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

Abstract

The specialized cholesterol/sphingolipid-rich membrane domains termed lipid rafts are highly dynamic in the cancer cells, which rapidly assemble effector molecules to form a sorting platform essential for oncogenic signaling transduction in response to extra- or intracellular stimuli. Density-based membrane flotation, subcellular fractionation, cell surface biotinylation, and co-immunoprecipitation analyses of bichalcone analog ((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4)-treated cancer cells showed dissociation between GRP78 and p85α conferring the recruitment of PTEN to lipid raft membranes associated with p85α. Ectopic expression of GRP78 could overcome induction of lipid raft membrane-associated p85α-unphosphorylated PTEN complex formation and suppression of GRP78PI3KAktGTP-Rac1-mediated and GRP78-regulated PERKNrf2 antioxidant pathway and cancer cell invasion by TSWU-BR4. Using specific inducer, inhibitor, or short hairpin RNA for ASM demonstrated that induction of the lipid raft membrane localization and activation of ASM by TSWU-BR4 is responsible for perturbing homeostasis of cholesterol and ceramide levels in the lipid raft and ER membranes, leading to alteration of GRP78 membrane trafficking and subsequently inducing p85α-unphosphorylated PTEN complex formation, causing disruption of GRP78PI3KAktGTP-Rac1-mediated signal and ER membrane-associated GRP78-regulated oxidative stress balance, thus inhibiting cancer cell invasion. The involvement of the enrichment of ceramide to lipid raft membranes in inhibition of NF-κB-mediated MMP-2 expression was confirmed through attenuation of NF-κB activation using C2-ceramide, NF-κB specific inhibitors, ectopic expression of NF-κB p65, MMP-2 promoter-driven luciferase, and NF-κB-dependent reporter genes. In conclusion, localization of ASM in the lipid raft membranes by TSWU-BR4 is a key event for initiating formation of ceramide-enriched lipid raft membrane platforms, which causes delocalization of GRP78 from the lipid raft and ER membranes to the cytosol and formation of p85α-unphosphorylated PTEN complexes to attenuate the GRP78-regulated oxidative stress balance and GRP78p85αAktGTP-Rac1NF-κBMMP-2-mediated cancer cell invasion.

Keywords: ASM; GRP78; PTEN; ROS; bichalcone analog; ceramide; lipid raft; p85α.

Figure 1

((E)-1-(4-Hydroxy-3-((4-(4-((E)-3-(pyridin-3-yl)acryloyl)phenyl)piperazin-1-yl)methyl)phenyl)-3-(pyridin-3-yl)prop-2-en-1-one (TSWU-BR4) inhibits the invasive activity of cancer cells. (A) The structure of TSWU-BR4. (B,C) The effect of TSWU- BR4 on cancer cell invasion. Cells were treated with vehicle (−) or the indicated concentrations of TSWU-BR4 for 36 h. The number of invaded cells was assessed using the Matrigel invasion assay. Cell viability was determined by the flow cytometric analysis of PI uptake. The values are presented as the means ± standard error (S.E.M.) of three independent experiments. * p < 0.05: significantly different from vehicle (−) or TSWU-BR4-treated cells. (D–F) The effect of TSWU-BR4 on the induction of cancer cell apoptosis. After a 36 h treatment with vehicle, TSWU-BR4 (300 nM), ActD (10 μM), or ActD and Ac-DEVD-CMK (8 μM), the percentage of Annexin V+ cells and caspase-3 activity were determined using flow cytometry. The values are presented as the S.E.M. of three independent experiments. * p < 0.05: significantly different from TSWU-BR4− or ActD-treated cells. The levels of PARP and caspase-3 in the cell lysates were determined by Western blot analysis with specific antibodies. β-Actin was used as an internal control for sample loading.

Figure 2

TSWU-BR4 changes the lipid raft membrane localization of GRP78 and PTEN. (A,B) Cells were treated with vehicle (−) or TSWU-BR4 (300 nM) for 36 h. Detergent-resistant membrane (DRM) and detergent-soluble (DS) fractions were prepared by flotation on a sucrose density gradient. Subcellular plasma membrane (M), cytosolic (C), and nuclear (N) fractions were separated by differential centrifugation. The levels of the indicated proteins in the lysates of vehicle- or TSWU-BR4-treated DRM, DS, M, C, and N fractions were determined by Western blot analysis using specific antibodies. Antibodies against cadherin, α-tubulin, and nucleolin were used as internal controls for the plasma membrane, cytosol, and nucleus, respectively. β-Actin was used as an internal control for sample loading.

