| Background and Objective:Malignant tumor is one of the most deadly diseases for human beings, and its incidence rate is still rising in recent years. While, at present, the usually used methods for cancer therapy including radiotherapy, chemotherapy, surgical therapy, etc., can not prevent and cure cancer effectively. Therefore, it needs to explore new approaches for cancer therapy, and which should be the hotspots in the fields of life sciences and medical research. In1989, Sonodynamic therapy (SDT) was firstly proposed by Umemura and colleagues to describe the synergistic effects of ultrasound and sono-sensitizers on tumor treatment. SDT is a relatively new approach for cancer treatment, which involves the administration of a sono-sensitizer, such as hematoporphyrin and its derivatives, then followed by local activation by ultrasound exposure to induce tissue or cell destruction and produce significant anti-tumor effects. A series of in vivo and in vitro experiments have demonstrated that the porphyrins alone had no or very low cytotoxicity and ultrasound, especially focused ultrasound, can be precisely focused on the target volume, which made it possible to effectively activate the cytotoxicity of sonosensitizers that preferentially accumulate in tumor sites while with minimal damage to peripheral healthy tissues, this indicates that SDT has potential value for cancer therapy. In particular, for some difficult surgery, deep tissue tumors, or some patients required intravenous chemotherapy, SDT has strong targeting and security. So, SDT has important theoretical significance and clinical application.SDT as a new cancer therapy, has the advantage of a number of interdisciplinary, involving biology, medicine, physics and chemistry fields, but this also increases the difficulty and complexity of the study. Recently, SDT has been widely investigated, mainly focusing on the mechanisms of killing effects by using different ultrasound parameters and different sonosensitizers. But until now, the exact mechanism about SDT is still unclear because of its multiple factors. Our previous study has found that SDT can induce cell apoptosis and necrosis. However, as the development of life sciences and the knowledge of cell death modes, the cytotoxicity of SDT protocols, cannot be totally explained by the induction of apoptosis or necrosis. Autophagy is a relatively newly described cellular response to various cancer therapies. Now, literature search indicates that no prior information on the potential role of autophagy in the efficacy of SDT. Therefore, in the current study, it is very interesting for us to evaluate whether autophagy occur following SDT at the experimental conditions, and to determine the function of SDT induced autophagy in the fate of tumor cells.We chose focus ultrasound at a relatively safe intensity with minimal damage to normal cells, and protoporphyrin IX (PpIX) known to have low cytotoxicity with special sub-cellular localizations, to study SDT induced cell death in tumor cell lines like S180, H-22, EAC and L1210. Following PpIX-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 role of autophagy in the SDT induced cell death was also evaluated. The available findings shed new insights into SDT induced cell death, and further propose some ideas about how to use the favorable factor of autophagy to enhance the anti-tumor effect of SDT, which provide useful information for SDT mediated cancer therapy from the clinical views.Methods and Results:1. Physical and chemical properties of sono-sensitizer. The ultraviolet-visible absorbance spectra and fluorescence emission spectra of PpIX before and after ultrasound exposure were recorded on a spectrophotometer and a spectrofluorimeter, respectively. The acoustic cavitation measurement was also studied.①The results showed that PpIX has different spectra pattern in different solvents. In PBS and1640medium, PpIX has five distinct absorption peaks, the maximum (max) peak was about at365nm; in complete cultured medium and methanol-water (v/v,9:1), the max peak was nearly at402nm when PpIX concentration was≤20μg/ml, while the absorbance pattern became irregular when PpIX concentration was up to40μg/ml.②Ultrasound treatment had different effects on PpIX absorption pattern in different solvents.In PBS buffer, ultrasound treatment decreased the max absorption peak of PpIX, but had no influence on other peaks. In1640, ultrasound treatment not only significantly reduced the max absorption value of PpIX, but also increased the peak at542nm, which may be due to some secondary products have strong absorption in this wavelength after sonication. In complete culture medium, ultrasound treatment did not produce any effect on PpIX, this may be due to PpIX has great affinity with serum and the higher viscosity in the solution reduced ultrasonic cavitation.③In PBS and1640, ultrasound treatment decreased the maximum fluorescence emission intensity, and which was more obvious in1640 medium.