Avaliação do quimioterápico doxorrubicina como agente fotossensibilizador em culturas de células MDA-MB-231 irradiadas com LED azul

Abstract

Adverse effects are common among triple negative breast cancer patients. Photodynamic therapy could complement their treatment combining a chromophore to low power non-ionizing radiation able to promote oxidative stress in cancer cells. Doxorubicin utilization as a photosensitizer and its combination with low power non-ionizing radiation at optimal wavelength could reduce doxorubicin doses and adverse effects, improving patient s quality of life. Thus, the aim of this work was to evaluate doxorubicin effects as a photosensitizer to low power blue LED on human breast cancer cell cultures. MDA-MB-231 cell cultures were cultivated and submitted to combination of different doxorubicin concentrations and low power blue LED fluences to evaluate: (i) localization of doxorubicin into cell by fluorescence microscopy, (ii) cell viability by WST-1, (iii) reactive oxygen species (ROS) generation by flow cytometry, (iv) NF-κB protein expression by imunocytochemestry, and (v) IL1B, IL6 and IL8 relative mRNA levels by quantitative Polymerase Chain Reaction (qPCR). Results obtained by fluorescence microscopy showed that doxorubicin localizes in MDA-MB-231 cell nuclei 2 hours after incubation, being nuclear doxorubicin staining observed also 24 hours after treatment. Compared to control, cell cultures incubated with doxorubicin (25nM) alone presented a reduction of 23% in cell viability, while doxorubicin combined to blue LED (640 J/cm²) reduced cell viability at 40% 24h after treatment. Cell viability 48h after treatment in cultures incubated with doxorubicin alone was reduced in 40%, while doxorubicin combination with blue LED reduced cell viability in 55%. EROs generation 30 minutes after treatment was 22% higher in cell cultures incubated with doxorubicin and irradiated with blue LED compared to other conditions. Cell cultures incubated with doxorubicin alone and cell cultures incubated with doxorubicin and irradiated with blue LED presented higher NF-κB protein staining at different periods of time (30 min, 12h and 24h). Compared to control not incubated with doxorubicin and not irradiated with blue LED, or compared to cell cultures irradiated with blue LED alone, Il1B, IL6 and IL8 mRNA levels were elevated in cell cultures incubated with doxorubicin alone and in cell cultures incubated with doxorubicin and irradiated with blue LED. These results demonstrate that low power blue LED irradiation increases doxorubicin cytotoxicity in MDA-MB-231 cell cultures, promotes ROS generation and induces pro-inflammatory cytokine expression.