Figure 3

TSWU-BR4 induces p85α–unphosphorylated PTEN complex formation (A,B) Coimmunoprecipitation of p85α, p110α, Rac1, and PTEN was performed using the DRM and M fractions prepared from the cells treated as described above. The antibody used for coimmunoprecipitation is indicated at the top. The proteins from the immunoprecipitated complexes were detected using Western blotting with specific antibodies. Normal IgG was used as a control for antibody specificity.

Figure 4

Ectopic expression of GRP78 overcomes TSWU-BR4-induced formation of p85α–unphosphorylated PTEN and inhibition of GRP78-mediated oxidative stress balance and cancer cell invasion. At 12 h after transfection with the GRP78-expressing plasmids or empty vector control, the cells were treated with TSWU-BR4 (300 nM) or TSWU-BR4 (300 nM) plus N-acetyl-L-cysteine (NAC) (100 μM) for an additional 36 h. (A) The GRP78 cell surface (cs) and cytosolic (c) levels were determined by flow cytometry. (B) Biotinylated proteins were pulled down using streptavidin agarose beads. The biotin-streptavidin complexes were immunoblotted with GRP78, p85α, and Rac1 antibodies. (C) The levels of the indicated proteins in the lysates of vehicle- or TSWU-BR4-treated GRP78 transfected cells were determined by Western blot analysis using specific antibodies. β-Actin was used as an internal control for sample loading. The values above the figures represent relative density of the bands normalized to β-actin. (D) The GRP78 used for co-immunoprecipitation is indicated at the top of the figure. The biotin-streptavidin complexes in the lipid rafts were immunoblotted with indicated antibodies. (E) The antibodies used for co-immunoprecipitation are indicated at the top of the figure. The proteins in the immunoprecipitated complexes from the DRM were analyzed by Western blot using specific antibodies. (F) The total cholesterol levels in the whole lysates of the treated cells were measured using the Amplex Red Cholesterol Assay Kit. (G) The generation of ROS was monitored by measuring increased fluorescence of 2,7-dichlorodihydrofluorescein by flow cytometry. (H) The invaded cell numbers were assessed by the Matrigel invasion assays. The values are presented as the S.E.M. of three independent experiments. *Significantly different at p < 0.05.

Figure 5

Lipid raft membrane-associated ASM activity is involved in TSWU-BR4-induced inhibition of GRP78-modulated oxidative stress balance and cell invasion. (A) Cells were treated with vehicle (−) or TSWU-BR4 (300 nM) for 36 h. DRM and DS fractions were prepared by flotation on a sucrose density gradient. The levels of the indicated proteins in the lysates of vehicle- or TSWU-BR4-treated DRM and DS fractions were determined by Western blot analysis using specific antibodies. At 12 h after transfection with an empty vector, GFP shRNA or ASM shRNA cells were treated with either vehicle (−), TSWU-BR4 (300 nM), DOPC (15 nmole), C2-ceramide (6 μM), imipramine (5 μM), TSWU-BR4 (300 nM) plus imipramine (5 μM), amitriptyline (8 μM), or TSWU-BR4 (300 nM) plus amitriptyline (8 μM) for 36 h. (B) The levels of the indicated proteins in the cell lysates were determined by Western blot analysis with specific antibodies. β-Actin was used as an internal control for sample loading. (C) ASM activities were determined by Acidic Sphingomyelinase Assay Kit. Cell viability was determined by the flow cytometric analysis of PI uptake. (D) The lipids were extracted from the DRM or ER fractions, and cholesterol, sphingomyelin, and ceramide were quantitated by Amplex Red Cholesterol Assay Kit and thin-layer chromatography, respectively. Ceramide concentrations were normalized to phospholipid phosphate. (E,F) The antibodies used for co-immunoprecipitation are indicated at the top of the figure. The proteins in the immunoprecipitated complexes from the DRM were analyzed by Western blot using specific antibodies. (G) The invaded cell numbers were assessed by the Matrigel invasion assays. (H) The levels of the indicated protein in the extracts of M, ER, and C fractions prepared from the cells treated as described above and determined by Western blot analysis using specific antibodies. The values are presented as the S.E.M. of three independent experiments. * p < 0.05: significantly different from empty vector-transfected TSWU-BR4-treated cells.

