Screening And Identifying Of L-OHP-resistant Proteins In Colorectal Cancer Cell Lines

Objective To screen and identify L-OHP-resistant proteins to find candidateprotein markers for tailored therapy of CRC.Method L-OHP-resistant human CRC cell lines HT-29/L-OHP were establishedby gradually increasing the concentration of L-OHP. The cytotoxicities in HT-29andHT-29/L-OHP cells induced by L-OHP were detected by MTT assay. The totalproteins of human CRC cell lines HT-29and HT-29/L-OHP were extracted, and thenthe expressions of P-gp and MRP were measured by Western blot. The differentiallyexpressed proteins in the HT-29and HT-29/L-OHP cells were separated andidentified by2-DE and MALDI-TOF/TOF. PCBP1、TUBB2A、ANXA3and STIP1obtained through proteomics were validated by Western blot in the cell lines.Result The HT-29/L-OHP cell lines were successfully established. The IC50induced by L-OHP in HT-29cell lines and HT-29/L-OHP cell lines were (4.15±0.17)μg/ml and (32.01±1.87) μg/ml respectively (P<0.05). They were significantlydifferent. The resistance index was7.71. Compared with HT-29cell lines, theexpressions of P-gp and MRP in HT-29/L-OHP cell lines were higher (P<0.05).2-DEmaps of HT-29and HT-29/L-OHP total proteins with high-resolution andreproducibility were established. The average spots of HT-29and HT-29/L-OHP celllines were2073±13and2077±18respectively. There were38differentially expressedspots identified (±over twofold, P<0.05) between the two cell lines, and there were37spots identified by MALDI-TOF/TOF analysis.17spots were up-regulated and20were down-regulated. The functions of these proteins analyzed by bioinformationwere related to transcription factors, cell structure, cell apoptosis, metabolism, and soon. The results of Western blot analysis showed that PCBP1and TUBB2A were up-regulated, but ANXA3and STIP1were down-regulated in HT-29/L-OHP celllines. The results of Western blot were in consistence with proteomics.Conclusions The expression levels of PCBP1、TUBB2A、ANXA3and STIP1were significantly different in HT-29cell lines and HT-29/L-OHP cell lines. Thesemay be candidate protein markers in further research on predictingoxaliplatin-resistance in CRC.