Study On The Killing Effect Of Chlorin E6 Mediated Acousto-optic Combination Therapy On Mouse Breast Cancer 4T1 Cells

Breast cancer has been one of the most fatal cancers threatening female health throughout the world for decades.On the basis of the traditional surgery,radiotherapy and chemotherapy,there are endocrine,biological and physical therapy,et al.Surgical therapy could improve the survival rate of patients to certain extent,but with the deficiency such as high risk of local recurrence,more severe trauma to patients' form,it tends to affect beauty and cause long term psychological shadow.Similarly,radiotherapy and chemotherapy have limitations with lots of side effects.There are serious complications of endocrine and biological therapy,with less security and need more complex and particular nursing after treatment,et al.Therefore,explore new methods and techniques for clinical treatment of breast cancer is one of the major biomedical issues currently.Photosensitizers can be selectively gathered in tumor tissues,activated locally by light with appropriate wavelength,and generate reactive oxygen species(ROS)with both strong biological and chemical activity,kill tumor cells and inhibit the growth of tumor tissue ultimately,that is photodynamic therapy(PDT).Since its proposal in the late 1970s,PDT has developed rapidly into a novel treatment for tumor in recent years,and has been approved by various regulatory agencies to cure a wide range of cancers.PDT is mainly applied in the theatment of the superficial tumors in clinic,and has certain restrictions for the inability of visible light to penetrate deep in biological tissue.So excitation source of strong penetrability are more and more attention by people.Sonodynamic therapy(SDT)referring to photosensitizers gathered at the tumor site being activated by ultrasound with a certain frequency and intensity,and generate anti-tumor factors to inhibit tumor growth or kill tumor cells.For some patients who are difficult to commit deep tissue tumor surgery or need intravenous chemotherapy,SDT made up for the inadequacy of PDT,and has various merits such as strong targeting property,security,and minimally invasive because of ultrasonic's characteristic of focusing,penetrating and selectivity of irradiation area.Therefore,SDT has important theoretical significance and potential'clinical value.With the continuous development and improvement of both PDT and SDT,there comes Sono-Photodynamic therapy(SPDT)in order to strive for breakthroughs in the treatment of tumor,which combines light and ultrasound to activate photosensitizer,and kill tumor cells by both photochemical and sonochemical activities.Although few reports and information concerning the responses to SPDT has been reported,the combined therapy has shown more remarkable anti-cancer effects than either monotherapy.Chlorin e6(Ce6)is one of the compounds with four pyrrole monomer derived from natural chlorophyll.Ce6 has been reported to accumulate more effectively in tumors,absorb more strongly at longer wavelengths(650 and 670 nm)and clear faster from the organism.Toxicological examinations have shown that Ce6 is basically nontoxic for experimental cells or animals,but it is characterized by a high sensitizing efficacy,being activated by both light and ultrasound to induce obvious inhibitory effect on cancers.With its advantages of non-toxic,good solubility in water,selective enrichment in tumor and higher clearance rate in non tumor tissues,Ce6 is developed into a new type of efficient photosensitizer.Supported by national scientific research project,on the basis of our previous work,this study selected murine breast cancer 4T1 cells as cell model,Ce6 as photosensitizer,flat field ultrasonic with the frequency of 1.0 MHz and a semiconductor laser with excitation wavelength of 650 nm,output power density of 10.4 mW/cm2 as irradiation source to research the cell-killing effect and its mechanism induced by Ce6 mediated Sono-Photodynamic therapy(Ce6-SPDT),the obtained findings may provide some experimental data and information for the study of cell biology mechanism of SPDT therapy