| Malignant tumors are common diseases characterized by abnormal cell proliferation, and early diagnosis and timely treatment is the key to reduce cancer mortality. But early diagnosis and effective control of malignant tumor is still a tricky problem of the medical community. Surgery, radiation therapy and drug treatment are still the current conventional methods for the treatment of malignant tumor. But the traditional treatments are often difficult to obtain the ideal effect, and the medical community continues to explore new methods for the treatment of tumor. Esophageal cancer is a common digestive tract malignant tumor, and in the world about 300 thousand people die each year from esophageal cancer. The incidence and mortality among each country and racial is very different. Radiotherapy and chemotherapy still is a routine method in treatment of esophageal cancer, especially for advanced esophageal cancer patients. The traditional treatment is often difficult to get the ideal effect, we are still continuously exploring new methods for the treatment of esophageal cancer. Photodynamic therapy (PDT) developed with the laser medicine and it is a new method for the treatment of tumor. It can absorb certain wavelength of laser energy, produce optical physical and chemical reactions in the tumor site and then kill tumour cells. During the past 20 years of clinical practice in the medical, photodynamic therapy in the treatment of malignant tumor made remarkable achievements, and the therapy has been recognized by many countries in the world. It has many advantages, such as high tumor specificity, remarkable treatment effect, small injury to normal tissue, and so on. The application of photodynamic therapy in the treatment of malignant tumor has a history of more than 100 years. In 1996, the FDA first approved PDT used in the treatment of advanced esophageal cancer. Now it has been applied to treat tumor of skin, head and neck, digestive tract, lung, bladder, liver, cervical, vaginal and other parts.The core of PDT is photosensitizer.5-aminolevulinic acid (ALA) is the second generation of photosensitizer, a kind of endogenous photosensitizer, and itself does not have photosensitive activity. In the body, glycine first synthesis protoporphyrin IX (PpIX), which is transformed into heme. ALA is the intermediate product in the process. PpIX has strong photosensitive activity, and the best absorption wavelength is at 635 nm. It has the advantages of low toxicity, fast metabolism and short time of avoiding light. Its aggregation concentration at the tumor cell is about 10 times higher than the surrounding normal tissues.In recent years, many studies in vitro and in vivo have found photodynamic therapy of tumor can cause two different ways of death, including apoptosis and necrosis.PDT can also stimulate variety of signaling pathways in tumor cells. EGFR tyrosine kinase receptor is one of the important members of the HER family,and many studies have shown that there are a variety of solid tumors with high EGFR expression or mutation. PI3K/Akt and Ras/Raf/MAPK are two important downstream signal pathways. These signaling pathways are associated with proliferation, apoptosis, angiogenesis, invasion, metastasis and concurrent chemoradiation antagonism and other malignant biological behavior in tumor cells. PI3K activation mechanism include:the activation of EGFR and combination with the adjusting p85 subunits and phosphorylation, changing conformation of PI3K protein, making p85 weaken the inhibitory effect to p110catalytic subunit. Akt is a molecular, weight of about 57 ku silk/threonine protein kinase. Akt in mammals is proto-oncogenes,homologue with virus. In a variety of human tumor such as ovarian cancer, pancreatic cancer, breast cancer, non-small cell lung cancer and esophageal cancer, Akt often showed high expression. The study by Jianghong, etc. found that stage-Ⅱ AKT protein expression is lower in well-differentiated esophageal tissue of stageⅡ-Ⅲ period without lymph node metastasis, than in less-differentiated esophageal tissue of stage Ⅲ b-Ⅳ, with lymph node metastasis. This reveals that AKT probably can promote invasion and metastasis of esophageal cancer cell.There are many drugs targeting EGFR, and all belong to two categories:the first kind is monoclonal antibodies, such as cetuximab and panitumumab, and so on. These drugs combine with the outer membrane of EGFR, and inhibit the EGFR signaling pathway activation. The others are small molecules of tyrosine kinase inhibitors(TKIs), such as gefitinib, erlotinib and lapatinib. Those drugs combine with EGFR tyrosine kinase region in the tumor cells, then block EGFR signaling pathway. Small molecules of EGFR tyrosine kinase inhibitor tyrphostin AG1478, is