AIM: Lysyl oxidase-like 2 (LOXL2) has emerged as a biomarker for oral squamous cell carcinoma (OSCC). Its overexpression is associated with poor prognosis in patients, however, the reason behind it is not well understood. The aim of this study is to determine the target for LOXL2 on human gingival fibroblasts (HGF) treated with cancer-conditioned media.
MATERIAL AND METHODS: 30 large (150 x 20mm) plates of human gingival fibroblast cells (HGF) were cultured. For serum depletion, HGF or cancer cells were washed two times with PBS and treated with serum-free DMEM for 24 h. Condition media (CM) was produced by growing cancer cells till they were confluent. Then, 10 plates of HSC3 cells were washed twice with PBS, and were treated with serum-free DMEM for 24 h and the conditioned media was collected. Three (A – CMI, B - CM, C- SF) groups of 10 plates each respectively were made. After incubation for 6 hours, HGFs were washed with ice cold PBS and scraped in about and equal volume of glass beads were added and were subjected to dialysis. Proteins from human fibroblasts were extracted and bioyinylated with biotin hydrazide followed by sodium cyanoborohydride reduction. For affinity binding to Neutravidin, 200 microliters of suspended Neutravidin-agarose was then added to a Neutravidin column at room temperature and equilibrated with 1 ml binding buffer. . The bound proteins were collected and subjected to SDS Page western blot and probed for PDGF Beta, Integrin Alpha V and Beta Actin.
RESULTS: PDGFR beta in fibroblasts displayed marked reduction in oxidation when PSX-S1C inhibitor of LOXL2 was added to cancer cell condition media.
CONCLUSION: The reduction of affinity of PDGF-BB for PDGFR in both the samples indicates the priming role of LOXL2 on PDGFR, which was evident when the inhibitor was added to the samples.
| Identifer | oai:union.ndltd.org:bu.edu/oai:open.bu.edu:2144/41795 |
| Date | 09 December 2020 |
| Creators | Singh, Varun |
| Contributors | Trackman, Phillip C., Bias, Manish V. |
| Source Sets | Boston University |
| Language | en_US |
| Detected Language | English |
| Type | Thesis/Dissertation |
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