ABSTRACT
Nasopharyngeal carcinoma (NPC) is of high prevalence in Hong Kong and southern China. The pathogenesis of NPC is closely associated with Epstein-Barr virus (EBV) infection via regulation of viral oncoprotein latent membrane protein 1 (LMP1). The conventional treatment for NPC is chemo-radiotherapy, but the prognosis remains poor for advanced stage, recurrent and metastatic NPC. Photodynamic therapy (PDT) is a therapeutic approach to combat tumors. PDT effectiveness depends on the interaction of photosensitizers, light and molecular oxygen. 5- aminolevulinic acid hexyl derivative (H-ALA) is one of the photosensitizers derived from 5-ALA. H-ALA with improved lipophilic properties by adding a long lipophilic chain (hexyl group) to 5-ALA, resulted in better penetration into cell cytoplasm. In this study, the effect of H-ALA-PDT on NPC cells (EBV positive C666-1 and EBV negative CNE2) was investigated. The H-ALA mediated cellular uptake and cytotoxicity was revealed via flow cytometry analysis and MTT assay respectively. H-ALA PDT mediated protein modulation was analysed by western blot analysis. Our finding reported that the cellular uptake of H-ALA in C666-1 and CNE2 cells was in a time dependent manner. H-ALA PDT was effective to C666-1 and CNE2 cells. EBV LMP1 proteins was expressed in C666-1 cells only and its expression was responsive to H-ALA PDT in a dose dependent manner. This work revealed the potential of H-ALA PDT as a treatment regiment for EBV positive NPC cells. Understanding the mechanism of H-ALA mediated PDT could develop improved strategies for the treatment of NPC.
Subject(s)
Aminolevulinic Acid/analogs & derivatives , Low-Level Light Therapy/methods , Nasopharyngeal Neoplasms/drug therapy , Photochemotherapy/methods , Photosensitizing Agents/pharmacology , Viral Matrix Proteins/drug effects , Aminolevulinic Acid/pharmacokinetics , Aminolevulinic Acid/pharmacology , Cell Line, Tumor , Cell Survival , Herpesvirus 4, Human/drug effects , Humans , Lasers, Semiconductor/therapeutic use , Photosensitizing Agents/pharmacokinetics , Protoporphyrins/pharmacokineticsABSTRACT
Multidrug resistance (MDR) refers to the ability of cancer cells to develop cross resistance to a range of anticancer drugs which are structurally and functionally unrelated. P-glycoprotein (P-gp) is the best studied MDR phenotype in photodynamic therapy (PDT) treated cells. Our pervious study demonstrated that FosPeg® mediated PDT is effective to NPC cell line models. In this in vitro study, the expression of MDR1 gene and its product P-gp in undifferentiated, poorly differentiated and well differentiated human nasopharyngeal carcinoma (NPC) cells were investigated. The influence of P-gp efflux activities on photosensitizer FosPeg® was also examined. Regardless of the differentiation status, PDT tested NPC cell lines all expressed P-gp protein. Results indicated that FosPeg® photoactivation could heighten the expression of MDR1 gene and P-gp transporter protein in a dose dependent manner. Up to 2-fold increase of P-gp protein expression were seen in NPC cells after FosPeg® mediated PDT. Interestingly, our finding demonstrated that FosPeg® mediated PDT efficiency is independent to the MDR1 gene and P-gp protein expression in NPC cells. FosPeg® itself is not the substrate of P-gp transporter protein and no efflux of FosPeg® were observed in NPC cells. Therefore, the PDT efficiency would not be affected even though FosPeg® mediated PDT could induce MDR1 gene and P-gp protein expression in NPC cells. FosPeg® mediated PDT could be a potential therapeutic approach for MDR cancer patients.
