Study Of 5-aminolevulinic Acid-mediated Photodynamic Therapy On Drug Resistant Or Non-drug Resistant Human Nasopharyngeal Carcinoma

Nasopharyngeal carcinoma (NPC) is highly prevalent in Southern China.Radiotherapy, chemotherapy and operation are the primary treatments of NPC, buttumor recurrence and metastasis are the main reasons causing treatment failure.Chemotherapy is the most important adjunctive therapy for NPC, however, multidrugresistance (MDR) presents a major obstacle for the successful chemotherapy ofcancer and new approaches needed to be explored to overcome MDR. Photodynamictherapy (PDT) is a novel treatment modality, which is based on a selectiveaccumulation of a certain photosensitizing agent in neoplastic rather than in healthytissue. Once the photochemical compound is activated by light of a specificwavelength, cytotoxic singlet oxygen and other reactive oxygen species (ROS) areproduced to kill tumor cells selectively. Because photosensitizers preferentiallyaccumulate in the tumor region and there is the possibility of killing MDR cells, PDTcan be promoted as an alternative treatment for MDR tumors.Photosensitizer is the kernel of PDT. Hematoporphyrin derivative (HpD), a kindof traditional photosensitizer, has been used almost exclusively in clinical PDT trials.Although favorable results have been reported, this photosensitizer has severaldrawbacks that may limit its applicability in certain situations. For example, the action spectrum of HpD is around 630nm, which leads to poor penetration ability.Cutantest is required before administration and there is a long interval beforeillumination. Furthermore, the uncommonly long period of cutaneousphotosensitization (lasting up to several weeks, even 18 weeks) was observed inpatients tbllowing administration of HpD.Due to the shortcomings of those traditional photosensitizers, otherphotosensitizers, such as 5-aminolevulinic (5-ALA) are currently being evaluated foruse in PDT. 5-ALA is not a photosensitizer itself and it serves as the biologicalprecursor in the heme biosynthetic pathway. In ALA-induced endogenousphotosensitization, the heme biosynthetic pathway is used to produce protoporphyrinⅨ(PpⅨ)-a potent photosensitizer. The short period of skin photosensitization (24-48h) and the possibility of oral administration and injection, makes ALA a promisingphotosensitizer for PDT treatments of cutaneous cancer, esophageal carcinoma andso on.5-ALA mediated Photodynamic therapy has been used in treatment NPC lately.The purpose of this study was to investigate the potential use of 5-aminolevulinic acidfor photodynamic therapy of cell line CNE2 and CNE2/DDP cultured in vitro, and theeffect of 5-ALA-PDT on human NPC xenografts was also observed in vivo so as toprovide experiment support for clinical use. Then the tumorcidal mechanism of5-ALA-PDT on drug resistant or non-drug resistant human NPC was explored. In theend, the role of verapamil enhancing the killing effect of 5-ALA-PDT on CNE2/DDPcells was evaluated. These results may be advisory in NPC combined therapy.Capter oneEffect of 5-aminolevulinic acid (5-ALA) mediated photodynamic therapy onhuman Nasopharyngeal Carcinoma cell line CNE2 and CNE2/DDP In vitro Objective: To investigate the potential use of 5-aminolevulinic acid forphotodynamic therapy of NPC cell line CNE2 and CNE2/DDP cultured in vitro andto explore the optimal 5-ALA concentration, proper incubation time and suitable laserdosage.Methods: Cellular morphology of CNE2 and CNE2/DDP were observed, cell cycle,P-gP, ABCG2 expression and rhodamine effluence were detected through flowcytometry. Drug resistant indexes of CNE2 and CNE2/DDP to 5-Fu, VCR, DDP werecalculated through MTT. Cofocal laser scanning microscopy were used to detect theintracellular localization of PpⅨ.Effect of different incubation time and 5-ALAconcentration on the the cellular fluorescence intensities of PpⅨin CNE2 orCNE2/DDP were measured with a luminescence spectrometer and flow cytometry.Effect of various incubation time, 5-ALA concentration and laser dosage on thesurvival rates of CNE2 or CNE2/DDP cells were investigated with MTT.Results: