| Background:Esophageal cancer is one of the most common cancers. The morbidity and mortality of esophageal cancer in China is very high, and it ranks the fourth killer among all cancer death in China. More than 90% of esophageal cancer in China is squamous cell carcinoma. The incidence of this cancer is increasing rapidly in the Western countries, however, there are considerably many adenocarcinoma of esophagus, most of which are developed from Barrett's esophagus. A great many of patients with this cancer are diagnosed in an advanced stage, with distant metastasis and esophageal obstruction. Many patients often lost the optimal chance for surgery, or even could not undergo chemotherapy or radiotherapy. It is highly demanded for an effective medical treatment such as photodynamic therapy (PDT). PDT has been found to be a promising treatment modality for esophageal cancer.PDT process involves the photosensitiser localized in tumor tissue and irradiation of the tumor site by visible light of specific wavelength, to produce singlet oxygen and other reactive oxygen species, leading to photodamage in cancer cells and cancer destruction. As contrast to conventional therapy such as operation and chemotherapy, PDT has the advantages as minimally invasive, little toxicity, high selectivity, wide applicability, well repeatability, be used as pamper treatment, maintain features and important organ function and can cure precancerous lesion. Nowadays, PDT has been used in curing some malignant tumor, and gradually to become a new method for tumor therapy. The first PDT used in esophageal carcinoma treatment was to cure esophageal precancerous lesion such as Barrett's of esophagus and early esophageal cancer, and now began to be used in diminish treatment of advanced stage. By properly chosen optimal parameters of PDT, complete cure of early esophageal cancer and a good therapeutic result in advanced stage of the disease can be frequently achieved. However, in clinical application, the optimal parameters have not been established that the effects for esophageal patients were instability.The tumor cell death by PDT is induced via apoptosis and/or necrosis, depending on various conditions, such as tumor cell type, laser irradiation dose, photosensitizer concentration and subcellular localization, et.al. It has been reported that PDT with mitochondria-localizing photosensitizers, such as 5-aminolevulinic acid (ALA), can induce rapid cell death via apoptosis and change the expression of apoptotic related gene and protein. Expression of some genes related with apoptosis pathway, such as Bcl-2 family, is involved and plays an important role in this photodynamic process. It has been reported that up-regulation or down-regulation some genes of Bcl-2 family can enhance the effect of PDT. However, their roles in ALA-PDT for esophageal cancer have still not been fully investigated.ALA is one of the second-generation photosensitizers, and has been approved to be used in clinical trial by the USA FDA since 2000. ALA is an endogenous material and a precursor of heme in living cells, thus the induced protoporphyrinⅨ(PpⅨ) can be employed as a photosensitizer in PDT. The ALA-produced PpⅨ(ALA-PpⅨ) induces a short-lasting phototoxicity about 24 hours, much shorter compared with that induced by photofrin which was used generally in clinical treatment。Thus, we choosed the second-generation photosensitizers ALA, and investigated the effects of ALA-PDT on esophageal cancer cells in vitro and vivo, to explore optimal parameters in PDT for esophageal cancer and provided some evidence for PDT optinal clinical premeters established. Also, we investigated the role of cell apoptosis, cell cycle and mitochondria membrane potential changes, subcellular localization of ALA-PpⅨ, and some molecular changes to understand the mechanism of ALA-PDT for esophageal cancer.Objectives:1. Investigate ALA-PDT effect in the human esophageal carcinoma both in intro and vivo to explore optimal parameters in PDT for esophageal cancer;2. Investigate the subcellular localization of ALA, the role of cell apoptosis, cell cycle and mitochondria membrane potential changes by ALA-PDT on Eca-109 cells to understand apoptotic mechanism for esophageal carcinoma;3. Investigate the expression changes in the apoptoptic related genes of Bcl-2, Bax,Bcl-xL, Bad, Bak-1, FADD