Oridonin induces apoptosis and senescence in colorectal cancer cells by increasing histone hyperacetylation and regulation of p16, p21, p27 and c-myc

Abstract

Background: Oridonin, a tetracycline diterpenoid compound, has the potential antitumor activities. Here, we evaluate the antitumor activity and action mechanisms of oridonin in colorectal cancer.

Methods: Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Cell cycle distribution was determined by flow cytometry. Apoptosis was examined by analyzing subdiploid population and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay. Senescent cells were determined by senescence-associated β-galactosidase activity analysis. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc. The concomitant changes of protein expression were analyzed with Western blot. Expression of AcH3 and AcH4 were examined by immunofluorescence staining and Western blots. Effects of oridonin on colony formation of SW1116 were examined by Soft Agar assay. The in vivo efficacy of oridonin was detected using a xenograft colorectal cancer model in nude mice.

Results: Oridonin induced potent growth inhibition, cell cycle arrest, apoptosis, senescence and colony-forming inhibition in three colorectal cancer cell lines in a dose-dependent manner in vitro. Daily i.p. injection of oridonin (6.25, 12.5 or 25 mg/kg) for 28 days significantly inhibited the growth of SW1116 s.c. xenografts in BABL/C nude mice. With western blot and reverse transcription-PCR, we further showed that the antitumor activities of oridonin correlated with induction of histone (H3 and H4) hyperacetylation, activation of p21, p27 and p16, and suppression of c-myc expression.

Conclusion: Oridonin possesses potent in vitro and in vivo anti-colorectal cancer activities that correlated with induction of histone hyperacetylation and regulation of pathways critical for maintaining growth inhibition and cell cycle arrest. Therefore, oridonin may represent a novel therapeutic option in colorectal cancer treatment.

Figure 1

Oridonin inhibits cell proliferation of colorectal cancer cells. (A) Chemical structure of oridonin. (B), (C) and (D) HCT116, HT29 and SW1116 cells were treated with 0, 6.25, 12.5, 25, 50 100 μM oridonin for 1, 2 and 3 days. Effects of oridonin on cell proliferation were determined by using a CCK-8 Kit. Error bars represent SD of experiments.

Figure 2

Oridonin induces cell cycle arrest and apoptosis. HCT116 and SW1116 cells were treated with 0, 12.5, 25 or 50 μM Oridonin for 24 hours. Cell cycle distribution was determined by flow cytometry and the representative graphs are shown in (A) and (B). Apoptosis was examined by analyzing subdiploid population (A and B, black arrow) and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assay (D and F), in which at least 1,000 cells from randomly selected high-power fields were counted. Symbol * represents P < 0.05 compared with control group. The indicated proteins were detected by western blot (C and E).

Figure 3

Oridonin induces cellular senescence. HCT116 and SW1116 cells were treated with 0, 12.5 or 25 μM oridonin for 72 hours. Morphology of control cells and oridonin-treated cells were examined by phase-contrast microscopy (A and C) (×200). Senescent cells were determined by senescence-associated β-galactosidase activity analysis (B and D) (×200).

Figure 4

Oridonin regulates the expression of p21, p16, p27 and c-myc in vitro. SW1116 cells were treated with 25 μM Oridonin for 0, 24, 48 and 72 hours. Semi-quantitative RT-PCR was used to examine the changes of mRNA of p16, p21, p27 and c-myc (A). (B) The graph shows densitometric analyses of the p16, p21, p27 or c-myc/GAPDH the ratios from (A). The concomitant changes of protein expression were analyzed with Western blot (C). (D) The graph shows densitometric analysis of the p16, p21, p27 or c-myc relative to GAPDH. Symbol * represents P < 0.05 compared with control group.

Figure 5

Induction of hyperacetylated histone H3 (AcH3) and H4 (AcH4) during the treatment with oridonin. SW1116 cells were treated with 25 μM oridonin for 72 hours. (A) Heterochromatin formation was shown by DAPI staining. Distribution of AcH3 and AcH4 were examined by immunofluorescence staining with the indicated antibodies (×200). (B) Protein levels of AcH3 and AcH4 were detected by Western blots with the indicated antibodies. H3 , H4 and GAPDH were used as loading control. (C) The graph shows densitometric analysis of the expression of AcH3 and AcH4 relative to GAPDH. Symbol * represents P < 0.05 compared with control group.

Figure 6

Oridonin inhibits colony formation of SW1116 in vitro and tumor growth in vivo. SW1116 cells were treated with 0, 6.25, 12.5, 25, 50, 100 μM oridonin for 14 days. (A) The CFE was defined as the percentage of plated cells that formed colonies relative to an untreated control (untreated cells = 100%). The means ± standard deviations of six independent experiments per oridonin concentration are shown. The symbol * represents P < 0.05 compared with control group. SW1116 cells (1 × 10⁷ cells in 100 μl of PBS) were subcutaneously injected into the right flank of BALB/c nude mice. The mice were then administered intraperitoneally with 0.2 ml of PBS or DMSO (1% in PBS) or Oridonin (6.25, 12.5 and 25 mg/kg respectively) daily when tumors reached a volume of 50-100 mm³. (B) Tumor dimension was periodically measured using calipers over a 4-week period. Each point represents average volume calculated from ten mice. (C) One day after the last treatment, tumors were excised from the animals, and tumor weight was measured. The data are representative of three independent experiments. Symbol * represents P < 0.05 compared with control group. (D) Changes in body weight of mice treated with PBS or DMSO (1% in PBS) or oridonin.

Figure 7

Oridonin inhibits tumor growth in nude mice through induction apoptosis and senescence. The mice were administered intraperitoneally with 0.2 ml of PBS or DMSO (1% in PBS) or indicated dose of oridonin for 28 days, then the tumors were excised for pathological examination. (A). Tumor section from PBS or DMSO (1% in PBS) or oridonin treated mice were stained with hematoxylin-eosin (HE) (×200). (B). Apoptotic colorectal cells were assessed by fluorescent TUNEL assay (×100). (C). Senescent colorectal cells were examined by Senescence-associated-β-galactosidase (SA-β-Gal) staining (×200).