Study On Photodynamic Effect Of A Novel Photosensitizer DTPP And Observation Of Cytoskeleton On Tumor Cells

Objective To investigate the killing effects of the novel photosensizer DTPP with650nm laser based photodynamic therapy on human breast cancer cell line MCF-7invitro. To study the mechanism of DTPP mediated photodynamic therapy.Methods MTT assay were performed to examine human breast cancer cell line MCF-7cells treated with different concentrations of DTPP(0,2,4,6,8,10,12,15,20,25,30μg/mL) and different intensities of laser(0,0.6,1.2,2.4,4.8,7.2,9.6J/cm2), to explorecell proliferation in different different concentrations of DTPP and different intensities oflaser. Different intensities of laser(1.2,2.4,4.8J/cm2) treated MCF-7cells with8μg/mLof DTPP. The morphological changes of MCF-7cells were observed by DAPI staining bymicroscope.The cellular apoptosis and cell cycle phase distribution of the MCF-7cellswere measured by a Flow cytometry (FCM). MCF-7cells were treated by6μg/mL DTPPand the intensities of laser at7.2J/cm2. Then the OD values and survival rates of MCF-7cells were measured by MTT assay at different time (0.5,1,3,6,12h) after DTPP-PDT.The cellular apoptosis rate of MCF-7cells were measured by a Flow cytometry (FCM)at different time (0.5,1,3,6,12h) after DTPP-PDT. The morphological changes of MCF-7cells were observed by comet assay at different time (0.5,1,3,6,12h) after DTPP-PDT.Western blotting test was used to examine the relative proteins expressions,such asBcl-2, β-Catenin, β-Actin, β-Tubulin and other factors to further clarify the mechanism ofinhibition human breast cancer cell line MCF-7cells proliferation.Results Significant differences in the inhibitory was observed in DTPP mediatedphotodynamic therapy(P<0.05). DTPP mediated PDT inhibit the proliferation of MCF-7cells depended on laser power and time after PDT. The morphological study found thatthe multinucleate giant cells existed in the apoptotic cells after DTPP mediatedphotodynamic therapy. The results of cell cycle analysis showed a significant increase ofthe number of cells in G0/G1phase. The FCM assay of Annexin/PI double-labeledstaining showed that the apoptosis rates were obvious and the killing effect improveddepended on time after PDT. The comet figure significantly observed the comettail.Microfilament was stained using Actin-Tracker-Green and microtubule was stainedusing beta-tubulin mouse antihuman monoclonal antibody and FITC-labeled Goat Anti- Mouse IgG after DTPP-PDT0.5h,1h,3h,6h and12h, with DAPI staining. Obviouschange were observed like shrinking and dispersing of microfilament and microtubule.Western blotting analysis found that a number of important proteins of cytoskeletonchanges during apoptosis progression were observed for cytoskeleton morphology. Theexpression of protein β-actin decreased more than β-tubulin with the increasingphotodynamic reaction, β-tubulin seemed more stable if reference to GAPDH. In addition,β-catenin,which was the main catenin directly being contacted to actin cytoskeleton,showed decreased expression during apoptosis progression.Conclusion Photosensitizer DTPP has low toxicity and efficient features. DTPPmediated photodynamic therapy can kill human breast cancer cell line MCF-7in vitroeffectively and show a dose-time-dependent effect. DTPP-PDT can induc typicalapoptotic cells. Although photodynamic effect occurs in a short time after laserirradiation of the photosensitizer, the induction of apoptosis of tumor cells is a gradualprocess. Cytoskeleton is an important component of the cells.