| Leukemia is a clonal malignant disease of hematopoietic stem cell. In China, the annual incidence of leukemia is about 3-4/100000 and increases year by year. Currently, the mainstream therapy strategy of leukemia is chemotherapy, in addition to bone marrow or stem cell transplantation if possible. These treatments bring not bad recovery and remission rate; however, there are still many defects. Chemotherapy is with serious toxic side effects and sometimes high recurrence rate, which obviously limit its application and increase the suffering of the patients; transplantation therapy is with limited application because of the transplant matching difficulties; besides, the high medical expenses bring a heavy burden to the family as well as the community. Therefore, to find out a more simple, safe and effective treatment, whether applied alone or in combination with other medical measures, is of great importance.Photodynamic therapy (PDT) is a cytotoxic treatment based on the interactions between light, cell or tissue molecular oxygen and photosensitizing molecule, termed photosensitizer (PS). Researches show once irradiated by visible light, PS can react through two photo-oxidative pathways classified as type I and type II, both leading to oxidative damage and ultimate lethal cell damages, including apoptotic, autophagic, and/or necrotic cell death. Compared with the conventional cancer treatments, PDT offers several advantages, including high sensitivity to tumor tissue, few side effects, the possibility of repetitive cycles of treatments, and the combination with other therapies such as chemotherapy and radiotherapy. It has been applied for the treatment of various malignancies such as bladder cancer, Barrett’s esophagus, and unresectable cholangio carcinoma, and skin cancers since the late 1970s.Hypericin (Hyp), with a typical naphthodianthrone structure, is the main active component of Hypericum species, commonly known as Hypericumperforatum or St. John’s wort. Hyp has been used as a drug against depressions and viruses for decades. And it is also one of the most powerful PSs, which has several advantages over other PSs, such as widely recognized security, strong light -dependent anti- tumor activity while low toxicity in the darkness. Hyp demonstrates intense absorption to the visible light of high wavelength and is with strong penetrativity. This feature is quite helpful in killing the leucocytes which are Often eclipsed by red blood cells in the blood flow. Besides, Hyp shows a selective accumulation in spheroids, which is exactly the cell type of the normal human white blood cells and leukemia cells. All the above characters decide Hyp an ideal photosensitizer for PDT in leukemia treatment.Although it has not yet received approval for clinical application, a great number of studies have demonstrated that Hypericin-mediated photodynamic therapy (Hyp-PDT) has high tumor-specific cytotoxicity and minimal side-effects. After gathering in tumor tissue, blood vessels or the matrix and activated by visible lights, Hyp is demonstrated to induce apoptosis secondary to the increase in ROS. And both the mitochondrial-mediated (intrinsic) pathway and the death receptor-mediated (extrinsic) pathway are involved in Hyp-PDT induced apoptosis. In addition, oxidative stress secondary to Hyp-PDT can not only induce apoptosis, but also affect autophagy of tumor cells. In fact, Autophagy has a two-way effect toward tumor cells: On one hand, autophagy may inhibit tumor growth and development by preventing intracellular protein synthesis; One the other hand, autophagy can help the tumor cells adapti to hypoxia and nutritional deficiencies, clear intracellular reactive oxygen species and thus protect tumor cells.Since the all the blood cells including leukemia cells flow within a closed vascular system in the body and can not be irradiated directly, people are confused how to conduct Hyp-PDT into the blood system. This is also the problem which limits the research of Hyp-PDT in diseases of the blood system. At present, research at home and abroad mainly focuses on the use of PDT in leukemia cells purification in vitro, trying to solve the residual of leukemia cells in autologous bone marrow transplantation. And most studies stop at in vitro experiments because of irradiation limitation. Some researchers have proposed to insert an optical fiber into the tail vein of a mouse for the implementation of intravascular PDT, and the results showed that tumor cell growth was inhibited. However, the intravascular PDT may result in severe damage on the vessel wall and surrounding tissue when acting on the targeting cells. Besides, the resource of irradiation light also limits the use of intravascular PDT. This study therefore innovatively proposes draining the blood of rats to an etracorporeal blood loop and then carries out irradiation to induct photodynamic therapy, trying to avoid vascular phototoxic damage surrounding tissue when killing target