Effects Of Curcumin Derivative C213on Mitochondrial Hsp90and Leukemia

Objective To study the effect of curcumin derivative C213on mitochondrial Hsp90and leukemia.Methods1. The MTT assay was used for detection the activity of proliferation inhibition ofC213on K562, HL60, MCF-7, SMMC-7721, HT-29, SW620, CNE2, SGC7901and KB tumor cells lines.2. Western blotting was applicated to the comparison between C213and Cur on theaction to heat shock proteins and Hsp90client proteins, to find out the effect ofC213to the leukemia Hsp90chaperon function3. JC-1staining and flow cytometry detection of K562and HL60cells to analysisthe impact of C213on mitochondrial membrane potential; Using western blottingto study the effect of C213on caspase apoptosis pathway; Using Annexin V/FITCapoptosis kit and flow cytometry detection to investigate cell state distribution.4. Application of mitochondrial protein extraction kit to get mitochondrial proteinsfrom K562and HL60, using each organelle marker proteins as refer for purity;Application of immunoprecipitation of Hsp90, with the refer of CypD inhibitorCyclosporine (CsA) and traditional cytoplasm Hsp90inhibitor17AAG, to investthe impact of C213on Hsp90-CypD compound.5. Using westen blotting to detect the impact of C213on K562and HL60cell cycleproteins; PI staining and flow cytometry detection on cell cycle distribution.Results1. C213could inhibit the proliferation of K562, HL60, MCF-7, SMMC-7721, HT-29,SW620, CNE2, SGC7901and KB tumor cells lines in vitro. After48h treatmentof C213with cells, MTT assay showed the IC50value was between1-10μM, andK562and HL60showed the best effect, with IC50of1.01and0.87μM.2. After24h treatment of C213（0.5,1,2μM）and Cur（2.5,5,10μM）, westernblotting showed Hsp70expression increased, Hsp90, p23and p60HOPhad nodistrict change, while Hsp90client Bcr-Abl, Akt and Raf-1decreased. The resultshowed C213had stronger inhibition on Hsp90chaperon function than Cur. 3. After24h treatment of C213（0.5,1,2μM）24h, the ratio of mitochondrialmembrane potential change of K562were14.0%，21.6%，37.0%，43.7%，and14.3%,22.5%,42.4%,51.7%on HL60;2μM C213treated K562(0,15,30,60min) with mitochondrial membrane potential change were17.0%,21.8%,30.3%,39.7%, and15.7%,19.3%,31.5%,40.2%on HL60. After24h treatmentof C213（0.5,1,2μM）24h, caspase3/9, Bcl-2and ParP decreased,with thecaspase cleaved fragment and p21increased. According to the increment of C213,early apoptotic cells proportion grew.4. Western blotting with each organelle marker proteins showed the extractedmitochondrial protein excluded the interference of other organelles proteins.Immunoprecipitation of K562and HL60mitochondrial Hsp90, C213coulddistinctively decreased the bound CypD, while CsA and17AAG had no effect.5. With the increment of C213, cell cycle protein CDK4/6had no prominent change,Cdc2decreased and p21increased. After C213treated for24h, K562and HL60showed G2/M arrested with the detected of flow cytometry.Conclusions1. C213had apparent proliferation inhibition on a dozen of tumor cells.2. C213could interfere the chaperon function of Hsp90, with the similar effect onheat shock proteins and Hsp90client proteins as Cur.3. C213could induce mitochondrial apoptosis on leukemia K562and HL60, withmitochondrial membrane protential dissipation, caspase cascade reations and earlyapoptotic.4. C213could specificly disturb mitochondrial Hsp90, and block Hsp90-CypDcompound.5. C213could obstruct K562and HL60cell cycle, and cause G2/M arrest.