Figure 1.
Effect of TQ on cell viability and apoptosis in oral cancer cells.
(A) SCC-4, SAS, SASVO3, OC2, and (B) S-G cells were treated with various TQ concentrations for 24 h. Cell viability was analyzed by MTT assay. (C) SASVO3 cells were treated with TQ for 24 h. Viable cells were then collected and counted using a hemocytometer. (D) Equal numbers of SASVO3 cells were plated and stained using colony formation assay as described in the text. (E) SASVO3 cells were treated with TQ (0, 20, 40, and 60 µM), and cell cycle distributions were assessed by flow cytometry using PI staining. (F) Flow cytometry analysis of annexin V/PI double staining was conducted to determine the number of apoptotic cells. The statistical significance of the results was analyzed by one-way ANOVA and post hoc Dunnett's test (*p<0.05, **p<0.01, ***p<0.001).
Figure 2.
Apoptotic patterns of SASVO3 cells treated with TQ.
Cells cultured with various TQ concentrations for 24 h were examined for apoptosis. (A) Cells were stained with DAPI and observed under a UV-light microscope to examine the nuclear morphology of the SASVO3 cells. The arrows show the areas with intense fluorescence staining and condensed nuclei (at a magnification of 200×). (B) Changes in ΔΨm were assessed using fluorescent lipophilic cationic JC-1 dye. JC-1 is selectively accumulated in intact mitochondria to form multimer J-aggregates that emit fluorescence at 590 nm (red) with a high membrane potential, upper. Monomeric JC-1 emits light at 527 nm (green) with a low membrane potential, lower. Western blot analysis was conducted on (C) caspase-9, Bcl-2, Bax, Bid, p-Histone H2A.X, (D) caspase-3, caspase-8, and (E) PARP. β-actin was used as a loading control. A result representing three separate experiments is shown.
Figure 3.
TQ induces autophagy in SASVO3 cells.
(A) Cells were treated with TQ for 24 h and then stained with MDC visualized at magnification of 200× under a fluorescence microscope. Cells were treated with TQ for 24 h and Western blot analysis was performed to evaluate (B) autophagy-related protein, (C) LC3-I, LC3II, p62, (D) total mTOR, and p-mTOR (Ser2481 and Ser2448). β-actin was used as an internal control. Cells were treated with TQ for an indicated period of time, and Western blot analysis was conducted on (E) cleaved caspase-9, Bax, (F) LC3A-I, and, LC3AII. β-actin was used as a loading control. (G) SASVO3/GFP-LC3 cells were treated with 100 nM bafilomycin-A1 (Baf A1) and TQ (40 and 60 µM) for 24 h. The GFP-LC3 dots induced by TQ and bafilomycin-A1 in SASVO3/GFP-LC3 cells were observed. A result representing three separate experiments is shown.
Figure 4.
TQ induces cell death in SASVO3 cells via two distinct pathways that can induce apoptosis and autophagy.
(A) Cells were pre-treated with Baf A1 (autophagy inhibitor) for 1 h and then exposed to TQ (20 and 40 µM) for 24 h. Cell viability was analyzed by MTT assay. (B) Cells were pre-treated with Baf A1 for 1 h and/or TQ for 24 h. Flow cytometry analysis of annexin V/PI double staining was performed to determine the number of apoptotic cells. (C) Western blot analysis was conducted on LC3-I and LC3-II, and β-actin was used as a loading control. (D) TQ and/or Baf A1 treatment increased the number of MDC-labeled vesicles. (E) Cytoplasmic vacuolization was observed in SASVO3 cells after Baf A1 and/or TQ treatment. (F) TQ-mediated cytotoxicity was analyzed in SASVO3 shLuc (vector control) and SASVO3 shLC3 cells by MTT assay. (G) Expression of LC3A-I and LC3A-II were analyzed in SASVO3 shLuc and SASVO3 shLC3 cells by Western Blot. (H) TQ-mediated apoptosis was analyzed in SASVO3 shLuc and SASVO3 shLC3 cells by flow cytometry analysis. (I) Cells were pre-treated with caspase-9 I (caspase-9 inhibitor) for 1 h and then exposed to TQ (20 and 40 µM) for 24 h. Cell viability was analyzed by MTT assay. (J) Cells were pre-treated with caspase-9 inhibitor for 1 h and then exposed to TQ (40 µM) for 24 h. The number of apoptotic cells was analyzed by flow cytometry. (K) Caspase-9 activity was measured using a caspase-9 activity assay kit. The statistical significance of the results was analyzed using one-way ANOVA with post hoc Dunnett's test (*p<0.05, **p<0.01, ***p<0.001; #p<0.05, ##p<0.01).
Figure 5.
TQ elicits in vivo anti-tumor effects.
BALB/c nude mice (N = 5 for each group) were treated with either olive oil or TQ after SASVO3 cells were subcutaneously implanted (s.c). Tumor growth was then analyzed. (A) Bioluminescence over time after s.c. SASVO3-cell inoculation. (B) Morphological characteristics of the control group treated with 10 and 25 mg/kg/day TQ. (C) Average body weight of the mice. (D) Average tumor weight and (E) tumor volume. Shown are means and standard errors, and the results were statistically evaluated using one-way ANOVA with post hoc Dunnett's test (*p<0.05, **p<0.01, ***p<0.001).
Figure 6.
TQ reduces tumor growth and induces apoptosis and autophagy in vivo.
(A) Ki-67 (cell proliferation marker) immunohistochemistry in SASVO3 tumors. (B) Bax level and LC3 expression and conversion in SAS tumors were determined by western blot analysis. β-actin was used as a loading control.
Figure 7.
Proposed molecular targets in TQ anti-cancer efficacy in SASVO3 cells.
TQ induced cell death in SASVO3 cells via two distinct antineoplastic activities that can induce apoptosis and autophagy. TQ induced autophagosome accumulation, resulting in autophagic cell death and increased DNA damage, potential ΔΨm collapse, chromosome condensation, and caspase-9 activation-dependent apoptosis.