Capsaicin-induced apoptosis of FaDu human pharyngeal squamous carcinoma cells

Abstract

Purpose: To investigate the anti-tumor effect of capsaicin on human pharyngeal squamous carcinoma cells (FaDu).

Materials and methods: The expression of apoptosis/cell cycle-related proteins (or genes) was examined by reverse transcriptase- polymerase chain reaction, western blotting and ELISA methods, while the apoptotic cell population, cell morphology and DNA fragmentation levels were assessed using flow cytometry, fluorescence microscopy and agarose gel electrophoresis.

Results: Capsaicin was found to inhibit the growth and proliferation of FaDu cells in a dose- and time-dependent manner. Apoptotic cell death was confirmed by observing increases in nuclear condensation, nuclear DNA fragmentation and sub-G1 DNA content. The observed increase in cytosolic cytochrome c, activation of caspase 3 and PARP (p85) levels following capsaicin treatment indicated that the apoptotic response was mitochondrial pathway-dependent. Gene/protein expression analysis of Bcl-2, Bad and Bax further revealed decreased anti-apoptotic Bcl-2 protein and increased pro-apoptotic Bad/Bax expression. Furthermore, capsaicin suppressed the cell cycle progression at the G1/S phase in FaDu cells by decreasing the expression of the regulators of cyclin B1 and D1, as well as cyclin-dependent protein kinases cdk-1, cdk-2 and cdk-4.

Conclusion: Our current data show that capsaicin induces apoptosis in FaDu cells and this response is associated with mitochondrial pathways, possibly by mediating cell cycle arrest at G1/S.

Fig. 1

Morphologic changes and growth inhibition in FaDu cells following exposure to capsaicin. (A) As the incubation time increases, the cell density is reduced, reflecting the inhibitory effects of capsaicin on cell growth. Untreated FaDu cells at 12 h (a) and 24 h (b) and FaDu cells treated with 200 µM capsaicin for 12 h (c) and 24 h (d). (B) The cells were treated with various concentrations of capsaicin for 24, 48 and 72 h. Cell viability was determined by MTT assay. The IC₅₀ of capsaicin against FaDu cells was measured at around 150 µM. The vertical bars in (B) indicate the means and standard errors (n=3).

Fig. 2

The effects of capsaicin on the mitochondrial cytochrome c release into cytoplasm and caspase activity. (A) Analysis of mitochondrial cytochrome c release: cells were treated with 50, 100 and 200 µM capsaicin for 12 h. The level of cytochrome c in the cytosol was detected by using ELISA kit, BS263 (Bender MedSystem, Wien, Austria). (B) Caspase activity was assayed by using a colorimetric substrate DEVD-pNA after treating cells with 200 µM capsaicin for indicated time intervals. Caspase activity was expressed as pmol cleaved/min/µg of protein. Each value is the mean of triplicate experiments. (C) Expression of PARP cleaved form (p85) after treating cells with 200 µM capsaicin for 24 h. The bar graphs represent arbitrary units of relative density after normalized with beta actin. Vertical bars indicate means and standard errors (n=3).

Fig. 3

The effects of capsaicin on apoptosis related gene/protein expression. (A) mRNA expression of Bcl-2 and Bax was assessed by RT-PCR. (B) Protein expression of Bcl-2, Bad and Bax was evaluated by western blotting. FaDu cells were treated with 200 µM capsaicin for 24 h and processed for RT-PCR and western blotting. The bar graphs indicate arbitrary units of relative density that have been normalized using beta actin. The vertical bars indicate the means and standard errors (n=3). RT-PCR, reverse transcriptase-polymerase chain reaction; GAPDH, glyceraldehydes-3-phosphate dehydrogenase.

Fig. 4

Capsaicin-induced DNA condensation and damage in FaDu cells. (A) DAPI staining indicating apoptotic nuclei. FaDu cells treated with capsaicin for 24 h were observed by fluorescence microscopy using a 488 nm filter. (B) Nuclear DNA fragmentation assessed on a 1.8% agarose gel. FaDu cells (1.2×10⁶/well) were incubated with capsaicin for 24 h or untreated. Lane 1, untreated control cells; lane 2, FaDu cells treated with 100 µM capsaicin; lane 3, FaDu cells treated with 300 µM capsaicin. DAPI, 4, 6-Diamidino-2-phenylindole.

Fig. 5

Flow cytometric assay. Cells were untreated or treated with 100 or 200 µM capsaicin for 24 h and then harvested for PI staining. The cellular DNA contents were monitored by flow cytometry. M₁ indicates apoptotic bodies in the sub-G₁ population. (A) Control cells. (B) 100 µM capsaicin treated cells. (C) 200 µM capsaicin treated cells.

Fig. 6

The effects of capsaicin on cell cycle proteins. FaDu cells were treated with 200 µM capsaicin for 24 h. Capsaicin was found to affect the protein expression of cdk-1, cdk-2, cdk-4, cyclin B1 and cyclin D1. The bar graphs represent arbitrary units of relative expression normalized to beta actin. The vertical bars indicate the means and standard errors of three to five independent experiments.