Basic Experimental Research Of New Photodynamic Therapy Combined With Adriamycin On Breast Carcinoma

Objective:To investigate the new photosensitizer NK01mediated photodynamic therapy (PDT) plus adriamycin (ADM) on4T1breast carcinoma cells in vitro and in vivo, and its underlying mechanism.Methods:1. The experimental groups are divided into four groups:A, blank, the control group; B, ADM (adriamycin) group; C, PDT (photodynamic therapy) group; D, combined treatment gro up (ADM with PDT)2. Detecting cell survival rate. The chemotherapy group were given different concentration ADM incubated for24h; photodynamic therapy group were added different concentration of methylene blue incubated for3h, then635nm wavelength light power for treatment; the combination group first give ADM incubation24h, then given different concentration of methylene blue incubated for3h, then the laser irradiation, continue to incubation24h, with MTT method to determine the cell OD value.3. Cell apoptosis measuring. Cell apoptosis was measured by flow cytometry with Annexin V-FITC/PI double staining.4. Detecting mitochondrial membrane potential. Rhodaminel23was used to test the mitochondrial membrane potential change of mice breast cancer cells4T1.5. Tumor growth curve and tumor growth inhibition rate. BALB/c mice tumor bearing model was established with breast cancer cell line4T1,24tumor-bearing mice were randomly divided into four groups. Tumor volume was measured before treatment and every2days after treatment, and then draw the grow curve. On the21day after treatment, mice were sacrificed, and the tumors were removed and weighted and then inhibition rate was calculated.6. Detecting microvessel density. Microcessel density was detected by immunohistochemical staining of CD34. 7. Statistical analysis:Data were expressed as mean±SD. Statistically evaluated by Student’s t-test when only two value sets were compared, and one-way ANOVA followed by Dunnett’s test when the data involved three or more groups. P<0.05was considered statistically significant.Results:1. Cell survival rate. The50%inhibiting concentration (IC50) of ADM to4T1was0.9μg/ml, so we selected0.5μg/ml for the combination concentration. In the PDT group and combination group, with the photosensitizer concentration increasing, cell survival rate decreased gradually. Comparing with PDT group, cell survival rate of the combination group was lower (P<0.05).2. Cell apoptosis rate. ADM group showed low apoptosis rate, PDT mainly showed early and late apoptosis, and the combination group showed rate apoptosis. The mortality rate of the combination group was87.1%, which was higher than others (P<0.05).3. Mitochondrial membrane potential. Fluorescence microscope showed:there was no obvious change in ADM group; some cells in PDT group showed lowered mitochondrial membrane potential; most cells of combination group showed lowered mitochondrial membrane potential. Flow cytometry showed:compared with the control group, MMP of ADM group showed no significant change (P>0.05), while PDT group and combination group showed significant changes (P<0.05); MMP of combination group decreased most (P<0.05).4. Tumor growth curve. On7th,9th,15th day, There was no significant changes of tumor volume between all groups (P<0.05); on11th and13th day, the tumor volumes of the PDT group and combination group decreased significantly compared with the control group (P<0.05); on17th and19th day, the tumor volumes of the PDT group and combination group were smaller than the control and ADM group (P<0.05).5. Tumor growth inhibition rate All treatment groups showed tumor-inhibiting effects. There was no obvious difference between PDT group and ADM group (P>0.05). The combination group showed significantly higher inhibition rate than ADM group (P<0.05), while no obvious difference compared with PDT group (P>0.05).6. Detecting microvessel density. Compared with the control group, the ADM group showed no significant change, while the PDT and combination groups decreased obviously, especially the combination group (P<0.05).Conclusions:1. NK01mediated PDT has a significant killing effect on mice breast caner cell4T1in vitro and in vivo, and the effect was strengthened through combining with ADM.2. The cancer cell killing effect of NK01mediated PDT is mainly through apoptosis pathway in vitro, and may be related to antiangiogenesis. Antiangiogenesis and apoptosis were the important mechanism responsible for the increasing killing effect of PDT plus ADM.