a synthetic, but specific inhibition of EGFR activation. It can be competitive with ATP locus, combine with EGFR tyrosine kinase area, and then block EGFR and its downstream signaling pathways. AG1478 block PI3K/Akt signal transduction pathway, by inhibiting EGFR activation, and inhibit tumor growth. LY294002 is the first synthetic PI3K alpha/delta/beta inhibitor. LY294002 make PKB/Akt deactivation, so it can inhibit cell proliferation and induce cell apoptosis. The studies of LY294002 using in lung cancer cell lines, found that the activity of Akt decreased, lung cancer cells apoptosis accured, cells growth restrained, sensitivity of chemotherapy drugs increased.This research was main to detect the effect of photodynamic therapy to esophageal cancer in vivo and in vitro on the growth and migration of tumor. The photosensitizer was ALA, the second generation. The study revealed ALA-PDT decreased the ability of the cell growth and migration ability,and its mechanism, was by the regulatoryof EGFR/PI3K/AKT signal pathway. Then the study combined ALA-PDT with EGFR inhibitors AG1478, and PI3K/AKT-inhibitor LY294002, and revealed changes of growth and migration on the Eca-109 cell, changes of gene and protein expression on EGFR/PI3K/AKT signaling pathway. Based on the mechanism, the purpose of this study is to develop a new treatment using ALA-PDT combined with small molecule inhibitors of signal channel. This new anticancer therapeutics might provide a new modality for clinical practice. Maybe it can change the defects of PDT, such as easy to relapse, poor long-term curative effect, improve the effect of tumor therapy, and can promote the development of photodynamic therapy and its application on cancer therapy.Objective:1. The inhibition of different doses ALA-PDT on Eca-109 cell growth;2. The inhibition of ALA-PDTon Eca-109 cell migration in vitro;3. The inhibition of ALA-PDT on nude mice tumor growth;4. The change of gene and protein expression in EGFR/PI3K/AKT signaling pathway after ALA-PDT on the nude mice tumor tissue;5. The inhibition of EGFR tyrosine kinase inhibitor AG1478 combined ALA-PDT on Eca-109 cell growth;6. The inhibition of PI3K/AKT inhibitor LY294002 combined ALA-PDT on Eca-109 cell growth;7. The inhibition of EGFR tyrosine kinase inhibitor AG1478 combined ALA-PDT on Eca-109 cell migration in vitro;8. The inhibition of PI3K/AKT inhibitor LY294002 combined ALA-PDT on Eca-109 cell migration in vitro;9. Identify the change of the gene and protein expression in EGFR/PI3K/AKT signaling pathway after EGFR inhibitor AG1478 combined ALA-PDT on Eca-109 cell;10. Identify the change of the gene and protein expression in EGFR/PI3K/AKT signaling pathway after PI3K/AKT inhibitors LY294002 combined ALA-PDT on Eca-109 cell.Contents and methods:1. The impact of ALA-PDT on Eca-109 cell growth1)The inhibition of Eca-109 cell proliferation after ALA-PDTMTT method is used to test the inhibition of Eca-109 cell proliferation by different doses of ALA photosensitizer mediated PDT.2) The effects of Eca-109 cell migration after ALA-PDT in vitro.Transwell experiment revealed the change of Eca-109 cell migration movement after ALA-PDT in vitro.2. The impact of ALA-PDT on nude mice tumor growth and its molecular mechanism1) Construction of GFP-Eca-109 cell lineThe lentivirus carrying GFP transfected Eca-109 cells, and had the transfection efficiency of more than 90%, by flow cytometry instrument detecting and CELLQUEST software, testing and analysising its transfection efficiency. Then photographed with fluorescence microscope.2) Construction of tumor-burdened nude mice modelThe Eca-109 cells, GFP-Eca-109 cells were respectively subcutaneous injected at different nude mice posterior hip skin. The mice were raised for 1 to 2 weeks. The esophageal nude mice model was established and used in subsequent experiment.3) The fluorescence imagingTake fluorescence imaging of the tumor-burdened nude mice before and after ALA-PDT at different time to observe the tumor shape, size, and the presence of metastases tumor.4) The inhibition of ALA-PDT on nude mice tumor growthThe size of the mice tumor was measured before and after ALA-PDT at different time, calculated the tumor volume, and revealed the inhibition of ALA-PDT on nude mice tumor growth.5) The change of EGFR/PI3K/AKT gene expression of ALA-PDT on Eca-109-burdened nude mice.The gene expression in mice tumor tissues was detected after ALA-PDT by real-time Q-PCR, and was compared to control group.6) The change of EGFR/PI3K/AKT protein expression of ALA-PDT on Eca-109-burdened nude mice.The protein expression in mice tumor tissues was detected after ALA-PDT by Western blot, andwas compared to control group.3. The impact of ALA-PDT combination with AG1478 and LY294002 on Eca-109 