177Lu-EDTMP (Ethylenediamine tetramethylene phosphonic acid) is the most used radioactive agent for pain palliation in bone cancer patients. The present study aims to study the impact of relaxin-2 on the 177Lu-EDTMP associated cell toxicity and death in osteosarcoma cells. MG63 and Saos-2 cells were cultured with 177Lu-EDTMP (37 MBq) for 24 h with and without pretreatment of recombinant relaxin 2 (RLXH2) for 12 and 24 h. 177Lu-EDTMP associated cellular deterioration and death was determined by LDH, MTT, and trypan blue dye assays. ELISA-based kit was used to determine apoptotic DNA fragmentation. Western blotting was used to determine expression levels of apoptotic-related signalling pathway proteins like bcl2, poly(ADP-ribose) polymerase (PARP), and MAPK (mitogen-activated protein kinase). Our results found that RLXH2 counters 177Lu-EDTMP associated cellular toxicity. Similarly, RLXH2 was able to counter 177Lu-EDTMP induced cell death in a concentration and time--dependent manner. Furthermore, it was found that RLXH2 treatment prevents apoptosis in 177Lu-EDTMP challenged cells through activation of the notch-1 pathway in a concentration- and time-dependent manner. We reported that RLXH2 significantly declined cellular toxicity and apoptosis associated with 177Lu-EDTMP in MG63 and Saos-2 cells through the notch-1 pathway.

Osteosarcoma is a highly prevalent type of primary bone tissues in children and young adolescents. Micro-RNA (miR) dysregulation has been linked to osteosarcoma tumorigenesis. The role of miR-196-5p was investigated in modulating the growth and metastatic behaviour of human osteosarcoma cells, along with exploring its mechanism of action. As shown by RT-qPCR expression analysis, osteosarcoma cell lines exhibited prominent (P<0.05) transcriptional repression of miR-196-5p. The latter was thus transiently overexpressed in osteosarcoma cells, which resulted in the loss of cell viability and colony formation via induction of autophagy. The western blot analysis of the autophagy marker proteins revealed that the expression of Beclin 1 and LC3B II proteins was induced by miR-196-5p, whereas that of p62 and LC3BI was repressed. Moreover, osteosarcoma cells overexpressing miR-196-5p showed significantly (P<0.05) lower migration and invasion concerning the control osteosarcoma cells. According to the results of the in-silico analysis, Derlin-1 participates in the regulation of miR-196-5p in osteosarcoma, and this prediction has been validated using a dual luciferase assay. The results indicated that miR-196-5p exerted its molecular role by targeting Derlin-1 at the post-transcriptional level. Summing up, the study revealed the modulatory potential of miR-196-5p/Derlin-1 on osteosarcoma cells and provided insights into the possible implications for the treatment and prognosis of the disease.