④Using TA dosimetry method, it was possible to evaluate the efficiency of irradiation parameters on the cavitation activity in ultrasound fields by monitoring hydroxyl radicals. The findings suggest cavitation dependents on the ultrasound intensity, and PpIX at different concentrations can cause different results on OH-radical, the max content of OH· was obtained when PpⅨ was at1μg/ml PpIX. The ultrasound exposure produced OH·radicals could be inhibited by NaN3, histidine, mannitol, EDTA and catalase, but not by SOD.2. Different sensitivities of ascites tumor cells to ultrasound exposure. A direct comparison among the different types of tumor cells (S180, H-22and EAC) was made, and the effects of ultrasound on cellular responses were evaluated, and the potential mechanism underlying different senstivities was also investigated.①The results showed that there were similar trends for three cell types exposed in vitro to potentially sonolytic ultrasound. The relative cell survival decreased as ultrasound intensity increased, which was very obvious in S180cells and H-22cells, and the sonication threshold was approximately at3W/cm2.②There also appeared to be a common dependency of lysis on density among different cell types, at higher cell concentrations with no obvious cell death, whereas at lower densities, most of cells were damaged. The density threshold seemed to be around1×106cells/ml.③The relative cell lysis was in an order of S180>H-22>EAC. There are several possible explanations for this apparent discrepancy in relation to the results:cell types are different, raising the potential structural membrane dissimilarities and hence, different sonolytic potentials.④Different cellular responses to a given ultrasound exposure were measured. The data implied that mitochondria may act as sensitive indicators for cell injury after irradiation, and the plasma membrane can be the critical target for ultrasound induced cell death. S180exhibited the most sensitive response to ultrasound induced cell damage.3. The pharmacokinetics and localization patterns of endo-or exo-generous PpIX in S180cells. The5-aminolaevulinic acid (ALA)-derived endogenous PpIX and exogenous PpIX pharmacokinetic profiles were determined by the fluorescence intensity of cell extractions with a fluorescence spectrophotometer based on the standard curve. The changes of their sub-cellular localization patterns with prolonged incubation time were evaluated by laser scanning confocal microscope. The cytotoxic effects of5-ALA mediated sonodynamic therapy (ALA-SDT) and exogenous PpIX mediated sonodynamic therapy (PpIX-SDT) were also evaluated by MTT assay.①Results showed that for exogenous PpIX, the pharmacokinetic was in a dose dependent manner and a plateau was found in intra-and extracellular content after45min of administration, followed by slightly decreasing, and saturated after60min.②The amount of ALA-derived endogenous intracellular PpIX showed a linear accumulation with incubation time, which was independent of ALA concentration, so did the extracellular PpIX level.③Fluorescent images revealed that the exogenous PpIX was mainly accumulated in plasma membrane, whereas the ALA-derived PpIX was initially localized in the mitochondria and released from mitochondria to cytosol at later time points.④In order to compare the sonodynamic cytotoxicity induced by PpIX and ALA, the similar amount of endogenous PpIX induced by ALA and exogenous PpIX was applied to cells under the same incubation conditions, then exposed to ultrasound treatment. Cell survival was evaluated upon irradiation by the MTT assay as described. The result showed exogenous PpIX has more potential to enhance the ultrasound induced cytotoxicity than ALA derived endogenous PpIX.4. SDT anti-tumor effect and the biological changes of cell membrane and mitochondrial. We performed some initial experiments to evaluate the ultrasound activation requirements of PpIX. After PpIX-SDT, several potential sensitive targets such as cell membrane and mitochondria were studied from the biological views.①After sonication, FD500fluorescent molecules in the cell and LDH released into the extracellular medium were increased, indicating the cell membrane integrity was damaged and PpIX efficiently mediated the ultrasound induced cytotoxicity.②Immediately after SDT, the activities of Na+-K+-ATPase and Ca2+-ATPase and the sialic acid content were obviously decreased, thus indicating the membrane proteins were seriously injured.③Ultra-structural observations confirmed the membrane had morphological changes after SDT treatment.④The results displayed intracellular Ca2+concentration instantly increased after exposure and resumed to normal level at2h post-treatment; the plasma membrane potential significantly decreased immediately after sonication, then quickly come to a nearly control value within1h. We speculate that on the one hand, SDT enhanced the cell membrane permeability and thus damaged its selective capacity to transport ions, causing intracellular Ca2+increased; on the other hand, SDT damaged the membrane proteins such as decreasing SA content and Na+-K+-ATPase and Ca2+-ATPase activities, promoting the plasma membrane depolarization.⑤Intracellular ROS generation increased quickly immediately after treatment and reached maximum at1h post-irradiation, then decreased gradually to normal level. And, cellular glutathione level also decreased remarkably, implying free radicals produced by acoustic cavitation play important role in S180cell damaging process.⑥The mitochondrial membrane potential and cytochrome c oxidase activity were significantly declined at8h post SDT treatment, and the cell viability decreased to its lowest level at12h post SDT treatment, this indicated that the mitochondria structural and functional damage may play important role in SDT induced cell death.5. SDT treatment on DNA damage and cell cycle arrest.