title: Efficacy of chlorin e6-mediated sono-photodynamic therapy on 4T1 cells.
abstract: OBJECTIVE
  The present study aims to investigate the antitumor effect and possible mechanisms of chlorin e6 (Ce6)-mediated sono-photodynamic therapy (Ce6-SPDT) on murine 4T1 mammary cancer cells in vitro.
  
  
  METHODS
  Cellular uptake and intracellular distribution of Ce6 in 4T1 cells were detected by flow cytometry and confocal microscope. Cells after loading with 1 μg/mL Ce6 were exposed to ultrasound at 1.0 MHz for up to 1 minute with an intensity of 0.36 W/cm2 and laser light with total radiation dose of 1.2 J/cm2. Cell viability and clonogenicity were determined by MTT assay and colony formation assay. Apoptosis was analyzed by DAPI staining, Western blots were used to detect the activity of Caspase-3. DNA damage, mitochondrial membrane potential (MMP), and intracellular reactive oxygen species (ROS) of 4T1 cells were also evaluated by flow cytometry. FD500 was employed to detect changes of membrane permeability after ultrasound.
  
  
  RESULTS
  Ce6 rapidly entered 4T1 cells within 4 hours after it has been added and displayed a mitochondria-localization pattern. Compared with sonodynamic therapy (SDT) and photodynamic therapy (PDT) alone, the combined SPDT treatment further enhanced cell viability loss, DNA damage, and clonogenicity inhibition. DAPI staining and western blots analysis reflected that cells with apoptotic morphological characteristics and the activity of Caspase-3 were apparently increased in the combined group. Besides, SPDT caused obvious MMP loss and intracellular ROS generation at early 1 hour post treatment. Interestingly, the SPDT induced cell viability loss and cell apoptosis was greatly inhibited by pre-treatment with ROS scavenger N-acetylcysteine and Caspase inhibitor z-VAD-fmk. FD500 detection showed that ultrasound enhanced cell membrane permeability, implying much higher uptake of Ce6 might be involved in PDT therapy by pre-ultrasound treatment.
  
  
  CONCLUSIONS
  The findings demonstrated that Ce6-mediated SPDT enhanced the antitumor efficacy on 4T1 cells compared with SDT and PDT alone, a Caspase-dependent apoptosis and loss of MMP, generation of ROS may be involved.
authors.affiliation: 1 Key Laboratory of Medicinal Resources and Natural Pharmaceutical Chemistry, Ministry of Education, National Engineering Laboratory for Resource Developing of Endangered Chinese Crude Drugs in Northwest of China, College of Life Sciences, Shaanxi Normal University , Xi'an, China .
authors.forename: Qing
authors.initials: Q
authors.lastname: Li
authors.forename: Xiaobing
authors.initials: X
authors.lastname: Wang
authors.forename: Pan
authors.initials: P
authors.lastname: Wang
authors.forename: Kun
authors.initials: K
authors.lastname: Zhang
authors.forename: Haiping
authors.initials: H
authors.lastname: Wang
authors.forename: Xiaolan
authors.initials: X
authors.lastname: Feng
authors.forename: Quanhong
authors.initials: Q
authors.lastname: Liu
mesh_terms: D000818:Animals; D017209:Apoptosis / Q000187:drug effects; D045744:Cell Line, Tumor; D002735:Chlorophyllides; D003131:Combined Modality Therapy; D005260:Female; D006801:Humans; D008325:Mammary Neoplasms, Experimental / Q000188:drug therapy / Q000378:metabolism / Q000473:pathology / Q000628:therapy*; D010778:Photochemotherapy / Q000379:methods*; D011166:Porphyrins / Q000493:pharmacokinetics / Q000494:pharmacology*; D011838:Radiation-Sensitizing Agents / Q000493:pharmacokinetics / Q000494:pharmacology*; D014464:Ultrasonic Therapy / Q000379:methods*
chemical_list: D002735:Chlorophyllides; D011166:Porphyrins; D011838:Radiation-Sensitizing Agents; C062985:phytochlorin