2 domains. We found that Tyr31/118-phosphorylated paxillin competes with p190RhoGAP for binding to p120RasGAP, and provides evidence that p190RhoGAP freed from p120RasGAP efficiently suppresses RhoA activity during cell adhesion. We conclude that Tyr31/118-phosphorylated paxillin serves as a template for the localized suppression of RhoA activity and is necessary for efficient membrane spreading and ruffling in adhesion and migration of NMuMG cells.     

KDR stimulates endothelial cell migration through heterotrimeric G protein Gq/11-mediated activation of a small GTPase RhoA

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor tyrosine kinases, Flt-1 (VEGFR-1) and KDR (VEGFR-2), both of which are selectively expressed on the primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell (EC) proliferation and migration, whereas Flt-1 down-modulates KDR-mediated EC proliferation. Flt-1 mediates down-regulation of EC proliferation through pertussis toxin-sensitive G proteins, betagamma subunits, small GTPase CDC42, and partly by Rac-1. However, the molecular mechanism by which KDR mediates EC migration is not clear yet. Here we show for the first time that activation of RhoA and Rac1 is fully and partially required for KDR-mediated human umbilical vein endothelial cell (HUVEC) migration, respectively, and that CDC42, however, is not involved. Furthermore, overexpression of the RhoA dominant negative mutant RhoA-19N does not affect VPF/VEGF-stimulated KDR phosphorylation, intracellular Ca(2+) mobilization, and mitogen-activated protein kinase phosphorylation. Utilizing the receptor chimeras (EGDR and EGLT) in which the extracellular domain of the epidermal growth factor receptor (EGFR) was fused to the transmembrane domain and the intracellular domains of KDR and Flt-1, respectively, we demonstrate that RhoA activation is mediated by EGDR, not by EGLT, and that EGDR mediates activation of Rac1, not CDC42. Furthermore, the EGDR-mediated RhoA and Rac1 activation is regulated by G proteins Gq/11, Gbetagamma, and phospholipase C independent of phosphatidylinositol 3-kinase and intracellular Ca(2+) mobilization. Interestingly, the RhoA activation can be partially inhibited by overexpression of Rac1-17N, but overexpression of RhoA-19N has no effect on Rac1 activation. Finally, Gq/11 and Gbetagamma subunits are also required for VPF/VEGF-stimulated HUVEC migration. Taken together, our results indicate that KDR stimulates endothelial cell migration through a heterotrimeric G protein Gq/11 and Gbetagamma-mediated RhoA pathway.     

MEK1 restores migration of polyamine-depleted cells by retention and activation of Rac1 in the cytoplasm

We previously showed that polyamines are required for proliferation and migration both in vivo and in a cultured intestinal epithelial cell (IEC-6) model. Wounding of the IEC-6 monolayer induced transient ERK activation, which was further enhanced by EGF. EGF stimulated migration in control and polyamine-depleted cells, but the degree of stimulation was significantly less in polyamine-depleted cells. Inhibition of MEK1 inhibited basal as well as EGF-induced ERK activation and migration. Expression of constitutively active (CA)-MEK and dominant-negative (DN)-MEK had significant effects on F-actin structure. CA-MEK increased stress fiber and lamellipodia formation, while DN-MEK showed loss of stress fibers and abnormal actin cytoskeletal structure. Unlike EGF, CA-MEK significantly increased migration of both control and polyamine-depleted cells. The most important and significant finding in this study was that polyamine depletion caused localization of Rac1 and RhoA to the nuclear as well as perinuclear regions. Interestingly, CA-MEK completely reversed the subcellular distribution of Rac1 and RhoA proteins in polyamine-depleted cells. Polyamine depletion increased Rac1 in the nuclear fraction and decreased it in the cytoplasmic and membrane fractions of vector-transfected cells. CA-MEK prevented accumulation of Rac1 in the nucleus. Polyamine depletion significantly decreased Rac1 activity during 6-h migration in vector-transfected cells. Cells transfected with CA-MEK had almost identical levels of activated Rac1 in all three groups. These results suggest that polyamine depletion prevents activation of Rac1 and RhoA by sequestering them to the nucleus and that expression of constitutively active MEK reverses this effect, creating the cellular localization required for activation. epidermal growth factor; extracellular signal-regulated kinase; IEC-6 cells     