cell migration ability and its molecular mechanism1) The inhibition of ALA-PDT combined with AG1478 on Eca-109 cell proliferationMTT method was used to test the inhibition on the Eca-109 cell proliferation by ALA-PDT combined with different concentrations of EGFR inhibitor AG1478. Selected the best dose of AG1478 combined with PDT.2) The inhibition of ALA-PDT combined with LY294002 on Eca-109 cell proliferationMTT method was used to test the inhibition on the Eca-109 cell proliferation by ALA-PDT combined with different concentrations of PI3K/AKT inhibitor LY294002. Selected the best dose of AG1478 combined with PDT.3) The change of Eca-109 cell migration after ALA-PDT combined AG1478 in vitroTranswell experiment revealed the change of Eca-109 cell migration movement after ALA-PDT combined AG1478 in vitro.4) The change of Eca-109 cell migration after ALA-PDT combined LY294002 in vitroTranswell experiment revealed the change of Eca-109 cell migration movement after ALA-PDT combined LY294002 in vitro.5) The change of EGFR/PI3K/AKT gene expression of ALA-PDT combined AG1478 or LY294002 on Eca-109-burdened nude mice.The gene expression in mice tumor tissues was detected after ALA-PDT combined AG1478 or LY294002 by real-time Q-PCR, and was compared to control group.6) The change of EGFR/PI3K/AKT protein expression of ALA-PDT combined AG1478 or LY294002 on Eca-109-burdened nude mice.The protein expression in mice tumor tissues was detected after ALA-PDT combined AG1478 or LY294002 by Western blot, and was compared to control group.Results:1. Different concentration of photosensitizer ALA mediated PDT of esophageal cancer cells in vitro had different growth inhibition rate, the difference showed statistical significance (P<0.05). In a certain range, the higher of ALA photosensitizer concentration, the great inhibition.2. ALA-PDT could decrease the Eca-109 cells migration ability in vitro, the difference was statistical significance (P<0.05)3. Tumor growth of carrying GFP nude mice was inhibited 8 days to 36 days after ALA-PDT, when compared to the blank control group and GFP group, the difference was statistical significance (P<0.05), and the difference was no statistical significance between the blank control group and the GFP group (P>0.05)4. The gene and protein expression on the EGFR/PI3K/AKT pathway of mice tumor tissue was downregulated after ALA-PDT. This revealed the molecular mechenism of ALA-PDT decreasing tumor ability of growth and metastasis.5. Different concentrations of EGFR inhibitor AG1478 had different inhibition to Eca-109 cell growth, the difference was statistical significance(P<0.05).When 0.5 mmol/L ALA-PDT combined different concentrations of AG1478 for Eca-109 cell growth, the inhibition rate was different, the difference was statistical significance between groups (P<0.05)6. Different concentrations of PI3K/AKT inhibitor LY294002 had different inhibition to Eca-109 cell growth, the difference was statistical significance(P<0.05). When 0.5 mmol/L ALA-PDT combined different concentrations of LY294002 for Eca-109 cell growth, the inhibition rate was different, the difference was statistical significance (P<0.05)7.0.5 mmol/L of ALA-PDT combined 20 umol/L AG1478 could reduce Eca-109 migration ability of cells in vitro.8.0.5 mmol/L of ALA-PDT combined 20 umol/L LY294002 could reduce Eca-109 migration ability of cells in vitro.9. EGFR gene and protein in Eca-109 cells decreased after ALA-PDT combined AG1478, the difference was statistical significance compared to control group (P<0.05)10. AKT gene and protein in Eca-109 cells decreased after ALA-PDT combined LY294002, the difference was statistical significance compared to control group (P<0.05)Conclusion:The study first revealed that ALA-PDT could inhibit the growth and migration ability of Eca-109 cell, and then successfully constructed GFP-Eca109 burdened nude mice model, which were treated with ALA-PDT. The results showed that ALA-PDT could decrease the nude mouse tumor growth, and downregulated the expression of EGFR/PI3K/AKT signal pathway genes and proterns.This study further applicated a new method that ALA-PDT respectively combined with EGFR inhibitor AG1478, PI3K/AKT inhibitor LY294002, which could further reduce the growth and migration ability of the Eca-109 cell in vitro and down-regulate the gene and protein expression of EGFR/PI3K/AKT signaling pathway.To sum up, this study revealed the molecular mechanism of ALA-PDT and developed a new photodynamic therapy, by combining with EGFR/PI3K/AKT signaling pathway small molecule inhibitors, which enhanced the lethality of ALA-PDT, helped to promote new modality application of therapy for better effect in the clinical practice. |
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