CNE2 with comparative larger cell body than that of CNE2/DDP. Furthermore, CNE2 cells were well distributed and CNE2/DDP grouped in the culture flasks.The heteroploid percentage of CNE2/DDP was 58% while the diploid percentage ofCNE2 was 100%. CNE2/DDP cells in G0/G1 stage increased and in S stage fell down.The drug resistant index of CNE2/DDP to DDP,5-Fu and VCR was 24.56,224.21 and52.65 respectively. The P-gP expression of CNE2 and CNE2/DDPwas(75.84±1.14)%,(2.78±0.70)% and the expression of ABCG2 in CNE2 orCNE2/DDP was (54.96±1.20)%, (2.75±0.55)%.The cellular rhodamine fluorescenceintensity was much higher in CNE2 than that in CNE2/DDP. The fluorescence imagedemonstrated the ALA induced PpⅨwas localized in the cytoplasm except nuclearregion of the two kinds of cells. The cellular fluorescence intensities of PpⅨincreased with the 5-ALA incubation times but saturated for 6 hours and increasedwith the 5-ALA concentration, saturated at 1 mmol/l in CNE2 and CNE2/DDP cells.Under the same conditions, the cellular fluorescence intensities of PpⅨwere lower inCNE2/DDP than that in CNE2 cells. Unlike sole incubation with 5-ALA or sole laser irradiation, the combination of both factors lead to a significant, concentration-,energy- and time-dependent decrease of cell survival rates up to a threshold of 0.5-1mmol/L 5-ALA, 40J/cm~2 and 6h, above which a platform of cell survival ratesoccurred both in CNE2 and CNE2/DDP. Under the same conditions, the cell survivalrates of CNE2/DDP were lower than that in CNE2 cells.Conclusions: CNE2 and CNE2/DDP cell have distinct bionomics. With thepathognomonic features, CNE2/DDP cell line has been a fine MDR model for NPC.Incubated with 5-ALA, PpⅨcan be generated in the cytoplasm of the two kinds ofneoplasm cells. The cellular fluorescence intensities of PpⅨin CNE2 andCNE2/DDP show a 5-ALA concentration and incubation time dependent increase upto a threshold of 1 mmol/L 5-ALA and 6h. The cellular fluorescence intensities ofPpⅨin CNE2 are always higher than that of CNE2/DDP under the same condition.The optimal concentration of 5-ALA might be 0.5-1mmol/L, the proper incubationtime be 6hours and the suitable laser dosage be 40J/cm2 for 5-ALA-PDT on CNE2and CNE2/DDP cells. Neither 5-ALA nor laser irradiation itself has cytotoxicity onCNE2 and CNE2/DDP cells. However, though 5-ALA-PDT may effectively killCNE2 and CNE2/DDP cells cultured in vitro, CNE2/DDP cells are less sensitive to5-ALA-PDT than CNE2 cells. The cross-resistance of CNE2/DDP might beovercome through increasing 5-ALA concentration and radiation dosage.Chapter TowTherapeutic effect of 5-ALA-PDT on human Nasopharyngeal Carcinomaxenografts in nude miceObjective: To investigate the effect of 5-ALA-PDT on human NasopharyngealCarcinoma xenografts in vivo. Methods: Cultured CNE2 or CNE2/DDP human Nasopharyngeal Carcinoma cellswere injected into the dorsum and below the skins of the nude mice to develop thetumor model. The nude mice were divided into control group, 5-ALA group,5-ALAoPDT group and light group. The tumor volumes and weights of the nude micein 1,3,7,14,21 days after treatment was measured, and their histology changes werealso studied.Result: Cultured CNE2 or CNE2/DDP cells injected into the nude mice cansuccessfully develop the tumor model. Either in CNE2 or CNE2/DDP xenograft mice,the tumor volumes and weights in 5-ALA-PDT groups were significant lower thanthose in other three groups. There were no significant differences in tumor volumes orweights among the control group, 5-ALA group and light group in CNE2 orCNE2/DDP nude mice. 7,14,21 days after 5-ALA-PDT, the tumor volumes of CNE2nude mice was significantly lower than that of CNE2/DDP nude mice. 21 days after5-ALA-PDT, the tumor weights of CNE2 nude mice was significantly lower that ofCNE2/DDP nude mice. The histology examine demonstrates that much tumor bloodsinus was ruined and cell necrosis developed after 5-ALA-PDT while suchmanifestation not observed in the control group, light group and 5-ALA group.Conclusions: 5-ALA-PDT has therapeutic effect on human NasopharyngealCarcinoma xenografts in nude mice.The inhibition effect on