4'-Chloro-3,5-dihydroxystilbene, a resveratrol derivative, induces lung cancer cell death

Abstract

Aim: To examine the antitumor effect of 4'-chloro-3,5-dihydroxystilbene, a resveratrol derivative, on lung adenocarcinoma A549 cells.

Methods: The cytotoxic IC(50) was determined by direct cell counting. Flow cytometry, monodansylcadaverine (MDC) staining, transfection, Western blot and a proteasome activity assay were used to study the cellular mechanism of 4'-chloro-3,5-dihydroxystilbene. A xenograft nude mouse model was used to analyze the antitumor effect in vivo.

Results: 4'-Chloro-3,5-dihydroxystilbene induced a rapid and persistent increase in the intracellular reactive oxygen species in the cells, but the cell death could not be inhibited by two antioxidant agents. The derivative caused sub-G(1) formation, a decrease in the mitochondria membrane potential and poly (ADP-ribose) polymerase degradation, and the caspase inhibitor Z-VAD-FMK could partially prevent cell death. It also induced a significant increase in intracellular acidic vacuoles, LC3-II formation and intracellular GFP-LC3 aggregation. An autophagic inhibitor partially reversed cell death. Additionally, 4'-chloro-3,5-dihydroxystilbene induced the accumulation of ubiquitinated conjugates and inhibited proteasome activity in cells. In an in vivo study, 4'-chloro-3,5-dihydroxystilbene retarded tumor growth in nude mice.

Conclusion: These data suggest that the resveratrol derivative 4'-chloro-3,5-dihydroxystilbene could be developed as an anti-tumor compound.

Figure 1

The cytotoxic IC₅₀ and morphological changes in resveratrol- and 4′-chloro-3,5-dihydroxystilbene-treated A549 cells. (A) The cytotoxicity IC₅₀ of resveratrol and 4′-chloro-3,5-dihydroxystilbene. A549, NCI-H23, and NCI-H1299 cells were treated with various concentrations of resveratrol or 4′-chloro-3,5-dihydroxystilbene for 48 h. Cell numbers were calculated by the trypan blue dye exclusion method using a hemocytometer. The cell number of untreated cells was 100%, and the other data were presented as the mean±SD from three independent experiments. (B) The morphological changes of A549 cells treated with resveratrol or 4′-chloro-3,5-dihydroxystilbene. A549 cells were treated with DMSO, 100 μmol/L resveratrol or 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 24 h or 48 h. The cells were photographed with an Axio Observer A1 phase-contrast microscope. Magnification ×200.

Figure 2

Effect of ROS on 4′-chloro-3,5-dihydroxystilbene-induced A549 cell death. (A) 4′-Chloro-3,5-dihydroxystilbene-induced ROS production. A549 cells were either untreated or treated with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 1, 3, 6, 24, and 48 h. Thirty minutes prior to harvesting, cells were incubated with 10 μmol/L DCF-DA. After incubation, the cells were harvested for analysis by FACS. The data in each panel represent the DCF fluorescence intensity within the cells. At each time point, the mean intensity of the control cells was at 100 and the other data were measured in this set condition. The values shown are the mean±SD of three determinations. (B) Effect of antioxidants on 4′-chloro-3,5-dihydroxystilbene-induced ROS. 10 mmol/L NAC or 10 mmol/L glutathione was added 1 h before treatment with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 3 h. Cells were harvested for analysis by FACS. The values shown are means±SD of three independent experiments. ^bP <0.05 vs the 4′-chloro-3,5-dihydroxystilbene-treated group. (C) Effect of antioxidants on 4′-chloro-3,5-dihydroxystilbene-induced damage of cellular membrane integrity. The cells were exposed to 10 mmol/L NAC or 10 mmol/L glutathione for 1 h before treatment with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 36 h. A549 cells were harvested and stained with propidium iodide and analyzed by FACS.

Figure 3

Effect of 4′-chloro-3,5-dihydroxystilbene and resveratrol on cell cycle distribution. (A) The cell cycle distribution is altered by 4′-chloro-3,5-dihydroxystilbene or resveratrol treatment. A549 cells were treated with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene or 100 μmol/L resveratrol for 24, 36, or 48 h. Cells were harvested, fixed with ethanol and stained with PI. The DNA content was analyzed by flow cytometry. A representative FACS scan chart of propidium iodide-stained A549 cells shows the percentages of G₀/G₁, S, G₂/M, and sub-G₁ cell cycle phases in each plot. The values shown are the mean of three determinations. (B) Time course of the 4′-chloro-3,5-dihydroxystilbene-induced degradation of cyclins. A549 cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 12, 24, 36, and 48 h. Cell lysates were harvested and analyzed by Western blotting with antibodies against cyclin E, cyclin D1, cyclin D3, cyclin B1 and p21.

Figure 4

Effect of 4′-chloro-3,5-dihydroxystilbene on mitochondria membrane potential. (A) Time course of the 4′-chloro-3,5-dihydroxy-stilbene-induced degradation of Bcl-2 and Bax. A549 cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 12, 24, 36, and 48 h. Cell lysates were harvested and analyzed by Western blotting with antibodies against Bcl-2, Bax and β-actin. (B) 4′-Chloro-3,5-dihydroxystilbene decreased the MMP. A549 cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 24 or 48 h. Cells were harvested and analyzed by FACS. A representative FACS scan chart shows the percentages of low MMP in each plot. The values shown are the mean±SD of three determinations.

