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Experimental Study on Photodynamic Therapy Effect Factor and Mechanism in the Human Esophageal Carcinoma

Author: ChenXiaoHua
Tutor: LuoRongCheng
School: First Military Medical University
Course: Oncology
Keywords: Hematoporphyrin derivative incubating concentration incubating time cell survival rate Photodynamic therapy PDT mechanism
CLC: R735.1
Type: Master's thesis
Year: 2007
Downloads: 57
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Abstract

Photodynamic Therapy (PDT) is a kind of photochemistry therapy. Its mechanism is that the resident photosensitizer in tumor tissue is activated by certain wavelength laser and takes action on oxygen in tumor tissue and produce some radical oxygen species (ROS), then attack the tumor cell through peroxidization. This may make the cellular membrane or oxidative protein damage. And the tumor cell will apoptosis after the oxidative damage get to the leak point. Also, it can damage to the tumor capillary. As contrast to conventional therapy such as operation and chemotherapy, PDT has the advantages as follows:(1) minimally invasive; (2)little toxicity;(3)high selectivity; (4)wide applicability; (5)well repeatability; (6)be used as pamper treatment; (7) can cure precancerous lesion; (8)maintain features and important organ function. Nowadays, PDT has been used in curing some malignant tumor, and gradually to become a new method for tumor therapy.Esophageal carcinoma is a kind of common human alimentary canal tumor and it has high death rate in our country. As the development of the new type photosensitizers and semiconductor laser generator, PDT has made great progression in curing esophageal carcinoma especially in the earlier period and precancerous lesion. But in clinical curing, we find it is common that the photodynamic therapy effect is instability. Some human esophageal carcinoma patients can be clinical curative, while some is negative. The reason may be as follows: the different content of oxygen in tumor tissue, the different concentration of photosensitizers, the different sensitive of the phot0sensitizer to the tumor. So, in this experimental study, we use the first generation photosensitizers Hematoporphyrin derivative (HPD) and Photofrin to contribute the human esophageal carcinoma cell line Eca-109, so as to investigate the influence of different incubating concentration, incubating time of the two photosensitizers and different laser energy density of PDT in the Eca-109 in vitro. Meanwhile, construct the human esophageal carcinoma nude mouse model. And then some of the nude mouse model were treated by HPD-PDT、Photofrin-PDT、5-ALA-PDT. And after the therapy, kill some models and test the content of MDA and the expression of survivin and caspase-3 protein and HE dyeing in the tumor tissue, meanwhile monitor the volume of the tumor and observe the change of the tumor skin. The objective of this part of experiment is to investigate the main factor of PDT effect and the mechanism of PDT in human esophageal carcinoma in vivo.The first part: Study on the main PDT effect factor of the human esophageal carcinoma cell line Eca-109 in vitroMethods:1) Use the MTT essay to monitoring the effect of different concentration HPD-PDT on the growth of the human esophageal carcinoma cell line Eca-109 in vitro. And use the OD value which were tested by Bio-Rad550 to draw the growth curve of different concentration HPD-PDT on Eta-109.2)The experiment of PDT on the human esophageal carcinoma cell line Eca-109: human esophageal carcinoma cells were incubated in vitro with two photosensitizers HPD and Photofrin with different incubating concentration (0ug/ml, 0.1ug/ml, 0.75ug/ml, 1.5ug/ml, 3.0ug/ml, 6.0ug/ml, 10.0ug/ml, 20.0 ug/ml) and different incubating time (for 4, 6, 12, 24hour), and then irradiated in DIOMED630PDT systems under 20 J/m2 saturated light energy density, meanwhile make other tests that human esophageal carcinoma cells were incubated in vitro with different HPD incubating concentration (0ug/ml, 0.1 ug/ml, 0.75 ug/ml, 1.5 ug/ml, 3.0 ug/ml, 6.0 ug/ml, 10.0 ug/ml, 20.0 ug/ml) and Photofrin incubating concentration (0ug/ml, 0.05 ug/ml, 0.375 ug/ml, 0.75 ug/ml, 1.5 ug/ml, 3.0 ug/ml, 5.0 ug/ml, 10.0 ug/ml) and then irradiated in DIOMED630PDT systems under three different light energy density(10 J/cm~2, 30 J/cm~2, 50 J/cm~2), and survival rate of each concentration was measured by MTT assay after 24 hours’ incubation. The software of statistics is SPSS13.0. The results of experiment express by the way of mean±SD. Survival rate of each group apply the way of General Linear Models of Univariate, and the relation between the concentration and cell survival rate use the way of curve estimation.3) Human esophageal carcinoma cells were incubated with different HPD incubating concentration (1.5 ug/ml, 3.0 ug/ml, 6.0 ug/ml, 10.0 ug/ml) for 4 hours’, and then departed culture solution and added deionized water, and freezed thawing for times, and then detected the intracellular photosensitizer on the model of RT-5000 fluorescence spectrometer. Using the cell freezed water to make HPD concentration as 0ug/ml, 0.008 ug/ml, 0.016ug/ml, 0.064ug/ml, 0.128 ug/ml, 0.150 ug/ml, then detected the intracellular photosensitizer on the model of RT-5000 