against breast cancer and its clinical application:1.Cellular uptake and sub-cellular localization of Ce6 in 4T1 cells.The intracellular uptake changes of Ce6 at different time points after addition to 4T1 cells were evaluated by mean fluorescence intensity using flow cytometry.The results showed that the uptake of Ce6 increased as its dose increased with the same incubation time;At the same Ce6 dose,the intracellular Ce6 increased quickly for the first few hours immediately after administration,then slightly increased and reached a relatively high level at 4 hour,and almost maintained the stable level after that.The result suggested that 4 hour may be the optimal incubation time of Ce6 for our follow-up ultrasound or light irradiation experiments.To assess the sub-cellular localization pattern of Ce6 in 4T1 cells,we co-loaded cells with the mitochondrial specific dye Mito Tracker Green(MTG)and Ce6.The fluorescence distributions of MTG and Ce6 were captured by laser scanning confocal microscopy,the co-staining images of MTG and Ce6 showed a good overlapping of both fluorescent signals indicating a higher concentration of Ce6 in the mitochondria,which indicated Ce6 mainly localized to the mitochondria of 4T1 cells.The mitochondria-localization pattern in 4T1 cells implying mitochondria may be one of the dominating targets organelle of SDT or PDT.2.Cytotoxicity of Ce6 Sono-Photodynamic therapy(Ce6-SPDT)on 4T1 cells.Cell-killing effects of Ce6 dose and irradiation time of light and ultrasound,as well as the intensity of ultrasound on 4T1 cells were evaluated by MTT assay.Results showed:Ce6 have no cytotoxic effect on 4T1 cells at the selected dose range;the relative cell survival rate decreased with Ce6 dose increased or laser irradiation time prolonged;when the total dose of 1.2J/cm2 as light exposure parameters,1?g/mL might be the destruction threshold concentration of Ce6 for Ce6-PDT on 4T1 cells.Similarly,the relative cell survival of 4T1 cells was certain irradiation time and ultrasound intensity depended,under the condition of fixed ultrasonic frequency of 1.0MHz,the longer the irradiation time or the higher the intensity,the more obvious damage to cells;and the addition of Ce6 enhanced the cytotoxicity induced by ultrasound.We chose 1 minute irradiation and intensity of 0.36W/cm2 with little damage of Ce6-SDT to 4T1 cells as the ultrasound irradiation conditions.Under the selected ultrasound and light processing parameters,the combination of ultrasound and light mediated by Ce6 showed significant proliferation inhibition effects on 4T1 cells,displaying a certain synergistic anti-tumor effect of Ce6-SPDT.3.Cell-killing effect of 4T1 cells induced by Ce6-SPDT.Cellular apoptotic,autophagic responses to Ce6-SPDT induction with the presence 1?g/mL Ce6,exposure to ultrasound with the frequency of 1.0 MHz,intensity of 0.36 W/cm2 for up to 1 minute and light with total radiation dose of 1.2J/cm2 were explored.? SEM observation confirmed the ultra-structural changes of membrane belebbing after treatment.?Apoptosis of 4T1 cells was analyzed using fluorescence microscope,flow cytometer,laser scanning confocal microscope and western blotting analysis.Obvious nuclear condensation after Ce6-SPDT was found with DAPI staining;More serious DNA damage induced by Ce6-SPDT than either mono-therapy group was detected by flow cytometry;The flow cytometry with Annexin V-PE/7-ADD staining indicated more apoptotic cells upon Ce6-SPDT;Significant mitochondria membrane potential(MMP)loss of was detected by flow cytometry with Rhol23 staining;.The apoptotic features were examined by laser scanning confocal microscopy,such as Bax re-localized from cytoplasm to mitochondria and Cytochrome C released from mitochondria into cytoplasm post Ce6-SPDT;The activity of key apoptosis related protein were evaluated by western blotting analysis 4 hours post treatment,more obvious increased level of cleaved Caspase-3,PARP and decreased level of Bcl-2 were detected after Ce6-SPDT,while Bax remained stable,the ratio of Bax to Bcl-2 in Ce6-SPDT group increased significantly.These suggested Ce6-SPDT could induce