Nonspecifically enhanced therapeutic effects of vincristine on multidrug-resistant cancers when coencapsulated with quinine in liposomes

Abstract

The use of vincristine (VCR) to treat cancer has been limited by its dose-dependent toxicity and development of drug resistance after repeated administrations. In this study, we investigated the mechanism by which quinine hydrochloride (QN) acts as a sensitizer for VCR. Our experiments used three kinds of multidrug-resistant cancer cells and demonstrated that QN worked by inducing intracellular depletion of adenosine triphosphate, increasing adenosine triphosphatase activity, and decreasing P-glycoprotein expression. Based on these results, we designed and prepared a VCR and QN codelivery liposome (VQL) and investigated the effect of coencapsulated QN on the in vitro cytotoxicity of VCR in cells and three-dimensional multicellular tumor spheroids. The antitumor effects of the formulation were also evaluated in multidrug-resistant tumor-bearing mice. The results of this in vivo study indicated that VQL could reverse VCR resistance. In addition, it reduced tumor volume 5.4-fold when compared with other test groups. The data suggest that VQL could be a promising nanoscaled therapeutic agent to overcome multidrug resistance, and may have important clinical implications for the treatment of cancer.

Keywords: P-glycoprotein; combination therapy; liposome; multidrug resistance reversal; quinine; vincristine.

Figure 1

Capacity to stimulate P-glycoprotein ATPase activity of verapamil and quinine hydrochloride. Abbreviation: ATPase, adenosine triphosphatase.

Figure 2

Effect of quinine on cellular adenosine triphosphate levels in A549/T, MCF-7/A, and HCT-8/V cells. Note: Cells were incubated with quinine in a series of concentrations at 150, 100, 50, 25, 10, and 5 µg/mL for 4 hours.

Figure 3

Ability of quinine to inhibit expression of P-gp in A549/T, MCF-7/A, and HCT-8/V cells using the Western blot assay. Note: Concentrations of quinine were 5 µg/mL, 10 µg/mL, 25 µg/mL, 50 µg/mL, and 100 µg/mL. Abbreviation: P-gp, P-glycoprotein.

Figure 4

Release profiles of VCR and QN from FVCR, FQN, VCRL, QNL, L1:1, L1:2 and VQL1:1, VQL1:2 in phosphate-buffered saline of pH 7.4 and 5.5 at 37°C. Notes: (A) pH 7.4 VCR, (B) pH 7.4 QN, (C) pH 5.5 VCR, and (D) pH 5.5 QN. Each condition was tested in triplicate. The standard deviation is presented as error bars. Abbreviations: VCR, vincristine; QN, quinine; FVCR, free vincristine; FQN, free quinine; VCRL, VCR liposome; QNL, QN liposome; L1:1, VCR liposome + QN liposome =1:1; L1:2, VCR liposome + QN liposome =1:2; VQL1:1, VCR and QN codelivery liposome with a ratio of 1:1; VQL1:2, VCR and QN codelivery liposome with a ratio of 1:2.

Figure 5

Cellular uptake of VCR for various groups in (A) A549/T, (B) MCF-7/A, and (C) HCT-8/V cells. Notes: Formulations were added at a VCR concentration of 50 µg/mL, and then incubated for 1, 2, 4, and 8 hours at 37°C. The data represent the mean ± standard deviation of three replicates. Abbreviations: VCR, vincristine; FVCR, free vincristine; F1:1, free vincristine + free quinine =1:1; F1:2, free vincristine + free quinine =1:2; VCRL, VCR liposome; L1:1, VCR liposome + QN liposome =1:1; L1:2, VCR liposome + QN liposome =1:2; VQL1:1, VCR and QN codelivery liposome with a ratio of 1:1; VQL1:2, VCR and QN codelivery liposome with a ratio of 1:2.

Figure 6

Morphological changes in A549/T multicellular tumor spheroids and MCF-7/ADR multicellular tumor spheroids. Notes: Multicellular tumor spheroids were treated with various drugs and formulations with the same concentration (150 µg/mL) of VCR for 7 days. The scale bar represents 500 μm. Abbreviations: VCR, vincristine; QN, quinine; FVCR, free vincristine; FQN, free quinine; F1:2, free vincristine + free quinine =1:2; VCRL, VCR liposome; QNL, QN liposome; L1:2, VCR liposome + QN liposome =1:2; VQL1:2, VCR and QN codelivery liposome with a ratio of 1:2.

Figure 7

Therapeutic efficacy in A549/T tumor-bearing mice (n=5 per group). Notes: Eight groups of drugs and formulations were injected intravenously into the lateral tail vein of each mouse at a VCR dose of 1.0 mg/kg on days 0, 3, 6, and 9. (A) Tumor growth curves for different groups with various treatments. (B) Photographs of the tumors collected from different groups of mice at the end of treatments (day 21). (C) Change in average body weight in mice with various treatments. (D) Average weights of tumors in each treatment group on day 21. The standard deviation is presented in error bars. Abbreviations: VCR, vincristine; QN, quinine; FVCR, free vincristine; FQN, free quinine; F1:2, free vincristine + free quinine =1:2; VCRL, VCR liposome; QNL, QN liposome; L1:2, VCR liposome + QN liposome =1:2; VQL1:2, VCR and QN codelivery liposome with a ratio of 1:2.

Figure 8

Histopathology of tumors. Notes: Hematoxylin/eosin staining of tumors treated with various groups at a VCR concentration of 1 mg/kg and a QN concentration of 2 mg/kg. Abbreviations: VCR, vincristine; QN, quinine; FVCR, free vincristine; FQN, free quinine; F1:2, free vincristine + free quinine =1:2; VCRL, VCR liposome; QNL, QN liposome; L1:2, VCR liposome + QN liposome =1:2; VQL1:2, VCR and QN codelivery liposome with a ratio of 1:2.