The Effects Of Autophagy Inhibition On The Drug Sensitivity And DEPTOR Gene On The Autophagy And Apoptosis In Human Multiple Myeloma RPMI-8226Cells

DEPTOR is an mTOR-interacting protein that normally functions to inhibit themTOR complex1(mTORC1) and activate the mTOR complex2(mTORC2)pathways. The level of expression of DEPTOR has been studied in many humantumors and it has been found to be of low expression in most of them. However,DEPTOR has been found to be overexpressed in a subset of multiple myeloma (MM)cells, e.g. human myeloma RPMI-8226cells, where it can mediate activation of thephosphoinositide3-kinase (PI3K)/Akt pathway. This indirect mode of PI3K/Aktactivation is important for the survival of myeloma cells. PI3K/Akt activation haslong been associated with cell proliferation, differentiation, and apoptosis.Downstream effectors of the PI3K Akt pathway include caspase-3and caspase-9.On the other hand, mTORC1and PI3K/Akt have been reported to inhibit theinduction of autophagy. In addition, the relationship between autophagy and apoptosishas been extensively studied in the past decade. Some studies found that inhibition ofautophagy may induce apoptosis. We tries to determine the role of DEPTOR in theautophagy. We hypothesize that interfering with the DEPTOR gene of MM cellsmight have therapeutic value for MM. The present study was designed to determinethe role of DEPTOR in the proliferation, apoptosis and autophagy and elucidate themechanisms by which DEPTOR contributes to the chemosensitivity in myeloma. PART1. Autophagy inhibition potentiates antimyeloma activity ofDNA-damaging chemotherapyObjective：This study was designed to investigate the effects and mechanism of3–methyladenine (3-MA, an autophagy inhibitor) on the viability change of humanmyeloma RPMI-8226cells under the treatment of doxorubicin (DOX). Methods：The viability change of RPMI-8226cell under the treatment of DOX were analyzedusing MTT before and after autophagy inhibition by3-MA. Western blot was used toexamine the apoptosis-associated proteins expression. The LC-3and Atg5proteinswere detected by western blot. The autophagy was measured by transmission electronmicroscopy. Results: DOX could induce autophagy in RPMI-8226cell and increasethe expression of autophagy-associated protein.This autophagy response could beinhibited by3-MA. The combined effect of3-MA and DOX may enhance theapoptosis of the RPMI-8226cell line. Conclusion：Autophagy is a protectivephenomenon during the RPMI-8226cell line apoptosis induced by DOX. Theapoptosis process could be apparently enhanced by the autophagy inhibitor,3-MA.3-MA may enhance the sensitivity of multiple myeloma towards chemotherapy.PART2. Construction and identification of short hairpin RNA lentiviral vectortargeting human DEPTOR gene in multiple myelomaObjective: To construct short hairpin RNA (shRNA) lentiviral vectors targetinghuman DEPTOR gene, and detect its effect of gene silence in human myelomaRPMI-8226cells. Methods: Four human DEPTOR siRNA sequences were designedand then cloned into hU6-MCS-CMV-EGFP (GV115)-shRNA vector. The aboverecombinants were transfected into293T cells by means of lipofectamine mediation.The gene silencing efficacy of these4siRNA sequences was compared in Westernblot analysis. The GV115-shRNA was co-transfected into293T cells. The mosteffective GV115-shRNA was screened out by Western blot. GV115-shRNA andlentiviral packaging plasmid were co-transfected into packging cell line293T. Culturesupernatants were harvested and concentrated to generate the lentivirus encodingDEPTOR-RNAi. The lentivirus particles were packaged and DEPTOR specificshRNA was transmitted into RPMI-8226cells after screening for the valid shRNA. DEPTOR silencing efficiency was determined by real-time PCR at mRNA level andwestern blot at protein level. Results: The constructed revealed shRNA plasmidswere proved to be correct by PCR and sequencing. shRNA plasmid (GV115-shRNA2),which efficiently silenced DEPTOR, was screened out. GV115-shRNA2and lentiviralpackaging plasmid were successfully packaged with a titer of1×109TU/ml. EGFPexpression could be detected in RPMI-8226after infection of the recombinantlentivirus. Real-time PCR and western blot showed that the expression of DEPTORdecreased obviously. Conclusion: The DEPTOR shRNA recombinant lentivirusvectors were successfully constructed, and the shRNA can significantly inhibit theexpression level of DEPTOR in RPMI-8226cells. This finding could provide anexperimental basis for further functional assay in tumour gene therapy.PART3. Knockdown of DEPTOR inhibits cell proliferation, induces apoptosis,and suppresses autophagy in human multiple myeloma RPMI-8226cells in vitroObjective: the aim of study was to investigate the effect of DEPTOR on theproliferation, apoptosis and autophagy in human multiple myeloma RPMI-8226cellsand the mechanism of its activity. Methods: DEPTOR small hairpin RNA weretransfected into human multiple myeloma RPMI-8226cells to knock down DEPTORgene. MTT method was used to detect the proliferation of these cells. Flow cytometrywas used to detect the cell apoptosis. Changes of cleaved caspase-3and cleaved PARPwere analyzed by western blot. Autophagy was measured by monodansylcadaverine(MDC) and transmission electron microscopy. Results: Down-regulation ofDEPTOR expression distinctly lowered the proliferation rates of MM cells (P <0.05),induced tumor cell apoptosis (P <0.05). DEPTOR shRNA could increase expressionof cleaved caspase-3and cleaved PARP and Bax. The expression of protein Bcl-2wasdecreased, while the PI3K/Akt signaling pathway was blocked (P <0.05). Moreover,the downregulation of DEPTOR suppressed autophagy in RPMI-8226cells.Conclusion: DEPTOR shRNA might inhibit MM cells proliferation，induce apoptosis,and suppress autophagy. PI3K Akt pathway may be involved in the process ofapoptosis and autophagy. PART4. Effects of DEPTOR shRNA combined with DNA-damagingchemotherapy on proliferation and apoptosis of multiple myeloma RPMI-8226cellsObjective: To investigate the effect of DEPTOR shRNA and DNA-damagingchemotherapy on human multiple myeloma RPMI-8226cells and its mechanisms.Methods: DEPTOR small hairpin RNA were transfected into human multiplemyeloma RPMI-8226cells to knock down DEPTOR gene. These cells were treatedwith chemotherapeutics. MTT method was used to detect the proliferation of the cells.Flow cytometry was used to detect the cell apoptosis. The expression ofapoptosis-associated proteins, autophagy-associated proteins, and the activation of thephosphoinositide3-kinase (PI3K)/akt signaling pathway were detected by westernblot analysis. Autophagy was also measured by MDC and transmission electronmicroscopy. Results: Knockdown of DEPTOR intensified chemotherapy-inducedgrowth inhibition and apoptosis, increased levels of apoptosis-associated proteins(cleaved caspase-3、cleaved PARP)(P <0.05), and suppressed autophagy-associatedproteins (P <0.05). Moreover, the downregulation of DEPTOR inhibited theactivation of the PI3K/Akt signaling in RPMI-8226cells (P <0.05). Conclusion:The down-regulation of DEPTOR might intensify chenmothrapy effects ofproliferation inhibition and apoptosis induction on human multiple myelomaRPMI-8226cells possibly via inhibiting PI3K/Akt activity.