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

Breast cancer is one of the most deadly diseases for females' health and more and more people pay attention to the treatment of it.At present,the commonly modalities being used in clinical for breast cancer therapy mainly include surgery,chemotherapy,radiation therapy,endocrine and biological therapy,etc.,but these methods all have their shortcomings,even strong toxic side effects and complication.Thus,explore new methods and techniques for clinical treatment of breast cancer is the biomedical major issues currently.Sono-Photodynamic therapy(SPDT)is a new modality for cancer treatment developing on the basis of photodynamic therapy(PDT)and sonodynamic therapy(SDT).The present studies show that the anti-tumor effect of SPDT is significant than PDT or SDT alone,but the specific mechanism hasn't been clarified,so further studies are needed.Chlorin e6,a chlorophyll derivative,is a second generation photosensitizer with advantages of non-toxic,good solubility in water,high tumor affinity and higher clearance rate in normal tissues.Researches have proved that Ce6-mediated both PDT and SDT have certain anti-tumor effect.This research is supported by the National Natural Science Funds of China.Based on our previous work,this paper selected human breast cancer MDA-MB-231 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).And we used focused ultrasound with the frequency of 1.90 MHz and a semiconductor laser with excitation wavelength of 650 nm,output power density of 26mW/cm2 as irradiation source to study the anti-tumor effect of Ce6-SPDT on 4T1 xenografts.The expression level of VEGF and MMP9 in tumor tissues was detected by immunohistochemistry.Present experimental results obtained are as follows:1.The results of MTT showed that Ce6 have no cytotoxic effect on MDA-MB-231 cells at the selected concentration range;when treated with Ce6-SDT,the relative cell survival rate decreased with Ce6 concentration increased;at the selected total light dose range,light irradiation alone have no cytotoxic effect on MDA-MB-231 cells,while in the presence of 1?g/mL Ce6,the relative cell survival rate decreased with total light dose increased and 1.2J/cm2 might be the destruction threshold dose of light for Ce6-PDT on MDA-MB-231 cells.Under the selected experimental parameters,the combination of ultrasound and light mediated by Ce6 showed significant proliferation inhibition effects on MDA-MB-231,MCF-7 and 4T1 cells,displaying a certain synergistic anti-tumor effect of Ce6-SPDT.2.The results of DAPI staining showed that SDT alone had no significant effect on MDA-MB-231 cell nucleus;PDT has slight damage to the MDA-MB-231 cell nucleus,while obvious apoptotic changes of MDA-MB-231 cell nuclear were caused by Ce6-SPDT treatment,including nucleus swelling and chromatin condensation.3.By flow cytometry,Ce6-SPDT significantly increased the MDA-MB-231 cell apoptosis rate and significant mitochondria membrane potential(MMP)loss of MDA-MB-231 cells was also detected after Ce6-SPDT.4.By flow cytometry,Ce6-SPDT could increase the level of intracellular reactive oxygen species(ROS).Moreover,the decreased cell viability caused by Ce6-SPDT was partially rescued by NAC,suggesting that a more enhanced generation of ROS has an important role in the synergistic effect of combined SDT and PDT.5.The results of pharmacoskinetic study of Ce6 in 4T1 tumor bearing mice by in vivo optical imaging indicated that Ce6 targeted the tumor in a very short time after intravenous injection;the tumor uptake reached a maximum at 2 h and sustained a relatively high level until 8 h p.i.,showed the retention effect of the tumor.The results of in vivo of some tissues of 4T1 tumor-bearing mice after 2h adminstration of Ce6 demonstrates that the fluorescence photons in tumor was the highest,then followed by lung,skin,liver,stomach,heart,muscle,intestine,kidney and spleen.6.The antitumor effect of combination of ultrasound and light mediated by Ce6 on 4T1 Xenografts showed that PDT or SDT alone inhibited the tumor growth to some extent,while the tumor volume inhibitory effect in the combination of ultrasound and light was more marked than that in the PDT or SDT groups,and the tumor size was constantly about a half of the untreated group 4 days after SPDT and PSDT treatment.The tumor weight inhibition rate of the SPDT and PSDT were 40.37%and 39.98%,higher than the other three groups.There was no statistically significant difference in body weight among different treatment groups.7.The tumor cell structure after Ce6-SPDT treatment was observed by HE staining.The results showed that PDT and SDT alone had some antitumor effect in vivo,and the synthetic antitumor effect of PDT and SDT was more significant than any monotherapy.8.The expression of vascular endothelial growth factor(VEGF)was detected by immunohistochemistry.The results indicated that the expression levels of VEGF after PSDT and SPDT treatment was significant decrease,indicating that the combination therapy may influence the capability of tumor tissue to form new vessels by down-regulation the level of VEGF and thus inhibit the tumor growth.9.After different treatments the lungs were removed and fixed in Bouin's solution,and the gross appearance of pulmonary nodules were counted.The results showed that the extent of metastasis to the lungs in the PSDT and SPDT groups were slighter than that in the other three groups.The expression levels of MMP-9 in the combination groups were significant decrease,indicating that Ce6-SPDT could restrain tumor metastasis.In this paper,we have investigated the combination of PDT and SDT induced cytotoxicity effect of Ce6 both in vitro and in vivo,and then preliminarily explored the possible mechanism about Sono-Photodynamic therapy,which may accumulate related information for SPDT applied in the clinical treatment of tumor as soon as possible.