and Bid to explore the main apoptosis pathway in Eca-109 cells after ALA-PDT;4. Investigate the expression changes in the proteins of Bcl-2 and cytochrome c (cytc) to explore the final effect pathway in Eca-109 cells after ALA-PDT.Methods:1. Use the MTT assay to detect ALA-PDT effect on the human esophageal carcinoma cell line Eca-109:The experiment was divided into two groups:the control group and PDT group. Cells of PDT group were incubated in vitro with ALA incubating concentration (0.01 mM,0.05 mM,0.1 mM,0.25 mM,0.5 mM,1 mM,2 mM) and different incubating time (for 4,6,12 and 24 h, respectively), and then irradiated in DIOMED 630PDT systems under 30 J/m2 saturated laser dose, meanwhile make other tests that cells were incubated in vitro with different ALA incubating concentration(0.01mM,0.05 mM,0.1 mM,0.25 mM,0.5 mM,1 mM and 2 mM) and then irradiated in DIOMED 630PDT systems under three different laser dose (10 J/cm2,30 J/cm2 and 50 J/cm2, respectively), and survival rate of each concentration was measured by MTT assay after 24 hours'incubation.2, The inhibition of tumor growth in the human esophageal carcinoma Eca-109 cells bearing mice:Raise the Eca-109 cells bearing mice model, and monitor the tumor volumes every three days. PDT therapy to the model was initiated.when the tumor volume reached 150-350 mm3. The mice were randomly assigned into four groups: control group, PDT group (given ALA and light irradiation), lighted group (given light irradiation alone) and ALA group (given ALA alone) (6 nude mice in each group). The PDT and ALA group were injected i.p. with ALA in a dose of 100 mg/kg. The mice of PDT and lighted groups were irradiated with 120 J/cm2 630 nm-light from the Diomed 630-PDT system 4 h after the photosensitizer injected. Then continue to monitor the tumor volume and observe the change of the tumor skin. Kill mice of each group 21 days after PDT. Then dislodged the tumor tissue and detected the H&E staining in the tumor tissue.3. Subcellular localization of ALA and Mito-Tracker Green by confocal laser scanning microscope:After the Eca-109 cells were incubated with ALA (to the final concentration of 2 mM) for 4 h, followed by stained with 100 nM of MitoTracker Green for 30 min, the cells were examined by confocal laser scanning microscope FV1000 to detect the subcellular localization of ALA and Mito-Tracker Green.4. Apoptosis determined by AnnexinV-FITC/PI binding assay:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the treatment, cells were further cultured for 24 h. Then cells were harvested and stained with 5μl of Annexin V-fluorescein isothiocyanate (AnnexinV-FITC) and 5μl of propidium iodide (PI) for 10 min in dark. The cell apoptosis were analyzed by fluorescence activated cell sorting (FACS) with flow cytometry(FCM)5. Cell cycle analysis by PI:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05,0.1 and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. Cells were harvested when were further cultured for 24 h after treatment and fixed with 70% ethanol at least 12 h at 4℃and then stained with propidium iodide (PI) (to final concentration of 1 mg/ml) for 10 min in dark. The cell clycle were analyzed by fluorescence activated cell sorting (FACS) in aFACS-SCAN system. 6. Determine of morphologic changes by Hoechst 33342/PI staining:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 mM,0.1 mM and 0.5 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the PDT treatment, the cells were further cultured for 24 h. Then the cells were harvested and stained with Hoechst 33342 and propidium iodide (PI) for 15 min in dark. The cells were observed under the fluorescence microscope BX51 to detect apoptosis in the Eca-109 cells.7. Determine of mitochondria membrane potential by rhodamine123 staining:The experiment was divided into two groups:the control group and PDT group. After culture for 24 h, the cells of PDT group were incubated with ALA to the final concentration of 0.05 mM,0.1 mM and 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. After the treatment, the cells were further cultured for 1 h. Then the cells were harvested and stained with rhodaminel23 for 30 min in dark. The cells were resuspended in medium and after the cells were incubated for 60 min, observed under the fluorescence microscope BX51.8. The gene expression changes of