| Part Ⅰ Expression and Significance of SCD1 and Lipid Droplets in Multiple MyelomaObjective: Previous studies have confirmed that lipid homeostasis can be destroyed by inhibiting the expression of stearyl coenzyme A desaturase 1(SCD1)in several types of tumor,but the role of SCD1 in the development of multiple myeloma(MM)remains unclear.This part aims to evaluate the expression of SCD1,a key enzyme regulating fatty acid composition,and its role in lipid metabolism and cell survival of MM.Methods: Bone marrow samples from 16 patients with multiple myeloma and peripheral blood from 5 healthy persons were collected from Union Medical College Affiliated to Tongji Medical College of Huazhong University of Science and Technology from October2016 to May 2018.Mononuclear cells were isolated by density gradient centrifugation.The expression of lipid synthesis gene SCD1 was detected by real-time PCR.The correlation between SCD1 expression and clinical features of MM was analyzed.The expression of SCD1 in CD138+MM cells from 126 newly treated patients and CD138-CD19+B cells from 18 myeloma patients were analyzed and compared by bioinformatics.Two myeloma cell lines,RPMI8226 and ARH77,were selected to detect the level of SCD1 protein by immunoblotting and lipid droplets(LDs)by BODIPY 493/503 fluorescence staining.Si RNA-SCD1 was transfected into MM cells to knock down the expression of SCD1.The effects of SCD1 on lipid droplet content and MM cell activity were detected.The changes of lipid droplet content and MM cell activity were observed by supplementing exogenous oleic acid(OA)or palmitic acid(PA)respectively.Results: In patients with MM,the expression of SCD1 was significantly higher than that of healthy donors,and its expression was not related to patient,age or gender.Bioinformatics analysis showed that the SCD1 expression of CD138 + MM cells in newly treated patients was higher than that of CD19 + B cells in bone marrow.Compared with normal B cells,the expression of SCD1 in MM cell lines RPMI8226 and ARH77 increased significantly,and the LDs content in MM cells was higher than that in normal B cells.Compared with the control group,the apoptotic rate of si RNA-SCD1 group increased,and the content of LDs decreased significantly.We found that the percentage of apoptotic cells in OA+si RNA-SCD1 group was lower than that in si RNA-SCD1 group,and the content of LDs in cells was increased.Conclusion: The data in this part indicate that there are high expression of SCD1 and increased lipid storage in multiple myeloma cells,and the expression of SCD1 is related to the clinical stage of MM.The expression of SCD1 affects the lipid droplet content in MM cells and the viability of MM cells,suggesting that SCD1 may be an important target to interfere with lipid metabolism and promote MM cell apoptosis.Part Ⅱ 20(S)-propanaxatriol induces endoplasmic reticulum stress and promotes apoptosis of multiple myeloma cells by down-regulating SCD1 expressionObjective: 20(S)-protopanaxatriol(PPT)is a kind of ginseng extract,which can regulate metabolic disorders and inhibit lipid synthesis.It plays an anti-tumor role in several types of tumor,but its effect on proliferation and apoptosis of MM cells is still unclear.In this part,the effects and mechanisms of 20(S)-protopanaxatriol on the biological functions of MM cells and the regulation of SCD1-mediated abnormal lipid metabolism in MM were discussed.Methods: RPMI8226 and ARH77 multiple myeloma cell lines were selected and treated with different concentrations of 20(S)-protopanaxatriol.CCK8 was used to detect cell viability and proliferation,and IC50 was calculated.Flow cytometry was used to detect cell apoptosis and cell cycle.Western blotting was used to detect apoptotic protein and cell cycle-related protein expression.PPT intervention group and DMSO group were set up,the expression of SCD1 was detected by q RT-PCR,and the expression of endoplasmic reticulum stress-related genes XBP-1,GRP78 and ATF4 was detected;the expression of SCD1 and endoplasmic reticulum stress apoptotic pathway-related proteins p-PERK,PERK and CHOP was detected by Western blotting.Supplementing oleic acid or palmitic acid,detect the expression of these proteins by flow cytometry,q RT-PCR and western blotting.The intracellular LDs levels of PPT group and control group were detected by immunofluorescence,and the intracellular LDs levels of each group were detected after supplementation of exogenous oleic acid or palmitic acid.Results: PPT inhibited cell viability and proliferation in a dose-dependent manner in RPMI8226 and ARH77 cell lines.Flow cytometry analysis showed that the apoptotic effect of PPT increased with the increase of PPT concentration.Cell cycle assay showed that PPT induced MM cell cycle arrest at G0/G1 phase.The results of q RT-PCR and Western blotting showed that PPT significantly decreased the expression of SCD1 in MM cells,while PA+PPT supplementation significantly increased the expression of SCD1 in MM cells,while the expression of SCD1 in OA+PPT cells did not change significantly.PPT decreased LDs levels in MM cells,while supplementation of oleic acid or palmitic acid increased LDs levels in MM cells.Cell viability and apoptosis assay showed that oleic acid supplementation not only remedied the viability of PPT-treated MM cells,but also partially reversed PPT-induced MM cell apoptosis.PPT induced high expression of ER stress-related proteins P-PERK and CHOP,and supplementation of oleic acid reversed PPT-induced endoplasmic reticulum stress.PPT and ER stress inhibitor TUDCA combined treatment group had lower apoptotic rate than PPT treatment group,which further confirmed that PPT could induce ER stress-related apoptosis in MM cells.Conclusion: The results of this study indicate that 20(S)-protopanaxatriol can inhibit the proliferation of multiple myeloma cells and induce apoptosis.SCD1 is a key regulator for the transformation of saturated fatty acids into unsaturated fatty acids.PPT inhibits the expression of SCD1,down-regulates LDs content in cells,interferes with lipid homeostasis,and further induces the lipotoxicity-related endoplasmic reticulum stress induced apoptosis of MM cells.Part Ⅲ In vivo experiments to verify the anti-myeloma effect and mechanism of 20(S)-protopanaxatriolObjective: In vitro experiments have proved that 20(S)-protopanaxatriol inhibits proliferation and activity of multiple myeloma cells by interfering with fatty acid synthesis mediated by SCD1.In vivo experiments further verify the killing effect and mechanism of20(S)-protopanaxatriol on multiple myeloma.METHODS: MM subcutaneous tumors of mice were established by subcutaneous injection of RPMI8226 cells into NOD/SCID mice.The survival status,tumor size and weight of mice were observed in PPT treatment group and PBS control group.Tumors were taken from mice,paraffin-embedded sections were taken,Ki67(a marker of proliferation),the apoptotic protein cleaved caspase3 and the SCD1 expression were detected by immunohistochemical staining.Immunofluorescence was performed to detect the expressions of SCD1 and CHOP.Results: Compared with PBS injection control group,the growth of tumors in PPT treatment group was moderately inhibited,and the weight of mice was significantly lower than that in control group.Tumor volume of mice was measured and calculated.The results showed that the tumor volume of PPT treatment group was significantly smaller.The levels of SCD1 and CHOP in tumors were detected by immunofluorescence.We observed a significant inverse relationship between SCD1 and ER stress.IHC staining was used to detect the expression of SCD1 in mouse tumors.The results showed that the expression of SCD1 in PPT treated mice tumors was lower than that in control group.In addition,the expression of Ki-67 decreased and the expression of cleaved caspase3 increased in PPT-treated tumors.Conclusion: PPT can inhibit the growth of MM tumors,inhibit the expression of lipid desaturase SCD1,and induce ER stress to promote MM cell apoptosis in mice. |
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