Study On Mechanism Of Apoptosis And Cell Autophagy Induced By Proto - Porphyrin IX Induced By Low Intensity Focused Ultrasound In Human Leukemia Cells

Cancer is defined as a malignant neoplasm or unregulated cell growth which is a matter of serious concern. The routes generally applying to treat the cancer cells include surgery, radiotherapy, chemotherapy, hormone therapy, biological therapy and photodynamic therapy, etc. The selection of the approach, depends on whether the tumor is at an early or advanced stages.Sonodynamic therapy (SDT), which is developed from photodynamic therapy (PDT), is a promising new approach for cancer treatment. It functions by activating tumor-localizing sonosensitizer such as porphyrins through acoustic cavitation by ultrasound. Sonodynamic cancer therapy based on the sonochemical effects of a combination of low-intensity ultrasound and a sonosensitizer (e.g., anti-cancer drugs or certain chemicals), which involves preferential uptake and/or accumulation in tumor tissues and subsequent activation by ultrasound irradiation. Accumulating evidences indicate that SDT has a great potential in cancer therapy. It has been shown that ultrasound can induce a range of biological effects on cellular level such as cell destruction, apoptosis etc. Exposure of tumor cells to SDT leads to cell death through multiple pathways. Over the past decades, apoptosis induction has been the major consideration in anti-cancer drug development. However, cancer cells trigger multiple pathways to escape from apoptosis. Autophagy is a degradative process in eukaryotic cells that results in the breakdown of intracellular material within lysosomes. It can be stimulated by various stress situations, including external stimuli (e.g. hypoxia, energy depletion, pharmacological agents), as well as internal stressors. Because of its internal needs to maintain cellular homeostasis, autophagy is essential for the survival, differentiation and development. Recent studies have shown that some chemotherapeutics known to induce apoptosis also activate autophagy. However, depending on different stimulation manners and cell types, autophagy acts not only as a protector-it prevents cells from undergoing apoptosis, but also as a promoter-it promotes cell apoptosis.Based on previous studies and the Natural Science Foundation support, the project intends to study SDT induced cell death in tumor cell lines like K562 and K562/DOX, through selected the optimal experimental parameters of the autophagic and apoptotic responses to PpIX-SDT in leukemia cells, and, compare the sensitivity of different cell death mode of the same kind of cells involved in the SDT process; The critical threshold of relevant physical and chemical factors, target, influence factor, cell signaling pathway and key gene regulatory of autophagy in the SDT induced cell death were evaluated; With the chosen SDT parameters, relationship between autophagy and apoptosis in leukemia cells was investigated by using pharmacological inhibitors and shRNA; Empirical analysis showed that the leukemia cells had different cell death modes after SDT, and took full advantage of autophagy in cancer research, clarify the molecular mechanism of SDT induced different leukemia cell death mode. The results are as follow:1. Cellular uptake and sub-cellular localization of PpIX in leukemia cells K562: The intracellular concentration changes of PpIX at different time points after addition to K562 cells were evaluated by mean fluorescence intensity as determined by flow cytometry. The results showed that the cell uptake of PpIX increased as PpIX dose increased under the same incubation time.At the same PpIX concentration, the intracellular PpIX increased quickly for the first few hours immediately after administration, then slightly increased and reached a relatively high level at 2 h after incubation which almost sustained the same level after that. To assess the sub-cellular localization pattern of PpIX in K562 cells, we co-loaded cells with the MTG/LTG and PpIX. After 2 h of incubation, the fluorescence distributions of MTG/LTG and PpIX were captured using laser scanning confocal microscopy, the co-staining images of MTG/LTG and PpIX showed a good overlapping of both fluorescent signals indicating a higher concentration of PpIX in mitochondria in K562 cells, which indicated PpIX mainly localized to the mitochondria of K562 cells. The result suggested that 2 h may be the optimal incubation time of PpIX for our follow-up ultrasound and laser light irradiation experiments and PpIX displayed a mitochondria-localization pattern in K562 cells suggesting mitochondria and --- may be one of the dominating targets of SDT.2. The molecular mechanism of SDT induced different leukemia cell death mode. In the study, it is very interesting for us to evaluate the autophagic and apoptotic responses to PpIX-SDT in human leukemia K562 cells. Following SDT, hallmarks of apoptosis and autophagy were detected by morphological observation, biochemical analysis and molecular measurements. The relationship between autophagy and apoptosis was further obtained by applying pharmacological inhibition studies. The potential mechanisms of SDT induced cellular responses were also evaluated. The main research results are as follows:① We detected the cell apoptosis rate with Annexin V-PE/7-AAD labeling in FCM analysis, these results suggested that SDT triggered apoptosis in K562 cells. DNA fragment of cells were detected by flow cytometry following PI staining, DNA fragments were increased in SDT, while no obvious DNA fragmentation was detected in PpIX alone or ultrasound alone group. The SDT induced cell viability loss and cell apoptosis were all differently prevented by pre-treatment with the caspase inhibitor z-VAD. In addition, following SDT, Cyto c release from mitochondria to cytosol was observed at early 0.5 h post SDT. Moreover, the ratio of Bcl-2/Bax obviously decreased with a time-dependent manner. These suggested SDT could induce mitochondrial-dependent apoptotic cell death of K562 cells. ② Autophagy of SDT treated cells was examined by using gene transfection and laser scanning confocal microscope. After SDT, eGFP-LC3 transfected