Suberoylanilide hydroxamic acid induces apoptosis and sub-G1 arrest of 320 HSR colon cancer cells

Abstract

Background: Histone deacetylases and histone acetyl transferases covalently modify histone proteins, consequentially altering chromatin architecture and gene expression.

Methods: The effects of suberoylanilide hydroxamic acid, a HDAC inhibitor, on 320 HSR colon cells were assessed in 320 HSR colon cancer cells.

Results: Concentration and time-dependent inhibition of 320 HSR cell proliferation was observed. Treatment of 320 HSR cells with 5 μM SAHA for 72 h significantly inhibited their growth by 50% as compared to that of the control. Fluorescence-activated cell sorting analysis demonstrated significant inhibition of cell cycle progression (sub-G1 arrest) and induction of apoptosis upon various SAHA concentrations after 48 h. In addition, the anti-apoptosis proteins, survivin and Bcl-xL, were significantly inhibited by SAHA after 72 h of treatment. Immunocytochemistry analysis revealed that SAHA-resistant cells were positive for cyclin A (85%), ki-67 (100%), p53 (100%), survivin (100%), and p21 (90%) expression. Furthermore, a significant increase cyclin A-, Ki-67-, p53-, survivin-, and p21-positive cells were noted in SAHA-resistant tumor cells.

Conclusion: Our results demonstrated for the first time in 320 HSR colon adenocarcinoma cells that SAHA might be considered as an adjuvant therapy for colon adenocarcinoma.

Figure 1

Morphology of 320 HSR colon cancer cells after 48 h treatment with 5 μM SAHA. Original magnification ×400.

Figure 2

Effects of SAHA on 320 HSR cell growth using the MTT assay. 320 HSR cells were treated with various concentrations of SAHA for 24, 48, and 72 h.

Figure 3

SAHA induces apoptosis in colon cancer cell lines. 320 HSR cells were stained with Annexin V (FITC) and propidium iodide (PI) after treatment with SAHA. Fluorescence-activated cell sorting analysis of 320 HSR cancer cell line at 48 h following treatment with 0, 1, 2.5, and 5 μM SAHA (A, B, C, D, respectively). Percentages represent Annexin V-positive/PI-negative (early apoptotic) and Annexin V-positive/PI-positive cells (apoptotic).

Figure 4

Fluorescence activated cell sorting (FACS) analysis revealed SAHA-induced sub-G1 arrest in 320HSR cells. Cells were harvested 48 h after stimulation in the absence or presence of SAHA (0.1 μM to 5 μM). Intracellular PI fluorescence intensities of cells are presented in the upper panels. The percentage of cells in the G0/G1 phase was significantly inhibited by SAHA treatment after 24 or 48 h. The percentage of cells in the sub-G1 phase was significantly increased in response to SAHA treatment.

Figure 5

SAHA alters survivin, C-PARP, and Bcl-xL protein levels in 320 HSR cells. Western blot analysis of cells treated with or without SAHA (1 or 3 μM) for 24, 48, and 72 h.

Figure 6

Histogram values are means and standard error of band densitometry data measured in figure 5. *P <0.05 vs. control.

Figure 7

Effects of SAHA on VEGF secretion by 320 HSR cells. Cells were treated with different SAHA concentrations for 24 h and 48 h. VEGF concentrations in the conditioned medium were determined by ELISA and normalized to 1 × 10⁵ viable cells.

Figure 8

Immunocytochemical analysis of cyclin A and Ki-67 expression in 320 HSR cells. Cells were treated with or without SAHA (1 or 5 μM) for 48 h. Original magnification ×400.

Figure 9

Immunocytochemical analysis of p53, survivin, and p21 expression in 320 HSR cells. Cells were treated with or without SAHA (1 or 5 μM) for 48 h. Original magnification ×400.