A Study Of Tumor Multidrug Resistance Reversal Activity By Tanshinone Ⅱ A Microemulsion

ObjectiveTo study the Multidrug Resistance (MDR) reversal activity and it's mechanism of TanshinoneⅡA microemulsion.Methods1. Measure the cytotoxic activity of TanshinoneⅡA microemulsion by MTT assay. Non-toxic dosage (Dosage for 95% cell viability, IC95) and low-toxic dosage (Dosage for 85% cell viability, IC85) can be measured with this measurement. Doxorubicin resistance K562/ADM cells are incubated with TanshinoneⅡA microemulsion of several concentrations'range for 48h. When incubated 44h, MTT was added into each well, and the effect was terminated by termination solution. Origin package is used to draw the cell viability curve, and IC95 and IC85 were measured.2. Measure the MDR reversal activity of TanshinoneⅡA microemulsion by MTT assay. K562/ADM cells were incubated with doxorubicin at several concentration level.0.11μg/ml and 0.17μg/ml TanshinoneⅡA microemulsion are added into each well to measure the MDR reversal activity. TanshinoneⅡA solution, empty microemulsion, Verapamil group were defined as control. Doxorubicin single used group is defined as blank. Origin package is used to draw cell viability curve and doxorubicin concentration of 50% cell viability (IC50) is calculated. Reversal Index= IC50 of blank/IC50 of TanshinoneⅡA microemulsion group or control.3. Fluorescence spectrophotometry and fluorescent microscopy was used to determine intracellular concentration of doxorubicin. K562/ADM cells were incubated with 0.17μg/ml TanshinoneⅡA microemulsion for 24h, and cells were washed and gathered. Intracellular doxorubicin is determined by fluorescence spectrophotometry. TanshinoneⅡA solution, empty microemulsion, Verapamil group were defined as control, doxorubicin single used group is defined as blank. At the same time, fluorescent microscopy was used to observe intracellular doxorubicin accumulation caused by TanshinoneⅡA microemulsion treatment for different time.4. HPLC assay was used to determine intracellular Tanshinone II A. K562/ADM cells were treated with 15μg/ml TanshinoneⅡA microemulsion and TanshinoneⅡA solution individually, after cells were dealt, intracellular TanshinoneⅡA is determined by HPLC. At the same time, standard curve, specificity, sensitivity, recovery, and RSD between samples' measurements were determined.5. Fluorescence spectrophotometry and fluorescent microscopy was used to determine intracellular concentration of Rhodamin 123. K562/ADM cells were treated with 0.17μg/ml TanshinoneⅡA microemulsion and 1μg/ml Rhodamin 123. After defined time, cells were dealt, and intracellular doxorubicin is determined by fluorescence spectrophotometry. And TanshinoneⅡA solution, empty microemulsion, Verapamil group were defined as control, Rhodamin 123 single used group is defined as blank. At the same time, fluorescent microscopy was used to observe intracellular Rhodamin 123 accumulation of each group.6. P-glycoprotein and Bcl-2 protein expression were determined by Flow Cytometry. K562/ADM cells were incubated with 0.17μg/ml TanshinoneⅡA microemulsion, and cells were washed and incubated with PE-conjugated anti-P-glycoprotein antibody and FITC-conjugated anti-Bcl-2 antibody individually, and samples were dealt, fluorescence intensity was determined by Flow Cytometry. TanshinoneⅡA solution, empty microemulsion were defined as control, Untreated K562/ADM and K562 cells were defined as blank.Results1. TanshinoneⅡA microemulsion's non-toxic and low-toxic dosage for K562/ADM cell line were 0.11μg/ml and 0.17μg/ml.2. The Reversal Index of non-toxic TanshinoneⅡA microemulsion was 12.63±1.20, and low-toxic TanshinoneⅡA microemulsion was 20.35±1.65. Reversal Index for TanshinoneⅡA solution, empty microemulsion, Verapamil group were 2.07±0.07,6.87±0.08,4.29±1.36 individualy. The 72h Reversal Index of non-toxic TanshinoneⅡA microemulsion and low-toxic TanshinoneⅡA microemulsion were 16.54±1.65,25.22±0.59 individually.3. The intracellular doxorubicin concentration of TanshinoneⅡA microemulsion group was 0.502±0.012μg/ml, and TanshinoneⅡA solution, empty microemulsion, Verapamil group as control were 0.406±0.008μg/ml, 0.461±.003μg/ml,0.551±0.010μg/ml, and doxorubicin group as blank was 0.285±0.009μg/ml. Intracellular doxorubicin accumulation was increased as TanshinoneⅡA microemulsion treated time extended, which was observed by fluorescent microscopy.4. Intracellular TanshinoneⅡA for 2h,4h,8h were 3.50±0.10μg/ml, 4.07±0.13μg/ml,3.69±0.13μg/ml individually for TanshinoneⅡA microemulsion group, which was 2.84±0.01μg/ml,3.37±0.18μg/ml, 3.07±0.08μg/ml for TanshinoneⅡA solution group. Intracellular TanshinoneⅡA for each time for TanshinoneⅡA microemulsion group were higher than TanshinoneⅡA solution group.5. The intracellular Rhodamin 123 concentration of TanshinoneⅡA microemulsion group was 3.86±0.14μg/ml, and concentration for TanshinoneⅡA solution, empty microemulsion, Verapamil group as control were 2.29±0.02μg/ml, 3.17±0.15μg/ml,2.44±0.21μg/ml, Rhodamin 123 group as blank was 2.13±0.03μg/ml. There's high concentration of Rhodamin 123 in cells treated with Tanshinone II A microemulsion.6. P-glycoprotein measurement by Flow Cytometry indicated TanshinoneⅡA microemulsion can significantly reduced P-glycoprotein expression level, its fluorescence intensity was 1.20. TanshinoneⅡA, empty microemulsion also have this effect, thier fluorescence intensity were 2.64 and 1.49 individually. Fluorescence intensity for K562/ADM and K562 cells'suspension were 3.24 and 1.98 individually. TanshinoneⅡA microemulsion can't significantly reduce the level of Bcl-2 expresson.ConclusionsTanshinoneⅡA microemulsion can induce cell uptake of TanshinoneⅡA and inducing apoptosis, reducing P-glycoprotein level of K562/ADM cell line. This may the mechanism of the MDR reversal activity by TanshinoneⅡA microemulsion.