Inhibition Of Curcumin And Its Derivative C086 On Proliferation Of Human Leukemia Cells Involves Disruption Chaperon Function Of Hsp90

Hsp90 is a molecular chaperone with over 200 identified client proteins. Because many of its client proteins are involved in proliferation signaling pathways, and these pathways are often important in many types of cancers, Hsp90 is promised as a target for anti-cancer drugs.Curcumin is a yellow-colored polyphenol, which has been shown to suppress multiple signaling pathways and inhibit tumor cell proliferation, invasion, metastasis, and angiogenesis. Various molecular targets modulated by this agent including Bcr/Abl、EGFR、Src、Erb-B2、VEGF and telomerase are also the client proteins of Hsp90. Our previously published work was the first to show that curcumin inhibited the proliferation of K562 cells and the inhibition effect was correlated with down-regulation of p210 Bcr/Abl protein level,And the down-regulation of p210 Bcr/Abl involved dissociating the binding of p210 Bcr/Abl with complex and increasing the binding of p210 Bcr/Abl with complex, which was similar to geldanamycin, a Hsp90 N-terminal inhibitor. Curcumin was able to disrupt the molecular chaperone functions of Hsp90.But its poor bioavailability may be a problem that limits the clinical application of curcumin. To improve the bioavailability of curcumin, numerous approaches have been undertaken. C086 was a new derivant of curcumin synthesized in our laboratory. In this study, we tried to confirm the effects of C086 on human leukemia cells, and the relationship between these effects and the molecular chaperone functions of Hsp90.Part I Inhibitory effect of C086 on human leukemia cell linesObjective: To evaluate the effect of C086 on the proliferation viability and induction of apoptosis on K562, HL60 and HL60（Bcr/Abl） cell lines and to investigate their possible mechanisms.Methods:⑴MTT method was used to observe the cell growth inhibition; ⑵Wright-Giemsa staining was utilized to investigate cell morphological change;⑶AO-EB fluorescent staining was used to observe apoptosis;⑷Cello-immunofluorescence was used to mesure the contents of Bcr/Abl;⑸Chromometry was to used to mesure the activation of Caspase-3 and Caspase-9;⑹Western blotting analysis was used to analyze Erk_1/2, p-Erk, Akt, p-Akt, PKC, Raf, C-myc, Src, p-Src, Bcl-2, Bax, Fas, Fasl, Caspase3, Caspase9 and p53 protein level;⑺Benzdine staining was utilized to investigate cell differentiation.The resutls showed:⑴C086 inhibited K562, HL-60 and HL60（Bcr/Abl）cells proliferation. And the IC50 was less than curcumin;⑵C086 down-regulated p-Erk, p-Akt, PKC, Raf, C-myc, p-Src level in K562, HL-60 and HL60（Bcr/Abl）cells;⑶C086 down-regulated Bcr/Abl level in K562 and HL60（Bcr/Abl）cells;⑷C086 was able to reduce the expression of Bcl-2 and induce the expression of Bax in K562, HL-60 and HL60（Bcr/Abl）cells;⑸C086 induced K562 cells apoptosis and up-regulated Fas、Fasl、Caspase3、Caspase9 and p53 level in K562;⑹C086 induced differentiation of K562 toward mature erythroid cells.PartII The effect of curcumin and C086 on the molecular chaperone functions of Hsp90Objective: In an effort to confirm the effect of curcumin and C086 on the molecular chaperone functions of Hsp90. Methods:⑴Western blotting analysis was used to analyze Hsp90、Hsp70、p23和p60^Hop protein level;⑵Immunohistochemistry was used to investigate the Hsp70 level;⑶HSP90 and HSP70 mRNA level were detected by Realtime PCR;⑷Coimmunoprecipitation was used to investigate the composition of multimolecular chaperone complexes;⑸Fluorescence spectrum method was used to test the interaction between the drug and Hsp90 or NHsp90 heterogenously expressed from E coli;⑹Piper Phosphate Assay Kit was applied to detect the influence on the activity of Hsp90 ATPase.The resutls showed:⑴Both curcumin and C086 were able to induce the expression of Hsp70 in K562, HL-60 and HL60（Bcr/Abl）cells. While neither of them could affect the protein level of p23 and p60^HOP. The tendency was the same as GA. C086 could reduce the expression of Hsp90, which was different from curcumin and GA;⑵Hsp90 mRNA lever was down regulated by C086, while Hsp70 mRNA lever was up regulated by C086 in K562 cells;⑶Lysates of leukeia cells were immunoprecipitated with Hsp90 antibody. Coprecipitation of members of Hsp90 multimolecular complexes was detected by immunoblotting with the Hsp70、p60^Hop or p23 antibody. Curcumin, C086 and GA decreased the binding of Hsp90 with p23, increased the binding of Hsp90 with Hsp70. Lysates of leukeia cells were immunoprecipitated with Abl antibody. Coprecipitation of members of Abl multimolecular complexes was detected by immunoblotting with the Hsp90, Hsp70, p60^Hop or p23 antibody. Curcumin, C086 and GA decreased the binding of Bcr/Abl with Hsp90 and p23, increased the binding of Bcr/Abl with Hsp70;⑷Both curcumin and C086 could combine with Hsp90 or NHsp90 heterogenously expressed from E coli;⑸Cur and GA showed the inhibitory effect on the activity of Hsp90 ATPase.PartIII The effect of C086 on K562 cell xenograft in nude mice Objective: Since C086 inhibited K562 cell growth and induced cell apoptosis in vitro as shown in the first part, we were interested if C086 could take the same effect in a murine K562 xenograft model.Methods:⑴Mice were inoculated subcutaneously with K562 cells in order to establish a murine K562 xenograft model;⑵Mice bearing growing tumors were randomly divided into four groups（with 6 mice/group）: control group, C086 50mg/kg group, C086 100mg/kg group and C086 200mg/kg group;⑶Volume of the tumors were measured per 3 days and the tumor formation rates were calculated;⑷At the end of time points, the mice were killed, the tumors were removed and measured;⑸Blood samples obtained from mice were used to detect the biochemical markers of liver or renal injury and the metabolism of Na⁺, K⁺ and Cl?;⑹H&E staining was used to study the pathological change of tumor tissue;⑺Immunofluorescence was used to mesure the contents of Bcr/Abl;⑻The protein was extracted from the tumor tissues. And Western blotting analysis was used to analyze Erk_1/2, p-Erk, C-myc, Hsp70 and Hsp90 expression.The resutls showed:⑴Growth velocity of K562 cell xenograft in C086 200mg/kg group was lower than that of control group. And its tumor inhibitory rate was 44.2%;⑵The results of the biochemical markers of liver or renal injury and the electrolyte metabolism indicated that there were no difference among the four groups （compared to the control group, p>0.05）;⑶The results of immunofluorescence showed decreased Bcr/Abl expression in groups treated with C086 compared with control group; （7） The results of Western blotting showed Erk_1/2, p-Erk, C-myc and Hsp90 protein level of groups treated with C086 were lower than that of control group.While Hsp70 protein level was higher than control group.Conclusions: These studies demonstrated for the first time the inhibitory effect of curcumin derivative C086 against human leukemia cells in vitro and suggested that destruction of Hsp90 chaperon function may be an important mechanism of curcumin and C086 inhibitory effects. C086 also showed its inhibitory effect in a murine K562 xenograft model.