Figure 5

The apoptotic parameters of 4′-chloro-3,5-dihydroxystilbene-treated A549 cells. (A) Time course of the 4′-chloro-3,5-dihydroxystilbene-induced degradation of PARP and procaspase-3. A549 cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 12, 24, 36, and 48 h. Cell lysates were harvested and analyzed by Western blotting with antibodies against two forms of PARP, pre-cleavage caspase-3 and β-actin. (B) Effect of Z-VAD-FMK on 4′-chloro-3,5-dihydroxystilbene-induced sub-G₁ formation. Z-VAD-FMK was added 1 h before treatment with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 48 h. Cells were harvested, fixed and stained with propidium iodide and analyzed by FACS. The values shown are the mean±SD of three determinations (C) Effect of Z-VAD-FMK on 4′-chloro-3,5-dihydroxystilbene-induced PARP cleavage. Z-VAD-FMK was added 1 h before treatment with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 24 h. Cell lysates were harvested and analyzed by Western blotting with antibodies against two forms of PARP and β-actin. (D) Effect of NAC and glutathione on 4′-chloro-3,5-dihydroxystilbene-induced sub-G₁ formation. NAC or glutathione was added 1 h before treatment with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene or 100 μmol/L resveratrol for 48 h. Cells were harvested, fixed and stained with propidium iodide and analyzed by FACS. The values shown are the mean±SD of three determinations. (E) Effect of Z-VAD-FMK on 4′-chloro-3,5-dihydroxystilbene-induced damage of cellular membrane integrity. Z-VAD-FMK was added 1 h before treatment with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 48 h. A549 cells were harvested and stained with propidium iodide and analyzed by FACS. The values shown are the mean±SD of three determinations. ^bP<0.05 vs the 4′-chloro-3,5-dihydroxystilbene-treated group.

Figure 6

The autophagic parameters of 4′-chloro-3,5-dihydroxystilbene-treated A549 cells. (A) 4′-Chloro-3,5-dihydroxystilbene increased acidic vacuoles as determined by the MDC staining method. A549 cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 24 h. Cells were then stained with MDC for photography. Magnification ×200. (B) 4′-Chloro-3,5-dihydroxystilbene increased acidic vacuoles as determined by the acridine orange staining method. A549 cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 24 h. Cells were then stained with acridine orange for flow cytometry analysis. ^bP<0.05 vs control. (C) 4′-Chloro-3,5-dihydroxystilbene induces LC3-II formation. A549 cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene or 100 μmol/L resveratrol for 24 h and 48 h. Cell lysates were harvested and analyzed by Western blotting with antibodies against LC3 and . (D) Time course of the 4′-chloro-3,5-dihydroxystilbene-induced aggregation of GFP-LC3 in A549 cells. A549 cells were transiently transfected with the GFP-LC3 plasmid. After transfection, the cells were treated with or without 80 μmol/L 4′-chloro-3,5-dihydroxystilbene for 24 h and 30 h. The cells were photographed with an Olympus IX70 fluorescent microscope. Magnification ×200. (E and F) Effect of autophagy inhibitor (3-MA), protease inhibitors (AEBSF, Z-FA-FMK, TPCK, TLCK, CA074-Me), caspase inhibitor (Z-VAD-FMK) and proteasome inhibitor (MG132) on 4′-chloro-3,5-dihydroxystilbene-induced cell death. A549 cells were individually treated with DMSO or various inhibitors for 1 h, then 80 μmol/L 4′-chloro-3,5-dihydroxystilbene or 100 μmol/L resveratrol was added for 36 h. A549 cells were harvested, stained with propidium iodide, and analyzed by FACS. The values shown are the mean±SD of three determinations.

Figure 7

4′-Chloro-3,5-dihydroxystilbene inhibited proteasome activity in A549 cells. (A) Morphological changes induced by 4′-chloro-3,5-dihydroxystilbene and MG132. A549 cells were treated with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene or 10 μmol/L MG132 for 48 h and the cells were photographed. Magnification ×200. (B) Effect of 4′-chloro-3, 5-dihydroxystilbene on the accumulation of ubiquitinated conjugates. A549 cells were treated with various concentrations of 4′-chloro-3,5-dihydroxystilbene or MG132 for 60 min. Cell lysates were harvested and analyzed by Western blotting with antibodies against ubiquitin and β-actin. (C) Effect of 4′-chloro-3,5-dihydroxystilbene on proteasome activity. A549 cells were treated with 80 μmol/L 4′-chloro-3,5-dihydroxystilbene or 10 μmol/L MG132 for 30 min to 90 min. Cell lysates were harvested and analyzed with proteasome fluorogenic peptide substrates.

Figure 8

Anti-tumor effect of 4′-chloro-3,5-dihydroxystilbene in vivo. Five-week-old female nude mice were injected sc with 1×10⁷ A549 cells in 100 μL of Matrigel in one rear flank. When the tumor volume reached 100 mm³, mice were given vehicle (n=7, control) or 50 mg/kg of 4′-chloro-3,5-dihydroxystilbene (n=7) ip once daily from day 1 to 4 and day 7 to 10. Tumor volume was calculated by the formula mm³=(the long axis)×(the short axis)²/2. ^bP<0.05 vs control.