Comparison Of Photodynamic Therapy For Damage And Death Response Of MCF - 7 And MCF - 7 / ADR Cells In Breast Cancer Cells

BackgroundPhotodynamic Therapy (PDT) mainly depends on different affinity of photosensitizer between tumor cells and normal cells. Photosensitizer, which was absorbed and reserved by tumor tissue, would be excited by particular light. Then it induces photochemical reaction with oxygen in tissue to generate singlet oxygen, which damages macromolecules in cells and tissues and eventually induces cell death in tumor. As PDT has many advantages, like effective, safe, low side effects and cost and collaborative etc., it has been approved for anti-tumor and non-tumorous diseases in Canada, the United States, France and the Netherlands.Photodynamic therapy requires two basic elements:photosensitizer and light. An ideal photosensitizer should meet several criteria:(1) chemical stability, (2) water-solubility, (3) high quantum yield of 1O2 generation, (4) tumor selectivity, (5) short retention time. Photosensitizers used in photodynamic therapy include hematoporphyrin derivatives, hypocrellin derivatives. Another key factor of PDT is light, photosensitizer’energy state changed after it absorbs certain wavelengths of light, switch from ground state to excited triplet sensitizer, which can transit electron to oxygen and generate singlet oxygen. Apart from light and photosensitizer, another element that can affect photodynamic therapy is the amount of photosensitizer in cell. Studies about photosensitizers show that ABCG2 overexpression can significantly reduce the amount of PhA, Ce6, MPPa, HpIX in bronchioloalveolar carcinoma; Relative resistance to PhA, MPPa, Ce6, ALA were 10,29,3 and 6 times higher in ABCG2 overexpression HEK-293 cells. However, the mechanism of affecting photodynamic therapy via decrease the amount of photosensitizer in cell by ABC family proteins remains unknown.So this paper was on the basis of previous work, and breast cancer cells MCF-7 and its subline MCF-7/ADR, which overexpresses ABCG2 and P-gp, was used. In order to provide theoretical basis for photodynamic therapy, different sensitivity, cell damage and cell death response to Ce6-PDT were studied in MCF-7 and MCF-7/ADR.Methods and Results:Part 1 Compared the cell growth inhibition between MCF-7 and MCF-7/ADR by Ce6-PDT In this part, growth curve of MCF-7/ADR was drew, and appropriate cell density was chose to investigate the cell growth inhibition between MCF-7 and MCF-7/ADR by different chemotherapeutic drugs, also by Ce6-PDT with different doses of laser light and different concentrations of Ce6. ① MTT was used to demonstrated that doxorubicin, cisplatin and paclitaxel caused cell viability loss in a concentration-dependent manner, and MCF-7 was more sensitive to these chemotherapeutics than MCF-7/ADR (p<0.05). ② Cell viability of MCF-7 and MCF-7/ADR was also evaluated through MTT assay after treated with different concentrations of Ce6 (0.1,0.2,0.5 μg/ml) and different doses of laser light (1.4, 2.8,5.6 J/cm2). Results showed cell viability loss of MCF-7 and MCF-7/ADR were in Ce6 concentration and doses of laser light-dependent manner, and MCF-7 is more sensitive to Ce6-PDT at the same treatment (p<0.05).Part 2 Ce6-PDT lead severely nuclear damage in MCF-7 In this part,ROS generation, nuclear damage, DNA damage and the function of ABCG2 and P-gp in MCF-7/ADR response to Ce6-PDT were investigated after treated with different doses of laser light (1.4 J/cm2、2.8 J/cm2、5.6 J/cm2) combined with 0.5 μg/ml Ce6.①Wound healing assay showed both cells experienced significantly less migratory activity, and migratory activity of MCF-7/ADR was more significant than MCF-7 at selected dose of laser light (p<0.05). ②DCFH-DA combined with flow cytometry showed that MCF-7 and MCF-7/ADR cells produced large amount of ROS after Ce6-PDT and ROS generation in MCF-7 was higher than that in MCF-7/ADR after same treatment. ③ Hochest33342 staining and analysis of the comet tail length found that DNA damage in MCF-7 cells was much more severe than that in MCF-7/ADR cells at the same laser dose (p<0.01). ④The resistance to PDT in MCF-7/ADR was reversed (p<0.01) after co-treatment with the special inhibitors of ABCG2 (Fumitremorgin C) and P-gp (LY335979), also the fluorescence intensity of intracellular Ce6 in MCF-7/ADR cells was obviously higher (p<0.05) after co-incubation with FTC or LY335979. ⑤Scanning electron microscope observation showed that the shape of both cells was altered, the volume of cells were reduced, meanwhile, the number of microvilli was sharply decreased and pseudopodia structure were damaged.Part 3 Different cell death response in MCF-7 and MCF-7/ADR In this part, percentage of apoptosis in MCF-7 and MCF-7/ADR after treated with Ce6-PDT were studied by flow cytometry, also cell death response of MCF-7/ADR was investigated. The result showed that ①Annexin V-FITC/PI combined with flow cytometry showed that Ce6-PDT treatment induced MCF-7 to apoptosis and no significant apoptotic cell death in MCF-7/ADR cells. ② When incubated with autophagy inhibitors 3-MA and bafilomycin A1, cell viability increased after Ce6-PDT (5.6 J/cm2 + 1μg/ml Ce6) treatment (p<0.01).③ MDC staining found that Ce6-PDT induced MCF-7/ADR to autophagy; Western blot showed that Ce6-PDT treatment enhanced the accumulation of LC3 Ⅱ in MCF-7/ADR. All these results indicated that Ce6-PDT induced MCF-7 to apoptotic response, but autophagic response in MCF-7/ADR.ConclusionTaken together, the mechanism of different sensitivities to Ce6-PDT was studied via investigating ROS generation, nucleus and DNA damage in MCF-7 and MCF-7/ADR. This study suggested ABCG2 and P-gp overexpression significantly decreased the amount of Ce6 in MCF-7/ADR, Ce6-PDT induced MCF-7 to apoptotic response, but induced MCF-7/ADR to autophagic response.