fluorescence spectrometer, and draw the standard curve according to the HPD concentration and the value of fluorescence. The software of statistics is also SPSS13.0.The relation between the incubation concentration and intracellular photosensitizer concentration use the way of linear regression. Results1)From the OD value curve, we can see that the OD value descent as the HPD incubating concentration increase, this means that as the incubating concentration increase, the survival rate of Eca-109 cells decrease, as also means that the killing effect of HPD-PDT on the Eca-109 cells enhance. But when the concentration is increased from 10 ug/ml to 20 ug/ml, the OD value dose not decent obviously.2) The cell survival rate between different Photofrin and HPD incubating concentration and three, different light energy density and under the same light energy density with the two different photosensitizers incubating concentrations are significant different and also significant interaction, all P＜0.01.While under the same concentration, the cell survival rate is also significant different with different light energy density except that the concentration is 10.0ug/ml of Photofrin and 10.0ug/ml of HPD. The cell survival rate is the lowest as Photofrin concentration is 10.0ug/ml and the light density was 10J/m~2. While Photofrin concentration increases from 5.0ug/ml to 10.0ug/ml, the cell survival rate is not decrease as the light energy density increase from 10J/m~2 to 50J/m~2. The killing effect is the best as HPD concentration is 20.0ug/ml and the light density was 30J/m~2. While HPD concentration increases from 6.0ug/ml to 10.0ug/ml, the cell survival rate is not decrease as the light energy density increase from 10J/m~2 to 50J/m~2.3) The cell survival rate between different Photofrin and HPD incubating concentration and four different incubating time and under the same incubating time with the two different photosensitizers incubating concentrations are significant different and also significant interaction, all P＜0.01.While under the same concentration, the cell survival rate is also significant different with different incubating time except that the concentration is 0ug/ml and 0.1 ug/ml of Photofrin and 0ug/ml of HPD. The cell survival rate is the lowest as Photofrin concentration is 10.0ug/ml and the incubating time is 24 hours. While Photofrin concentration increases from 10.0ug/ml to 20.0ug/ml, the killing effect is not enhance as the incubating time increase from 12 hours to 24 hours. The killing effect is the best as HPD concentration is 10.0ug/ml and the incubating time is 12 hours. While the HPD concentration increases from 6.0ug/ml to 20.0ug/ml, the killing effect is not enhance as the incubating time increase from 12 hours to 24 hours.4) The curve equation between different Photofrin incubating concentration and cell survival rate is Y=1.042-0.241X+0.058X~2-0.005X~3,under the incubating time of 4 hours and Y=1.120-0.305X+0.054X~2-0.003X~3 under the incubating time of 6 hours and Y=0.837+0.017X+0.034X~2+0.003X~3 under the incubating time of 12 hours and Y=0.920-0.019X-0.041X~2+0.004X~3 under the incubating time of 24 hours. The curve equation between different HPD incubating concentration and cell survival rate is Y=0.771+0.029X-0.005X~2-0.001X~3 under the incubating time of 4 hours and Y=1.012-0.247X+0.059X~2-0.005X~3 under the incubating time of 6 hours and Y=0.712+0.119X-0.062X~2+0.005X~3 under the incubating time of 12 hours and Y=0.953-0.153X+0.006X~2+0.002X~3 under the incubating time of 24 hours. Then calculate Photofrin half inhibition concentration is 0.935ug/ml and 0.981ug/ml and 0.837ug/ml and 0.901ug/ml and HPD half inhibition concentration id 0.784ug/ml and 0.903ug/ml and 0.757ug/ml and 0.875ug/ml according to the curve equation5)The standard curve regression equation is Y=0.86X-0.083,R=0.963, F=50.482 P=0.002, The intracellular photosensitizer concentration was calculated according to the intracellular fluorescence and the standard curve regression equation, and then made regression with the incubating concentration and got the regression equation: Y=12.251+0.001X, and the coefficient of correlation R=0.998, F=451.724, P=0.003. The above result is to describe that the intracellular photosensitizers incubation was related to the incubating concentration, and this also describe that the reason why the killing effect enhance as the incubating concentration increase is because the increase of intracellular photosensitizer concentration.Conclusions1)The killing or inhibiting effect of HPD-PDT to Eca-109 cell is enhance as the incubating concentration of HPD, but as the concentration increase from 10ug/ml to 20ug/ml, the killing effect is not enhance, that meas the killing effect is come to the platform stage.1) The best PDT killing effect point of ligt energy density and incubating concentration in Eca-109 cell in vitro is that Photofrin incubating concentration of 10ug/ml under 10J/cm~2 while HPD incubating concentration of 20ug/ml under 30 J/cm~2, and the best cell killing effect point of incubating time and incubating concentration is that Photofrin incubating concentration of 10ug/ml under the incubating time of 24 hours, while incubating concentration of 10ug/ml under the incubating time of 12 hours. The half