Curcumin-Mediated Degradation of S-Phase Kinase Protein 2 Induces Cytotoxic Effects in Human Papillomavirus-Positive and Negative Squamous Carcinoma Cells

Abstract

S-phase kinase-associated protein2 (Skp2), a proto-oncoprotein, plays an important role in development and progression of human malignancies. Skp2 is frequently overexpressed in many human malignancies. It targets cell cycle progression through ubiquitin mediated degradation of G1-checkpoint CDK inhibitors-p21 (CDKN1A) and p27 (CDKN1B). We investigated the role of Skp2 and its ubiquitin-proteasome pathway in head and neck squamous cell carcinoma (HNSCC) using a panel of cell lines with and without human papillomavirus (HPV⁺, HPV^-). Treatment of HNSCC cell lines with curcumin, a natural compound isolated from rhizomes of the plant Curcuma longa, or transfection of small interfering RNA of Skp2, causes down-regulation of Skp2 with concomitant accumulation of p21 and p27 in HPV⁺, HPV^- cells. Furthermore curcumin inhibits cell viability and induces apoptosis in a dose-dependent manner. Treatment of HPV⁺ and HPV^- cells with curcumin induced apoptosis via mitochondrial pathway and activation of caspases. In addition, treatment of HPV⁺ and HPV^- cell lines with curcumin down-regulated the expression of XIAP, cIAP1, and cIAP2. Interestingly, co-treatment of HNSCC cells with curcumin and cisplatin potentiated inhibition of cell viability and apoptotic effects. Altogether, these data suggest an important function for curcumin, acting as a suppressor of oncoprotein Skp2 in squamous cell carcinoma cells in both HPV⁺ and HPV^- cells; raise the possibility that this agent may have a future therapeutic role in squamous cell carcinoma.

Keywords: HPV; Skp2; apoptosis; cancer; head and neck squamous cell carcinoma.

Figure 1

Curcumin suppresses dose-dependent cell proliferation in HNSCC cells. Curcumin inhibits the cell viability of HNSCC cells. (A) SCC25 (B), FaDu, and (C) SCC090 cells were incubated with 5, 10, 20, 40, and 80 μM curcumin for 24 h. Cell proliferation assay was performed using CCK8 as described in Materials and Methods. The graph displays the mean ± S.D. (standard deviation) of three independent experiments with replicates of six wells for all the doses. ^*p < 0.05, ^***p < 0.001. Real time cell proliferation (cell index) analysis of HNSCC cells. (D) SCC25 (E) FaDu, and (F) SCC090, cell were grown in monolayer on top of the electrodes and treated with indicated concentration of curcumin. The real time cell analyzer was used to determine cell index as described in method section.

Figure 2

Curcumin-induced apoptosis in HNSCC cells. Curcumin mediated annexin/PI staining in HNSCC cells. (A) SCC25, (B) FaDu, and (C) SCC090 cells were treated with 10, 20, 40 μM curcumin for 24 h and cells were subsequently stained with flourescein-conjugated annexin-V and propidium iodide (PI) and analyzed by flow cytometry. Curcumin mediated apoptosis in HNSCC cell line. (D) SCC25, (E) FaDu, and (F) SCC090 cell line were treated with curcumin and apoptosis was measured by flow cytometry after staining with annexin-V and PI. Percentage of apoptosis relative to untreated cells was calculated as described previously (26). The graph displays the mean ± S.D. of three independent of experiments. ^*p < 0.05, ^**p < 0.01, ^***p < 0.001. Curcumin mediated phosphorylation of H2AX in HNSCC cell lines. (G)SCC25, (H) FaDu, and (I) SCC090 cells were treated with 10, 20, 40 μM curcumin for 24 h and cells were lysed. After lysis proteins were separated by SDS–PAGE, transferred to PVDF membrane and immunoblotted with p-H2AX and GAPDH antibodies as indicated.

