Suppression of phospho‐p85α–GTP‐Rac1 lipid raft interaction by bichalcone analog attenuates cancer cell invasion

Abstract

The p85α subunit of phosphatidylinositol 3-kinase (PI3K) acts as a key regulator of cell proliferation and motility, which mediates signals that confer chemoresistance to many human cancer cells. Using small interfering RNAs against matrix metalloproteinase-2 (MMP-2) and the MMP-2 promoter-driven luciferase assay, we showed that the new synthetic bichalcone analog TSWU-CD4 inhibits the invasion of human cancer cells by down-regulating MMP-2 expression. Treatment with TSWU-CD4 inhibited MMP-2 expression and cell invasion, which were restored by ectopic wild type (wt) p85α or a constitutively active form of MAPK kinase 3 (CA MKK3), CA MKK6, or CA p38α mitogen-activated protein kinase (MAPK). The attenuated formation of lipid raft-associated phospho (p)-p85α-GTP-Rac1 complexes, protein kinase B (Akt) Ser 473 phosphorylation, and cell invasion by TSWU-CD4 was reversed by overexpression of wt p85α or the p85α Brc-homology (BH) domain. The ectopic expression of CA Rac1 (but not wt Rac1) could overcome the suppression of Ser 473 phosphorylation, lipid raft association of Akt, the interaction between GTP-bound Rac1 and p85α in lipid rafts, and cell invasion by TSWU-CD4. The involvement of Akt activity in the functions of NF-κB-mediated MMP-2 was further confirmed through the attenuation of Akt phosphorylation signaling using the Akt-specific inhibitor MK-2206 and ectopic expression of NF-κB p65. Collectively, the inhibitory effect of TSWU-CD4 on cancer cell invasion was likely to suppress the p-p85α-GTP-Rac1 interaction in lipid rafts by targeting the p85α BH domain, which resulted in the suppression of MMP-2 expression via the PI3K-Akt-mediated ERK-MKK3/MKK6-p38 MAPK-NF-κB signaling pathway. © 2016 Wiley Periodicals, Inc.

Impairment of Membrane Lipid Homeostasis by Bichalcone Analog TSWU-BR4 Attenuates Function of GRP78 in Regulation of the Oxidative Balance and Invasion of Cancer Cells

“…Both assays were performed as previously described [35]. Briefly, the pGL3-basic (vector), pGL3-control (control), or MMP-2 promoter promoter plasmids were co-transfected with a β-galactosidase expression vector (pCH110) (10:1) into cells using Lipofectamine 2000 following the manufacturer's protocols (Invitrogen Life Technology, Carlsbad, CA, USA).…”

“…The β-galactosidase expression vector pCH110 was included as an internal control. After 12 h of transfection, the cells were treated with vehicle, TSWU-BR4, or C2-ceramide for an additional 36 h. The cell lysates were harvested, and the protein expression and luciferase activities were assessed as previously described [35].…”

“…The in vitro invasion assay was performed in triplicate as previously described [35] using transwell chamber units with 8 µm pore polycarbonate membranes. Briefly, approximately 1 × 10 4 cells were resuspended in 200 µL of serum-free DMEM, seeded into the upper chambers of the Matrigel-coated transwell plates and treated with vehicle or TSWU-BR4.…”

“…The assay was performed as previously described [35]. Briefly, pTAI-SEAP, pNF-κB-SEAP, or pSEAP2 plasmids were co-transfected into cells with an EGFP expression vector (at a 10:1 ratio).…”

“…At 8 h post-transfection, the cells were incubated with medium containing vehicle (-), TSWU-BR4 (300 nM), C2-ceramide (6 µM), TPCK (10 µM), or PDTC (10 µM) for the indicated periods. For the overexpression assay, overnight cultured cells were co-transfected with the pNF-κB-SEAP and EGFP expression vectors and an NF-κB p65 expression plasmid (10:10:1) for 24 h. The SEAP activity in the medium was evaluated as previously described [35].…”