Figure 1.
EF25-(GSH)2 inhibited proliferation of tumor cells in vitro.
(A) The structures of curcumin, EF25 and EF25-(GSH)2. (B) a and b, EF25-(GSH)2 showed similar toxicity towards six human tumor cells (BEL-7402, HCT116, HepG2, A549, SMMC-7721 and Hela) (a) and the toxicity of curcumin was much lower than that of EF25-(GSH)2 (b). c, cells were incubated with increasing doses of indicated compounds for 24-, 48-, and 72-h periods and analyzed by MTT assay. The IC50 of each agent at each time period was calculated and compared using SPSS. The IC50 of EF25-(GSH)2 is much lower than that of curcumin and essentially equivalent to that of cisplatin. d, the cytotoxicity of EF25-(GSH)2 to HL-7702 cells was much lower than that of cisplatin and similar to curcumin after 48-hour incubation as determined by MTT assay (*, p<0.01, **, p<0.001). (C) Cells were incubated with 0.5 µmol/L of the indicated compound for 24 h and subsequently allowed to grow into colonies (2 weeks). EF25-(GSH)2 totally inhibited colony formation leading to clean plates, while curcumin and cisplatin did not. Results are representative of three independent experiments.
Figure 2.
EF25-(GSH)2 suppressed HepG2 xenograft growth in vivo.
(A) HepG2 cells were injected into the left flank of nude mice and tumors were allowed to grow to a size of about 100 mm3. Subsequently, EF25-(GSH)2 (dissolved in PBS, 1.5 mg/kg body weight) was injected daily i.p. for 30 d (n = 6). The cisplatin group (dissolved in PBS, 0.5 mg/kg body weight) was injected every other day i.p. (n = 4), and the control group was injected with the same volume of PBS daily i.p. (n = 6). Tumor growth was significantly suppressed in the EF25-(GSH)2-treated group compared to either control (**, p<0.001) or cisplatin-treated group (*, p<0.01). (B) At the end of the treatment, tumor volume in the EF25-(GSH)2-treated group was much smaller than that of the control group. (C) The EF25-(GSH)2-treated group maintained normal weight gain while the cisplatin-treated group suffered a remarkable weight loss throughout the treatment (*, p<0.001). (D) At the end of the treatment, EF25-(GSH)2 treatment resulted in significantly lower tumor weight when compared with control group.
Figure 3.
The morphological appearance of EF25-(GSH)2-treated HepG2 cells.
(A) HepG2 cells treated with increasing concentrations of EF25-(GSH)2 for 16 h were observed under a light microscope and representative images were visualized. EF25-(GSH)2-treated cells underwent vacuolization, the extent of which varied when treated with different concentrations of EF25-(GSH)2. At 20 µmol/L, apoptotic-like cell membrane blebbing was observed (arrowheads). (B) A representative transmission electron microscopy (TEM) image of untreated HepG2 cells. (C) In 5 µmol/L EF25-(GSH)2-treated cells, most vacuolated cells regained normal morphology at 32 h post-treatment (arrows, 1-4) while some did not (arrow heads, 5 and 6). (D) Representative TEM images of cells treated with 10 µmol/L EF25-(GSH)2 for 16 h. *, large empty vacuoles with varying size. (E) Representative TEM images of cells treated with 20 µmol/L EF25-(GSH)2 for 16 h. *, large empty vacuoles; arrows, autophagic vacuoles.
Figure 4.
Morphology of autophagosomes in EF25-(GSH)2-treated HepG2 cells.
HepG2 cells were treated with 20 µmol/L EF25-(GSH)2 for 16 h and observed under transmission electron microscopy. (A) and (B), multimembranous autophagic vacuoles engulfing cytoplasmic components are indicated with black arrowheads. (C) and (D), autophagic vacuoles containing a mitochondrion are indicated with black asterisk.
Figure 5.
EF25-(GSH)2 induced autophagy in HepG2 cells.
(A) Western blot analysis of the LC3B expression in HepG2 cells treated with EF25-(GSH)2 at varying concentrations for 12 to 48 h with or without chloroquine (CQ, 100 µmol/L). (B) The cellular distribution of mCherry-GFP-LC3B in HepG2 cells treated with EF25-(GSH)2 at different concentrations for 24 h was examined under a laser confocal microscope. (C) Lysates from HepG2 cells incubated with 10 µmol/L EF25-(GSH)2 for 12 or 24 h pretreated with or without wortmannin (Wm, 100 nmol/L, pretreated for 2 h) were analyzed by Western blotting for LC3B expression level.
Figure 6.
The apoptosis in HepG2 cells triggered by EF25-(GSH)2 in the presence or absence of CQ/Z-VAD-FMK.
