| Sonodynamic therapy(SDT) is a approach for cancer treatment basing on ultrasound(Us) could be precisely focused on the target volume,which made it possible to effectively activate the cytotoxicity of sonosensitizers(Hemtoporphyrin and Hemtoporphyrin Derivatives) that preferentially accumulate in tumor sites.Currently,there are lots of conventional treatments for tumor as surgery,radiation therapy and chemotherapy.However,these methods all have their pros and cons.Surgery,surgical excision of tumor,is better in early treatment but advanced ineffective;Radiation therapy is failed on widely spread or partial tumor invasion,and it also brings some near or long-term side effects; Chemotherapy is the main cancer treatment using chemical means of antineoplastic agents,which have strong side effects,mainly including nausea,vomiting,alopecia,leukopenia losing,and so on. In 1978,American scholar Doughtery put forward Photodynamic Therapy(PDT),which was already applied to the clinical treatment of tumors.Because of the undesirable penetration of the light, photodynamic therapy was used mostly to treat tumors located in human skins,but not those in the deep part of human bodies.Ultrasound which can penetrate deep into the tissue and have little harm to natural tissue,at the same time,hematoporphyrin,can specifically accumulate in tumor cells.So, SDT is a promise therapy for cancer treatment.Since SDT was proposed in 1989,it has been widely studied by many researchers,and they also proposed many cell killing mechanism including sono-cavitation,free radicals,lipid peroxidation,and so on.But until now,we still do not have a unanimous conclusion about SDT.Cytoskeleton,the important cell component,plays a crucial role in numerous cell functions, such as proliferation,morphology,signal transduction,cancer metastasis,etc.Literatures repoted the photodynamic damages of PDT on cell cytoskeleton which affect the functions of cells.For example, Zinc(Ⅱ)-phthalocyanine can induced photodamage to microtubule,actin,α-actinin and keratin of HeLa Cells,and suppresses cell cycle and change cell shape;ALA-PDT-induced changes in adenocarcinoma WiDr and glioblastoma D54Mg cells cytoskeleton,cell shape,and adhesion; ALA-PDT can also affect the cytoskeleton organization in K562 cells.Cytoskeleton constitutes a very important objective for all cancer therapies.However,so far,the effect of SDT on cytoskeleton has not been reported.So it is very important to identify whether the sonodynamic effect have some impacts on cytoskeleton.and also interesting to compare the effects between protoporphyrin IX (PPIX) and hematoporphyrin(Hp) on cytoskeleton of Ehrlich ascites carcinoma(EAC) cells under the same ultrasound exposure conditions.Basing on the international development and earlier experiment results,this paper has done some work about the National Natural Science Foundation of China(Grant No.39870240 and No. 30270383).The sonodynamical effect was investigated on EAC cells exposed to the combination of 20μmol/L PPIX and Hp and focused ultrasound at the frequency of 1.34 MHZ,and the present results can be explained as follows:1.When observed under SEM,untreated cells showed a typical surface morphology,with numerous randomly distributed microvilli.The number of microvilli on cells in ultrasound alone group decreased with time.After 2 h of incubation,the surface of many cells became relatively smooth with no obvious microvilli,several small craters were also seen at 4 h.In the synergistic treatment groups,the extent of cell damage increased in a time dependent manner.In both Uh and Up groups,the number of microvilli remarkably decreased at 2 h after treatment and had apparent deformation 4 h later.Cells in Up group with some irregular blebs were seen in the surface where the cytoplasm seemed to have extruded through the membrane boundary.Hp and Pp groups had only a slight effect on the surface of the cells until 4 h post-treatment and had no obvious difference with CT group.2.The ultrasonically induced F-actin damage increased rapidly with ultrasound intensity and the sensistizer concentration increased.The fluorescence intensity of FITC-Phalloidin labeled cells was detected by flow cytometry.The fluorescence intensity of F-actin decreased in ultrasound treated group cells.The fluorescence intensity of F-actin in Hp-SDT and Pp-SDT decreased as time prolonged.3.The fluorescence study revealed restructuring of microtubule and intermediate filament networks represented byβ- tubulin and vimentin elements.MT and VF polymerization state in Hp and Pp groups cells had no obvious changes as time passed.In Us alone group,cells MT and VF fluorescence intensity partially decreased as time prolonged.Changes of MT and VF were more obvious in Uh and Up groups.Immediately after the treatment,cells MT and VF became diffuse, and the fluorescence intensity of MT and VF decreased.Severe alterations in morphology and distribution of MT and VF could be detected in most Up group ceils 4h later,which exhibited alterations of the plasma membrane,and numerous blebs reacted positively toβ-tubulin and vimentin antibodies could be seen on the surface of cells.4.The IOD value of nuclear lamina proteins- LaminB was examined by the method of the immunocytochemistry which indicated that the IOD value remained no visible difference among control groups,Hp and Pp groups.In ultrasound alone group,the IOD value of LaminB proteins decreased at 4 h after the sonication.The IOD value of LaminB proteins signifficantly decreased in both Uh and Up groups as time prolonged,which was more obvious in Up groups.5.Hoechst 33258 staining was used to detect the apoptosis of EAC cells after SDT treatment. The control cells were uniformly blue,and had no visible nuclei changes with time.Hp and Pp groups had no obvious difference with CT group.The apoptotic cells were blue and contain bright blue dots in their nuclei,representing the nuclear fragmentation in both Uh and Up groups as time prolonged.In this paper,we study the sonodynamic effect of SDT on the cytoskeleton of EAC cells to find out the biology effect of SDT. |
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