Hydrogen peroxide/ATR-Chk2 activation mediates p53 protein stabilization and anti-cancer activity of cheliensisin A in human cancer cells

Abstract

Chiliensisine A (Chel A) as a novel styryl-lactone isolated from Goniothalamus cheliensis Hu has been indicated to be a chemotherapeutic agent in Leukemia HL-60 cells. However, its potential for cancer treatment and the underlying mechanisms are not deeply investigated to the best of our knowledge. Current studies showed that Chel A could trigger p53-mediated apoptosis, accompanied with dramatically inhibition of anchorage-independent growth of human colon cancer HCT116 cells. Further studies found that Chel A treatment resulted in p53 protein stabilization and accumulation via the induction of its phosphorylation at Ser20 and Ser15. Moreover, Chel A-induced p53 protein accumulation and activation required ATR/Chk2 axis, which is distinct from the mechanism that we have most recently identified the Chk1/p53-dependent apoptotic response by Chel A in normal mouse epidermal Cl41 cells. In addition, our results demonstrated that hydrogen peroxide generation induced by Chel A acted as a precursor for all these signaling events and downstream biological effects. Taken together, we have identified the Chel A as a new therapeutic agent, which highlights its potential for cancer therapeutic effect.

Fig 1. Chel A inhibited cell viability and anchorage-independent growth via induction of apoptosis in human colon cancer HCT116 cells

(A) HCT116 cells were treated with Chel A for cell proliferation assay as described in “Material and Methods”. After treated for 48 h, cell proliferation was measured by using Cell Titer-GloLuminescent Cell Viability Assay kit. The results are expressed as relative luminescence signal to medium control (proliferation index). Each bar indicates the mean and SD of triplicate assays. The symbol (*) indicates a significant decrease as compared with that of medium control (P<0.05). (B&C) HCT116 cells were exposed to indicate concentrations of Chel A in soft agar as described in Material and Methods. After being cultured in 37°C with 5% CO₂ for 3 weeks, the colony formation was observed under inverted microscope and photographed (B). The number of colonies was scored, and presented as colonies per 10⁴ seeded cells (C). The symbol (*) indicates a significant decrease as compared with that of vehicle control (P <0.05). Each bar indicates the mean and SD from three independent experiments. (D-F) HCT116 cells (D & E) or U5637 cells (F) were cultured in each well of a six-well plate with McCoy's 5A medium containing 10% FBS at 37°C overnight. After synchronization of cells by culturing in McCoy's 5A medium containing 0.1% FBS for 24 hours, the cells were treated with various concentrations Chel A as indicated, for 48 hours (D) or with 4.0 ¼M Chel A for indicated time periods (E) or for 36 hrs (F). The cells as indiacted were collected and subjected to flow cytometry assay (D) and Western blot assay (E & F). The result was representative one from three independent experiments.

Fig 2. p53 induction mediated Chel A-induced apoptosis

(A) HCT116 cells were treated with Chel A for indicated time periods and cell extracts were subjected to Western blotting for the detection of phosphorylated and total p53 protein expression. β-Actin was used as a control for protein loading. (B&C) HCT116 p53+/+ and p53−/− cells were seeded into six-well plates. The cells were treated with Chel A for 48 hrs and then subjected to flow cytometry assay (B) or treated with Chel A for 48 hrs and then subjected to Western blotting to determine the expression and cleavages of PARP and Caspase 3. β-Actin was used as a control for protein loading (C). The result showing was a representative one from three independent experiments. (D&E) HCT116 p53+/+ and p53−/− cells were exposed to indicate concentrations of Chel A in soft agar as described in Material and Methods. After being cultured in 37℃ with 5% CO₂ for 3 weeks, the colony formation was observed under inverted microscope and photographed (D). The relative colony formation was presented as the colony in Chel A-treated group relative to the vehicle control in HCT116 p53+/+ and p53−/− cells, respectively (E). The symbol (*) indicates a significant increase in HCT116 p53−/− cells with treatment of Chel A as compared with that in HCT116 p53+/+ cells treated with Chel A (p <0.05). Each bar indicates the mean±SD of three independent experiments.

Fig 3. p53 protein induction by Chel A was via the inhibition of p53 protein degradation

(A) HCT116 cells were treated with Chel A for indicated time periods, and p53 mRNA was measured by RT-PCR. (B) HCT116 cells were pretreated with MG132 for 4 hrs, followed by exposure with CHX combined with Chel A or CHX alone as indicated. Then cell extracts were subjected to Western Blotting and β-Actin protein expression was used as a protein loading control. (C) HCT116 cells were exposed to Chel A for indicated time periods, and cell extracts were subjected to Western Blotting and β-Actin protein expression was used as a protein loading control. (D) HCT116 cells were treated with Chel A for indicated time periods, and ATR mRNA was evaluated by RT-PCR.

