Korean J Ophthalmol.  2015 Dec;29(6):424-432. 10.3341/kjo.2015.29.6.424.

Effects of Bevacizumab on Bcl-2 Expression and Apoptosis in Retinal Pigment Epithelial Cells under Oxidative Stress

• Kim S
• Kim YJ
• Kim NR
• Chin HS

• KMID: 2363851
• DOI: http://doi.org/10.3341/kjo.2015.29.6.424

Abstract

PURPOSE
To evaluate the effects of bevacizumab on expression of B-cell leukemia/lymphoma (Bcl)-2 and apoptosis in retinal pigment epithelial (RPE) cells under oxidative stress conditions.
METHODS
RPE cells were treated with H2O2 (0, 100, 200, 300, and 400 microM) and bevacizumab at or above the doses normally used in clinical practice (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cell apoptosis was measured using flow cytometry with annexin V-fluorescein isothiocyanate. The expression of Bcl-2 mRNA was determined using reverse transcription polymerase chain reaction.
RESULTS
Under low oxidative stress conditions (H2O2 100 microM), cell apoptosis was not significantly different at any concentration of bevacizumab, but Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL). Under moderate oxidative stress conditions (H2O2 200 microM), Bcl-2 mRNA expression decreased with increasing concentration of bevacizumab (0.33, 0.67, 1.33, and 2.67 mg/mL), but cell apoptosis increased only at 2.67 mg/mL of bevacizumab. Under high oxidative stress (300 microM) conditions, cell apoptosis increased at high concentrations of bevacizumab (1.33 and 2.67 mg/mL), but it did not correlate with Bcl-2 expression.
CONCLUSIONS
Withdrawal of vascular endothelial growth factor can lead to RPE cell apoptosis and influences the expression of anti-apoptotic genes such as Bcl-2 under oxidative stress conditions. Since oxidative stress levels of each patient are unknown, repeated injections of intravitreal bevacizumab, as in eyes with age-related macular degeneration, might influence RPE cell survival.

Keyword

Apoptosis; Bcl-2; Bevacizumab; Retinal pigment epithelial cell

MeSH Terms

Angiogenesis Inhibitors/*pharmacology
Apoptosis/*drug effects
Bevacizumab/*pharmacology
Cell Line
Enzyme-Linked Immunosorbent Assay
Flow Cytometry
Gene Expression Regulation/physiology
Humans
Hydrogen Peroxide/toxicity
Oxidative Stress/drug effects
Proto-Oncogene Proteins c-bcl-2/*genetics
RNA, Messenger/genetics
Real-Time Polymerase Chain Reaction
Retinal Pigment Epithelium/*drug effects/metabolism/pathology
Vascular Endothelial Growth Factor A/antagonists & inhibitors
Angiogenesis Inhibitors
Bevacizumab
Hydrogen Peroxide
Proto-Oncogene Proteins c-bcl-2
RNA, Messenger
Vascular Endothelial Growth Factor A

Figure

• 

  Fig. 1 Influence of bevacizumab on apoptosis of retinal pigment epithelial cells in the absence of H2O2. The retinal pigment epithelial cells were treated with bevacizumab for 16 hours (0, 0.33, 0.67, 1.33, and 2.67 mg/mL). Cellular apoptosis was not significantly different at control, clinical (0.33 and 0.68 mg/mL) and high (1.33 and 2.67 mg/mL) doses of bevacizumab in the absence of H2O2 (p = 0.121, 0.439, 0.221, and 0.063, respectively). Each bar shows the mean ± standard deviation of results of three or more independent experiments.

• 

  Fig. 2 Apoptosis and expression of vascular endothelial growth factor (VEGF)-A and B-cell leukemia/lymphoma (Bcl)-2 after treatment of retinal pigment epithelial (RPE) cells with H2O2. (A) Effects of H2O2 on apoptosis of RPE cells. The RPE cells were treated with various concentrations of H2O2 for 16 hours. Cell apoptosis was 3.26% for RPE at 100 µM H2O2 compared to 3.82% for the control, but the difference was not significant. Cell apoptosis decreased at 200 µM H2O2 (2.65%) but increased at 300 and 400 µM of H2O2 (10.42%, 17.99%). Means were significantly different than the control (p < 0.05). Each bar shows the mean ± standard deviation of results of three or more independent experiments. The asterisk indicates a statistically significant difference within the group (*increased, **decreased, p < 0.05). (B) Expression of VEGF-A after exposure to H2O2. VEGF-A excretion into the medium was measured using enzyme-linked immunosorbent assay. VEGF-A expression increased after addition of 50, 100, or 200 µM H2O2 to RPE cells. However, after being treated with 300 and 400 µM H2O2, VEGF-A expression decreased. Data are expressed as the mean ± standard deviation of the results of three or more independent experiments. Means were statistically significant compared to the control at all concentrations of H2O2 (p < 0.05). (C) Expression of Bcl-2 mRNA after exposure to H2O2. Cells were cultured with various concentrations of H2O2 for 16 hours (0, 100, 200, and 300 µM). Expression of Bcl-2 mRNA decreased as oxidative stress increased. Data are expressed as the mean ± standard deviation of the results of three or more independent experiments. Means were statistically significant compared with the control at all concentrations of H2O2 (p < 0.05).