Figure 3

Curcumin mediated accumulation of ubiquitinated proteins via suppression of F-box protein Skp2 in HNSCC cell lines (A) Curcumin-mediated ubiquitination of various proteins. SCC25, FaDu, and SCC090 cells were treated with indicated doses of curcumin for 24 h. Equal amounts of protein lysates were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies of anti-ubiquitin and GAPDH as indicated (B) Curcumin treatment down-regulated the expression of Skp2 and enhanced the level of p27 and P21. SCC25, FaDu, and SCC090 cells were treated with various doses of curcumin for 24 h. After cell lysis, equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immuno-blotted with antibodies against Skp2, p27, p21, and GAPDH as indicated (C) Skp2 siRNA transfection downregulates Skp2 and accumulated p27 and p21. FaDu and SCC090 cells were transfected with Scrambled siRNA (100 pmol) and Skp2 siRNA (50 and 100 pmol) using Lipofectamine 2000 as described in Materials and Methods. After 48 h of transfection, cells were lysed and equal amounts of proteins were separated by SDS-PAGE, transferred to PVDF membrane, and immunoblotted with antibodies against Skp2, p27, p21, and GAPDH as indicated.

Figure 4

Curcumin-induced activation mitochondrial apoptotic pathway. (A) Curcumin-mediated upregulation of Bax expression and downregulation of Bcl2 in HNSCC cells. SCC25, FaDu, and SCC090 cells were treated with indicated doses of curcumin for 24 h. Equal amounts of protein lysates were separated by SDS–PAGE, transferred to PVDF membrane and immunoblotted with Bax, Bcl2, and GAPDH antibodies as indicated. (B) Curcumin treatment causes the loss of mitochondrial membrane potential in HNSCC. SCC25, FaDu, and SCC090 cells were treated with indicated doses of curcumin for 24 h. After JC1 staining cells were analyzed by flow cytometry as described in Materials and Methods. The graph displays the mean ± S.D. of three independent of experiments. ^*p < 0.05, ^***p < 0.001. (C) The curcumin-induced release of cytochrome c. SCC25, FaDu, and SCC090 cells were treated with and without curcumin for 24 h. Cytoplasmic fraction was isolated as described in Materials and Methods. Cell extracts were separated on SDS-PAGE, transferred to PVDF membrane, and immunoblotted with an antibody against cytochrome c and GAPDH.

Figure 5

Curcumin-mediated activation of caspases in HNSCC cells: (A) SCC25, (B) FaDu, and (C) SCC090 cell lines were treated with indicated doses of curcumin for 24 h. Equal amounts of protein lysates were separated by SDS–PAGE, transfered to PVDF membrane, and immunoblotted with antibodies of caspases, and GAPDH as indicated.

Figure 6

Curcumin augments the antitumor effect of cisplatin in HNSCC cells. (A) Combination treatment of curcumin and cisplatin potentiated inhibition of cell proliferation of HNSCC cells. FaDu cells were treated either with 10 μM curcumin and 10 μM cisplatin alone or with a combination of 10 μM curcumin and 10 μM cisplatin for 24 h. Cell proliferation assays were performed using CCK8 as described in Materials and methods. The graph displays the mean ± S.D. of three independent experiments with replicates of four wells for all the doses and vehicle control for each experiment. ^*p < 0.05, ^***p < 0.001. (B) Combination treatment of curcumin and cisplatin induced shrinkage (apoptosis) of HNSCC cells. (C) Combination treatment of curcumin and cisplatin potentiates activation of caspase, PARP and p-H2AX in HNSCC cell. FaDu cells were treated either with 10 μM curcumin or 10 μM cisplatin alone or with a combination of 10 μM curcumin and 10 μM cisplatin for 24 h. Cells were subsequently lysed, equal amounts of proteins were separated by SDS–PAGE and immunoblotted with antibodies against PARP, cleaved capsase-3, p-H2AX and tubulin as indicated.