Focused Ultrasound Combined With Protoporphyrin Ix Inhibition Of H22 Tumor

Ultrasound is a kind of mechanical wave which frequency is above 20 KHz,Ultrasound has good characters such as having precise directionality,penetrating deep place and easying to focus power.It has been used in medicine,military,industry,agriculture and so on.Especially in ultrasound medicine,ultrasound diagnosis has used to forecast and examine many different diseases. In ultrasound therapy,the cavatition effect,thermal effect,mechanical effect and sound pressure are the main rsearch aspects.And sonodynamic therapy(SDT) is one kind of the tumor therapy mothed, the synergistic effect of ultrasound and drugs on tumor cells,is a promising modality for antitumor. Ultrasound can be focused in a small region and can penetrate deeply in tissues,then activate the sonosensitizers.The studies of SDT had persisted nearly twenty years when first reported the papers of ultrasound combining hematoporphyrin(HP) in 1989.But the exactly mechanism of SDT is still unknown until now.It seems that many factors including the sonosensitizer used,the acoustic parameters and the biological mode being irradiated affect the specific mechanism of SDT.Previous work had done many about SDT in our laboratory.This study has done a part of work about the Excellent Doctor Project of Shaanxi Normal University and National Natural Science Foundation of China(No.30270383):the biological mechanism of inducing tumor cell apoptosis by ultrasound activating hematoporphyrin.The antitumor effect was investigated on H22 tumor exposed to the combination of protoporphyrin IX(PPIX) and focused ultrasound at the frequency of 1.43MHZ,and the results be explained as follows:1.The character of protoporphyrin IX has been studied for our research.The maximal excition spectrum(405 nm) and emission spectrum(604 nm) of PPIX were scanned by fluorescence photometer in our experimental system.Then the pharmacokinetics of PPIX in H22 cells was detected by the fluorescence intensity to determine the optimum time for ultrasonic irradiation.This study implied that the cell damage increased as the ultrasound intensity strength.PPIX dose also affected the damage effect of ultrasound.The intensity threshold for ultrasonically induced cell damage was observed to be 1～2 W/cm~2 when exposure 1 min.When plus PPIX,the ultrasound had more serious killing effect on H22 cells.The threshold concentration of PPIX was about 20μM.For to study the detail mechanism of damage effect,exposure 1 min ultrasound at 1 W/cm~2 plus 20μM PPIX was to be decided as the research parameter of our experiments.2.Hematoxylin and Eosin(HE) staining was used to observe the morphological changes of H22 cells after PPIX-SDT.The ultrastructure of H22 cells were observed by scanning electron microscope(SEM) and transmission electron microscope(TEM) after SDT treatment.The morphological observation indicated that the membrane system of cells,especially the membrane bound organelles such as mitochondria and endoplasm was the action point of SDT.H&E observation found that the morphological structure of H22 cells was severely damaged by PPIX-SDT as the time delayed.Ultrasound alone had little effect on the surface of H22 cells,but when plus PPIX,the H22 cells showed significantly damaged in ultrasound combined PPIX group. And as time delayed,the morphological changes of apoptosis were presented.We observed that the membrane-bound organelles and membrane systems were the important sites to be destroyed in treatment groups.As time delayed,the apoptosis was observed in ultrasound combined with PPIX group after 3 h by ultrastructure observation.3.The product of lipid peroxidation(LPO) in tumor cells was measured by the thiobarbituric acidx(TBA) method.The concentrations of calcium ion in cytoplasm of different groups were assayed by Fluorescence Labeling Technique.The results indicated that PPIX-SDT can enhance the LPO and damage the membrane system of tumor cells.Then the calcium ion flow into cytoplasm, then the concentration of calcium ion escalating in cytoplasm may lead into a series of physiological and biochemical changes to cells.4.The bearing Hepatoma22(H22) mice were employed as the stable experiment model.After administering 5.0 mg/kg PPIX,the pharmacokinetics of PPIX in the plasma,tumor,skin and muscle were assayed.From 8 h to 36 h,the PPIX concentration in the tumor was at least 24 times,6 times and 1.7 times higher than in the plasma,muscle and skin,respectively.Because the major goal of SDT treatment was to avoid severe side effect to healthy tissues and to obtain the best anti-tumor effect,the period between 8 h and 36 h may be the suitable treatment time in our study.During this period,we applied three times interval treatment mode(at 8,12,24 h after administration, respectively) to gain the best anti-tumor effect of PPIX-SDT on H22 tumor.5.The tumors were exposed to the 3 W/cm~2 focused ultrasound for 3 min,respectively exposed to ultrasound at 8 h,12 h and 24 h after 5.0 mg/kg PPIX be administrated,according to the result of pharmacokinetics.The tumor inhibitory effects were evaluated by the tumor volume and weight inhibition ratios.After fifteen days' observation,tumor growth in the PPIX plus ultrasound group was significantly retarded in comparison with that in the Control,PPIX and Ultrsasound groups.The synergistic antitumor effects of PPIX and ultrasound were always significant as time delayed.The volume inhibition ratios and weight inhibition ratios respectively were 53.84%and 45.86%in the Ultrasound plus PPIX group.In the PPIX group there was no any inhibition effect compared with Control group.The weight curve of mice also indicated the curing effect of PPIX-SDT.At fifteenth day the vigor of mice almost lost,a few died before fifteenth day.PPIX-SDT manifested obvious inhibitory effect to H22 tumor and therapeutic effect to ICR mice.6.The morphological changes of H22 tumor tissues were evaluated by H&E and TEM observations immediately after SDT therapy.Tumor tissues appeared a few agglomerate necrosis sections and the tumor cells were separated from each other when exposed three times ultrasound at the intensity of 3 W/cm~2 for 3 min.The histological examination showed that the damage level of PPIX-SDT is more serious than ultrasound alone.The structure of tumor tissue was seriously damaged,obvious vacant sections were dilated and there were a lot of cell fragments.The ultrastructure showed that tumor cells in PPIX plus ultrasound group experienced cellular membrane injury,mitochondria swollen,cytoplasm lost and chromatin condensation.7.The peroxidation test and activities of anti-oxidative enzymes assay were conducted immediately after the third treatment.These results suggestated oxidative stress might be involved in the PPIX-SDT treatment effect.The activities of three important antioxidant enzymes in tumor tissues were evaluated instantly after SDT treatment.The activities of SOD and GSH-PX in the PPIX plus ultrasound group were lower than ultrasound alone group.Although the activity of CAT had no statistical difference between PPIX plus ultrasound and ultrasound groups,it was also significant comparing with the untreated group.The activity of Caspase-3 was detected through immunohistochemical method.After SDT therapy,the strongly positive of Caspase-3 was found at the around of the necrosis position of tumor tissues.Basing the before works of our laboratory,this work studies the biological effect of PPIX-SDT on H22 tumor.The damage effect and inhibitory tumor effect of PPIX-SDT to H22 was investigated. The biological responses of H22 solid tumor to PPIX-SDT treatment were analyzed.Therefore,this paper may provide a potential biological mechanism for sonodynamic therapy from the point of in vivo study and a few materials to clinical research in the future.