Fig 1.
PCT analogs reduced viability of human head and neck cancer cells (SCC25 and SCC104) in a dose-dependent manner.
(A-C): 1×104 cells of each type were treated with TM-025 and TM-026 for 24, 48 and 72 h (A, B and C, respectively) in increasing concentrations (1, 10, 100, 500, 1000 and 5000 nM). Percentage viability of SCC104 and SCC25 cells (estimated by MTT assay) were significantly (P<0.05) reduced with higher doses of both analogs. (D): Effect of 48 h treatment of the parent compound PCT in SCC25 and SCC104 cell lines. All experiments have been performed in triplicates, and results obtained have been plotted as the data means (±SEM).
Fig 2.
PCT analogs did not inhibit protein synthesis, which is a capstone property of PCT.
Detection of protein synthesis was performed using the Click-iT azide-alkyne streptavidin HRP reaction model with Western blot. SSC104 cells were incubated in L-methionine-free medium containing 50 μM Click-iT AHA and subsequently treated with 100 nM of TM-025, TM-026 and PCT for 30 min and 90 min. Immunoblot shows the newly synthesized proteins (bands) in TM-025 and TM-026-treated cells, and a lack of such synthesis in PCT-treated cells.
Fig 3.
Dose-dependent inhibition of proliferation in SCC25 and SCC104 cells after treatment with PCT analogs.
(A-D):48-h treatment with TM-025/TM-026 showed significant (P<0.0001; depicted with *) dose-dependent decrease in the levels of Ki67, a cell proliferation marker, in SCC25 (A and B) and SCC104 (C and D) cells. All experiments were performed in five replicates, and the results are presented as means (±SEM). (E-F): 48 h treatment with either of the PCT analogs exhibited a dose-dependent (0, 1, 10 and 50 nM) decrease in the percentage of actively proliferating (BrdU+) cells in SCC25 (E) and SCC104 (F) cells. Results were significant at P<0.0001 (n = 5), and depicted with *.
Fig 4.
PCT analogs did not cause apoptosis or autophagy.
(A): TUNEL assay: 24-h treatment with increasing concentrations (100 and 500 nM) of TM-025 and TM-026 did not show a significant (p<0.05) number of TUNEL+ apoptotic cells (green fluorescence) marked with arrow-heads in SCC25 and SCC104 cells. DNase I (100 ul; 10 U/ml; positive control) treated cells were used as a positive control. (B-C): Autophagy was estimated as a measure of ratio of cellular LC3-II to LC3-I. (B) Western blot depicting levels of LC3-I and LC3-II in PCT analog-treated cells. (C) The ratio of LC3-II:LC3-I did not show a significant (P<0.05) induction (represented by *) of autophagy in the PCT analog-treated cells, in comparison to the positive control (cells treated with 100 μl of 50 ng/ml rapamycin). All sample groups were found to be identical to the vehicle-treated group at the same level of significance (i.e., P<0.05).
Fig 5.
PCT analogs caused senescence in SCC25 and SCC104 cell lines by upregulation of p53, p21Cip1/WAF1 and p27kip21.
(A): SCC25 and SCC104 cells were treated with DMSO (vehicle), 100 nM of TM-025 and TM-026 for 3 days, followed by SA-β-gal staining. A prominent onset of senescence (blue) was observed in both cell lines. (B): Western blots showing activation of p53, p21Cip1/WAF1 and p27kip21 by 50 and 100 nM of the PCT analogs. Quantifications are shown in S4 Fig Part A.
Fig 6.
Treatment with PCT analogs altered cell cycle progression in HNSCC cells.
Flow cytometry-based cell cycle analysis of SCC104 and SCC25 cells upon treatment with PCT analogs. Dose kinetics (A–D) was determined for a period of 24 h (with 1, 10 and 50 nM doses). A significant decline in the percentage of G2 cells was observed, along with a considerable augmentation in the percentage of S-phase cells. Time kinetics (E–H), on the other hand, was investigated for 24, 48 and 72 h for a dose of 1 nM. A time-dependent inhibition of cell cycle progression was observed. All results are significant at P<0.01.
Fig 7.
Altered expression of key cell cycle regulators, silencing of Cdc2 and inhibition of p53 in SCC25 cells by the PCT analogs.
(A): Immunoblotting showed mild dose-dependent (50 and 100 nM) reduction in the expression of cyclin D1, moderate induction of cyclin E, and no or negligible effect on cyclin B. Both PCT analogs did not cause any change in the levels of Cdk2 and Cdk4, but induced the expression of Cdc2 (Cdk1) and Cdc25C. The analogs significantly augmented phosphorylation of Cdc2 at the Tyr15 residue, thus upregulating the level of phospho-Cdc2(Tyr15), which is an inactive form of Cdc2. Induction was also observed in the expression of phospho-Cdc25C(Ser216) in samples treated with TM-025/TM-026. Equal loading was confirmed by β-actin. Quantifications are shown in S4 Fig Part B. (B): siRNA mediated silencing of Cdc2. Cells were transfected with Si-Cdc2 and si-Con (control), and 24h later 50 nM of PCT analogs were added and incubated for 24h and harvested for protein extraction. Immunoblots showed knock down efficiency of the Cdc2 siRNA. Briefly, the gel showed untransfected cells, Cdc2 siRNA-transfected cells, Cdc2 siRNA or control siRNA-transfected cells treated with 50 nM PCT analogs. (C): Bar graph shows % S-phase cells after cell cycle analysis of propidium iodide (PI) stained cells. NS: not significant. (D): Pharmacological inhibition of p53 by PFT. Cells were treated with vehicle, PCT analogs, and PCT analogs with multiple doses of 50 nM PFT. Western blots showing inhibition of p53 by PFT at 72 h in TM-025 and TM-026 (50 nM) treated SCC25 cells. (E) Bar graph shows % S-phase cells after cell cycle analysis of PI-stained cells. Results showing significant changes are denoted by * (P<0.05) and not significant denoted as ns. β-Actin was used as loading control for A, B and D.
Fig 8.
Schematic representation of mechanisms of action of the PCT analogs.
Treatment of SCC25/SCC104 cells with TM-025 or TM-026 induced S-phase cell cycle arrest (S/G2-checkpoint) and senescence, but did not induce apoptosis or autophagy. At the molecular level, TM-025 and TM-026 induced expression of p53, its downstream target p21Cip1/WAF1, and also p27kip21, which are known to arrest cell cycle progression. Inhibition of p53 signaling by PFT abrogated TM-025/TM-026-induced S-phase arrest, thereby confirming a direct role of p53 in these processes. PCT analogs also enhanced phosphorylation of phosphatase Cdc25C (at Ser216 residue) and induced expression of total and Phsopho-Cdc2 (at Tyr15 residue). Inhibition of Cdc2 signaling by siRNA-mediated knockdown of Cdc2 did not relieve the drug-induced S-phase arrest. Solid arrows indicate confirmed mode of action of TM-025/TM-026 mediated via p53, while dotted arrows indicate additional downstream effectors of TM-025 and TM-026.