cells in vivo. In the present study, after Hyp was given, the rat blood was drained into an slide sandwich to form a thin layer of etracorporeal blood loop through which the irradiation was performed. We aim to observe the impact of Hyp-PDT on white blood cells through etracorporeal blood loop and find out whether this procedure would do harm to the other blood cells in the blood flow as well as the other organs of the rat. Secondly, Hyp-PDT was perform on cultured human leukemia cell line K562 in vitro to explore its relating mechanisms and possible pathway. Finally, we conduct cultured human leukemia cell line K562 and U937 suspension flowing through the slide sandwich to imitate an etracorporeal blood loop of leukemia rats and perform Hyp-PDT, in order to verify the probability of implement Hyp-PDT through etracorporeal blood loop in leukemia rats. Through the above experiment, we hope to find out a new therapeutic strategy for leukemia and thus be helpful in improving the health status of patients with leukemia.Part Ⅰ Cytotoxicity of Hypericin-mediated photodynamic therapy to normal white blood of rats through etracorporeal blood loop and the impact on other blood cells in the blood flow.Objective:To preliminarily explore Cytotoxicity of Hypericin-mediated photodynamic therapy to normal white blood of rats through etracorporeal blood loop and the possible side effects, and to verify the effectiveness of Hyp-PDT implementation through etracorporeal blood loop.Methods:All the experimental animals were randomly divided into four groups: Sham group, etracorporeal blood loop grouop (EBL group), etracorporeal blood loop and hyperin without irradiation grouop (EBL+Hyp group), etracorporeal blood loop and Hypericin-mediated photodynamic therapy grouop (EBL+Hyp-PDT group), n=5. In vitro SD rat model of etracorporeal blood loop was established by carotid artery and femoral vein intubation before connected to the slide sandwich. Rats in EBL +Hyp-PDT group was given Hyp 3 mg/kg by intraperitoneal injection 4 h before the incubation operation,1.5 h after the operation the blood was conducted in to the slide sandwich for 1 h irradiation with the wavelength of 595 nm to perform Hyp-PDT.; EBL+Hyp group was performed 1 h of extracorporeal circulation but without irradiation, and other managements were the same as EBL+Hyp-PDT group; Rats of EBL group was given the same amount of DMSO saline by intraperitoneal injection 4 h before incubation, and 1.5 h after the operation the blood was conducted in to the slide sandwich for of extracorporeal circulation; The Sham group was given the same amount of DMSO saline by intraperitoneal injection at the same time point, and the incubation operation was performed as other groups without being connected to the extracorporeal slide sandwich system. Blood of all rats undergoing extracorporeal circulation was taken for routine blood test before administration(Tl), before surgery(4h after administration) (T2),0.5h after surgery (T3), before irradiation(1.5h after surgery) (T4),1.5h after irradiation (T5),3 h after irradiation (T6) respectively. Sham group were also detected at the corresponding time points. Renal function of all the groups was detected before Hyp administration and 3 h after irradiation, or at the corresponding time points respectively. Blood smears were made before administration, before irradiation (1.5h after surgery) and 3 h after irradiation respectively for observation. And neutrophils were collected from the blood before administration,3 h after administration,6 h after administration and 9 h after administration for observation under the fluorescence microscope to verify the uptake of hypericin by the cells.Results:1) Weak fluorescence could be observed in some of the leukocytes 3 h after administration, and 6 h and 9 h after administration this orange fluorescence became very strong and could be found in all of the leukocytes under fluorescence microscope.2) The routine blood test showed the white blood cells in all the groups showed a significant rise and then fell back to preoperative levels 1 h later, but there was no significant difference at the same time point between different groups. In the EBL+Hyp-PDT group, the number of the leukocyte continued to decline 1.5 h and 3 h after irradiation (all P<0.05). decreasing by about 30% and 47% respectively when compared with that before the administration; In comparison with those of the control groups at the same time points after irradiation, the leukocyte also decreased obviously (all P<0.01). Blood smears showed reduced white blood cell after Hyp-PDT, and leukocyte with deformation could be observed under high power microscope.3) Compared with those before the administration, red blood cell count decreased in each group after surgery (all P<0.05). However, no significant difference of the red blood cell number was found neither before and after irradiation, nor before and after extracorporeal circulation in each group. There