Isozyme-specific stimulation of phospholipase C-gamma2 by Rac GTPases

The regulation of the two isoforms of phospholipase C-gamma, PLCgamma(1) and PLCgamma(2), by cell surface receptors involves protein tyrosine phosphorylation as well as interaction with adapter proteins and phosphatidylinositol 3,4,5-trisphosphate (PtdInsP(3)) generated by inositol phospholipid 3-kinases (PI3Ks). All three processes may lead to recruitment of the PLCgamma isozymes to the plasma membrane and/or stimulation of their catalytic activity. Recent evidence suggests that PLCgamma may also be regulated by Rho GTPases. In this study, PLCgamma(1) and PLCgamma(2) were reconstituted in intact cells and in a cell-free system with Rho GTPases to examine their influence on PLCgamma activity. PLCgamma(2), but not PLCgamma(1), was markedly activated in intact cells by constitutively active Rac1(G12V), Rac2(G12V), and Rac3(G12V) but not by Cdc42(G12V) and RhoA(G14V). The mechanism of PLCgamma(2) activation was apparently independent of phosphorylation of tyrosine residues known to be modified by PLCgamma(2)-activating protein-tyrosine kinases. Activation of PLCgamma(2) by Rac2(G12V) in intact cells coincided with a translocation of PLCgamma(2) from the soluble to the particulate fraction. PLCgamma isozyme-specific activation of PLCgamma(2) by Rac GTPases (Rac1 approximately Rac2 > Rac3), but not by Cdc42 or RhoA, was also observed in a cell-free system. Herein, activation of wild-type Rac GTPases with guanosine 5'-(3-O-thio)triphosphate caused a marked stimulation of PLCgamma(2) but had no effect on the activity of PLCgamma(1). PLCgamma(1) and PLCgamma(2) have previously been shown to be indiscriminately activated by PtdInsP(3) in vitro. Thus, the results suggest a novel mechanism of PLCgamma(2) activation by Rac GTPases involving neither protein tyrosine phosphorylation nor PI3K-mediated generation of PtdInsP(3).     

RhoA inhibits the nerve growth factor-induced Rac1 activation through Rho-associated kinase-dependent pathway

The Rho family of small GTPases has been shown to be involved in the regulation of neuronal morphology, and Rac and Rho exert antagonistic actions in neurite formation. In this study, we have examined the cross-talk between Rac and Rho in relation to the nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. NGF induced a rapid activation of Rac1 and suppression of RhoA activity. Constitutively active RhoA, RhoA(V14), or constitutively active Galpha(12)-induced endogenous RhoA activation inhibited the NGF-induced Rac1 activation without any effect on the NGF-induced extracellular signal-regulated kinase activation. Moreover, Y-27632, an inhibitor of Rho-associated kinase, completely abolished the RhoA-induced down-regulation of the NGF-induced Rac1 activation. We also revealed that NGF induced a rapid recruitment of Rac1 to the cell surface protrusion sites and formed filamentous actin-rich protrusions. Activation of RhoA and Rho-associated kinase formed a thick ringlike structure of cortical actin filaments at the cell periphery and then inhibited the NGF-induced recruitment of Rac1 to protrusions. These results indicate that RhoA down-regulates the NGF- induced Rac1 activation through Rho-associated kinase, inhibiting the neurite formation.     

Collapsin response mediator protein switches RhoA and Rac1 morphology in N1E-115 neuroblastoma cells and is regulated by Rho kinase

The formation and directional guidance of neurites involves dynamic regulation of Rho family GTPases. Rac and Cdc42 promote neurite outgrowth, whereas Rho activation causes neurite retraction. Here we describe a role for collapsin response mediator protein (Crmp-2), a neuronal protein implicated in axonal outgrowth and a component of the semaphorin 3A pathway, in switching GTPase signaling when expressed in combination with either dominant active Rac or Rho. In neuroblastoma N1E-115 cells, co-expression of Crmp-2 with dominant active RhoA V14 induced Rac morphology, cell spreading and ruffling (and the formation of neurites). Conversely, co-expression of Crmp-2 with dominant active Rac1 V12 inhibited Rac morphology, and in cells already expressing Rac1 V12, Crmp-2 caused localized peripheral collapse, involving Rho (and Cdc42) activation. Rho kinase was a pivotal regulator of Crmp-2; Crmp-2 phosphorylation was required for Crmp-2/Rac1 V12 inhibition, but not Crmp-2/RhoA V14 induction, of Rac morphology. Thus Crmp-2, regulated by Rho kinase, promotes outgrowth and collapse in response to active Rho and Rac, respectively, reversing their usual morphological effects and providing a mechanism for dynamic modulation of growth cone guidance.     

Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein

Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.     

Integrin-mediated adhesion regulates cell polarity and membrane protrusion through the Rho family of GTPases

Integrin-mediated adhesion is a critical regulator of cell migration. Here we demonstrate that integrin-mediated adhesion to high fibronectin concentrations induces a stop signal for cell migration by inhibiting cell polarization and protrusion. On fibronectin, the stop signal is generated through alpha 5 beta 1 integrin-mediated signaling to the Rho family of GTPases. Specifically, Cdc42 and Rac1 activation exhibits a biphasic dependence on fibronectin concentration that parallels optimum cell polarization and protrusion. In contrast, RhoA activity increases with increasing substratum concentration. We find that cross talk between Cdc42 and Rac1 is required for substratum-stimulated protrusion, whereas RhoA activity is inhibitory. We also show that Cdc42 activity is inhibited by Rac1 activation, suggesting that Rac1 activity may down-regulate Cdc42 activity and promote the formation of stabilized rather than transient protrusion. Furthermore, expression of RhoA down-regulates Cdc42 and Rac1 activity, providing a mechanism whereby RhoA may inhibit cell polarization and protrusion. These findings implicate adhesion-dependent signaling as a mechanism to stop cell migration by regulating cell polarity and protrusion via the Rho family of GTPases.     

G alpha 13 signals via p115RhoGEF cascades regulating JNK1 and primitive endoderm formation

The heterotrimeric G-protein G(13) mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this G alpha(13)-mediated pathway, whereas that between G alpha(13) and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active G alpha(13) was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated G alpha(13). Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of G alpha(13). Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated G alpha(13)-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from G alpha(13) to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm.     

Deamidation of RhoA glutamine 63 by the Escherichia coli CNF1 toxin requires a short sequence of the GTPase switch 2 domain

CNF1, a toxin produced by pathogenic Escherichia coli strains, deamidates the RhoA GTP-binding protein glutamine 63 and impairs RhoGAP-mediated GTP hydrolysis resulting in RhoA permanent activation. Using peptides derived from the RhoA sequence, we found that DTAGQEDYDRL (corresponding to RhoA 59-69 residues) was the minimum RhoA-derived peptide which could be deamidated in vitro by the CNF1 catalytic domain (CNF1-Cter). Site-directed mutagenesis outside the RhoA 59-69 sequence had no influence on glutamine 63 deamidation by CNF1-Cter. RhoA proteins with substitutions L57G, D65G, Y66G, or R70G were not affected in their ability to be deamidated by CNF1-Cter, whereas this was abolished by the R68G substitution. Arginine 68 is part of the DYDRL motif that is strictly conserved in Rho, Rac, and Cdc42 but not in other small GTP-binding proteins consistent with the observation that only Rho, Rac, and Cdc42 can be modified by CNF1.     

Disruption of RhoGDI and RhoA regulation by a Rac1 specificity switch mutant

Rho family GTPases are important regulators of the actin cytoskeleton. Activation of these proteins can be promoted by guanine nucleotide exchange factors containing Dbl and Pleckstrin homology domains resulting in membrane insertion of a Rho family member, whereas the inactive GDP-bound form is sequestered primarily in the cytoplasm, bound to the guanosine dissociation inhibitor RhoGDI. Dominant interfering variants of Rac1, but not Cdc42, inhibit beta1 integrin-promoted uptake of Yersinia pseudotuberculosis. Unexpectedly, we found that the Rac1(W56F) guanine nucleotide exchange factors specificity switch mutant blocked invasin-promoted uptake as well as Cdc42-dependent uptake of enteropathogenic Escherichia coli. Fluorescence resonance energy transfer experiments demonstrated that Rac1(W56F) retained the ability to be loaded with GTP, bind a downstream effector, and interact with RhoGDI. Mutational analyses of intragenic suppressors and coexpression studies demonstrated that binding of the Rac1(W56F) mutant to RhoGDI appeared to play a role in the inhibition of uptake. As RhoGDI inhibits RhoA, overactivation of RhoA may account for the uptake interference caused by Rac1(W56F). Consistent with this model, a dominant interfering form of RhoA restored significant uptake in the presence of the Rac1(W56F) mutant but had no effect on another interfering Rac1 form. Furthermore, the cellular GTP-RhoA level was elevated by the presence of Rac1(W56F) mutant protein. These data are consistent with the proposition that Rac1(W56F) blocks invasin-promoted uptake by preventing RhoGDI from inactivating RhoA. We conclude that RhoGDI allows cross-talk between Rho family members that promote potentially antagonistic processes, and disruption of this cross-talk can interfere with invasin-promoted uptake.     

