HPMA copolymer-bound doxorubicin induces apoptosis in ovarian carcinoma cells by the disruption of mitochondrial function

Abstract

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-bound doxorubicin has showed greater potency than free doxorubicin in the treatment of ovarian cancer in vivo and in vitro. The promising activity of the conjugate demonstrated in clinical trials has generated considerable interest in understanding the mechanism of action of this macromolecular therapeutic. In this study, the involvement of the mitochondrial pathway in HPMA copolymer-bound doxorubicin-induced apoptosis in the human ovarian cancer cell line A2780 was investigated. Through a series of in vitro assays, including confocal microscopy, flow cytometry, and spectrofluorimetry, a significant decrease in mitochondrial membrane potential in A2780 cells treated with HPMA copolymer-bound doxorubicin was found. The most dramatic changes in mitochondrial membrane potential were observed between 2 and 12 h of continuous drug exposure. The potential of the mitochondrial membrane remained collapsed when drug treatment continued up to 24 h. For the first time, it was shown that HPMA copolymer-bound doxorubicin induces apoptosis in ovarian cancer cells by simultaneous activation of both caspase-dependent and caspase-independent pathways of DNA damage. This was determined by monitoring the translocation of the mitochondrial proteins cytochrome c and apoptosis-inducing factor to cytosol. The altered balance between anti-apoptotic and pro-apoptotic members of the Bcl-2 family of proteins was responsible for the mitochondrial function distraction. HPMA copolymer-bound doxorubicin induced a time-dependent decrease in the expression of the anti-apoptotic Bcl-2 and Bcl-xL proteins, which control cell survival. At the same time, the expression level of pro-apoptotic members (Bax, Bad) of the Bcl-2 family was increased under the chosen experimental conditions. Altogether, these results indicate that HPMA copolymer-bound doxorubicin induced apoptosis in ovarian cancer cells through the mitochondrial pathway.

Figure 1

Structure of HPMA copolymer-doxorubicin conjugate (P-GFLG-DOX). P is the HPMA copolymer backbone; GFLG (Gly-Phe-Leu-Gly) - a lysosomally degradable spacer. The conjugate contained 0.11 mmol doxorubicin/g polymer (6.4 wt.-%); molecular weight, M_w = 27 kDa.

Figure 2

An alteration of JC-1 staining in A2780 cells. Cells were cultured in 35-mm dishes and exposed to 2 × IC₅₀ DOX and P-GFLG-DOX for 24 h. After being incubated with drugs, cells were loaded with JC-1, washed, and analyzed under laser scanning confocal microscope. Incubation of A2780 cells with free DOX or P-GFLG-DOX resulted in the collapse of mitochondrial potential. It is manifested with decrease of red fluorescence (JC-1 aggregated form) and an increase of green fluorescence (JC-1 monomer form). A - control cells. B - DOX treated cells. C - P-GFLG-DOX treated cells. Scale bar = 10 μm.

Figure 3

Row cytometric analysis of A2780 cells loaded with JC-1 fluorescence dye. A2780 cells were treated with 2 × IC₅₀ DOX or P-GFLG-DOX for 24 h. After trypsinization, the resulting cell suspension was stained with 1 ug/ml of JC-1 for 15 min at 37°C, washed, and analyzed for green (FL-1) and red (FL-2) fluorescence by flow cytometry. A: Dot-plot data generated using Cell Qwest software. B: Distribution of gated cells in upper or lower quadrants as shown in A. Cells with altered mitochondrial membrane potential are located in the low right quadrant. The experiment was repeated at least three times with similar results.

Figure 4

Time course measurement of the effects of DOX and P-GFLG-DOX on mitochondrial membrane potential using JC-1 dye. All assays were performed using PerkinElmer LS55 spectrofluorometer equipped with plate reader. Cells were incubated with 2 × IC₅₀ DOX or P-GFLG-DOX for 24 h, washed, incubated with 1 μg/ml of JC-1 for additional 30 min at 37°C, washed again, and analyzed for the change of JC-1 fluorescence. JC-1 staining was performed using the kit purchased from Cell Technology, Inc. Data are shown as means of duplicate measurements of JC-1 fluorescence (A) or results of dye exclusion assays (B). The experiment was repeated twice with similar results.

Figure 5

Release of cytochrome c (A) and AIF (B) from mitochondria into cytosol after exposure of A2780 cells to 2 × IC₅₀ DOX or P-GFLG-DOX for 24 h. After treatment, A2780 cell lysates were fractionated and the cytosolic (CF) and the mitochondrial (MF) fractions were analysed by Western blot using relevant primary antibodies. Blots were developed with ECL. Lane 1 - untreated cells. Lane 2- cells treated with DOX. Lane 3 - cells treated with P-GFLG-DOX. C - COX IV served as mitochondrial fraction marker.

Figure 6

Expression regulation of anti-apoptotic proteins in A2780 cells treated with 2 × IC₅₀ DOX and P-GFLG-DOX. Proteins (50 μg) from cell lysates were separated by 15% SDS-PAGE, transferred to membranes, and probed with relevant primary antibodies. Blots were developed with ECL. A: Bcl-2 protein; B: Bcl-x_L protein, 24 incubation with drugs; C: densitometry analysis of Bcl-x_L expression. Lane C - untreated cells. Lane 1 - DOX treated cells. Lane 2 - P-GFLG-DOX treated cells. Data are representative of three independent experiments.

Figure 7

Expression regulation of pro-apoptotic proteins in A2780 cells treated with 2 × IC₅₀ DOX and P-GFLG-DOX for 24 h. Proteins (50 μg) from cell lysates were separated by 15% SDS-PAGE, transferred to membranes, and probed with relevant primary antibodies. Blots were developed with ECL. A: typical Western blots images: Lane 1 - untreated cells. Lane 2 - DOX treated cells. Lane 3 - P-GFLG-DOX treated cells. Data are representative of three independent experiments. B: densitometry analysis of protein expression.