Abstract
Although conventional clinical treatment with low LET (linear energy transfer) including gamma-ray and X-ray has been widely used for radiotherapy in various cancers, however, ineffective outcomes occur due to radioresistance caused by p53 mutation. High LET has become alternative since it is able to induce apoptosis regardless of p53 status. Indeed, the molecular mechanisms toward high LET have been suggested. Nevertheless, most studies have been done in monolayer culture system which cannot promptly represent solid tumor microenvironment. Here we applied in vivo mimic 3D spheroid to conduct microarray-based genomic expression and molecular signaling pathway analyses under neutron irradiation. As a result, 3D spheroid system was achieved using thermorevesible gel system. An effective apoptosis-inducible dose of neutron was determined by Acridine Orange (AO) staining in 3D spheroid. Differentially expressed genes in both unique and common responses to neutron were identified in the 3D spheroid compared to the monolayer cells. Total 95 and 169 genes were notably altered at transcription level toward neutron in monolayer and 3D spheroid system, respectively. Based on microarray data, putative apoptosis signaling was depicted using Pathway Studio software. In 3D-in vivo mimic model, the molecular networks interacted with ITGB1, MAP4K4, PAPPA, and SGK1 might be suggested as plausible molecular pathways. In conclusion, we demonstrate novel molecular signaling and corresponding targets of in vitro solid tumor following high LET exposure. This result might provide critical clues for clarification of neutron-induced apoptosis mechanism.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
U.S. Department of Health and Human Services Public Health Service National Toxicology Program. Report on carcinogens background document for X radiation & gamma radiation and neutron. 2003
Kantor, G. & Simon, J. M. Treatment of bone metastases. Bull Cancer Radiother 83:275–276 (1996).
Mori, E., Takahashi, A., Yamakawa, N., Kirita, T. & Ohnishi, T. High LET heavy ion radiation induces p53-independent apoptosis. J Radiat Res 50:37–42 (2009).
Debus, J., Jäackel, O., Kraft, G. & Wannenmacher, M. Is there a role for heavy ion beam therapy? Recent Results Cancer Res 150:170–182 (1998).
Nakano, T. et al. Carbon beam therapy overcomes the radiation resistance of uterine cervical cancer originating from hypoxia. Clin Cancer Res 12:2185–2190 (2006).
Matsufuji, N. et al. Specification of carbon ion dose at the National Institute of Radiological Sciences (NIRS). J Radiat Res 48:A81–A86 (2007).
Hamada, N. et al. Energetic heavy ions overcome tumor radioresistance caused by overexpression of Bcl-2. Radiother Oncol 89:231–236 (2008).
Masunaga, S. et al. Radiobiologic significance of response of intratumor quiescent cells in vivo to accelerated carbon ion beams compared with γ-rays and reactor neutron beams. Int J Radiat Oncol Biol Phys 70:221–228 (2008).
Takahashi, A. et al. Effects of accelerated carbon-ions on growth inhibition of transplantable human esophageal cancer in nude mice. Cancer Lett 122:181–186 (1998).
Takahashi, A. et al. High-LET radiation enhanced apoptosis but not necrosis regardless of p53 status. Int J Radiat Oncol Biol Phys 60:591–597 (2004).
Takahashi, A. et al. Apoptosis induced by high-LET radiation is not affected by cellular p53 gene status. Int J Radiat Biol 81:581–586 (2005).
Fujita, Y. et al. Role of p53 mutation in the effect of boron neutron capture therapy on oral squamous cell carcinoma. Radiat Oncol 4:63 (2009).
Wang, P. et al. Boron neutron capture therapy induces apoptosis of glioma cells through Bcl-2/Bax. BMC Cancer 10:661 (2010).
Wang, L. P. et al. Neutron-induced apoptosis of HR8348 cells in vitro. World J Gastroenterol 7:435–439 (2001).
Ferrante, A. et al. Increased cell compaction can augment the resistance of HT-29 human colon adenocarcinoma spheroids to ionizing radiation. Int J Oncol 28:111–118 (2006).
Kelm, J. M., Timmins, N. E., Brown, C. J., Fussenegger, M. & Nielsen, L. K. Method for generation of homogeneous multicellular tumor spheroids applicable to a wide variety of cell types. Biotechnol Bioeng 83:173–180 (2003).
Hirschhaeuser, F. et al. Multicellular tumor spheroids: an underestimated tool is catching up again. J Biotechnol 148:3–15 (2010).