K562 cells appeared more green fluorescent aggregation, suggesting LC3 protein redistribution occurred in autophagy. Western blotting were used to assess the processing of LC3-I to LC3-Ⅱ, the autophagy marker protein LC3, was obviously converted from its I type to II type at 0.5 h post SDT, after that, the phenomenon gradually weakened. Simultaneously, the occurrence of autophagosomes and AVOs stained with AO were measured. Furthermore, AVOs marker LC3 displayed increased fluorescence staining and co-localized well with LAMP2, showing the fusion of autophagosomes and lysosomes. Following SDT, the relationship between autophagy and apoptosis was analyzed using the autophagy inhibitor 3-MA and BaA1, apoptosis inhibitor z-VAD-FMK, found that autophagic vacuoles formed upstream and independently from caspase-dependent cell death. ③ Under SEM, the cells exposed to SDT were seriously damaged with apparent deformation, while the SDT induced K562 cell membrane damage was significantly enhanced by pre-treatment with the 3-MA and bafilomycin A1. Furthermore, the presence of 3-MA and Ba Al reduced the number of viable cells and enhanced SDT induced cell apoptosis.3-MA or Ba Al could additionally aggravate chromatin condensation. Ba A1 enhanced SDT induced caspase-3-and PARP-cleavage. These data demonstrated that autophagy inhibition enhanced SDT-induced cell apoptosis in K562 cells. ④ The regulating function of ROS in the apoptosi and autophagy effect induced by SDT. Obvious ROS formation immediately after SDT treatment, and the presence of ROS scavenger NAC (N-acetylcysteine) significantly decreased ROS generation. Under SEM, the SDT induced K562 cell membrane damage was significantly reduced by pre-treatment with the NAC. The SDT induced cell apoptosis was visibly prevented by NAC. Additionally, blockage of ROS production partially protected SDT induced PARP cleavage. These results were also in agreement with our previous studies that ROS generation played a vital role in SDT induced cell apoptosis. Moreover, NAC also greatly inhibited SDT induced autophagy as demonstrated by inhibition of beclin 1 expression, MDC and acridine orange punctate staining and co-localization of LC3 and LAMP2. These results suggested that ROS may participate in regulating autophagy induction by SDT treatment.3. The role of Beclin 1 in SDT induced apoptosis and autophagy in human leukemia cells. The relationship between autophagy and apoptosis was further illustrated by short hairpin RNA (shRNA) studies with beclin 1-targeted shRNA (Beclin 1 shRNA). The ViaCount assay was adopted to examine cytotoxicity of different treatments. Nuclear morphology changes were observed under a fluorescence microscopy with 4’-6-Diamidino-2-Phenylindole (DAPI) staining. Western blotting was used to analyze the expression of caspase-3, Beclin 1 and the conversion of LC3 Ⅱ/Ⅰ. Fluorescence microscope was used to identify the formation of autophagic vacuoles (AVO) during autophagy. Pre-treatment of cancer cells with Beclin 1 shRNA was shown to reduce the level of LC3-II accumulation and staining with punctate spots of MDC staining. Besides, the cytotoxic effect of SDT was significantly increased by Beclin 1 shRNA. Furthermore, studies showed a marked effect on the apoptosis of cells by Beclin 1 shRNA to sonodamage with increased DAPI staining and caspase-3 cleavage. These results demonstrated that Beclin 1 played a critical role in the regulation of apoptosis of cancer, and, the autophagy probably to protect the K562 cells from sonodamage.4. Influence of PpIX-SDT on phosphorylation of Bcr-Abl in K562 cells. First, the clone forming assay was adopted to examine cell proliferation of different treaments, Western blotting was used to analyze the expression of Bcr-Abl. The results show that, SDT significantly inhibited the proliferation of K562 cells and compared with the control group, the expression of BCR-ABL obviously decreased at 0.5 h post SDT. The inhibitory study showed that the cytotoxic effect of SDT was significantly increased by AP24534. These results suggest that Bcr-Abl played a critical role in the regulation of death of K562 cells.5. Combination of PpIX mediated SDT with doxorubicin synergistically induced cell death of a multidrug-resistant leukemia K562/DOX cell line. At the beginning of this study, data indicated that ultrasound inhibited the cell toxicity of K562/DOX cells in ultrasound intensity-dependent manner. Ultrasound alone at intensity less than 1 W did not produce significant inhibition of cell proliferation. The subsequent analyses focused on cell toxicity varied with different doses of PpIX at a given US intensity of LP= 1 W, and, PpIX inhibited K562/DOX cells proliferation depending on the drug dosage. Then, the studies focused on cell toxicity varied with different doses of DOX at a given US intensity of LP= 1 W and a chosen PpIX dose of 2 μg/ml. With the introduction of PpIX into the US field, a remarkable synergistic increment of DOX-induced cytotoxicity was detected under our experimental conditions. The effects of the combined therapy with DOX and US+PpIX were slightly DOX dosage-dependent, Therein, at a DOX dose of 100 ng/ml, the CIs rose at first. These show that the cytotoxicity of DOX might have been efficiently enhanced via PpIX-US with optimized parameters. US+PpIX+DOX treated K562/DOX cells were stained with Annexin V-PE and 7-AAD. These results suggest at the given experimental conditions, US+PpIX+DOX treatment could remarkably increase the percentage of apoptotic cells. Moreover, more DNA fragmentation was also detected in US+PpIX+DOX treatment by flow cytometry. According to the data from flow cytometry, a significant increase on intracellular reactive oxygen species production after treated with US+PpIX+DOX, implying that ROS may be involved in the US+PpIX+DOX induced cell injury. And, US+PpIX+DOX revealed the strongest P-gp inhibitory activity compared with other groups. The result indacated that US+PpIX+DOX exposure may influence the membrane drug efflux pumps. These results suggested that the molecular mechanism was potentially attributed to the reducing the expression of P-gp and increasing the concentration of DOX in K562/DOX cells.