5-aminolevulinic Acid-mediated Photodynamic Therapy Of Human Glioma

The most common neoplasms in neurosurgery are gliomas, which account for 50-60% of all the primary intracranial tumors. And most of these glial rumors are malignant gliomas. Due to the invasive nature of these malignant glial tumors, complete excision of tumor tissue is extremely difficult. Now there is no satisfactory treatment for these infiltrative neoplasms. The best available treatment using surgery, chemotherapy and radiation therapy results in only 15 months of survival for malignant gliomas. Five -year survival rates are still dismal.The shortcomings of standard treatment modalities in the management of glioma patients have led to a search for more aggressive focal treatments. Photodynamic therapy (PDT) has been used successfully in the treatment of a wide variety of localized malignancies and may prove useful as an adjuvant therapy in the treatment of resected margins after surgery. Some of the features that make PDT an effective adjuvant therapy in the treatment of brain tumors are: (1) it is a local form of treatment in which the treated volume is limited by high attenuation of light in brain tissues; (2) resistance to PDT has not been encountered during treatments of brain tumors; and (3) repeated applications of PDT is an option due to low long-term morbidity. PDT utilizes a photosensitizer that is selectively taken up and/or retained by neoplastic tissue. When absorbing light of an appropriate wavelength, thephotosensitizer produces cytotoxic oxygen product causing direct cell death, and/or vascular shutdown.Porphyrins, a kind of traditional photosensitizer, such as hematoporphyrin derivative and Photofrin have been used almost exclusively in clinical PDT trials of the brain tumors. Although favorable results have been reported by a number of clinicians, these photosensitizers have several drawbacks that may limit their applicability in certain situations. For example, the uncommonly long period of cutaneous photosensitization (lasting up to several weeks, even 18 weeks) was observed in patients following administration of these porphyrins photosensitizers. And furthermore, the relatively poor tumor-to-normal tissue localization observed by several groups may limit the effectiveness of these photosensitizers due to the potential of normal tissue complications.Due to the drawbacks of these traditional photosensitizers, other photosensitizers, such as 5-aminolevulinic acid (5-ALA) are currently being evaluated for use in PDT. 5-ALA itself is not a photosensitizer and it serves as the biological precursor in the heme biosynthetic pathway. In ALA-induced endogenous photosensitization, the heme biosynthetic pathway is used to produce protoporphyrin IX(Pp IX)—a potent photosensitizer. Heme is synthesized from glycine and succinyl CoA. The rate-limiting step in the pathway is the conversion of glycine and succinyl CoA to ALA, which is under negative feedback control by heme. Through the introduction of ALA, the regulatory feedback system becomes overloaded causing an accumulation of Pp IX, which may cause the photosensitizing effect for PDT following the activation. The combination of excellent tumor-to-normal brain tissue localization, short period of skin photosensitization (24-48 h) and the possibility of oral administration, makes ALA a promising photosensitizer for PDT treatments of glioma patients. Therefore ALA-PDT may have a great potential in the treatment ofbrain tumors.Several studies overseas have suggested that 5-aminolevulinic acid -mediated photodynamic therapy can kill the glioma cells in vitro, while domestic not. However, there are no in vivo articles released so far. Furthermore, the tumorcidal mechanism of this form of treatment is still obscure.The primary aim of this study is to investigate the response of human glioma to ALA-mediated PDT.Chapter OneObject: To investigate the effect of 