Bcl-2, Bax, Bcl-xL, Bad, Bak-1, FADD and Bid by quantitative real-time polymerase chain reaction ((QRT-PCR):The experiment was divided into two groups:the control group and PDT group. The cells of PDT group werer incubated with ALA to the final concentration of 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. RNA was extracted from Eca-109 cells at 24 h post-PDT.and synthesized cDNA.Then the cDNA was amplified and analyzed for Bcl-2, Bax,Bcl-xL, Bad, Bak-1, FADD, Bid andβ-actin on ABI 7500 PCR detector using QuantiTect SYBR Green kits.9. The protein expression changes of Bcl-2 and cytc (cytoplasm):The experiment was divided into two groups:the control group and PDT group. The cells of PDT group were incubated with ALA to the final concentration of 0.25 mM for 6 h in dark, and irradiated with a light dose of 30 J/cm2 (200 mW/cm2,150 s) under the 630 nm laser system. Total protein and cytoplasm protein were extracted from Eca-109 cells at 24 h after ALA-PDT. Then Western Blotting assay was used to detect the protein expression changes of Bcl-2 and cytc.10. Statistical analysis:All datas were analysised by SPSS 13.0 statistical softwire. The results of experiment were expressed by the way of mean±SD. Survival rate of each group was analyzed apply by the way of General linear Models of Univariate. The tumor tissue volume analysis used the way of General Linear Models of Repeated Measures. Use one-way ANOVA followed by LSD multiple comparison test when the data was equal variance to analysis cell apoptosis rate, cell cycle and the difference between the tumor volume of different groups. Multiple comparison test used F test (Welch) and Dunnett's T3 test if the data was not equal variance. The relative gene and protein expression difference between the control group and the PDT group were analysis using independent t test. P-Values were considered to be significant at<0.05.Results:1. ALA-PDT significantly inhibited the growth of Eca-109 cells The cell survival rate between different ALA concentration and three different laser dose and under the same laser dose with different incubating concentration are all significant different (all P<0.01). While under the same concentration, the cell survival rate is also significant different with different laser dose except that the concentration is 0.01 mM and 0.05 mM. The cell survival rate is the lowest as ALA concentration is 0.5 mM and the laser dose is 50 J/m2. While ALA concentration increases from 0.5 mM to 2.0 mM, the cell survival rate is not decrease as the laser dose increase from 30 J/m2 to 50 J/m2. The killing effect is the best as ALA concentration is 0.5 mM and the laser dose was 50 J/m2. The cell survival rate between different ALA concentration and four different incubating time and under the same incubating time with different ALA concentrations are all significant different (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 0.01 mM, 0.05 mM,0.1 mM and 1.0 mM. The cell survival rate is the lowest as ALA concentration is 2.0 mM and the incubating time is 24 hours. While ALA concentration increases from 0.5 mM to 2.0 mM, the killing effect is not enhanced as the incubating time increase from 6 hours to 24 hours.2. The tumor volume before and after PDT between the four groups were significant different (P<0.01) There were significant different between volumes of the four groups of the different period before and after therapy (P<0.01). The volumes before PDT in the four groups were not significant different (P>0.05).There are significant different between the four different time point after therapy (all P< 0.05), The tumor growth in the ALA-PDT group was more slowly in 2 weeks after PDT, and the best inhibited effect was at the seventh day after PDT. The tumor in ALA-PDT group began to increase faster at the 14 th day after PDT., while the tumor volumes were smaller than the other groups at the four time points after PDT.3. Under microscope from H&E staining, we can see allotype tumor cells and caner nest, the blood vessel endothelium is complete in the control group, ALA group and lighted group. While the PDT group, we can observed some homogen eryth-dyeing necrosis, and with some lymphocyte infiltrated.4. Under confocal laser scanning microscope, there was co-localization of MitoTracker Green and ALA-PpⅨ, that meaned ALA-PpⅨwas partially localized in