Figure 5

Lipid raft membrane-associated ASM activity is involved in TSWU-BR4-induced inhibition of GRP78-modulated oxidative stress balance and cell invasion. (A) Cells were treated with vehicle (−) or TSWU-BR4 (300 nM) for 36 h. DRM and DS fractions were prepared by flotation on a sucrose density gradient. The levels of the indicated proteins in the lysates of vehicle- or TSWU-BR4-treated DRM and DS fractions were determined by Western blot analysis using specific antibodies. At 12 h after transfection with an empty vector, GFP shRNA or ASM shRNA cells were treated with either vehicle (−), TSWU-BR4 (300 nM), DOPC (15 nmole), C2-ceramide (6 μM), imipramine (5 μM), TSWU-BR4 (300 nM) plus imipramine (5 μM), amitriptyline (8 μM), or TSWU-BR4 (300 nM) plus amitriptyline (8 μM) for 36 h. (B) The levels of the indicated proteins in the cell lysates were determined by Western blot analysis with specific antibodies. β-Actin was used as an internal control for sample loading. (C) ASM activities were determined by Acidic Sphingomyelinase Assay Kit. Cell viability was determined by the flow cytometric analysis of PI uptake. (D) The lipids were extracted from the DRM or ER fractions, and cholesterol, sphingomyelin, and ceramide were quantitated by Amplex Red Cholesterol Assay Kit and thin-layer chromatography, respectively. Ceramide concentrations were normalized to phospholipid phosphate. (E,F) The antibodies used for co-immunoprecipitation are indicated at the top of the figure. The proteins in the immunoprecipitated complexes from the DRM were analyzed by Western blot using specific antibodies. (G) The invaded cell numbers were assessed by the Matrigel invasion assays. (H) The levels of the indicated protein in the extracts of M, ER, and C fractions prepared from the cells treated as described above and determined by Western blot analysis using specific antibodies. The values are presented as the S.E.M. of three independent experiments. * p < 0.05: significantly different from empty vector-transfected TSWU-BR4-treated cells.

Figure 6

Ceramide generation is involved in TSWU-BR4-induced inhibition of GRP78−PI3K−Akt−NF-κB-mediated MMP-2 expression and cellular invasiveness. (A) At 12 h after transfection with the indicated plasmids, the cells were treated with the indicated compounds for an additional 36 h. The levels of the indicated proteins in the whole-cell, cytosolic, and nuclear extracts were then determined with specific antibodies. α-Tubulin and nucleolin were measured as internal controls for the cytosolic and nuclear fractions. The invaded cell numbers were assessed by Matrigel invasion assays. The values are presented as the S.E.M. of three independent experiments. * p < 0.05: significantly different from the vehicle-treated and vector-transfected cells or C2-ceramide-treated and vector-transfected cells. (B,C,D,E) At 12 h after co-transfection with the indicated reporter plasmids, the cells were treated with the indicated compounds for an additional 36 h. The SEAP, MMP-2 promoter, and invasive activities were then determined as described in Material and Methods. * p < 0.05: significantly different from the vehicle-treated, pNF-κB SEAP-transfected cells; the vehicle-treated, MMP-2 promoter-transfected cells; or the vehicle-treated cells.

Figure 7

A molecular model for the induction of the inhibition of cancer cell invasion by TSWU-BR4. (A) The selective interaction of clustered GRP78−PI3K−Akt−GTP-Rac1 signaling molecules in the lipid raft membranes constitutes a central element in the initiation of ER lipid raft membrane-associated GRP78−PERK−Nrf2-mediated oxidative stress balance and NF-κB−MMP-2-mediated cell invasion in response to survival signals. (B) Under the condition of cellular TSWU-BR4 uptake, TSWU-BR4 triggers the lipid raft membrane translocalization of ASM from lysosome to promote the generation of ceramide-rich lipid raft membranes, thus inducting the formation of p85α–unphosphorylated PTEN tetrameric complexes and thereby disturbing the interaction between GRP78 and p85α in the lipid raft membranes. The lipid raft membrane targeting of ASM also lead to the translocation of GRP78 from the ER into the cytosol by displacing cholesterol with ceramide in the ER lipid raft membranes, causing oxidative damage-induced inhibition of cell invasion. The resulting lipid raft membrane-associated p85α–PTEN complexes can negatively regulate Akt activity and attenuate Rac1 activation by dephosphorylating the 3-position of PIP₃ to PIP₂. Akt inactivation was accompanied by attenuation of NF-κB-mediated MMP-2 expression, which can result in the suppression of the invasive activity of cancer cells.