To Research The Anti-tumor Activity And Mechanism Of Curcumin Derivative C212

Objective: To study the antitumor activity of curcumin derivative C212 and its mechanismMethods:1. The MTT assay was used for detection the activity of proliferation inhibition of C212 on SW620、HT-29、HCT-116、MCF-7、K562、HL-60、Herp G-2 and SGC-7901 tumor cell lines. 2. To observe the inhibition of colony formation of C212 on colon cancer cell SW620. 3. Using FITC/PI staining to detect the apoptosis effect of C212 on leukemia cell lines K562 and HL-60; PI staining and flow cytometry detection on cell cycle distribution. JC-1 staining and flow cytometry detection of K562 and HL60 cells to analyze the impact of C212 on mitochondrial membrane potential. 4. Western Blotting was applicated to detect the effect of C212 on expression of cell cycle regulatory proteins, tumor-suppressor factors and related factors in apoptosis pathway. 5. Fluorescence quenching to detect the interaction of C212 with different fragments of Hsp90 protein. 6. The impact of C212 on expression of Hsp90 client proteins, detected by using Western Blotting. 7. Application of immunoprecipitation of Hsp90 to invest the impact of C212 on Hsp90/p-Akt compound. 8. Applying the proteasome inhibitor MG132 to explore the degradation pathway of p-Akt.Results:1. C212 could inhibit the proliferation of SW620、HT-29、HCT-116、MCF-7、K562、HL-60、Herp G-2 and SGC-7901 tumor cell lines in vitro. After 48 h treatment of C212 with cells, MTT assay showed the IC50 value was less than 10μM, and the effect is better than curcumin. 2. With the increase of drug concentration, colony number of SW620 also gradually reduced, and colony size was smaller than the control group. 3. Data showed the proportion of apoptosis of K562 and HL-60 cell were(59.80±1.14%) and(40.50±0.94%) in C212, respectively, which were significantly higher than control(11.30±0.65%) and(8.70±0.37%)(P<0.01). And G2/M phase cells relatively increased after C212 treated K562 and HL-60 for 24 h. Compared with the control group, mitochondrial membrane potential were differently lower than dosing group, and C212 could momently cause mitochondrial membrane potential dissipate in 60 min. 4. Western Blot assays showed that C212 has the effect of apoptosis induction of leukemia cells via activating caspase-9/caspase-3 mitochondrial pathway. And it could down-regulate the expression of G2/M phase correlation cycle regulatory protein Cdc2, p-Cdc2 and cyclin B1. In addition, C212 could increase the expression of tumor-suppressor factors p53, p21 and p27 in K562 cell. 5. C212 could combinate with different fragments of Hsp90 protein, and the combination between C212 with CHsp90 was the strongest. 6. The expression level of p-Akt, p-Raf, p-Mek and p-Erk was significantly higher in blank group than in C212 groups. 7. By immunoprecipitation of Hsp90, compared with the control group, C212 could drop Hsp90 customer protein p-Akt’s expression level. 8. Degradation of p-Akt caused by C212 was completely blocked by treatment with the proteasome inhibit or MG132.Conclusions:1. C212 had apparent proliferation inhibition on a dozen of tumor cells. 2. C212 could inhibit the colony formation of cancer cell SW620. 3. C212 could cause cell cycle G2/M phase arrest and induce apoptosis of leukemia cells via activating caspase-9/caspase-3 mitochondrial pathway. 4. C212 could combinate with different fragments of Hsp90 protein, and down-regulate the expression level of Hsp90 client proteins, such as p-Akt、p-Raf, p-Mek and p-Erk, etc. 5. C212 could directly affect Hsp90/p-Akt interaction. 6. MG132 could completely block the degradation of p-Akt caused by C212.