was also no significant difference observed between the red blood cell numbers of various groups at the same time point. To the blood platelet, there was a slight decline after surgery in all the groups except Sham group (all P<0.05). However, similar to the red blood cells, no significant difference was found neither before and after irradiation, nor before and after extracorporeal circulation in each group. There was also no significant difference observed between various groups at the same time point.4) There was no significant difference of serum creatinine and blood urea nitrogen of rats at different time points in each group. And no significant difference was observed between various groups at the same time point either.Conclusion:1) Intraperitoneal injection of 3mg/kg of hypericin DMSO saline solution does not result in hemolytic reaction in rats; 6 hours after administration, uptake of hypericin by the leukocyte in the blood has been quite adequate and could continue for some hours.2) With hypericin as the photosensitizer, undergoing 1 h irradiation through the extracorporeal blood flow loop with 595nmLED lights can effectively kill leukemia cells of the normal SD rats while the red blood cells and the blood platelets remain intact on the whole.3) The rats undergoing 1 h extracorporeal circulation through the thin-layer slide system designed by our group showed no significant damage of renal function.Part II Cytotoxicity of Photodynamic therapy with hypericin on K562 human leukaemia cells in vitroObjective:To investigate the cytotoxic effect of Hyp-PDT on K562 cells, find out the optimal conditions for Hyp-PDT on K562 cells, and discover the role of apoptosis and autophagy in the procedure.Methods:Human leukemia cell line K562 was cultured in vitro. Hyp-PDT was performed with different light intensities (0.1,0.3 and 0.5 mW/cm2), Hyp concentrations (0,0.2,0.4 and 0.8p.g/ml) and irradiation times (0,2,4 and 8min) in our present investigation to select the optimal protocol. The CCK8 analysis was applied and the results were analyzed. After the optimal conditions was decided, cell protein was collected immediately before irradiation,4 h,8 h and 16 h after irradiation respectively for western blot determination of apoptosis and autophagy related protein. And the Morphological changes of K562 were observed under phase contrast microscope and electron microscopy.Results:1) Hyp-PDT with the light intensity of both 0.3 and 0.5 mW/cm2 significantly decreased cell viabilities in K.562 post irradiation at different time points (all P<0.01). With the light intensity of 0.3 mW/cm2 or over, the decrease of the cell viabilities post irradiation became more markedly with the time and was relatively worsened with 0.5 mW/cm2. However, little decrease of cell viability could only be observed post irradiation with significant difference. According to the above results, Hyp-PDT with light intensity of 0.3 mW/cm2 which resulted in significant but moderate cell damages was selected for further study.2) On the whole, Hyp-PDT could affect K562 in a dose and irradiation time dependent manner and cell viability of the sample in the presence of 0.8μg/ml Hyp after 8min irradiation decreased to 5% in the end. However, the viability curves of K562 with 0.2μg/ml Hyp displayed low Hyp concentration with 2 min irradiation had little effect on cell viability and the cell viability even experienced a slight increase with 4 min irradiation.8 min irradiation could increase the reduction of 0.2μg/ml Hyp to below 70% yet still in a fluctuated way with the incubation time post irradiation.3) The morphological observation under the phase-contrast microscopy showed after Hyp-PDT, the cells begin to float in the culture medium and start to lose its ability to contact with each other. The cells also appeared to have undergone cell death and the cell intensity decreased obviously with the time as compared with healthy cells.4) 4 h after Hyp-PDT treatment, some cells presented typical characteristics of apoptotic cells, such as chromatin condensation, agglomerating in nuclear central or gathering at the periphery to form crescent, as well as the concentrated cytoplasm, bubble-like protrusions on cell surface and formation of apoptotic bodies. The changes of apoptosis are getting more frequently seen and more serious over time and loss of intracellular details could be clearly observed in K562 cells 16h after Hyp-PDT. There were several autophagies in K562 cells before irradiation.8 h after irradiation, the number of autophagies increased in cells treated with DMSO while that in the Hyp group decreased obviously or even disappeared.5) The analysis of western blot detection showed after irradiation, the expression of cleaved caspase-3 and cleaved caspase-9 increased significantly in the cells treated with Hyp while that in the DMSO group did not change.8 h and 16 h after irradiation, the expression of LC3 protein decreased while that in the DMSO group increased