Schematic representation of mechanisms of action of the PCT analogs.

<p>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 p21^Cip1/WAF1, and also p27^kip21, 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 <i>via</i> p53, while dotted arrows indicate additional downstream effectors of TM-025 and TM-026.</p

Treatment with PCT analogs altered cell cycle progression in HNSCC cells.

<p>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.</p

PCT analogs did not inhibit protein synthesis, which is a capstone property of PCT.

<p>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.</p