(A) HepG2 cells were treated with various concentrations of EF25-(GSH)2 for 24 h and 48 h with or without chloroquine (CQ, 100 µmol/L)/Z-VAD-FMK (30 µmol/L, pretreated for 2 h) and then analyzed for DNA content (propidium iodide, PI) and cell cycle distribution. Apoptosis was measured as the percentage of cells containing hupodiploid quantities of DNA (sub-G1-G0 peak). Percentage of cells within the sub-G1-G0 and G2/M stages is shown for each data point. Graphs are representative of data collected from three independent experiments. (B) HepG2 cells incubated with increasing concentrations of EF25-(GSH)2 for 48 h were stained with 4, 6-diamidino-2-phenylindole (DAPI) and examined by laser confocal microscopy. Untreated HepG2 cells showed uniformly stained nuclei, while EF25-(GSH)2-treated cells exhibited chromatin condensation in a concentration-dependent manner. (C) Lysates from HepG2 cells incubated with increasing concentrations of EF25-(GSH)2 for 24 or 48 h with or without chloroquine (CQ, 100 µmol/L) were analyzed by Western blotting for both full length and cleaved caspase-3 and caspase-8 expression levels.
Figure 7.
The effect of Wm, CQ and Z-VAD-FMK on the cytotoxicity and morphological changes induced by EF25-(GSH)2 in HepG2 cells.
(A) Cell viability was determined by the MTT assay after treatment with increasing concentrations of EF25-(GSH)2 for 24 h or 48 h in the absence or presence of CQ (100 µmol/L)/Wm (100 nmol/L, pretreated for 2 h)/Z-VAD-FMK (30 µmol/L, pretreated for 2 h). *, p<0.001, EF25-(GSH)2 plus Z-VAD-FMK vs. EF25-(GSH)2 alone. **, p<0.001, EF25-(GSH)2 plus CQ vs. EF25-(GSH)2 alone. (B) Representative light microscopic images of HepG2 cells treated with various concentrations of EF25-(GSH)2 for 24 h in the absence or presence of CQ (100 µmol/L)/Z-VAD-FMK (30 µmol/L, pretreated for 2 h). (C) Representative light microscopic images of HepG2 cells treated with 10 µmol/L EF25-(GSH)2 for 48 h in the absence or presence of Z-VAD-FMK (30 µmol/L, pretreated for 2 h).
Figure 8.
Knockdown of Atg5 and Beclin-1 expression does not rescue EF25-(GSH)2-treated HepG2 cells.
(A) HepG2 cells respectively transduced with shLacZ-, shBeclin-1-C2-, shBeclin-1-C3-, shAtg5-D8- and shAtg5-D9-lentivirus were mock-, or treated with 10 µmol/L EF25-(GSH)2 for 24 h. Cells lysates were analyzed by Western blotting with antibodies against Atg5, Beclin-1, LC3 or actin, as indicated. (B) For HepG2 cells respectively transduced with shLacZ-, shBeclin-1-C2-, shBeclin-1-C3-, shAtg5-D8- and shAtg5-D9-lentivirus, cell viability was determined by MTT assay after treatment with increasing concentrations of EF25-(GSH)2 for 48 h. (C) HepG2 cells respectively transduced with shLacZ-, shBeclin-1-C2- and shAtg5-D8-lentivirus were treated with 10 µmol/L EF25-(GSH)2 for 24 h and observed under the light microscope.
Figure 9.
Working model of the mechanisms of EF25-(GSH)2-induced cell death in HepG2 cells.
Stress induced by EF25-(GSH)2 promotes autophagy in HepG2 cells. When treated with EF25-(GSH)2 at concentrations of 5 µmol/L or lower, cells experienced full-scale autophagy that displayed moderate cytoplasmic vacuolization, ultimate recovery and partial rescue of cells from the resulting stress. In contrast, the protective autophagy was blocked in cells treated with EF25-(GSH)2 at concentrations of 10 µmol/L or higher which led to massive cytoplasmic vacuolization. The latter cells arrested in the G2/M phase succumbed to both caspase-dependent and caspase-independent cell death. EF25-(GSH)2 treatment alone led mainly to caspase-dependent apoptotic cell death, but also to a significant proportion of caspase-independent apoptosis. The action of EF25-(GSH)2 could be modulated by CQ (green) and Z-VAD-FMK (blue). Co-treatment of EF25-(GSH)2 with CQ promoted autophagy blockage and cytoplasmic vacuolization, which then enhanced apoptosis for both caspase-dependent and caspase-independent mechanisms. Co-treatment of EF25-(GSH)2 with Z-VAD-FMK inhibited caspase activation and subsequently blocked the caspase-dependent apoptotic death route. Thus, cells were trapped by cytoplasmic vacuolization and G2/M cell cycle arrest, which eventually led to non-apoptotic cell death.