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The in vitro and in vivo antitumor effects of clotrimazole on oral squamous cell carcinoma.

Wang J, Jia L, Kuang Z, Wu T, Hong Y, Chen X, Leung WK, Xia J, Cheng B - PLoS ONE (2014)

Bottom Line: Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro.In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax.Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Medicine, Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China.

ABSTRACT

Background: Clotrimazole is an antifungal imidazole derivative showing anti- neoplastic effect in some tumors, but its anticancer potential is still unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the antitumor effect of clotrimazole, and to investigate the possible mechanism of clotrimazole-mediated antitumor activity in OSCC.

Methodology: In vitro experiments, the cell viability and clonogenic ability of three human OSCC cell lines CAL27, SCC25 and UM1 were detected after clotrimazole treatment by CCK8 assay and colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the involvement of several mediators of apoptosis was examined by western blot analysis. Then, the in vivo antitumor effect of clotrimazole was investigated in CAL27 xenograft model. Immunohistochemistry and western blot analysis were performed to determine the presence of apoptotic cells and the expression of Bcl-2 and Bax in tumors from mice treated with or without clotrimazole.

Results: Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Clotrimazole caused cell cycle arrest at the G0/G1 phase. In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax. Notably, clotrimazole treatment inhibited OSCC tumor growth and cell proliferation in CAL27 xenograft model. Clotrimazole also markedly reduced Bcl-2 expression and increased the protein level of Bax in tumor tissues of xenograft model.

Conclusion: Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC.

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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.
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pone-0098885-g006: 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.

Mentions: To evaluate whether clotrimazole affects tumor growth in vivo, the CAL27 cell line was selected for the establishment of the OSCC xenograft nude mouse model. Ten days later, the mice were randomly assigned into control and treated groups. The tumor volume (mean±SD) was 52.7±2.1 mm3 in control group and 51.7±2.3 mm3 in clotrimazole-treated group. Then clotrimazole was injected intraperitoneally at a dose of 150 mg/kg/day 6 days a week for two weeks. During the 14 days of drug or vehicle treatments, all the mice appeared to be healthy and there was no obvious sign or symptom of drug toxicity. The animal weight was not significantly different between the groups at any time point (Fig. S1). There are no obvious abnormalities were detected upon gross observation of the heart, liver, spleen, kidney, and gastrointestinal tract in clotrimazole-treated animals (Fig. S2). Consistent with our in vitro results, intraperitoneal administration of clotrimazole strikingly decreased the tumor volume of CAL27 cell xenograft in nude mice by 57.9% (P = 0.042, Fig.6A,B), and the mean weights of the excised tumors were approximately 53.6% lower in clotrimazole-treated mice than in control mice (P = 0.035, Fig.6C). The proliferation rates of tumor cell were assessed in situ by PCNA antigen labeling index. Our findings showed that clotrimazole treatment decreased tumor cell proliferation (Fig.6D).


The in vitro and in vivo antitumor effects of clotrimazole on oral squamous cell carcinoma.

Wang J, Jia L, Kuang Z, Wu T, Hong Y, Chen X, Leung WK, Xia J, Cheng B - PLoS ONE (2014)

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.
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pone-0098885-g006: 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.
Mentions: To evaluate whether clotrimazole affects tumor growth in vivo, the CAL27 cell line was selected for the establishment of the OSCC xenograft nude mouse model. Ten days later, the mice were randomly assigned into control and treated groups. The tumor volume (mean±SD) was 52.7±2.1 mm3 in control group and 51.7±2.3 mm3 in clotrimazole-treated group. Then clotrimazole was injected intraperitoneally at a dose of 150 mg/kg/day 6 days a week for two weeks. During the 14 days of drug or vehicle treatments, all the mice appeared to be healthy and there was no obvious sign or symptom of drug toxicity. The animal weight was not significantly different between the groups at any time point (Fig. S1). There are no obvious abnormalities were detected upon gross observation of the heart, liver, spleen, kidney, and gastrointestinal tract in clotrimazole-treated animals (Fig. S2). Consistent with our in vitro results, intraperitoneal administration of clotrimazole strikingly decreased the tumor volume of CAL27 cell xenograft in nude mice by 57.9% (P = 0.042, Fig.6A,B), and the mean weights of the excised tumors were approximately 53.6% lower in clotrimazole-treated mice than in control mice (P = 0.035, Fig.6C). The proliferation rates of tumor cell were assessed in situ by PCNA antigen labeling index. Our findings showed that clotrimazole treatment decreased tumor cell proliferation (Fig.6D).

Bottom Line: Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro.In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax.Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC.

View Article: PubMed Central - PubMed

Affiliation: Department of Oral Medicine, Guanghua School of Stomatology, Guangdong Provincial Key Laboratory of Stomatology, Sun Yat-sen University, Guangzhou, Guangdong, China.

ABSTRACT

Background: Clotrimazole is an antifungal imidazole derivative showing anti- neoplastic effect in some tumors, but its anticancer potential is still unclear in oral squamous cell carcinoma (OSCC). The aim of this study was to evaluate the antitumor effect of clotrimazole, and to investigate the possible mechanism of clotrimazole-mediated antitumor activity in OSCC.

Methodology: In vitro experiments, the cell viability and clonogenic ability of three human OSCC cell lines CAL27, SCC25 and UM1 were detected after clotrimazole treatment by CCK8 assay and colony formation assay. Cell cycle progression and apoptosis were assessed by flow cytometry, and the involvement of several mediators of apoptosis was examined by western blot analysis. Then, the in vivo antitumor effect of clotrimazole was investigated in CAL27 xenograft model. Immunohistochemistry and western blot analysis were performed to determine the presence of apoptotic cells and the expression of Bcl-2 and Bax in tumors from mice treated with or without clotrimazole.

Results: Clotrimazole inhibited proliferation in all three OSCC cell lines in a dose-and time-dependent manner, and significantly reduced the colony formation of OSCC cells in vitro. Clotrimazole caused cell cycle arrest at the G0/G1 phase. In addition, clotrimazole induced apoptosis in OSCC cells, and significantly down-regulated the anti-apoptotic protein Bcl-2 and up-regulated the pro-apoptotic protein Bax. Notably, clotrimazole treatment inhibited OSCC tumor growth and cell proliferation in CAL27 xenograft model. Clotrimazole also markedly reduced Bcl-2 expression and increased the protein level of Bax in tumor tissues of xenograft model.

Conclusion: Our findings demonstrated a potent anticancer effect of clotrimazole by inducing cell cycle arrest and cellular apoptosis in OSCC.

Show MeSH
Related in: MedlinePlus