Fig 4. Chk2 mediated the biological effect of Chel A in HCT116 cells

(A) HCT116 Chk2−/− cells and its parental wild type cells were treated with Chel A as indicated, and the cell extracts were subjected to Western Blotting for determination of the protein expressions using specific antibodies. β-Actin was used as protein loading controls. (B) HCT116 Chk2−/− and its parental wild type cells were pretreated with MG132 for 4 hrs, followed by exposure with CHX combined with Chel A or CHX alone as indicated. Then cell extracts were subjected to Western Blotting and β-Actin protein expression was used as a protein loading control. (C&D) HCT116 Chk2+/+ and Chk2−/− cells were treated with Chel A (4.0 ¼M) for 48 hrs. Cells were then collected for flow cytometry assay (C) or subjected to Western blotting as indicated (D). The result showing was a representative one from three independent experiments. (E&F) HCT116 Chk2+/+ and Chk2−/− cells were exposed to indicate concentrations of Chel A in soft agar. After being cultured in 37°C with 5% CO₂ for 3 weeks, the colony formation was observed under inverted microscope and photographed (E). The relative colony formation was presented as the colony in Chel A-treated group relative to the vehicle control in HCT116 Chk2+/+ and Chk2−/− cells, respectively (F). The symbol (*) indicates a significant increase in HCT116 Chk2−/− cells with Chel A treatment as compared with that in HCT116 Chk2+/+ cells treated with Chel A (P<0.05). Each bar indicates the mean±SD of three independent experiments

Fig 5. ATR conditional knockout attenuated the biological effect of Chel A in HCT116 cells

(A) HCT116 ATR flox^/− cells and its parental wild type cells were treated with Chel A as indicated, and the cell extracts were subjected to Western blotting for determination of the protein expressions using specific antibosies. β-Actin was used as protein loading controls. (B&C) HCT116 ATR flox^/- and wild type cells were were treated with Chel A for 48 hrs. The cells were then collected and subjected to flow cytometry analysis (B), or subjected to Western blotting(C). The result showing was a representative one from three independent experiments. (D&E), HCT116 ATR flox/- and its parental wild type cells were exposed to indicated concentrations of Chel A in soft agar. After being cultured in 37°C with 5% CO₂ for 3 weeks, the colony formation was observed under inverted microscope and photographed (D). The relative colony formation was presented as the colony in Chel A-treated group relative to the vehicle control in HCT116 ATR^+/+ and ATR flox^/- cells, respectively (E). The symbol (*) indicates a significant increase in HCT116 ATR flox^/- cells with Chel A treatment as compared with that in HCT116 cells treated with Chel A (P<0.05). Each bar indicates the mean±SD of three independent experiments.

Fig 6. mCat overexpression blocked the biological effect of Chel A-induced hydrogen peroxide in HCT116 cells

(A&B) HCT116 cells were pretreated with 2',7'-dichlorfluorescein-diacetate (DCFH) (A) ordihydroethidium (HE) (B), respectively, for 0.5 hr, followed by being treated with Chel A for indicated time periods. Then cells were washed with PBS, and subjected to fluorescence intensity detection. The results were shown as the induction of each time point relative to 0 h(Relative fluorescence intensity). The symbol (*) indicates a significant increase as compared with that at 0 h (P <0.05). Each bar indicates the mean±SD from three independent experiments. (C&D) HCT116 mCat and its parental HCT116 (vector) cells were treated with Chel A for 0-6 hrs, and the cell extracts were subjected to Western blotting. β-Actin was used as protein loading controls. (E&F) HCT116 mCat and HCT116 (vector) cells were treated with indicated concentrations of Chel A in soft agar. After being cultured in 37°C with 5% CO₂ for 3 weeks, the colony formation was observed under inverted microscope and photographed (E). The relative colony formation was presented as the colony in Chel A-treated group compared with vehicle control in HCT116 mCat and HCT116 (vector) cells, respectively (F). The symbol (*) indicates a significant increase in HCT116 mCat cells treated with Chel A as compared with that of HCT116 (vector) cells treated with Chel A (p <0.05). Each bar indicates the mean±SD of three independent experiments. (G)The diagram indicates mechanisms responsible for Chel A-induced apoptosis in HCT116 cells.