Subject(s)
Gene Expression Regulation/drug effects , Liposomes/pharmacology , Mesoporphyrins/pharmacology , Photosensitizing Agents/pharmacology , ATP Binding Cassette Transporter, Subfamily B/genetics , ATP Binding Cassette Transporter, Subfamily B/metabolism , Carcinoma , Cell Line, Tumor , Drug Resistance, Neoplasm/drug effects , Gene Expression Regulation/radiation effects , Humans , Light , Liposomes/chemistry , Liposomes/therapeutic use , Mesoporphyrins/chemistry , Mesoporphyrins/therapeutic use , Nasopharyngeal Carcinoma , Nasopharyngeal Neoplasms/drug therapy , Nasopharyngeal Neoplasms/metabolism , Nasopharyngeal Neoplasms/pathology , Photochemotherapy , Photosensitizing Agents/chemistry , Photosensitizing Agents/therapeutic use , RNA, Messenger/metabolismABSTRACT
AIM OF STUDY: This study aimed to elucidate and compare the anti-metastatic mechanism of Tian-Xian liquid (TXL) and its bioactive components namely butanol (BU), ethyl-acetate (EA) and aqueous (WA) fractions on human colorectal cancer in vitro (HT-29 cancer cells) and in vivo (nude mouse xenografts). MATERIALS AND METHODS: The anti-proliferative effects of TXL and its bioactive components in HT-29 cells were determined by MTT assay. Their modulations on the potential angiogenic and metastatic marker expressions on HT-29 cells and xenografts were investigated by real-time PCR and Western blot at transcriptional and translational levels, respectively. For the in vitro study, migration abilities of HT-29 cells were determined using wound healing assay. For the in vivo study, daily measurements of the tumor size and volume of the xenografts were also performed. RESULTS: TXL, BU, EA and WA effectively inhibited the proliferation of HT-29 cells in a dose- and time-dependent manner. The IC(50) value of TXL on HT-29 cells was obtained after incubation with 1% (v/v) TXL for 4h; whereas IC(50) values were obtained for the following bioactive components: BU at 1.25% (v/v); EA at 5% (v/v); and WA at 0.3125% (v/v). It was found that 1% (v/v) TXL significantly down-regulated MMP2 and MMP7 expression at both transcriptional and translational levels and it reduced MMP9 and VEGF protein expression in vitro. TXL decreased the metastatic ability of HT-29 cells as demonstrated by wound healing assay. TXL and its bioactive fractions caused no significant changes in the body weight indicating lack of toxicity to the xenografts. CONCLUSIONS: In summary, TXL multi-targeted to down-regulate the metastatic markers in both in vitro and in vivo models. However, the effects of its bioactive fractions were not obvious. This study profoundly elucidated the anti-proliferative mechanism of TXL, which is vital for the development of future anti-cancer regime in Chinese medicinal formulations.
Subject(s)
Antineoplastic Agents, Phytogenic/pharmacology , Colorectal Neoplasms/drug therapy , Drugs, Chinese Herbal/pharmacology , Acetates/chemistry , Animals , Antineoplastic Agents, Phytogenic/chemistry , Blotting, Western , Butanols/chemistry , Cell Movement/drug effects , Cell Proliferation/drug effects , Chemical Fractionation , Colorectal Neoplasms/genetics , Colorectal Neoplasms/metabolism , Colorectal Neoplasms/secondary , Dose-Response Relationship, Drug , Drugs, Chinese Herbal/chemistry , Female , Gene Expression Regulation, Neoplastic/drug effects , HT29 Cells , Humans , Inhibitory Concentration 50 , Matrix Metalloproteinase 2/genetics , Matrix Metalloproteinase 2/metabolism , Matrix Metalloproteinase 7/genetics , Matrix Metalloproteinase 7/metabolism , Matrix Metalloproteinase 9/genetics , Matrix Metalloproteinase 9/metabolism , Mice , Mice, Nude , Neoplasm Invasiveness , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , Solvents/chemistry , Time Factors , Tumor Burden/drug effects , Vascular Endothelial Growth Factor A/genetics , Vascular Endothelial Growth Factor A/metabolism , Water/chemistry , Xenograft Model Antitumor AssaysABSTRACT