apoptotic cell death on 4T1 cells,which might be caspase dependent,and a mitochondria-dependent apoptosis might be involved in our experimental system.?Autophagic responses to Ce6-SPDT were examined by fluorescence microscope,gene transfection and western blotting analysis,laser scanning confocal.microscope,the results indicated that:Visible autophagic vacuoles(AVOs)were more significantly enhanced in Ce6-SPDT by vital staining with MDC,indicting the enhancement of cell autophagy activity;EGFP-LC3 transfected 4T1 cells appeared more bright green fluorescent aggregation region after Ce6-SPDT,suggesting LC3 protein redistribution occurred in autophagy;Western blotting analysis displayed that Ce6-SPDT resulted in more obvious conversion of LC3-I to LC3-?and enhanced Beclin-1 expression level;The mitochondria co-localized well with autophagosome markers Atg5 suggesting the mitochondria damage might be involved in the process of autophagy initiating;In addition,co-localizations of LC3(an AVOs marker)with Lamp2(the lysosomal marker protein)supporting the formation of autolysosomes.Taken together,the findings suggested that autophagy occurred in 4T1 cells following Ce6-SPDT at the experimental conditions.4.The other sensitive targets and function damage on 4T1 cells upon Ce6-SPDT treatment.? Increased FD500 fluorescent molecules in 4T1 cells after sonication,enhanced PI staining observed by HO-PI double staing after Ce6-SPDT,indicating ultrasound enhanced cell membrane permeability and the cell membrane integrity was damaged.? Cell cycle distribution analyzed by flow cytometry displayed obvious S phase arrest in Ce6-SPDT group 24 hours post treatment.? The impact of biological behaviors on 4T1 cells by Ce6-SPDT:Wound healing assay reflected that the migration of Ce6-SPDT treatments were notably lower than either mono-therapy,suggesting that Ce6-mediated SPDT significantly enhanced the migration inhibition effeciency;Clone formation assay displayed that Ce6-SPDT resulted in more significant inhibition of clonogenicity upon 4T1 cells compared with SDT and PDT.These results suggested that cell damage induced by Ce6-SPDT might have multiple effects.5.Different cell death modes and its potential mechanisms in 4T1 cells following Ce6-SPDT.The relationship between autophagy and apoptosis was further obtained by applying pharmacological inhibition studies to detecte the hallmarks of apoptosis and autophagy following Ce6-SPDT and the potential mechanisms were also evaluated.Data showed that the autophagy inhibitors 3-methyladenine(3-MA)suppressed the occurrence of autophagy,enhanced cell apoptosis such as nuclear damage observed by DAPI staining,activation of Caspase-3,strengthened the loss of MAP and the ultimate cell death induced by Ce6-SPDT;Whereas the pan-caspase inhibitor z-VAD-fink partially prevented Ce6-SPDT induced cytotoxicity and Caspase-3 activation,weakened cell apoptosis,but neither obviously improve the loss of MMP,the autophagy activity nor inhibit cell death ultimately,suggesting that there might be multitudinous patterns in cell death pathways in 4T1 cells induced by Ce6-SPDT.6.The regulating function of ROS in the apoptosis and autophagy effect induced by Ce6-SPDT.In the study,it is very interesting for us to evaluate the role of ROS in Ce6-SPDT on 4T1 cells,ROS scavenger NAC(N-acetylcysteine)was added to detecte the autophagic and apoptotic responses.The main results are as follows:Obvious ROS generated immediately after Ce6-SPDT treatment,and NAC significantly decreased ROS generation,blockage of ROS production partially protected Ce6-SPDT induced cell apoptosis rate and DNA fragmentation,prevented the mitochondrial membrane potential loss.These results suggested intracellular ROS levels might be associated with the changes in cell apoptosis activity.MDC staining revealed that NAC almost completely inhibited the autophagy activity in 4T1 cells after Ce6-SPDT,suggesting ROS also have a certain relationship with the changes of cell autophagy activity;Moreover,MTT assay showed NAC slightly protected the loss of cell viability induced by Ce6-SPDT,indicated the ultimate role of ROS.