5-aminolevulinic acid (ALA) - mediated photodynamic therapy (PDT) on U251 human glioma cells in vitro and the optimal incubation concentration and time of 5-ALA. Methods: U251 human glioma cells were routinely cultured and then treated with ALA (2 mmol/1) for 6hours, after that, fluorescene microscopy and cofocal laser scanning microscopy were used to measure the intracellular localization of Pp IX. Cells cultured with various concentrations or various time were measured with a luminescence spectrometer to detect the fluorescene intensity released by Pp IX. Cells of pure U251 group, pure 5-ALA group, light group and PDT groups were determined using a colony-forming assay to measure the anti-invasion effect. U251 human glioma cells were routinely cultured and then treated with ALA (2 mmol/1) for different time followed by irradiation of light. The PDT-induced phototoxicity of the cells was determined by MTT assay. Cells at various concentrations with ALA at a fixed time followed by irradiation of light, at a fixed concentration subjected to various doses of light, 5-ALA only and light only were also determined by MTT assay. Results: The fluorescence images demonstrated the ALA induced Pp IX was localized in the cytoplasm except nuclear region of the cells. By measuring the intensities of fluorescence, the Pp IX cellularamount increased with the drug incubation times but saturated for 6 hours and increased with the drug concentrations, saturated at 2 mmol/1. The colonies of pure U251 group, pure 5-ALA group, light group and PDT groups were 10.67 + 2.73,9.50 ±2.17,10.17 + 2.14,5.50+ 1.38, respectively (F=7.14, p=0.0019).With the same light dose (25.0J/cm2), the survival rates of cells were 70.16% + 5.02%, 50.19%±4.79%, 34.97% + 5.34%, 27.04% ± 4.34%, 24.26% ± 2.76% for ALA concentration of 0.25mM, 0.5 mmol/1, 1.0 mmol/1, 2.0 mmol/1, 4.0 mmol/1, respectively (F=279.88, p=0.0000).But the survival rates of the cells with 2.0 mmol/1 ALA compared to those with 4.0 mmol/1 ALA (27.04% ±4.34% vs 24.26% ±2.76%) had no significant difference (p=0.611). The survival rates of cells were 83.48%±6.79%, 68.09%± 6.02%, 33.75%±6.70%, 23.34%±5.08%, 15.14%±3.60% for light doses of 6.25, 12.5, 25.0, 50.0, 100J/cm2, respectively with the same ALA concentration (F=422.03,p=0.0000).Without exposure to light, however the survival rates of cells were 96.64%±6.56%, 97.71%±5.48%, 98.10%±6.25%, 99.44%±7.02%, 95.86% ±7.80% for ALA concentrations of 0.25 mmol/1, 0.5 mmol/1, 1.0 mmol/1, 2.0 mmol/1, 4.0 mmol/1, respectively (F=0.68, p=0.6085) while without ALA incubation, 98.74% ±6.20%, 96.49%±7.13%, 97.60%±5.94%, 95.70%±4.86%, 98.08%±6.26% for light doses of 6.25, 12.5, 25.0, 50.0, 100 J/cm2, respectively (F=0.6400 p=0.6368).Conclusions: U251 human glioma cells incubated with ALA can produce Pp IX in the cytoplasm of the neoplasm cells. ALA-PDT can markedly reduce the invasion of glioma cells. The PDT damaging to the U251 cells increased with ALA incubation concentration within a relative lower range, and then saturated at higher concentration and it is proportional to the doses of light irradiated. Without ALA, the light delivered cannot cause photodynamic therapy and ALA itself is nontoxic. The optimal incubation concentration of 5-ALA may be 2 mmol/1 and time, 6 hours. The ALA-induced PDT appears to be a promising therapy for glioma.Chapter TwoObject: To investigate the effect of 5-aminolevulinic acid (ALA) - mediated photodynamic therapy (PDT) on human glioma xenografts in vivo. Methods: Cultured U251 human glioma cells were injected into the dorsum and below the skins of the nude mice to develop the tumor model. When tumors grew to a size of 6-8 mm (the treatment size), the sensitizer (5-ALA, 100 mg/kg) was intraperitoneal injected. And after injection for 6 hours, the tumor-bearing nude mice were examined under the Leica LT-9MACIMSYSPULS to detect the fluorescence emitted by the Pp IX in the tumors. The tumor volume and