inhibition concentrations of HPD are less than Photofrin under the four different incubating time, so the Eca-109 cell is more intensive to HPD than Photofrin.2) Using the fluorescence spectrometer to detect the intracellular fluorescence, and the result show that the intracellular photosensitizer incubation are related to the incubating concentration, and this also describe that the reason why the killing effect enhance as the incubating concentration increase is because the increase of intracellular photosensitizer concentration.The second part: The study of effect of HPD-PDT、Photofrin-PDT、5-ALA-PDT inhibiting effect to the human esophageal carcinoma bearing nude mouseMethods: Raise the human esophageal carcinoma bearing nude mouse model, and monitor the tumor every three days. Give PDT therapy to the model when the tumor volume is between 150 and 300mm~3. This experiment is divided into two groups: the PDT groups and the control groups. The control groups included the blank control group which was not given any treatment and the illuminated group which was only given illuminated. The therapy groups were divided into three groups(the HPD-PDT group, the Photofrin-PDT group and the 5-ALA-PDT group) as given different photosensitizers. Each group contain seven nude mouse models. The photosensitizers were given through intraperitoneal injection. And HPD photosensitizer was given as 30mg/Kg, while Photofrin as 10mg/Kg, and 5-ALA as 100mg/Kg. Then after 4 hours (Photofrin for 24 hours), under the DIOMED630 PDT system and the light energy density of 120J/cm~2 were given to the nude models which has been injected photosensitizers. And continue to monitor the tumor volume and observe the change of the tumor skin. Kill three nude mouse of each group after 3 days since the therapy given, and 4 after 21 days. Then dislodge the tumor tissue and detect the content MDA, the express of survivin and caspase-3 protein and the HE dyeing in the tumor tissue. The software of statistics is SPSS13.0.The results of experiment express by the way of mean±SD. The tumor tissue volume use the way of General Linear Models of Repeated Measures. The content of tumor MDA and the masculine expression of surviving and caspase-3 protein between the blank control group and the therapy groups were use the way of one-way ANOVA.Results1)The change of tumor skin: The control group has not obviously changed in the tumor skin. But the tumor volume increase more quickly than the therapy groups. The tumor skin of the therapy groups flare between the first and the third day since the therapy given, and some even change as erythema, and some appear edema, bubble and wound surface effusion, and some even with necrosis and ulcer. And the tumor tissue atrophy in the 7th day since the therapy given, and the ulcer surface begin to form brown crust. Then in the 21st day, the neogenesis epithelium begin to form in the basal surface of ulcer as the crust ablate. 2)The tumor volume comparison between the control group and the therapy groups: The tumor volume of each therapy groups comparing to the control group are increase more slowly. The volume of the tumor are all significant different between different groups and in each group before and after therapy, all P＜0.01. There are significant different between the control group and the therapy group of the four different period after therapy, all P＜0.05, while before the therapy is not. The volume of the tumor are also significant different between each groups before and after therapy, all P＜0.01.3)Comparing the HPD-PDT group, Photofrin-PDT group and the 5-ALA-PDT group to the control, the result of one-way ANOVA shows that there are significant different of MDA tumor tissue content between them, all P＜0.05.4)HE dying result: From the control group we can see many allotype tumor cells and caner nest, and the blood vessel endothelium is complete. While from HPD-PDT and Photofrin-PDT groups, we can see some homogen eryth -dyeing necrosis. The blood vessel endothelium is widespread damaged, and the endothelial cells’ boundary were ambiguity, and the lumen of blood vessel disappeared. But the blood vessel endothelium are not damaged in the 5-ALA-PDT group, while there are many damaged tumor cells.5) The immunohistochemistry result: The masculine rate of caspase-3 protein expression is more in the HPD-PDT and 5-ALA-PDT group comparing to the blank control group. While survivin protein expression is less. There is significant different of caspase-3 and survivin protein expression between the therapy groups comparing to the blank control group except the HPD-control group of caspase-3 protein expression, using the way of Independent t Test, all P＜0.05.Conclusions:1) The PDT can make the tumor growth tendency to be torpidity.2) The main mechanism of HPD-PDT and Photofrin-PDT and 5-ALA-PDT is that it can produce singlet oxygen to damage the cell membrane and form the lipid peroxidatic reaction,and then generate MDA to damage the tumor cell.During HPD-PDT and Photofrin-PDT, it can also damage the tumor blood vessel.3) It may activate the capase-3 apoptosis pathway during 5-ALA-PDT.

Experimental Study on Photodynamic Therapy Effect Factor and Mechanism in the Human Esophageal Carcinoma

Abstract

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