In radiotherapy, cancer stem cells (CSCs) are well recognized as one of the radioresistant cell types. Even in a small subpopulation, CSCs may have an influence on tumor control probability, represented by cell killing after irradiation. However, the relationship between the percentage content of CSCs and the cell survival dose–response curve has not yet been quantitatively clarified. In this study, we developed a cell-killing model for two cell populations (CSCs and progeny cells) to predict the surviving fractions, and compared it with the conventional linear–quadratic (LQ) model. Three prostate cancer cell lines (DU145, PC3 and LNCaP) were exposed to X-rays at doses ranging from 0 to 10 Gy. After the irradiation, we performed clonogenic survival assays to generate the cell survival curves, and carried out flow-cytometric analyses to estimate the percentage content of CSCs for each cell line. The cell survival curves for DU145 cells and PC3 cells seemed not to follow the conventional LQ model in the high dose range (>8 Gy). However, the outputs of the developed model agreed better with the experimental cell survival curves than those of the LQ model. The percentage content of CSCs predicted by the developed model was almost coincident with the measured percentage content for both DU145 cells and PC3 cells. The experiments and model analyses indicate that a small subpopulation of radioresistant CSCs has lower radiosensitivity in the high-dose range, which may lessen the clinical outcome for patients with prostate cancer after high-dose radiation therapy.

Abstract. Hyaluronan (HA) is a major component of the extracellular matrix that is synthesized in excess in cancer tissues. 4-methylumbelliferone (MU) inhibits the synthesis of HA and is closely related to the invasion and metastasis of cancer. However, the effects of MU in conjunction with cancer radiotherapy remain unknown. The present study assessed the anti-tumor and anti-invasion effects of the concomitant use of ionizing radiation (IR) and 100 µM MU on human fibrosarcoma HT1080 cells. Cell viability and cellular invasion potency assays were performed. There was a greater decrease in the viability of cells cultured with a combination of 2 Gy IR and MU compared with untreated control cells. In addition, cell cycle distribution analysis demonstrated that a higher proportion of these cells were in the sub-G1 phase and higher fractions of annexin-V positive, propidium iodide positive cells (i.e., apoptotic cells) were observed. HA concentration in the 2 Gy irradiated culture was similar to that in the non-irradiated control culture, however, it significantly decreased following the administration of both MU alone and 2 Gy IR with MU. Furthermore, treatment with 2 Gy IR and MU resulted in a significant decrease in the invasion rate and matrix metalloproteinase (MMP)-2 and MPP-9 expression. Taken together, these results suggest that the administration of MU with 2 Gy IR is effective at reducing HA production, cell invasion and the metastatic potential of cancer cells.

Hypoxic cancer cells within solid tumours show radio-resistance, leading to malignant progression in fractionated radiotherapy. When prescribing dose to tumours under heterogeneous oxygen pressure with intensity-modulated radiation fields, intercellular signalling could have an impact on radiosensitivity between in-field and out-of-field (OF) cells. However, the impact of hypoxia on radio-sensitivity under modulated radiation intensity remains to be fully clarified. Here, we investigate the impact of hypoxia on in-field and OF radio-sensitivities using two types of cancer cells, DU145 and H1299. Using a nBIONIX hypoxic culture kit and a shielding technique to irradiate 50% of a cell culture flask, oxygen enhancement ratios for double-strand breaks (DSB) and cell death endpoints were determined. These in vitro measurements indicate that hypoxia impacts OF cells, although the hypoxic impacts on OF cells for cell survival were dose-dependent and smaller compared to those for in-field and uniformly irradiated cells. These decreased radio-sensitivities of OF cells were shown as a consistent tendency for both DSB and cell death endpoints, suggesting that radiation-induced intercellular communication is of importance in advanced radiotherapy dose-distributions such as with intensity-modulated radiotherapy.

Acquisition of radioresistance (RR) has been reported during cancer treatment with fractionated irradiation. However, RR is poorly understood in the prognosis of radiotherapy. Although radiotherapy is important in the treatment of prostate cancer (PCa), acquisition of RR has been reported in PCa with an increased number of cancer stem cells (CSCs), neuroendocrine differentiation (NED) and epithelial-mesenchymal transition. However, to the best of our knowledge, the mechanism underlying RR acquisition during fractionated irradiation remains unclear. In the present study, human PCa cell lines were subjected to fractionated irradiation according to a fixed schedule as follows: Irradiation (IR)1, 2 Gy/day with a total of 20 Gy; IR2, 4 Gy/day with a total of 20 Gy; and IR3, 4 Gy/day with a total of 56 Gy. The expression of cluster of differentiation (CD)44, a CSC marker, was identified to be increased by fractionated irradiation, particularly in DU145 cells. The expression levels of CD133 and CD138 were increased compared with those in parental cells following a single irradiation or multiple irradiations; however, the expression levels decreased with subsequent irradiation. RR was evidently acquired by exposure to 56 Gy radiation, which resulted in increased expression of the NED markers CD133 and CD138, and increased mRNA expression levels of the pluripotency-associated genes octamer-binding transcription factor 4 and Nanog homeobox. These data indicate that radiation-induced CSCs emerge due to the exposure of cells to fractionated irradiation. In addition, the consequent increase in the expression of NED markers is possibly induced by the increased expression of pluripotency-associated genes. Therefore, it can be suggested that cancer cells acquire RR due to increased expression of pluripotency-associated genes following exposure to fractionated irradiation.

Tumor recurrence and distant metastasis following radiotherapy, which can lead to poor prognosis, are caused by residual cancer cells that acquire radioresistance. Chemotherapy or a combination of targeted inhibitors can potentially enhance radiation sensitivity and prevent metastasis. It was previously reported that co-administration of the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU) enhanced the lethality of X-ray irradiation in HT1080 human fibrosarcoma cells and decreased their invasiveness to a greater extent than either treatment alone. To clarify the molecular basis of these effects, the present study conducted mRNA expression profiling by cDNA microarray to identify the signaling pathways that are altered under this combination treatment. The activation state of the signaling pathways was classified by z-scores in the Ingenuity Pathway Analysis. The results revealed that the pro-inflammatory cytokines interleukin (IL)-6 and IL-8 were activated by 2 Gy X-ray irradiation, an effect that was abolished by co-administration of 4-MU. Similar trends were observed for the upstream signaling component IL-1. These results indicate that the radiosensitivity of fibrosarcoma cells is improved by suppressing inflammation through the administration of 4-MU.