Mechanism Of CD147 Involved In Multidrug Resistance Of Human Oral Squamous Carcinoma Cells

BackgroundMulti-drug resistance(MDR) is a major obstacle to the effective treatment of malignant tumors and its complex mechanisms remain to be elucidated.Most tumors with high metastatic potential exhibit the MDR phenotype and vice versa.A functional linkage between drug resistance and metastasis has been proposed in tumor cells.Studies reported that switched-on genes in metastatic cancer cells affected their sensitivity to chemotherapeutic drugs.There is growing evidence that the human cell-surface molecule CD147,an integral plasma membrane glycoprotein belonging to the Ig superfamily,participates in tumor metastasis and MDR.CD147 is highly expressed on the surface of various tumor cells.It stimulates the production of multiple matrix metalloproteinases(MMPs) by tumor cells and fibroblasts,serving as a key regulator of tumor cell invasiveness and metastasis.In addition,studies have discovered that CD147 expression was upregulated in MDR cancer cells and CD147 stimulated the production of hyaluronan(HA) in mammary carcinoma cells and induced MDR in a hyaluronan-dependent manner.Thus CD147 represents a potent target for both tumor metastatic behavior and drug resistance.Methods and ResultsWe studied the expression of CD147 in sensitive human oral squamous carcinoma(SCC) KB and MDR derivative KB/V cells. Reverse transcription-PCR,western blotting and flow cytometric analysis revealed that KB/V cells expressed CD147 at significantly higher levels than their parental KB cells(2.41-fold).Using stable RNA interference, we succeeded in establishing three CD147 knock-down KB/V cell lines (KB/VsiCD147-Cla,Clb,and C2).Compared to the control,CD147 expression was downregulated by 50.63%,80.54%,and 86.50%in KB/VsiCD147-Cla,Clb,and C2,respectively.MTT colorimetric assay showed an increase in the chemosensitivity to vincristine(VCR),all transretinoic acid(ATRA),taxol,and 5-fluorouracil(5-Fu) of KB/VsiCD147-C2 cells.We performed a comparative proteomics study of KB,KB/V,and KB/VsiCD147-C2 cells.The 2-DE pattern of each cell line was highly reproducible and well-resolved,and approximately 1000 protein spots were visualized.Using image analysis,we compared the 2-D PAGE patterns of paired KB/V and KB cells or KB/V and KB/VsiCD147-C2 cells,the average number of differential protein spots that showed more than 2-fold changes in expression were 93±5.84,98±7.91,respectively.We converged those spots detected in both paired cells and found that 21 proteins were differentially expressed.In KB/V cells, 15 spots were specifically upregulated while 6 spots were downregulated as compared to KB and KB/VsiCD147-C2 cells.We performed automated MALDI-TOF-MS and database searches on the 21 differentially expressed protein spots.All 21 proteins were identified on MALDI-TOF-MS maps and by database query.The enhanced expression of representative active proteins,GRP75 and CyPA,were confirmed by western blotting and RT-PCR analysis,the protein expression were in accordance with previous proteomics assessment,while no changes in CyPA mRNA were detectable.In addition,pretreatment of KB/V cells with a CyPA-binding immunosuppressive drug,cyclosporine A(CsA), enhanced the chemosensitivity to VCR and 5oFu of KB/V cells.We also aimed to reveal the anti-apoptotic effect of CD147 on the MDR phenotype of KB/V cells and its possible pathways.RT-PCR analysis revealed that KB/V cells expressed equal levels of MDR related genes MDR1,MRP1,and LRP,whereas significantly higher leve of X-linked inhibitor of apoptosis(XIAP) than its sensitive counterpart KB cells. Western blotting analysis showed that XIAP expression level in KB was 39.44%of that in KB/V cells.Down-regulation of CD147 by transfection with CD147 siRNA resulted in decreased XIAP expression by 36.89% and 37.59%in KB/VsiCD147-C1b and C2 cell lines.We analyzed apoptosis in KB,KB/V and KB/VsiCD147-C1b and C2 cells by flow cytometry.The average%apoptosis in KB,KB/VsiCD147-C1b and C2 was 3.48%,5.48%,and 12.24%,respectively,significantly higher than those of KB/V(0.07%).Electron microscopic observation comfirmed the morphology changes of apoptosis related to CD147 expression. Additionally,chemo-sensitivity to 5-Fu of KB/V was increased by CD147 silencing as measured by MTT colorimetric assay.ConclusionWe propose that CD147 is at least partly involved in the MDR of KB/V cells by upregulating GRP75 and CyPA,CD147 may represent a therapeutic target to overcome MDR by oral SCC.Our results also suggest that inhibition of CD147 and subsequent XIAP depletion may have an anti-tumor effect through enhancing the susceptibility of cancer cells to apoptosis.