Bilberry Anthocyanins (Vaccinium myrtillus L.) Induced Apoptosis of B16-F10 Cells and Diminished the Effect of Dacarbazine.

Anthocyanins have been reported to have potential as dietary or pharmaceutical supplements in the application of cancer prevention and adjunctive treatment. However, there are few studies on the effect of anthocyanins on melanoma, which have only been performed in cell lines. The objective of this work was to investigate the anticancer effects and mechanisms of bilberry anthocyanin extract (BAE) on melanoma In Vitro and In Vivo. Moreover, a primary study was done to investigate how BAE influenced C57BL/6 mice bearing subcutaneous B16-F10 tumors treated with dacarbazine (DTIC). BAE-induced apoptosis in B16-F10 cells was associated with activation of the mitochondrial pathway induced by increased reactive oxygen species. More, In Vivo anticancer activity studies indicated that BAE attenuated melanoma growth, as identified by hematoxylin-eosin staining, Ki-67, and TUNEL assays. Further western blot results revealed higher phospho-Akt expression with the combination of BAE and DTIC, indicating no suppression of the PI3K/AKT signaling pathway. In summary, this study demonstrated the anti-melanoma activity of BAE and investigated its mechanism. Notably, it should be careful to use products enriching BAE for those melanoma patients treated with DTIC.

[Determination of dacarbazine in the urine of mice with melanoma by high performance liquid chromatography].

Dacarbazine (DTIC) is a first-line chemotherapy drug that is widely used in clinical practice for malignant melanoma. DTIC is metabolized by the liver in vivo. Some drugs are excreted in urine in the form of a prototype. Hence, DTIC in urine can be monitored to evaluate its utilization and conversion rate in the human body, and then to determine its therapeutic effect. Urine is the only body fluid that can be obtained in large quantities without damage, and it plays an important role in the analysis of body functions. However, the composition of urine is complex and there is large matrix interference, because of which trace analysis or trace component analysis is difficult. At present, the main analytical methods for DTIC are high performance liquid chromatography (HPLC) with/without mass spectrometry (MS). HPLC and HPLC-MS have the advantages of good separation effect, good selectivity, high detection sensitivity, automatic operation, and wide application range. Unfortunately, DTIC is a strongly polar and weakly basic compound; thus, it is difficult to achieve good separation and obtain good peak shapes by conventional reversed-phase chromatography. To overcome these defects, it is necessary to develop a novel method for the analysis of DTIC. In this study, mice were subjected to 12 h of fasting; then, blueberry anthocyanin was administered by gavage, and DTIC was administered by intraperitoneal injection. Then, morning urine was collected in a metabolic cage. Urine collection was continued every 4 days for a total of 5 times. Within 2 h, the collected urine was centrifuged (3000 g, 4℃) for 10 min to remove solids. The supernatant was stored in a refrigerator at-80℃. Before analysis, the urine samples were removed from the refrigerator and thawed naturally at room temperature. Then, the samples were treated by the acetone-sediment method, freeze-dried, dissolved in the mobile phase, and subjected to HPLC analysis with isocratic elution. The separation was performed on a Shimadzu-GL ODS column (250 mm×4.6 mm, 5 µm). The mobile phase was methanol/acetonitrile (1:1, v/v)-0.01 mol/L NaH2PO4 (pH 6.5; 20:80, v/v) at a flow rate of 1 mL/min. The detection wavelength, column temperature, and running time were 280 nm, 30℃, and 15 min, respectively. Under the optimized conditions, the retention time of DTIC was 5.3 min, and a good peak shape was obtained. The linearity ranged from 0.25 to 1000 µg/mL (r2=0.999). The limits of detection and quantification were calculated to be 0.12 µg/mL and 0.25 µg/mL based on signal-to-noise ratios of 3 and 10, respectively. At three spiked levels (50.0, 375, and 500 µg/mL), the average recoveries were 98.9%, 102%, and 99.1% with relative standard deviations (RSDs) of 3.2%, 1.3%, and 1.2% (n=5), respectively. The RSDs of the interday and intraday measurements were lower than 3.8% and 4.4%, respectively. The proposed method allowed for the accurate determination of DTIC in urine using a mixed organic solvent/phosphate buffer solution as the mobile phase, with equivalent elution for 15 min. This method was successfully applied to monitor the change in DTIC concentration in the urine of C57BL/6 mice in various stages of melanoma. The results demonstrate that the method is simple, reliable, and easy to apply.