Figure 1.
Clotrimazole inhibits OSCC cells proliferation.
OSCC cells (CAL27, SCC25, and UM1) were treated with clotrimazole (0–80 µM) for 24 h, 48 h and 72 h, and cell viability was detected using a Cell Counting Kit-8 assay. The results presented as mean ± standard deviation values for three independent experiments.
Figure 2.
Clotrimazole inhibits colony formation of OSCC cells.
OSCC cells (CAL27, SCC25, and UM1) grown in 6-well plates (1000 cells/well) were incubated with various concentrations of clotrimazole (0, 10, 20 and 30 µM) for two weeks. Cell colonies were stained and counted as described in the Methods section. The results presented as mean ± standard deviation values for three independent experiments. *P<0.05; **P<0.01 compared with solvent control.
Figure 3.
Clotrimazole induces G0/G1 cell cycle arrest in OSCC cells.
OSCC cells were exposed to various concentrations of clotrimazole (0, 30 and 40 µM) for 24 h. Cell cycle distributions were analyzed by flow cytometry with PI staining. *P<0.05, **P<0.01 as compared with the CAL27 control cells; # P<0.01 as compared with the SCC25 control cells; ‡P<0.01 as compared with the UM1 control cells.
Figure 4.
Clotrimazole induces apoptosis of OSCC cells.
CAL27, SCC25 and UM1 cells were incubated with various concentrations of clotrimazole (0, 30 and 40 µM) for 24 h and labeled with Annexin V and propidium iodide (PI). (A) Apoptosis of OSCC cells was analyzed by flow cytometry. The bottom right quadrant represents the percentage of early apoptotic cells (Annexin V+/PI−), whereas the top right quadrant is the percentage of late apoptotic cells (Annexin V+/PI+). (B) Percentages of cells in apoptosis at each clotrimazole concentration. The results are presented as the mean of three similar experiments. *P<0.05; **P<0.01 compared with solvent control.
Figure 5.
Clotrimazole regulates apoptotic protein levels in OSCC cells and tumor tissue.
(A) CAL27 and UM1 cells were treated with 40 µM clotrimazole or DMSO for 12 h, 24 h, and 48 h. The expression of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax was assessed by western blot. (B) The apoptosis-related protein expressions in tumors from control mice and clotrimazole-treated mice (150 mg/kg) were also analyzed by western blot analysis. Clotrimazole treatment decreased the expression of Bcl-2 and increased the level of Bax in clotrimazole-treated tumors compared with control animals. Data shows the representative of three independent experiments. Quantification of bands was performed using Image J software and the relative ratio was calculated by the density.
Figure 6.
Clotrimazole slows the growth of human OSCC xenograft tumors in nude mice.
A total of 5×106 CAL27 cells/mouse were injected subcutaneously into the back next to the right front limb. When a tumor became palpable, clotrimazole (150 mg/kg/body) was administered intraperitoneally for 2 weeks, 6 times per week, control mice treated with equal volume of peanut oil. (A) Representative photographs of the gross tumors from nude mice treated with clotrimazole or peanut oil. (B) Graphs represent the average tumor volumes of CAL27 xenografts in mice from the control and clotrimazole-treated groups. (C) Graphs represent the average weight of tumors from the control and clotrimazole-treated groups. (D) PCNA expression in tumor tissues was assessed by IHC. The bar graph shows PCNA labeling index (PCI) in twelve tumors per each experimental group. PCI (%) = positive tumor cells/total tumor cells×100%. (E) Cleaved caspase-3 expression in tumor tissues was assessed by IHC. The bar graph shows cleaved caspase-3 labeling index (CI) in twelve tumors per each experimental group. CI (%) = positive tumor cells/total tumor cells ×100%. *P<0.05; ** P<0.01 compared with control nude mice.