①DNA damage is an early event in cell damage. At1h after treatment, compared with control, PpIX alone, ultrasound alone and SDT can cause significant DNA damage in S180cells, and the combined group showed more seriously DNA damage than any alone treatment group.②There were obvious S phase arrest in both PpIX group and SDT group cells at20h after treatment, and which was relieved after a cell cycle (at40h after treatment), this may be due to the reduced intracellular PpIX content after longer incubation time. Compared with PpIX group, more remarkable S phase arrest in SDT group, this might be caused by more PpIX accumulation in S180cells after ultrasound exposure.6. SDT induced cell apoptosis and autophagy in S180cells. Based on previous study, this study was to determine whether autophagy may exist after PpIX-SDT in in vitro S180cells and to investigate its relationship with apoptosis.①Under the optimal SDT conditions, autophagy was indentified by transmission electron microscopy, immunoblot and immunofluorescence observations. Autophagy flux occurred in the early step of cell damage following SDT, and gradually decreased with the incubation time.②The apoptotic features such as Bax redistribution, cytochrome c release, caspase-3activation and chromatin condensation were prominent and time dependent, which required4-8hours.③The relationship between autophagy and apoptosis was studied by applying pharmacological inhibition of autophagy or apoptosis. Data showed the autophagy inhibitors either3-methyladenine (3-MA) or Bafilomycin Al (Ba Al) led to increased dissipation of mitochondria potential. SDT treatment combined with autophagy inhibitor especially Ba Al, significantly enhanced Caspase-3activity and the ultimate cell death. Whereas the pan-caspase inhibitor, z-VAD-fmk partially prevented SDT induced cytotoxicity and Caspase-3activation, but did not obviously improve the mitochondria depolarization, suggesting that the MMP loss was likely to occur upstream and independently from caspases. DAPI staining further confirmed that autophagy inhibitors enhanced SDT induced cell apoptosis; while, the pan-caspase inhibitor z-VAD-fmk weakened SDT induced cell apoptosis.7. Different cell death modes and its potential mechanisms in murine leukemia L1210cells following SDT. In the study, it is very interesting for us to evaluate the autophagic and apoptotic responses to PpIX-SDT in murine leukemia L1210cells. 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 methods and results are as follows:①Results show that SDT induced autophagy was a general phenomenon at a wide range of PpIX concentrations and ultrasound intensities. However, taken the viability assay together, we chose the optimal SDT does in which PpIX (1μg/ml) alone and ultrasound alone (1W/cm2) caused slight cytotoxicity, while the synergistic effect of them can produce significant anti-tumor effect (cell viability declined about40%).②Under the given exposure conditions, hallmarks of autophagy were confirmed. Autophagosome formation was examined by TEM observation and LC3-Ⅱ generation, furthermore, the extent of induction of autophagy was time dependent and occurred as early as0.5h post SDT. Additional markers of autophagy associated proteins Beclin1, UVRAG and Lamp2also showed enhanced expression levels following SDT.③Confocal microscopy also revealed co-localizations of LC3(an AVOs marker) with the lysosomal marker proteins Lamp2and Cathepsin B, supporting the formation of autolysosomes.④After6h of incubation, SEM observation provided the classical cellular shrinking and membrane belebbing following SDT. DAPI staining demonstrated the condensed chromatin by fluorescence observation. SDT could induce significant caspase-3activation compared with control, and was ensured by decreased level with the pan caspase inhibitor z-VAD-fink. Consistent with the findings, the cleavage assay of PARP, a classical caspase-3substrate, confirmed similar changes of89kDa PARP fragments in cells.⑤As the immunofluorescence and FACS analysis revealed, after SDT, apoptotic features such as dissipation of mitochondria potential, Bax/Bak redistribution, sonodamage of Bcl-2and Cyto c release were prominent and time dependent, which suggested mitochondria dependent apoptosis pathway was involved.⑥The inhibitor studies suggested that AVOs are formed upstream and independently of the caspase dependent death mechanism; the autophagy inhibitors either3-methyladenine (3-MA) or bafilomycin Al (Ba Al) enhanced the anti-tumor effect of SDT through induction of apoptosis and necrosis, while the pan-caspase inhibitor z-VAD-fmk decreased cell apoptosis but did not protect SDT induced cell death. The findings implied multiple cell death modes occurred following SDT.⑦Results demonstrated mitochondria damage was an early event following SDT, and the damaged mitochondria co-localized rapidly with autophagosome markers LC3and Atg5, which were inhibited by Ba Al, suggesting mitochondria damage might play a role in initiation of autophagy. In addition, pretreatment with Ba Al clearly enhanced SDT induced Bax redistribution onto mitochondria, indicating the increased cell apoptosis by inhibiting autophagy might be related with the more seriously damaged mitochondria.