mitochondria.5. The date of FCM analysis indicated that there was significant different apoptosis rate between the control group and the ALA-PDT (with ALA concentration of 0.05 mM and 0.5 mM) (P<0.05). When the concentration was 0.05 mM, there was not significant different between the PDT group and the control group (P>0.05), while when the concentration was up to 0.5 mM, the different between the control group and PDT group was significant (P<0.01).6. The result of cell cycle analysis indicated that there were significant different in the cell rate of G0/G1, S and G2/M phase between the control group and the PDT group (all P<0.05). The cell rate of G0/G1 phase was increase as ALA concentration increased, while the proportion of cells in S and G2/M phase was decreased.7. Fluorescence microscopy with Hoechst 33342/PI staining detected typical apoptotic changes (condensed chromatin, shrunken nuclei and high blue fluorescence by Hoechst 33342 staining) and necrotic cells (high red fluorescence) in the PDT-treated cells. In contrast, few apoptotic and necrotic cells were observed in the cells of control group.8. Fluorescence microscopy with rhodamine123 staining detected mitochondria membrane potential changes, with high flavovirens fluorescenc in all the ALA-PDT groups, and the fluorescenc enhanced as ALA concentration increased, while in the control group, the fluorescenc is thinness or even with no fluorescenc.9. The expression of Bcl-2, Bcl-xL and FADD genes were all significant down regulation at 24 h after ALA-PDT on the Eca-109 cell, while Bax was significant up-regulation (all P<0.05). Bad, Bid and Bak-1 were all slightly up-regulation, but the difference contrast to the control group were all not significant (all P> 0.05).10. The expression of Bcl-2 protein was significant down regulation at 24 h psot-PDT in the Eca-109 cell, while the expression of cytc (cytoplasm) was significant up-regulation (all P<0.05).Conclusion:1. ALA-PDT significantly inhibited the growth of Eca-109 cells. Under the special light source, the laser dose, the kinds of photosensitizers, the photosensitizers incubating concentration and incubating time, were the main effect to PDT. Under the specific incubating time (24 h), the cell survival rate is the lowest as ALA concentration is 0.5 mM and the laser dose was 50 J/m2. While ALA concentration increases from 0.5 mM to 2.0 mM, the cell survival rate reached a plateau as the laser dose increase from 30 J/m2 to 50 J/m2. Under specific laser dose (30 J/m2.), the cell survival rate is the lowest as ALA concentration is 2.0 mM and the incubating time is 24 hours. While ALA concentration increases from 0.5 mM to 2.0 mM, the killing effect also reached a plateau as the incubating time increase from 6 hours to 24 hours. These results indicated that it is demanded to choose the optimal premeters to achieve the best effect in ALA-PDT of esophageal carcinoma.2. ALA-PDT inhibited the tumor growth significantly. The best inhibited effect was at the seventh day after PDT. The tumor in ALA-PDT group began to grow faster at the 14 th day post-PDT., thus 14 th day after PDT can be choosen as the date to take the second treatment.3. The results of H&E and Hoechst 33342/PI staining indicated the morphologic changes in the human esophageal carcinoma after ALA-PDT.4. ALA-PpIX was partially localized in mitochondria. ALA-PDT changed the mitochondria membrane potential of Eca-109 cells and it maybe one of the mechanisms for ALA-PDT to induce cell death.5. ALA-PDT induced the Eca-109 cells to apoptosis, and the apoptosis rate was increase as ALA concentration increased.6. ALA-PDT arrested the Eca-109 cells at G0/G1 phase, and the cell rate of G0/G1 phase was increased as ALA concentration increased. The arrest of cell cycle maybe one of the mechanism that to induce apoptosis.7. The extrinsic apoptosis pathway was not the main apoptosis way in ALA-PDT, The main way was mitochondria-dependent apoptosis pathway. The protein and gene expression of Bcl-2 were all significant down regulation in Eca-109 cells at 24 h after ALA-PDT indicated that Bcl-2 maybe one of the target of photo-damage during the PDT process8. Release cytc from mitochondria to induce apoptosis maybe one of the final reaction in the mitochondria-dependent apoptosis pathway.
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