Low energy proton beam induces tumor cell apoptosis through reactive oxygen species and activation of caspases

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.

Amplification and Overexpression of c-erbB-2 in Gastric Cancer / 대한암학회지

PURPOSE: We compared c-erbB-2 oncogene amplification and oncoprotein expression, trying to identify the biologic and prognostic significance of c-erbB-2 in adendegrees Carcinoma of the stomach. MATERIALS AND METHODS: Formalin-fixed, paraffin-embedded tissue sections from 43 cases of gastric cancer were analyzed for amplification of c-erbB-2 by differential polymerase chain reaction and for overexpression of gene product by immunohistdegrees Chemistry. RESULTS: The amplification was detected in 13 cases (30%). Enhanced c-erbB-2 immunoreactivity was observed in 30% (13/43) of tumors. Tumors with gene amplification generally stained strongly (p=0.003). Although the frequency of amplification and overexpression of c-erbB-2 was increased with advanced gastric cancer and with lymph node metastasis, this difference was not statistically significant. c-erbB-2 gene amplification or protein overexpression showed a trend toward a better five year survival rate, but this did not reach a statistical significance. CONCLUSION: Amplification and/or overexpression of the c-erbB-2 may be of value in clarifying the biologic characteristics of the human gastric cancer. However, more sensitive and more speci fic methods of identifying gene amplification are needed and the standardization of the staining method as well as guidelines for interpreting the staining result are mandatory for this purpose.