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. 2012;4(2):99-107.
Epub 2012 Jun 25.

Selective toxicity of rose bengal to ovarian cancer cells in vitro

Steven B Koevary

Selective toxicity of rose bengal to ovarian cancer cells in vitro

Steven B Koevary. Int J Physiol Pathophysiol Pharmacol. 2012.

Abstract

Rose bengal (RB) has been utilized as a photodynamic agent for the targeted killing of cancer cells. Recent data suggest that intralesional RB alone may be effective in chemoablating locoregional and metastatic melanomas. The ability of RB to induce direct and bystander melanoma cell death led to the speculation that it may be similarly effective in the treatment of other neoplasms. The objective of this study was to determine whether RB can limit the growth, or kill, ovarian cancer cells in vitro. Ovarian carcinoma cells with or without a germline BRCA1 mutation were cultured with up to 800 μM RB for one hour or four days, after which their ability to proliferate was assessed using the MTT assay. Control cells included an embryonic kidney cell line transformed with adenovirus, and normal human fibroblasts. Ovarian cancer cells exhibited significant dose-dependent suppression of growth in response to RB; this suppression was similar to that seen with carboplatin. RB treated ovarian cancer cells appeared rounded, shrunken, and damaged. RB also inhibited the growth of kidney tumor cells but was much less effective in slowing the growth of normal human fibroblasts suggesting that RB-mediated growth suppression might be tumor cell specific. Ovarian cancer cells treated with RB displayed a significant increase in apoptosis that peaked at approximately four times the levels seen in untreated control cells. Furthermore, RB exposure resulted in the intracellular generation of reactive oxygen species (ROS) at levels that were significantly greater than in untreated cells and similar to levels seen in cells treated short term with H(2)O(2). These data suggest that RB may not only suppress ovarian cancer cell growth but also induce their apoptotic cell death, justifying the further investigation of the effects of RB in an animal model of ovarian cancer.

Keywords: BRCA1; MTT assay; Rose bengal; apoptosis; ovarian cancer; reactive oxygen species.

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Figures

Figure 1
Figure 1
A and B. Effects of RB on UWB and BRCA1 cell growth; cells were cultured with RB for four days (*p<0.0001, **p<0.001, and †p=0.0002 relative to untreated control cells). C. UWB cells cultured with carboplatin showed significantly reduced cell growth compared to untreated cells at all tested concentrations (p<0.0001). D. Relationship between UWB cell number and absorbance in the MTT assay.
Figure 2
Figure 2
A. Untreated UWB cells after five days in culture, as viewed under low power (10X objective) with a Nikon TMS phase contrast microscope. B. UWB cells treated with 200 μM RB for four days, same magnification as A. Cells appeared rounded, shrunken, and damaged, but were still attached to the plate.
Figure 3
Figure 3
Effects of a one hour incubation of UWB cells with RB on their ability to proliferate; the MTT assay was carried out four days later. *p<0.0001, **p=0.0003, and †p=0.012 relative to control.
Figure 4
Figure 4
Suppression of HEK-293 (A) and Detroit 551 (B) cell growth by RB. The doses at which HEK-293 growth was suppressed were similar to those seen with UWB and BRCA1 cells, while only the highest doses suppressed growth of normal Detroit 551 cells. *p<0.001 relative to controls.
Figure 5
Figure 5
Quantitation of apoptosis in RB-treated UWB cells. The positive control consisted of nuclease-treated cells. *p<0.0001 relative to positive control. **p=0.0029, †p=0.0031, and §p=0.024 relative to untreated control cells.
Figure 6
Figure 6
Generation of ROS in UWB cells exposed to various concentrations of H2O2 or 50 μM RB. Cells generated ROS in a dose-dependent manner in response to a 20 minute incubation with H2O2. Cells that were incubated with RB for 24 hours similarly generated ROS at levels that were significantly greater than untreated cells (*p<0.001); these levels were statistically similar to those seen in cells treated with 1000 μM H2O2 for 20 minutes.
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