High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation - PubMed (original) (raw)

High LET radiation enhances apoptosis in mutated p53 cancer cells through Caspase-9 activation

Nobuhiro Yamakawa et al. Cancer Sci. 2008 Jul.

Abstract

Although mutations in the p53 gene can lead to resistance to radiotherapy, chemotherapy and thermotherapy, high linear energy transfer (LET) radiation induces apoptosis regardless of p53 gene status in cancer cells. The aim of this study was to clarify the mechanisms involved in high LET radiation-induced apoptosis. Human gingival cancer cells (Ca9-22 cells) containing a mutated p53 (mp53) gene were irradiated with X-rays, C-ion (13-100 KeV/microm), or Fe-ion beams (200 KeV/microm). Cellular sensitivities were determined using colony forming assays. Apoptosis was detected and quantified with Hoechst 33342 staining. The activity of Caspase-3 was analyzed with Western blotting and flow cytometry. Cells irradiated with high LET radiation showed a high sensitivity with a high frequency of apoptosis induction. The relative biological effectiveness (RBE) values for the surviving fraction and apoptosis induction increased in a LET-dependent manner. Both RBE curves reached a peak at 100 KeV/microm, and then decreased at values over 100 KeV/microm. When cells were irradiated with high LET radiation, Caspase-3 was cleaved and activated, leading to poly (ADP-ribose) polymerase (PARP) cleavage. In addition, Caspase-9 inhibitor suppressed Caspase-3 activation and apoptosis induction resulting from high LET radiation to a greater extent than Caspase-8 inhibitor. These results suggest that high LET radiation enhances apoptosis by activation of Caspase-3 through Caspase-9, even in the presence of mp53.

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Figures

Figure 1

Figure 1

Surviving fraction after exposure to X‐rays or to heavy‐ion beams. (a) Radiation sensitivity; (b) RBE at D 10. Cells were irradiated with X‐rays (formula image) or C‐ion beams with LET values of 13 KeV/µm (Δ), 30 KeV/µm (formula image), 70 KeV/µm (formula image), and 100 KeV/µm (formula image), and Fe‐ion beams with LET values of 200 KeV/µm (formula image). The data are presented as the means and standard errors of three independent experiments.

Figure 2

Figure 2

Time‐ and dose‐dependent apoptosis induction after exposure to X‐rays or heavy‐ion beams. (a) Time course of apoptosis induced by X‐ray and heavy‐ion beam irradiation with 2 Gy. (b) The induction of apoptosis was investigated after irradiation with beams of various LET values at 48 h after a 2 Gy irradiation. (c) The induction of apoptosis as a function of dose. The data are presented as the means and standard errors of three independent experiments. (d) Relative biological effectiveness at a dose which results in 10% apoptosis induction. Cells were irradiated with X‐rays (formula image), C‐ion beams with LET values of 13 KeV/µm (Δ), 30 KeV/µm (formula image), 70 KeV/µm (formula image), 100 KeV/µm (formula image), and Fe‐ion beams with LET value of 200 KeV/µm (formula image). The data are presented as the means and standard errors of three independent experiments.

Figure 3

Figure 3

Caspase‐3 activity after exposure to X‐rays or heavy‐ion beams. Fragmentation of PARP and Caspase‐3 at 48 h after a 2 Gy irradiation is shown.

Figure 4

Figure 4

Caspase‐3 activity after exposure to X‐rays or Fe‐beams in viable cells. Caspase‐3 activity was analyzed at 48 h after irradiation. (a) without Caspase inhibitors, and (b) with Caspase‐3, Caspase‐8, and, Caspase‐9 inhibitors (at 20 µM concentrations). The data are presented as the means and standard errors of three independent experiments.

Figure 5

Figure 5

Effect of Caspase inhibitors on apoptosis induction. Induction of apoptosis was investigated at 48 h after D 10 irradiation in the presence of inhibitors for (a) Caspase‐3 (2–100 µM) (b) Caspase‐8 (5–200 µM), and (c) Caspase‐9 (2–100 µM). Cells were irradiated with C‐ion beams with LET values of 100 KeV/µm (formula image) and X‐rays (formula image). Relative apoptosis values were calculated as apoptosis induction with or without inhibitors. The data are presented as the means and standard errors of three independent experiments.

Figure 6

Figure 6

A model for high LET radiation‐induced _p53_‐independent apoptosis. Apoptotic pathways triggered by high LET radiation do not require p53. Any damage induced by high LET radiation is proposed to be a critical trigger for the activation of the Caspase‐9‐related apoptotic pathway, rather than the Caspase‐8 related pathway. Caspase‐3 is activated by Caspase‐9 leading to _p53_‐independent apoptosis. In addition, Caspase‐8 is not activated because p53 is defective and does not respond in a normal manner.

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