slightly. The expression of Beclin-1 decreased 16 h after Hyp-PDT and a relating decrease of p-Akt protein at the same time point was observed.Conclusion:1) Hyp-PDT in K562 could notably depress cell viability in a light intensity, dose and irradiation time dependent manner.2) The Hyp concentration of 0.4μg/ml with 5h drug light interval, as well as 4min irradiation and the light intensity of 0.3 mW/cm2 was selected the optimal conditions for further study.3) The increase of apoptosis observed under electron microscope and the up-grated activity of apoptosis related proteins caspase-3 and caspase-9 showed Hyp-PDT of K562 in vitro could notably induce mitochondria-caspase dependent apoptosis.4) The decrease of autophagy related protein LC3 and Beclin-1, together with the significantly reduced autophgosome observed under electron microscope, showed Hyp-PDT of K562 in vitro could promote cell death by inhibiting autophagy in K562 cells. And the above effect may be related to the inhibition of Akt pathway.5) Hyp-PDT may kill K562 cells probably by inhibiting autophagy and increasing apoptosis.Part III Hyp-PDT affected apoptosis and autophagy of human leukemia cell line K562 through M APK pathwayObjectives:To explore further the role of MAPK pathways, such as p38MAPK, ERK, JNK, in apoptosis and autophagy affected by Hyp-PDT on K562 leukemia cells.Methods:Cell protein was collected immediately before irradiation,4 h,8 h and 16 h after irradiation respectively for western blot determination of ERK、p-ERK、 JNK、p-JNK、P38 and p-P38 protein.Results:4 h after irradiation, the expression of p-ERK appeared in cells treated with Hyp, and the activity increased with the incubation time; But no ERK activation was observed in cells treated with DMSO. The expression of p-JNK increased significantly after Hyp-PDT and this expression was most obvious 4 h after irradiation. The expression of p-P38 was also up-grated significantly after Hyp-PDT. Total JNK and total P38 expression did not change at different time point in either Hyp group or DMSO group.Conclusion:1) The phosphorylated expression of ERK, JNK and P38 pathways increased after Hyp-PDT while there were no obvious changes in the level of total ERK, total JNK and total P38, which indicate that Hyp-PDT regulate the above pathways by increasing its degree of phosphorylation rather than the gene levels.2) Hyp-PDT may induce apoptosis of K562 cells by promoting phosphorylation of ERK, JNK, P38 pathways.3) Autophagy inhibition by Hyp-PDT on K562 cells may be associated with the increased phosphorylation of ERK, JNK, P38 pathways, although the specific relationship needs further exploration.Part IV Cytotoxicity of Photodynamic therapy with hypericin on human leukaemia cells K562 and U937 through the thin slide circulation systemObjective:To imitate an etracorporeal blood loop of leukemia rats and perform Hyp-PDT by conducting cultured human leukemia cell line K562 and U937 suspension flowing through the slide sandwich, and finally verify the probability of implement Hyp-PDT through etracorporeal blood loop in leukemia rats.Methods:Human leukemia cell lines K562 and U937 were cultured in vitro. After 0.4 μg/ml Hyp was added for 5 h incubation, the cell suspensions of K562 and U937 were respectively pushed through the thin slide circulation system by a micro-pumps at a speed of 9 μl/s according to blood flow speed of rats in the in vivo experiments of the part I. At the same time, Hyp-PDT was performed with a LED light with the wavelength of 595 nm and the light intensity was 1mW/cm2. The time for the leukemia cells flowing through this outer circulation system and receive irradiation was about 15 seconds. The CCK8 analysis was applied for the analysis of cell viability. And the Morphological changes of K562 and U937 after Hyp-PDT were observed under phase contrast microscope.Results:1) The results of CCK8 showed serious injury of K562 cells and U937 cell after Hyp-PDT. Compared with those before irradiation, the cell viability decreased 92.65% and 98.27% respectively.2) The observation under microscope showed there were obvious apoptosis in both K562 and U937 after Hyp-PDT, and necrotic cells and debris could also be found.Conclusion:Imitating an etracorporeal blood loop of leukemia rats and perform Hyp-PDT by conducting cultured human leukemia cell line K562 and U937 suspension flowing through the slide sandwich could effectively kill the leukemia cells through single cycle.In summary, the findings of this study not only confirms the effective cytotoxicity of Hyp-PDT for leukemia cells and discovers the possible pathways, but also raises an innovative ideas of applying systemic PDT through etracorporeal blood loop. This idea, once confirmed feasible in the clinic, may put forward a new idea and strategy for the treatment of hematologic malignancies, as well as the inhibition of the hematogenous metastasis of some non-hematologic tumor. |
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