BACKGROUND: 5-aminolevulinic acid (ALA) and its hexylester (ALA-H) are the drugs currently used in photodynamic therapy (PDT). The side effect, especially the long-term side effect of these drugs is a problem of concern in this field, which has not been clearly understood yet. PURPOSE: The normal lymphocytes and nasopharyngeal carcinoma (NPC) cells were used as the cell models to evaluate the side effects of ALA or ALA-H in the absence of light or under sub-lethal doses of light. METHODS: The cytotoxic and DNA-damaging effects of ALA or ALA-H on lymphocytes and NPC cells were studied by means of the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the alkaline comet assay. ALA at 0.75 mM concentration and ALA-H at 10-microM concentrations were selected in the studies. This is because under these concentrations, ALA- or ALA-H-mediated PDT can destroy most NPC cells in vitro. The intracellular distributions of the protoporphyrin IX (PpIX), induced by the ALA or ALA-H, were measured by the confocal laser scanning microscope to provide more information for understanding the DNA damage. RESULTS: The incubation of 0.75 mM ALA or 10 microM ALA-H alone (without light) did not cause DNA damage as well as the considerable cytotoxic effect on NPC cells. However, after ALA (0.75 mM) incubation and without light irradiation, the serious cytotoxicity and remarkable DNA damage were found in lymphocytes. When the lymphocytes were incubated with ALA-H (10 microM) alone (in the absence of light), no DNA damage could be detected and a slight cytotoxic effect was found. Both ALA and ALA-H induced PpIX in the lymphocytes. The fluorescence images of PpIX intracellular localization demonstrated that the PpIX diffused into the nuclear region in ALA-(0.75 mM)-incubated lymphocytes but not existed in the nucleus of ALA-H(10 microM)- incubated lymphocytes, providing an explanation for the facts that ALA (0.75 mM) induced the DNA damage while ALA-H (10 microM) did not. CONCLUSION: These results suggested that the genotoxic potential of lymphocytes seems high for ALA (0.75 mM) and could be excluded for ALA-H (10 microM).
Subject(s)
Aminolevulinic Acid/analogs & derivatives , DNA Damage , Lymphocytes , Mutagens , Photosensitizing Agents , Ultraviolet Rays , Aged , Aminolevulinic Acid/adverse effects , Aminolevulinic Acid/pharmacokinetics , Cell Line, Tumor , Cell Survival/drug effects , Cell Survival/radiation effects , Comet Assay , Humans , Lymphocytes/drug effects , Lymphocytes/metabolism , Lymphocytes/radiation effects , Male , Microscopy, Confocal , Mutagens/adverse effects , Mutagens/pharmacology , Nasopharyngeal Neoplasms/pathology , Photosensitizing Agents/adverse effects , Photosensitizing Agents/pharmacology , Protoporphyrins/metabolismABSTRACT
Nasopharyngeal carcinoma (NPC) is a prevalent cancer in Hong Kong and southern China. To explore a new modality of NPC treatment, 5-aminolevulinic acid (ALA) or its hexyl ester (ALA-H) mediated photodynamic therapy (PDT) was studied in vitro. The results show that NPC cells are sensitive to both ALA and ALA-H mediated PDT. However, ALA-H PDT is much more effective at cell inactivation than ALA-PDT, due to a higher efficiency of ALA-H on producing endogenous protoporphyrin (PpIX) in cells. Both apoptosis and necrosis are involved in cell death, but apoptosis plays a major role under the short time incubation of drugs. ALA and ALA-H mediated PDT not only destroy the cells directly, but also inhibit the expression of matrix metalloproteinase-2 (MMP2) in cells, a maker for tumor metastasis. The ALA-H shows promising PDT results on NPC in vitro; therefore it is worth investigating further in vivo for NPC treatment.
