| ObjectivesHistone deacetylase inhibitors(HDACi)--SAHA is a novel targeted anticancer drug for the treatment of leukemia. However, with the increase in the use of time and population, drug-resistance appeared inevitable. Multidrug resistance is still a major obstacle to the clinical treatment of hematologic malignancies. In order to explore the relationship between polylactosamine chains and leukemia cells SAHA-resistant and then find new therapeutic targets, we screened the drug-resistant cell line K562/SAHA, and then examined the expression difference of polylactosamine and β3GnT8 in K562/SAHA and K562 cells. After interfering gene β3GnT8 in K562/SAHA with siRNA, the expression of polylactosamine was detected as well as the apoptosis, cell cycle and the sensitivity to SAHA. Experiments above preliminary explain the relationship of N-glycan with drug-resistance of leukemia cells. Besides, we investigated the mechanism of Chinese medicine triptolide effect on invasion and migration of breast cancer drug-resistant strains MCF-7 / Adr.Methods(1) After K562 cells treated with SAHA, Western blot was used to detect the protein expression of β3GnT8, β3GnT2 and CD147; Lectin blot and flow cytometry were respectively used to detect the expression of polylactosamine on cell surface and in total cellular.(2) IC50 of K562 was detected by MTT assay and with stepwisely increasing the concentration of SAHA added to K562 cells, we got a drug-resistant cell line K562/SAHA.(3) Real-time PCR was used to detect the RNA expression of Pglycoprotein(P-gp), β3GnTs, ppGalNAcTs, MMPs and sialic acid in K562/SAHA. Western blot was used to detecte the protein expression of CD133, β3GnT8 and CD147 in K562/SAHA. Flow cytometry was used to detecte the apoptosis of K562/SAHA. Lectin blot and flow cytometry were respectively used to detect the expression of polylactosamine on cell surface and in total cellular in K562/SAHA.(4) After konocking down the gene β3GnT8, Lectin blot and flow cytometry were respectively used to detect the expression changes of polylactosamine on cell surface and in total cellular in K562/SAHA. Apoptosis and cell cycle changes of K562/SAHA were detected by flow cytometry. MTT assay was used to detecte the K562/SAHA sensitivity to drugs after konocking down the gene β3GnT8.(5) With a concentration gradient of Chinese medicine triptolide treated to adriamycin-resistant breast cancer cells MCF-7/Adr, MTT was used to detect the sensitivity of MCF-7/Adr to triptolide. Apoptosis of MCF-7/Adr was detected by Hoechst33258 staining. Lectin blot and flow cytometry were respectively used to detect the expression changes of polylactosamine on cell surface and in total cellular in MCF-7/Adr. RNA and protein expression changes of β3GnT8, β3GnT2, CD147 and MMP14 were detected by Real-time PCR and Western blot. Invasion and migration of MCF-7/Adr were detected by Transwell.Results(1) After K562 treated with SAHA, the protein expression of β3GnT8, β3GnT2 and CD147 was decreased, and the total polylactosamine expression was decreased. All these are time-dependent.(2) K562/SAHA was screened. Its resistance index(RI) was 14.74. P-gp mRNA expression level and tumor stem cell marker CD133 protein level in K562/SAHA were significantly higher than K562 cells. These results indicated that drug-resistant cells were screened successfully.(3) RNA and proteins expression of β3GnT8 in K562/SAHA cells were significantly higher than in K562 cells. Polylactosamine and high glycosylation CD147 were expressed significantly higher in K562/SAHA cells than in K562 cells.(4) After konocking down the gene β3GnT8 in K562/SAHA, expression of polylactosamine in cells was reduced. The rate of early apoptotic cells increased significantly. The cell cycle tended to arrest at G1/S phase. The sensitivity of K562/SAHA to SAHA significantly increased.(5) After triptolide treated to MCF-7/Adr, the apoptosis rate increased significantly. Polylactosamine expression on cell surface was decreased. β3GnT8 gene and protein expression were decreased. MMP14 gene expression was decreased. Invasion and migration of MCF-7/Adr were significantly weakened.Conclusion(1) Polylactosamine and glycosyltransferase β3GnT8 express higher in K562/SAHA, and the sensitivity of K562/SAHA to the drug SAHA is significantly reduced. After knocking down the gene β3GnT8 in K562/SAHA, the expression of polylactosamine fall down, and rate of apoptosis is significantly increased, and cell cycle is arrest, and drug sensitivity of cells are significantly increased. It is reasoned that high expression of β3GnT8 leads to aberrant glycosylation in K562 cells, thus forming the drug-resistant of K562/SAHA. While knocking down gene β3GnT8 can reverse the drug-resistance.(2) Triptolide may inhibit the synthesis of polylactosamine by inhibiting the transcription and expression of β3GnT8 and other glycosyltransferase genes, thus affecting the glycosylation of CD147. CD147 may change the regulation of MMPs, and ultimately weaken the invasion and migration of MCF-7/Adr. |
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