In Vitro Studies Of Abscopal Effect And Mechanisms On The Skeleton By Irradiation

[Background and Objectives]With advances in cancer treatment and diagnostic methods, significant increase has been observed in the survival rate of cancer patients. Radiation therapy is an important approach for cancer treatment worldwide. Radiation therapy can kill tumor cells at the same time can damage the normal tissue. Non-malignancy bone complications, frequently in the form of bone loss and in many cases diagnosed only after the occurrence of fractures, are common post-irradiation complications. The etiology of non-malignancy bone complications in cancer patients treated with radiotherapy remains unclear and seems to be multifactorial.Skeletal complications from radiation therapy have been reported for breast, brain, pelvic, and blood cancers. Cumulative evidence of clinical study and animal experiment suggests the role of abscopal effects in the development of injuries in normal tissues including the skeleton after local radiotherapy. In addition, kidney is one of the complications of abdominal radiotherapy that cannot be ignored, and abnormal renal function will lead to bone metabolism disorders. This may also be the occurrence mechanism of local and systemic bone damage including nonirradiated bone density decreased, osteoporosis and non-brittle fracture incidence increase in patients with abdominal radiotherapy. It remains unclear however, whether localized radiotherapy could result in systemic adverse effects on the unirradiated skeleton by an abscopal mechanism.First, osteoblast and NRK-52E were irradiated by 137Cs y-rays. Conditioned medium, osteoblasts-osteoblasts co-culture system, NRK-52E-osteoblasts co-culture system were established. To discuss abscopal bone damage and cellular mechanisms caused by ionizing radiation we observe the proliferation, differentiation, mineralization, and osteogenic related gene expressions and apoptosis rate of unirradiated osteoblasts. This study may provide a new target for therapeutic intervention and theoretical basis of clinical cancer radiotherapy-related bone injury.[Methods]Calvarias osteoblasts were prepared from calvarias of 1-or 2-day old SD rats. Osteoblastic cells were irradiated by different doses (0,1,2,5, lOGy) of 137Cs y-rays. Medium from irradiated OB/NRK-52E was harvested and transferred to unirradiated OB in 10 and 20 percentage. Cell morphology was observed with a microscopy, cell viability was analyzed by MTT assay, and ALP activity was analyzed by enzyme histochemistry and PNPP. Mineralization of the nodules in the cultures was assessed using alizarin red staining. Gene expressions were analyzed using RT-PCR for ALP, OPG and OC. Annexin V-FITC/PI staining was employed to determine the impact of ICCM on apoptosis.To further clarify the biological functions and mechanisms of irradiated osteoblast\NRK-52E for unirradiated osteoblasts, a transwell plate co-culture system was established. The first part of experiment, osteoblast/NRK-52E was treated with 137Cs γ-rays in advance and cultured for 2,24,48 and 72 h after irradiation, then co-cultured with osteoblast unexposed, and observe the biological changes of OB (NR). Osteoblast/NRK-52E were irradiated with different doses of y-rays and then assigned to co-culture groups as follows:① Single-culture group (OB);②Control group (OB/NRK-52E (OGy)+osteoblasts);③(OB/NRK-52E (1Gy)+osteoblasts);④(OB/NRK-52E (2Gy)+osteoblast;⑤ (OB/NRK-52E (5Gy)+osteoblast and (OB/NRK-52E (10Gy)+osteoblasts). A series of biological indicators such as proliferation, differentiation, mineralized nodule area, gene and protein levels of OB (NR) were measured to evaluate the indirect effects of ionizing radiation.[Results]1. ICCM may regulate osteoblast function, inhibiting viability and differentiation, resulting in apoptosis or cell death. ICCM (10% or 20%) from osteoblasts irradiated with 10Gy γ-rays significantly inhibited the proliferation of osteoblastic cells (p<0.01). Meanwhile, an increase in apoptosis of osteoblasts incubated with ICCM (40%) was noted with increasing radiation dose. In addition, ICCM (20%) inhibited the ALP activity and mineralization in both group, particularly of 10Gy ICCM (p<0.05). Meanwhile, ALP, OPG and OC mRNA expression of cells treated with ICCM (20%) were down-regulated significantly than those treated with medium from unirradiated cells.2. The proliferation and activity of ALP in 5Gy-2h group relative to the control group (OGγ) showed obvious inhibition. The 5, 10Gγ OB (IR) inhibited the proliferation of OB (NR) obviously. The activity of ALP was significantly reduced in 1,5, 10Gγ groups. The area of mineralization nodules were decreased in the 1, 2Gy groups. The expression levels of ALP, OC, OPG, RANKL mRNA of the co-culture group were significantly lower than those in control group (p<0.001); accompanied by up-regulation of caspase3 mRNA in 5, 10Gy (p<0.05). ALP, caspase 3 protein levels were also changed.3.10% NRK-52E conditioned medium had no obvious effect on the proliferation and ALP activity of OB (NR), and proliferation was decreased whereas ALP was increased in the 10Gγ ICCM group. In the 20% group, proliferation and ALP activity were both inhibited, and there is significant difference in 10,15Gγ ICCM group. As for the time-response, NRK-52E cultured 72h after 5Gy y-rays showed obvious inhibitory effect on osteoblastic proliferation and activity of ALP (F=14.212, p<0.01). The following dose response experiment used 72h time point. After co-culturing with NRK-52E (IR), the OB (NR) showed decrease in viability and ALP activity, both had statistical significance at 1Gγ. The activity of ALP were suppressed in 1,5, 10Gγ and there was dose-response relationship.[Conclusions]1. Using conditioned medium methods and osteoblast-osteoblasts co-culture system, we prove that irradiated-osteoblast may regulates function of non-irradiated -osteoblast, inhibit viability and differentiation, and cause apoptosis or cell death. Some cytokines produced by osteoblasts after exposure may be associated with non-target effects of ionizing radiation related.2. Proximal tubular epithelial cells after irradiation have a direct regulatory role on osteoblasts, there is a hint of possible mode of kidney-bone action in radiotherapy-induced bone loss in cancer.3. The established co-culture system can be used to study the further mechanism of radiation induced abscopal effect, including the mechanism and pathway of in vitro kidney-bone abscopal effect.