The Effect Of Quality Control Of Mitochondria On Bortezomib Resistance In Multiple Myeloma

Objective:To study the effect of quality control of mitochondria on bortezomib resistance in multiple myeloma(MM)and explore the effect of mitochondrial division inhibitor(Mdivi-1)and autophagy inhibitor(3-MA)reversing bortezomib resistance.Methods:1.The RPMI 8226 cell line resistant to bortezomib(named RPMI 8226BR)was constructed by stepwise increasing extracellular concentrations of bortezomib.CCK-8 was used to detect the effect of bortezomib inhibiting proliferation of RPMI 8226 BR and RPMI 8226 cells.The IC 50 and the resistance multiple were calculated.RT-PCR was used to detect the expression of multidrug resistance gene(MDR)1 mRNA in RPMI 8226 BR and RPMI 8226 cell.Fluorescence technique was used to detect the mitochondria-l reactive oxygen species(ROS)and Western blot for the expression of mitochondrial autophagy-related proteins(PINK1,Parkin),mitochondrial dynamics-related proteins(Drp1,Mfn1)and autophagy-related proteins(LC3B)in RPMI 8226 BR and RPMI 8226 cells.Both RPMI 8226 BR and RPMI 8226 cells were treated with bortezomib and its effect on the expression of apoptosis-related proteins(Bcl2,Bax)was measured by Western blot.2.Effects of Mdivi-1 and/or 3-MA combined with bortezomib on RPMI 8266 BR cells proliferation and apoptosis: RPMI 8266 BR cells were treated with different concentrations of Mdivi-1 or 3-MA and CCK-8 was used to detect the effect of bortezomib on the proliferation and the IC 50 was calculated.Then,cells were treated with Mdivi-1 group and/or 3-MA at the concentration near IC50.There were 4 groups: untreated group,Mdivi-1 group,3-MA group and Mdivi-1 + 3-MA group.The fluorescence technique was used to measure the effect of bortezomib on the production of mitochondrial ROS.Western blot was used to detect the expression level of mitochondrial autophagy-related proteins(PINK1,Parkin),mitochondrial dynamics-related proteins(Drp1,Mfn1),autophagy-related proteins(LC3B)and apoptosis-related proteins(Bcl2 and Bax).And flow cytometry was used to detect The effect of bortezomib on apoptosis.Results:1.The RPMI 8226 BR cell which resistant to bortezomib was successfully established by stepwise increasing extracellular concentrations of bortezomib.CCK-8 was used to detect the effect of bortezomib inhibiting RPMI 8226 BR and RPMI 8226 cells proliferation.Twenty-four treatment resulted in IC 50 of 1.78 ?mol/L and 0.07 ?mol/L,respectively,with the resistance multiples of 25.4.For forty-eight treatment the IC 50 were 0.81 ?mol/L and 0.03 ?mol/L,respectively,with the resistance multiples of 27.RT-PCR showed no significant difference in MDR1 mRNA expression between RPMI 8226 BR and RPMI 8226 cells;Mitochondrial ROS production of RPMI 8226 BR cells was lower than RPMI 8226 cells.Western blot results showed that the expression level of Drp1,PINK1,Pakin,LC3-II/LC3-I in RPMI 8226 BR cells was higher than that in RPMI 8226 cells,but there was no significant difference in the expression level of Mfn1.Treatment of bortezomib significantly increased the expression of anti-apoptotic protein Bax and decreased the expression of pro-apoptotic protein Bcl2 in RPMI8226 cells.However,there was no significant change in the expression of both proteins in RPMI 8226 BR cells.2.Both Mdivi-1 and 3-MA intensified the inhibition of bortezomib on RPMI 8226 BR cells proliferation and stronger suppression was seen when higher concentration of Mdivi-1 or 3-MA was applied.ROS production was higher in Mdivi-1 group,3-MA group,Mdivi-1 + 3-MA group than in untreated group.The flow cytometry results showed that the apoptotic rates of the untreated group,Mdivi-1 group,3-MA group and Mdivi-1 + 3-MA group were 10.2%,16.2%,17.5% and 24.9% respectively.Western blot results showed that the expression level of PINK 1,Parkin,LC3-II/LC3-I,Mfn1 and Bcl2 was lower in Mdivi-1,3-MA and 3-MA + Mdivi-1 groups than in untreated group.The expression level of Bax was higher in either treatment group than untreated group.The expression of Drp 1 was decreased in the Mdivi-1 group and the 3-MA + Mdivi-1 group.Conclusions:1.We have successfully established bortezomib resistance cell line RPMI 8226 BR by stepwise increasing extracellular concentrations of bortezomib.2.Possible way of bortrezomib resistance is the increase of mitochondrial division and mitochondrial autophagy which might remove damaged mitochondria,reduce mitochondrial ROS production and therefore facilitate cells survival.3.Mdivi-1 and 3-MA might reverse the resistance to bortezomib in RPMI 8226 cells.4.The mechanism of Mdivi-1 and 3-MA reversing bortezomib resistance in RPMI 8226 BR cells might be inhibiting mitochondrial division and autophagy which increases mitochondrial ROS production and therefore induce cells apoptosis.