Determination the effect of the combination treatment of photodynamic therapy and chemotherapy on triple negative breast cancer

Triple-negative breast cancer (TNBC) which is one of the subtypes of breast cancer that is one of the most common cancer types in the world, is lack of estrogen and progesterone receptors and human epidermal growth factor receptor 2 (HER2) and constitutes approximately 10-20 % of all breast cancer cases. Although different methods such as surgery, radiotherapy, and immunotherapy can be used, the primary treatment option is chemotherapy for the treatment of TNBC. However, the heterogeneous and aggressive nature of TNBC, its high recurrence and metastatic potential, and its resistance to existing cancer treatments should be paid attention to studies on new treatment strategies. One of the current approaches in cancer treatment is photodynamic therapy. Photodynamic therapy (PDT) is based on the production of reactive oxygen species (ROS) as a result of the interaction between molecular oxygen in cells and a photosensitizer (PS) substance that is stimulated by light in a specific wavelength. ROS are formed as a result of the induction of photochemical events also cause apoptotic, necrotic, or autophagic death in cancer cells. There are three components of PDT: photosensitizer (PS), light and oxygen. These components are harmless on their own, but when they are combined, they exhibit cytotoxic effects on cancer cells and thus, providing higher selectivity in the target tissue. The first of these three important elements, PS, is a photoactive molecule that catalyzes the formation of ROS, which absorbs light energy for effective PDT. In the light source activity in PDT, the right light dose, sufficient PS concentration and oxygen are needed to achieve the desired effect in the target tissue. Within this framework, different laser or non-laser light sources are used in PDT applications. The most important last parameter in PDT is oxygen. Since the concentration of oxygen in the tissues plays an important role in the production of ROS in PDT, the therapeutic effectiveness of PDT depends on the molecular oxygen concentration in the target tissues. 5-aminolevulinic acid (5-ALA) is a PS used in PDT applications as a precursor to protoporphyrin IX (PpIX). When 5-ALA is converted to protoporphyrin, it becomes an active PS. 5-ALA is not PS alone, but it becomes an active PS by rapidly metabolizing another Hem precursor, PpIX, with the Hem biosynthetic pathway. Then, 5-ALA is excited by light at 635 nm wavelengths, and damages the mitochondria of the cells due to the increase in the amount of ROS. In previous studies, it has been determined that 5-ALA-based PDT causes intracellular PpIX accumulation in cells, and increased ROS production leads to its cytotoxicity in different cancer cells. However, limitations such as the ineffectiveness of PS, especially 5-ALA alone, the penetration of light into the target tissue, problems of PS selectivity, and oxygen hypoxia restrict the effectiveness of PDT. In this context, studies on the combination of PDT and other treatment methods has drawn attention. In this context, it has been determined in the literature that the combined applications of 5-ALA-based PDT and docetaxel, cisplatin, fluorouracil, and gefitinib chemotherapy agents are more effective than the monotherapies in oral cell squamous carcinoma, breast cancer, melanoma, and lung cancer cells. However, there is no study evaluating the combined effect of 5-ALA/PDT and chemotherapy agents in TNBC cells. Cisplatin as a potent chemotherapeutic agent used in the treatment of various types of cancer forms inserts in the DNA of the cell. Thus, it shows antineoplastic activity by inhibiting DNA replication and chain elongation. However, the side effects caused by cisplatin and acquired resistance limit the effective use of cisplatin in treatment. From this viewpoint, it was aimed to determine the effect of cisplatin-based chemotherapy and 5-ALA/PDT combination treatment in vitro in TNBC and healthy breast cells for the first time to reduce the side effects caused by cisplatin and overcome the limitations in 5-ALA-based PDT applications. To determine the effect of the combined treatment of cisplatin and 5-ALA/PDT in MDA-MB-231 TNBC cells and MCF-10A healthy breast epithelial cells, the anti-cancer effects of two different cisplatin and 5-ALA/PDT combined treatment options, simultaneously and sequentially, were