• 

  Fig. 3 Influence of bevacizumab on apoptosis of retinal pigment epithelial (RPE) cells and B-cell leukemia/lymphoma (Bcl)-2 mRNA expression under low oxidative stress (100 µM H2O2). (A) Influence of bevacizumab on apoptosis of RPE cells under low oxidative stress (100 µM H2O2). The RPE cells were treated with various concentrations of bevacizumab (0, 0.33, 0.67, 1.33, and 2.67 mg/mL) under low oxidative stress (100 µM H2O2). Cell apoptosis was not significantly different at all concentrations of bevacizumab less than 100 µM H2O2. Each bar shows the mean ± standard deviation of results of three or more independent experiments. p < 0.05 vs. control. (B,C) Expression of Bcl-2 mRNA with bevacizumab under low oxidative stress (100 µM H2O2). Expression of Bcl-2 mRNA decreased under low oxidative stress (100 µM H2O2), and this decrease was proportional to the increase in bevacizumab dose. There was a statistically significant difference at all concentrations. The mRNA expression of Bcl-2 was normalized to glycerol-3-phosphate dehydrogenase (GAPDH, housekeeping gene). Each bar shows the mean ± standard deviation of results of three or more independent experiments. p < 0.05 vs. control.

• 

  Fig. 4 Influence of bevacizumab on apoptosis of retinal pigment epithelial (RPE) cells and B-cell leukemia/lymphoma (Bcl)-2 mRNA expression under moderate oxidative stress (200 µM of H2O2). (A) Influence of bevacizumab on apoptosis of RPE cells under moderate oxidative stress (200 µM H2O2). The RPE cells were treated with various concentrations of bevacizumab (0, 0.33, 0.67, 1.33, and 2.67 mg/mL) under moderate oxidative stress (200 µM H2O2). Cell apoptosis decreased at 200 µM H2O2 (2.65%) compared with the control (3.82%). Cell apoptosis did not show significant change until a bevacizumab concentration of at least 1.33 mg/mL. Apoptosis increased to 10.16% at high doses of bevacizumab (2.67 mg/mL) under the same oxidative stress conditions. Each bar shows the mean ± standard deviation of results of three or more independent experiments. The asterisk indicates a statistically significant difference within the group (*p <0.05). (B,C) Expression of Bcl-2 mRNA with bevacizumab under moderate oxidative stress (200 µM H2O2). Expression of Bcl-2 mRNA decreased under moderate oxidative stress (200 µM H2O2), and that decrease was proportional to the increase in bevacizumab dose. There was a statistically significant difference at all concentrations. The mRNA expression of Bcl-2 was normalized to glycerol-3-phosphate dehydrogenase (GAPDH, housekeeping gene). Each bar shows the mean ± standard deviation of results of three or more independent experiments. p < 0.05 vs. control.

• 

  Fig. 5 Influence of bevacizumab on apoptosis of retinal pigment epithelial cells under high oxidative stress (300 µM H2O2). The retinal pigment epithelial cells were treated with various concentrations of bevacizumab (0, 0.33, 0.67, 1.33, and 2.67 mg/mL) under high oxidative stress (300 µM of H2O2). As mentioned previously, cell apoptosis increased at 300 µM H2O2 (10.42%) compared with the control (3.82%). Cell apoptosis increased to 12.65% and 10.97% at clinical doses of bevacizumab (0.33, 0.67 mg/mL) under high oxidative stress, but there was no statistically significant difference between the increases. Cell apoptosis significantly increased to 19.83% and 24.85% at high doses of bevacizumab (1.33, 2.67 mg/mL) under the same oxidative stress. Each bar shows the mean ± standard deviation of results of three or more independent experiments. The asterisk indicates a statistically significant difference within the group (*p < 0.05).

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