Abstract
Proton beam is useful to target tumor tissue sparing normal cells by allowing precise dose only into tumor cells. However, the cellular and molecular mechanisms by which proton beam induces tumor cell death are still undefined. We irradiated three different tumor cells (LLC, HepG2, and Molt-4) with low energy proton beam (35 MeV) with spread out Bragg peak (SOBP) in vitro, and investigated cell death by MTT or CCK-8 assay at 24 h after irradiation. LLC and HepG2 cells were sensitive to proton beam at over 10 Gy to induce apoptosis whereas Molt-4 showed rather low sensitivity. Relative biological effectiveness (RBE) values for the death rate relative to gamma-ray were ranged from 1.1 to 2.3 in LLC and HepG2 but from 0.3 to 0.7 in Molt-4 at 11 d after irradiation by colony formation assay. The typical apoptotic nuclear DNA morphological pattern was observed by staining with 4'-6-diamidino-2-phenylindole (DAPI). Tiny fragmented DNA was observed in HepG2 but not in Molt-4 by the treatment of proton in apoptotic DNA fragment assay. By FACS analysis after stained with FITC-Annexin-V, early as well as median apoptotic fractions were clearly increased by proton treatment. Proton beam-irradiated tumor cells induced a cleavage of poly (ADP-ribose) polymerase-1 (PARP-1) and procaspases-3 and -9. Activity of caspases was highly enhanced after proton beam irradiation. Reactive oxygen species (ROS) were significantly increased and N-acetyl cysteine pretreatment restored the apoptotic cell death induced by proton beam. Furthermore, p38 and JNK but not ERK were activated by proton and dominant negative mutants of p38 and JNK revived proton-induced apoptosis, suggesting that p38 and JNK pathway may be activated through ROS to activate apoptosis. In conclusion, our data clearly showed that single treatment of low energy proton beam with SOBP increased ROS and induced cell death of solid tumor cells (LLC and HepG2) in an apoptotic cell death program by the induction of caspases activities.
Highlights
The use of proton beam in radiation therapy has increased considerably in the past few years (Levin et al, 2005; MacDonald et al, 2006)
We found that the doses we used at the position of cells plated were quite optimal to observe the cancer cell death with proton beam
Our experiment used in vitro cell culture systems and determined the relative biological effectiveness (RBE) of proton beam relative to γ-ray at the spread-out Bragg peak (SOBP) on which cells were plated and the cell death mechanisms induced by proton beam irradiation
Summary
The use of proton beam in radiation therapy has increased considerably in the past few years (Levin et al, 2005; MacDonald et al, 2006). Optimization of the doses to induce the cell death of various cancer cells at the Bragg peak is very important to reduce the side effects and maximize the therapy (Belli et al, 2000; Bettega et al, 2000). In the spread-out Bragg peak (SOBP), particles have a broad spectrum of energy with covering sample depth and the SOBP has a higher LET than the entrance beam. The relative biological effectiveness (RBE) of protons for cell survival varies with LET. Actual RBE value is important for the determination of the effective dose to the tumor region. RBE values for proton beam therapy with SOBP relative to γ-ray are evaluated in vitro and in vivo system; for various cells ranging from 0.7 to 2.1 (Paganetti et al, 2002)
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