compared with cisplatin alone and 5-ALA/PDT were determined. In this context, the cells treated with 1, 2.5 and 5 µM cisplatin alone or 1 mM 5-ALA and irradiated with 635 nm diode laser at energy densities of 1.5, 3, 6, 9 and 12 J/cm2 and the cell viability was determined by WST-1 analysis after 24 h incubation. In the simultaneous combined treatment application, 2.5 and 5 µM cisplatin was applied to the cells with 1 mM 5-ALA and after incubation for 4 hours, laser irradiation was performed at energy densities of 1.5, 3, 6, 9 and 12 J/cm2. In the sequential combination treatment, the cytotoxic effect of cells incubated with 1 mM 5-ALA for 4 hours and after laser irradiation at the indicated energy intensities, the cells were incubated with 2.5 and 5 µM cisplatin and the cell viability was determined for 24 hours. In this study, the cytotoxicity was determined by WST-1 analysis, and the total amount of apoptotic cell death was determined by Annexin V analysis. Additionally, acridine orange, MitoTracker, and DAPI staining were performed to observe the damage in the cell morphology, mitochondria and nucleus caused by cisplatin and 5-ALA/PDT alone and their combination treatment. After the application of 2.5 µM cisplatin and 1 mM 5-ALA to MDA-MB-231 and MCF-10A cells, they were visualized with a red filter, and it was observed that the amount of intracellular PpIX associated with ROS production increased. According to the WST-1 results, it was determined that the simultaneous application of 5-ALA/PDT and cisplatin (1.5 J/cm2: 121.89 %, 3 J/cm2: 113.34 %, 6 J/cm2: 55.77 %, 9 J/cm2: 43.83 %, 12 J/cm2: 58.02 %) was more effective in MDA-MB-231 cells compared to 2.5 µM cisplatin (73.96 %) and 5-ALA/PDT (6 J/cm2: 75.97 % and 9 J/cm2: 59.55 %) monotherapies. Besides, it was analyzed that the simultaneous combination therapy had more anti-proliferative effect on cells compared to the sequential combination therapy (1.5 J/cm2: 128.05 %, 3 J/cm2: 128.12 %, 6 J/cm2: 90.99 %, 9 J/cm2: 77.80 %, 12 J/cm2: 76.51 %) (p<0.05*). On the other hand, when MDA-MB-231 cells treated with 5 µM cisplatin and 5-ALA/PDT simultaneously, it showed an antagonistic effect compared to 2.5 µM cisplatin and 5-ALA/PDT combination, and was less effective on cell viability, especially at energy densities of 6 and 9 J/cm2 (6 J/cm2: 72.9 % and 9 J/cm2: 65.50 %). On the other hand, combined administration of cisplatin and 5-ALA/PDT (6 J/cm2: 48.10 % and 9 J/cm2: 47.25 %) compared to 5-ALA/PDT alone (6 J/cm2: 40.54 % and 9 J/cm2: 42.22 %) induced less toxic effects in MCF-10A cells. According to the results of the annexin V analysis, the percentage of total apoptotic death significantly increased in the simultaneous combined treatment (6 J/cm2 :46.78 % and 9 J/cm2: 53.6 %) compared with 2.5 µM cisplatin (37.88 %) and 5- ALA/PDT alone (6 J/cm2: 31.48 % and, 9 J/cm2: 37.78 %) in MDA-MB-231 cells (p<0.01**). In addition, it was determined that the simultaneous combination treatment of cisplatin and 5-ALA/PDT caused more apoptotic death and nuclear and mitochondrial damage in MDA-MB-231 cells. Differently from this, less apoptotic death and nuclear and mitochondrial damage were observed in MCF-10A cells treated with combination therapy compared to MCF-10A cells treated with cisplatin or 5-ALA/PDT monotherapies. In this study, the anti-cancer effects of cisplatin and 5-ALA/PDT combination treatment on TNBC cells were investigated for the first time. The findings showed that the simultaneous application of 2.5 µM cisplatin and 1 mM 5-ALA irradiated by 6 J/cm2 and 9 J/cm2 energy densities is more effective than 5-ALA/FDT and cisplatin alone and the simultaneous combination therapy exerts a synergistic effects (Combination index<1). However, further studies are needed to elucidate the combined effect of cisplatin and 5-ALA/PDT on TNBC cells at the molecular level. On the other hand, 5-ALA/PDT combination treatment with cisplatin exerts toxicity in MCF-10A normal cell line. Therefore, nanoparticulate drug delivery systems could be used for reducing the toxic effect of 5-ALA with cisplatin combination on MCF-10A cells and increasing the selectivity in the target tissue. Additionally, further studies can be carried out with advanced molecular analysis to optimize the PDT application protocol and to detect each FS in cancer cells after irradiation. Finally, the combined effects of different types of PS and cisplatin in different cancer types will be further analyzed.