CNE2 nude mice issignificantly stronger than that on CNE2/DDP mice. 5-ALA or light alone could notinhibit the tumor growth.Chapter ThreeMechanism of 5-ALA-PDT on human Nasopharyngeal CarcinomaObjective: To investigate the tumorcidal mechanism of 5-ALA-PDT on human NasopharyngeaI Carcinoma.Methods: Cultured CNE2 or CNE2/DDP cells were divided into control group,5-ALA group, 5-ALA-PDT group and light group. Hoechst, AO-BE andAnnexin-V-PI apoptosis detection were developed to examine the apoptosis cellsamong the four groups. Furthermore, the cells of the 4 groups were analyzed by flowcytometry with Annexin-V-PI apoptpsis detection kit and examined with theelectronmicroscope. The levels of MDA in four groups of CNE2 or CNE2/DDP wereanalyzed to illustrate the mechanism of the PDT. The tumor tissues of CNE2 orCNE2/DDP nude mice were also examined with the electronmicroscope and detectedapoptosis by TUNEL. Bax and Bcl-2 expression of CNE2 or CNE2/DDP cells in thecontrol group and 5-ALA-PDT group were analyzed by flow cytometry.Results: CNE2 or CNE2/DDP cells' apoptosis was observed only in 5-ALA-PDTgroup by Hoechest,AO-EB, Annexin-V-PI apoptosis detection andelectornmicroscope examination. CNE2 or CNE2/DDP cells' necrosis was alsoobserved by Annexin-V-PI in 5-ALA-PDT group only. Flow cytometry analysisshowed the rates of necrosis and/or apoptosis of CNE2 or CNE2/DDP cells in5-ALA-PDT group were much higher than that of the other three groups. And CNE2cells' necrosis rate in 5-ALA-PDT group was significantly higher than that ofCNE2/DDP cells. Conversely, the necrosis rate of CNE2/DDP cells in 5-ALA-PDTgroup was significantly higher than that of CNE2 cells. The level of MDA in tumortissues of 5-ALA-PDT group was higher than that of the other groups both in CNE2and CNE2/DDP nude mice. The level of MDA in CNE2 nude mice was significantlyhigher than that in CNE2/DDP nude mice. Apoptosis indexes were much higher in thetumor tissue of 5-ALA-PDT group than those of the other groups examined byTUNEL in CNE2 or CNE2/DDP nude mice. More over, apoptosis index was muchhigher in 5-ALA-PDT group of CNE2/DDP nude mice than that of CNE2 nude mice. Comparing with the control groups, Bax expressions were up-regulation while Bcl-2down regulation leading to Bax/ Bcl-2 down regulation both in CNE2 andCNE2/DDP cells.Conclusions: The results suggested that 5-ALA-PDT may induced apoptosis inCNE2 and CNE2/DDP cells, reactive oxygen species generation which were toxic tocells and tissues may also involve in the mechanism of 5-ALA-PDT. It seems toCNE2 cells, the cytotoxicity of reactive oxygen species play an important role inkilling effect of 5-ALA-PDT, while to CNE2 cells, apoptosis is likely preponderanceunder the same condition.Chapter fourEffect of verapamil on the results of CNE2 and CNE2/DDP cells after5-ALA-PDTObjective: To investigate whether verapamil may enhance the killing effect of5-ALA-PDT on CNE2/DDP cells.Methods: Effect of verapamil on the cellular fluorescence intensities of PpⅨinCNE2 or CNE2/DDP after 5-ALA-PDT were measured with a luminescencespectrometer and flow cytometry. Cultured CNE2 or CNE2/DDP cells were dividedinto control group, 5-ALA group, VER group and 5-ALA+VER group. Effect ofverapamil on the survival rates of CNE2 or CNE2/DDP cells after 5-ALA-PDT wasinvestigated with MTT.Results: the intercellular PpⅨfluorescence intensities of CNE2/DDP cells washigher in 5-ALA + VER group than that in 5-ALA group under various 5-ALAconcentrations. The intercellular PpⅨfluorescence intensities of CNE2 cells increased slightly in 5-ALA + VER group under various 5-ALA concentrations, butthere was no significant different comparing with 5-ALA group. The cell survival rateof CNE2/DDP was higher in 5-ALA + VER group than that in 5-ALA group undervarious 5-ALA concentrations. There was no significant difference in CNE2 cellsurvival rates between 5-ALA + VER group and 5-ALA group under different 5-ALAconcentration.Conclusions: Verapamil may increase the accumulation of PpⅨin CNE2/DDP cellswhile not in CNE2 cells and may enhance the killing effect of 5-ALA-PDT onCNE2/DDP cells while not in CNE2 cells.