weight of 1,3,7,14,21 days after treatment were measured, and its histology changes were also studied. Results: Cultured U251 human glioma cells injected into the dorsum and below the skins of the nude mice can successfully develop the tumor model. After injected 5-ALA for 6 hours, the tumor in the nude mice emit the red fluorescence characteristic of the Pp IX excited by blue light under the Leica LT-9MACIMSYSPULS. The tumor volumes are 0.202 ±0.011, 0.195 + 0.011, 0.198 + 0.020, 0.194 + 0.016 for the control group, light only group, 5-ALA only group, PDT group, respectively after 1 day of treatment(F=r0.41 p=0.748). For 3 days, The tumor volumes are 0.313±0.010, 0.299 + 0.014, 0.296 + 0.017, 0.195 + 0.015 for the control group, light only group, 5-ALA only group, PDT group, respectively (F=l 16.48 p=0.0000), while 7 days,0.435 + 0.017, 0.438 + 0.009, 0.432 ±0.017, 0.236 ±0.008 respectively (F=433.38 p=0.0000); 14 days,1.028± 0.058, 1.034±0.057, 1.039±0.033, 0.339±0.005 (F=494.89 p=0.0000); 21 days, 1.579±0.067, 1.601±0.064, 1.612±0.069, 0.471 ±0.042(F=669.26 p=0.0000). Nosignificant difference among the control group, light only group and 5-ALA only group after 21 days of treatment while PDT group is significantly different from any of the three groups. The histology examined demonstrates that most tumor blood vessels were congested and necrosis developed after PDT while not in the control group, light only group and 5-ALA only group. Conclusions: 5-aminolevulinic acid - mediated photodynamic therapy (PDT) could induce injury to human glioma xenografts and inhabit the tumor growth while light only and 5-ALA only could not.Chapter ThreeObject: To investigate the tumorcidal mechanism of 5-aminolevulinic acid (ALA) - mediated photodynamic therapy (PDT) on human glioma. Methods: Hoechst, AO-EB and Annexin-V —PI apoptosis detection were developed to examine the apoptosis cells among the control group, light only group, 5-ALA only group and PDT group. Furthermore, the cells of the 4 groups were analyzed by flow cytometry with Annexin-V — PI apoptosis detection kit and examined with the electronmicroscope. And the tissues of the tumors in nude mice of the control group, light only group, 5-ALA only group and PDT group were analyzed to investigate the of level of MDA to illustrate the mechanism of the PDT. The tissues were also examined with the electronmicroscope and detected apoptosis by TUNEL. The photobleaching was observed with different concentrations of Pp IX and NaN3, which is the inhibitor of singlet oxygen. Results: Apoptosis was observed in the cells of PDT group by Hoechst, AO-EB, Annexin-V — PI apoptosis detection andelectronmicroscope examination and necrosis was also observed by Annexin-V—PI apoptosis detection and electronmicroscope examination while no apoptosis or/and necrosis in the cells of the control group, light only group and 5-ALA only group. Flow cytometry analysis showed the rate of necrosis and/or apoptosis of the cells in the PDT group was much higher than that of the other three groups and the cells in the PDT group died mainly by necrosis. The level of MDA of the tumors in the PDT group was higher than that of the other groups, which indicated the mechanism of the ALA-PDT involving in reactive oxygen species generation which were toxic to cells and tissues. Necrosis and apoptosis were observed in the cells of the tumors of the PDT group examined by TUNEL and electronmicroscope while not in the cells of the tumors of the PDT the other groups. The inhibitor of singlet oxygen, NaN3, could obviously decrease the efficacy of the PDT. Conclusions: 5-aminolevulinic acid -mediated photodynamic therapy(ALA- PDT) appears to be a promising therapy for human glioma, which the mechanism involves in the destruction of the tumors mainly by I and II type cytotoxic reactive oxygen species(ROS) generated by Pp DC induced by 5-ALA after irradiation of light and partly by apoptosis.