Kwon, J. Y. and Seo, Y. R. Genome-wide profiling induced by ionizing radiation (IR) in non-small cell lung cancer (NSCLC) grown as three-dimensional spheroid. Mol Cell Tox 6:229–237 (2010).
Kwon, J. Y. and Seo, Y. R. Differential gene expression following ionizing radiation in multicellular spheroid depending on p53 status: identification of potential targets and prediction of responsive signaling pathways. BioChip J 5:280–288 (2011).
Mizukami-Murata, S. et al. Genome-wide expression changes in Saccharomyces cerevisiae in response to high-LET ionizing radiation. Appl Biochem Biotechnol 162:855–870 (2010).
Kojima, T. et al. Decreased expression of CXXC4 promotes a malignant phenotype in renal cell carcinoma by activating Wnt signaling. Oncogene 28:297–305 (2009).
Liu, A. W. et al. ShRNA-targeted MAP4K4 inhibits hepatocellular carcinoma growth. Clin Cancer Res 17:710–720 (2011).
van Ree, J. H., Jeganathan, K. B., Malureanu, L. & van Deursen, J. M. Overexpression of the E2 ubiquitinconjugating enzyme UbcH10 causes chromosome missegregation and tumor formation. J Cell Biol 188:83–100 (2010).
Gauci, S. et al. Lys-N and trypsin cover complementary parts of the phosphoproteome in a refined SCX-based approach. Anal Chem 81:4493–4501 (2009).
Jin, L., Williamson, A., Banerjee, S., Philipp, I. & Rape, M. Mechanism of ubiquitin-chain formation by the human anaphase-promoting complex. Cell 133:653–665 (2008).
Rajkumar, T. et al. Identification and validation of genes involved in cervical tumourigenesis. BMC Cancer 11:80 (2011).
Lin, J. et al. Expression and effect of inhibition of the ubiquitin-conjugating enzyme E2C on esophageal adenocarcinoma. Neoplasia 8:1062–1071 (2006).
Jang, E. R., Lee, J. H., Lim, D. S. & Lee, J. S. Analysis of ataxia-telangiectasia mutated (ATM)- and Nijmegen breakage syndrome (NBS)-regulated gene expression patterns. J Cancer Res Clin Oncol 130:225–234 (2004).
Scanlan, M. J. et al. Charaterization of human colon cancer antigens recognized by autologous antibodies. Int J Cancer 76:652–658 (1998).
Mao, X. W., Mekonnen, T., Kennedy, A. R. & Gridley, D. S. Differential expression of oxidative stress and extracellular matrix remodeling genes in low- or high-dose-rate photon-irradiated skin. Radiat Res 176:187–197 (2011).
Dragoni, I. et al. EDF-1, a novel gene product downregulated in human endothelial cell differentiation. J Biol Chem 273:31119–31124 (1998).
Brendel, C., Gelman, L. & Auwerx, J. Multiprotein bridging factor-1 (MBF-1) is a cofactor for nuclear receptors that regulate lipid metabolism. Mol Endocrinol 16:1367–1377 (2002).
Amundson, S. A. et al. Human in vivo radiationinduced biomarkers: gene expression changes in radiotherapy patients. Cancer Res 64:6368–6371 (2004).
Solito, E. et al. A novel calcium-dependent proapoptotic effect of annexin 1 on human neutrophils. FASEB J 17:1544–1546 (2003).
Petrella, A. et al. Induction of annexin-1 during TRAIL-induced apoptosis in thyroid carcinoma cells. Cell Death Differ 12:1358–1360 (2005).
Ruiz-Romero, C. et al. Hypoxia conditions differentially modulate human normal and osteoarthritic chondrocyte proteomes. J Proteome Res 9:3035–3045 (2010).
Zhao, Z. S., Li, L., Wang, H. J. & Wang, Y. Y. Expression and prognostic significance of CEACAM6, ITGB1, and CYR61 in peripheral blood of patients with gastric cancer. J Surg Oncol 104:525–529 (2011).
Koutros, S. et al. Pooled analysis of phosphatidylinositol 3-kinase pathway variants and risk of prostate cancer. Cancer Res 70:2389–2396 (2010).
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Kwon, J.Y., Kim, J.M., Ji, Y.H. et al. Genome-wide microarray investigation of molecular targets and signaling networks in response to high-LET neutron in in vivo-mimic spheroid of human carcinoma. Mol. Cell. Toxicol. 8, 9–18 (2012). https://doi.org/10.1007/s13273-012-0002-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s13273-012-0002-z