FAP和Rho 家族蛋白在卵巢癌侵袭迁移中的作用

目的：明确FAP 是否通过RhoA/ROCK、Rac1-GTP 通路发挥促增殖、侵袭和迁移作用。方法：用MTT 实验,Transwell 实验 和迁移实验检测FAP、RhoA/ROCK、Rac1-GTP 对卵巢癌细胞系HO-8910PM 的增殖,侵袭和迁移的影响。结果：1、MTT 法,迁移和 侵袭实验证实用Y-27632 抑制RhoA/ROCK 途径能够促进卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用时促进作用增强。 2、MTT 法, 迁移和侵袭实验证实NSC23766 抑制Rac1 途径能够抑制卵巢癌细胞的增殖、迁移和侵袭,与FAP 联合作用使FAP 的 促进作用减弱。结论：1、RhoA/ROCK 通路抑制HO-8910PM 细胞增殖、迁移和侵袭;Rac1-GTP 促进HO-8910PM 细胞增殖、迁移 和侵袭。2、FAP不是通过RhoA/ROCK而是通过Rac1-GTP 信号通路在HO-8910PM细胞发挥促增殖、迁移和侵袭作用的。     

Inhibitory and stimulatory regulation of Rac and cell motility by the G12/13-Rho and Gi pathways integrated downstream of a single G protein-coupled sphingosine-1-phosphate receptor isoform

The G protein-coupled receptors S1P2/Edg5 and S1P3/Edg3 both mediate sphingosine-1-phosphate (S1P) stimulation of Rho, yet S1P2 but not S1P3 mediates downregulation of Rac activation, membrane ruffling, and cell migration in response to chemoattractants. Specific inhibition of endogenous Galpha12 and Galpha13, but not of Galphaq, by expression of respective C-terminal peptides abolished S1P2-mediated inhibition of Rac, membrane ruffling, and migration, as well as stimulation of Rho and stress fiber formation. Fusion receptors comprising S1P2 and either Galpha12 or Galpha13, but not Galphaq, mediated S1P stimulation of Rho and also inhibition of Rac and migration. Overexpression of Galphai, by contrast, specifically antagonized S1P2-mediated inhibition of Rac and migration. The S1P2 actions were mimicked by expression of V14Rho and were abolished by C3 toxin and N19Rho, but not Rho kinase inhibitors. In contrast to S1P2, S1P3 mediated S1P-directed, pertussis toxin-sensitive chemotaxis and Rac activation despite concurrent stimulation of Rho via G12/13. Upon inactivation of Gi by pertussis toxin, S1P3 mediated inhibition of Rac and migration just like S1P2. These results indicate that integration of counteracting signals from the Gi- and the G12/13-Rho pathways directs either positive or negative regulation of Rac, and thus cell migration, upon activation of a single S1P receptor isoform.     

The Effect of Z-Ligustilide on the Mobility of Human Glioblastoma T98G Cells

Z-ligustilide (LIG), an essential oil extract from Radix Angelica sinensis, has broad pharmaceutical applications in treating cardio-vascular diseases and ischemic brain injury. Recently, LIG has been connected to Glioblastoma multiforme (GBM) because of its structural similarity to 3-n-alkyphthalide (NBP), which is specifically cytotoxic to GBM cells. Hence, we investigated LIG’s effect on GBM T98G cells. The study shows that LIG can significantly reduce T98G cells’ migration in a dose-dependent manner. Furthermore, the attenuation of cellular mobility can be linked to the activity of the Rho GTPases (RhoA, Rac1 and Cdc42), the three critical molecular switches governing cytoskeleton remodeling; thus, regulating cell migration. LIG significantly reduces the expression of RhoA and affects in a milder manner the expression of Cdc42 and Rac1.