⑧The current study demonstrated obvious ROS formation immediately after treatment, and the presence of ROS scavenger NAC (N-acetylcysteine) significantly decreased ROS generation. NAC also visibly reduced the LC3-Ⅱ levels and almost completely inhibited the co-localization of mitochondria and Atg5at0.5h post exposure, thus preventing the damaged mitochondria being enclosed by AVOs.⑨The comet assay showed intracellular DNA damage occurred early and repaired quickly within4hours following SDT, and the induced DNA damage could be mostly suppressed by cyclosporine A (an inhibitor for mitochondria permeability transition pore) and NAC. Otherwise, the autophagy inhibitor Ba Al slightly enhanced SDT induced DNA damage. Therefore, we speculate that autophagy may play a role in preventing DNA damage, presumably through its cellular housekeeping role in removing sources of oxidative stress such as defective mitochondria.⑩Blockage of ROS production partially protected SDT induced Caspase-3activation and PARP cleavage. The ultimate role of ROS in SDT induced cell death as determined by MTT assay, showed NAC slightly protected SDT induced loss of cell viability.Conclusion:1. The mechanism of ultrasound activating PpIX was related to acoustic cavitation. Cavitation destroyed PpIX molecules, and reduced its maximum absorption and fluorescence emission intensity, which was dependent on ultrasonic medium, ultrasound intensity and PpIX does. PpIX concentration has great relationship with OH-production, at lower dose, it potentiated OH·radicals, while at higher does, it can inhibite OH-radicals. Cavitation can produce high-temperature and high-pressure to split water molecules, and Fenten reaction, H2O2, OH·, O21were involved during the process.2. The results showed that cellular responses of different cells were distinct, of interest to note, the aggressive S180cells were much more sensitive than others, whereas EAC cells were relatively more resistant to ultrasound irradiation. The direct comparisons among different types of cells indicate that the sono-sensitization seems to depend on the physiological and chemical properties of tumor cells. Perhaps sections of cell membrane became destabilized following the initial radical attack and LPO reaction, which caused S180cells more susceptible to mechanical stresses during sonolysis.3. PpIX has great preferential accumulation in in vitro cultured S180cells. Sono-sensitization with PpIX involved a45min drug-loading incubation at37℃, allowing sufficient time for cell uptake of the sensitizer to reach a maximum level, then, cells were exposed to ultrasound. Exogenous PpⅨ was mainly accumulated in plasma membrane, whereas endogenous PpⅨ was initially localized in the mitochondria. Exogenous PpIX has more potential than ALA derived endogenous PpIX in SDT induced cytotoxicity in S180cells.4. PpIX mediated sonodynamic therapy can trigger a series of bio-effects by both direct mechanical stress and indirect chemical actions. The main damaging targets are the cell membrane proteins, membrane enzymes and cellular other organelles such as mitochondria, then eventually lead to cell death.5. PpIX-SDT led to DNA damage and cell cycle arrest, which to some extent, inhibited the rapid proliferation of tumor cells, reflecting the pleiotropic effect of SDT on tumor cells6. The experiments confirmed that, at optimal SDT dose, both apoptosis and autophagy occurred in a time-dependent manner. Autophagy occurred earlier than apoptosis. Autophagy by itself was not sufficient to induce cell death, which played a protective role, perhaps by promoting the rapid remove of sono-damaged mitochondria, and thereby preventing some apoptotic response following SDT. And, inhibition of the early stage of autophagy, using3-MA, sensitized cells to SDT-induced cell death through apoptosis and necrosis, and the latter was more than the former. Similarly, inhibition of the later stage of autophagy, using Ba Al, also enhanced SDT induced cell death, but was less efficient than3-MA, which mainly promoted cell apoptosis. Therefore, irrespective of the stage at which autophagy was inhibited, disabled autophagy accelerated SDT induced cell death. Therefore, the findings can be incorporated into a more general hypothesis suggesting the efficiency of killing cancer cells by sonodynamic therapy may be enhanced by the simultaneous treatment with autophagy inhibitors.7. Because different tumor cell lines have different sensitivities to ultrasound irradiation, this study firstly selected the optimal SDT parameters in which SDT treatment can reduce the cell viability of L1210cells and simultaneously induce autophagy occurrence. Some key proteins in the process of autophagy showed time dependent changes after SDT treatment.8. SDT can significantly inhibit the rapid proliferation of murine leukemia L1210cells, and the corresponding mechanism may be associated with the induced mitochondrial--dependent apoptosis pathway.9. Following SDT, autophagy flux well before cell apoptosis. The relative percentages of cells undergoing apoptosis and autophagy following SDT could be experimentally manipulated. Pre-incubation with autophagy inhibitors prior to SDT promoted the appearance of apoptosis and suppressed AVOs formation.10. ROS play important role in initiating cell autophagic and apoptotic responses. The mitochondria damage and DNA damage had great relationship with the occurrence of autophagy. |
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