Influence Of 5-aminolevulinic Acid-mediated Photodynamic Therapy On EGFR Signaling Pathway Of Esophageal Cancer

Background and Objection:Esophageal cancer is one of the most common cancers, with serious threat to human health. Esophageal cancer is at this moment the eighth cause of disease and the sixth cause of death for cancer worldwide.As one of the common malignant in China, esophageal cancer is at this moment the fourth cause death. More than 90% of esophageal cancer in China is squamous cell carcinoma. Different stages takes on various treatments. Surgery therapy is the treatment of choice for the early esophageal carcinoma, however the recrudescent rate is high.lt is urgent to find a more effective non-surgical treatment of esophageal cancer for the patients who are diagnosed advanced esophageal carcinoma and lose the opportunity of surgery. With the development of photosensitizer and illumination system,PDT has made considerable progress in the treatment of esophageal cancer, especially in early esophageal cancer and precancerous lesions such as Barret's esophagus.Evenly patients with early stage can be cured via PDT. For advanced cancer patients PDT can not only significantly improve the clinical symptoms (such as food obstruction),but also prolong the median survival period.PDT has been proven to be a promising treatment for esophageal cancer.Photodynamic Therapy(PDT)is a kind of photochemistry therapy.Its mechanism is that photosensitizer molecules produce some radical oxygen species(ROS) after light,then make damage to cell structure and function and lead to apotosis. It is a unique cancer treatment.Laser, photosensitizer and molecular oxygen compose of the three elements of PDT,and one single is harmless to cell. However several elements combined will produce a strong cytotoxic. Photodynamic reaction produce reactive oxygen species and make selective tissue damage to the target. Unlike traditional chemotherapy, surgical therapy and radiotherapy,PDT has several potential advantages:relatively non-invasive treatment;locating the target tumor accurately;repeated administration without limit dose;no scars or small in the whole process; relatively easy operation and fewer side effects. Because of tumor tissue selective retention of photosensitizer,this characteristic can make PDT reaction confine to tumor tissue and make no damage to normal tissue around the tumor tissue, so it can keep the integrity and continuity of diseased organs.In recent years, with the depth studing of PDT in cancer clinical treatment, photodynamic therapy may be following the surgery, radiotherapy and chemotherapy and then the fourth special type of cancer treatment.The protein encoded by epidermal growth factor receptor (EGFR) gene is a transmembrane glycoprotein, which is a member of the protein kinase superfamily. It consists of 1186 amino acid residues, a molecular weight of 170kD transmembrane glycoprotein. With tyrosine kinase activity,EGFR is mainly located in the cytoplasmic membrane, and belongs to typeⅠreceptor subfamily of tyrosine kinase. This protein is a receptor for the epidermal growth factor (EGF) family. EGFR is a protein that binds to EGF, activating the intracellular downstream signaling pathways.Activation of these pathways will eventually lead to cell proliferation, apoptosis escaping and invasion and metastasis of cancer cells. Overexpression of EGFR delivers continuely proliferation signal to cells, and interferes with the normal regulation state of cell differentiation, and leading to continued proliferation and the occurrence of malignant transformation. High expression of EGFR can promote tumor cell proliferation, angiogenesis, adhesion, invasion and metastasis.the PI3K/Akt pathway is very important in these signaling pathways. EGFR binds to EGF, activating the intracellular downstream PI3K/Akt signaling pathway, playing its regulating cell proliferation, anti-apoptosis and so on. EGFR expresses stably in a variety of epithelial tissue, and mesenchymal and neurogenic tissue. EGFR also expresses highly in solid tumors in different organs, such as head and neck cancer, ovarian cancer, cervical cancer, bladder cancer and esophageal cancer and so on. EGFR expresses highly in tumors and plays an important role in tumor cell growth, differentiation,and these characteristics make EGFR as a promising target for cancer diagnosis and treatment. As the increasing of degree of malignancy, invasion and metastasis of esophageal cancer, EGFR protein expression increases gradually; EGFR can be used to monitor the occurrence of early esophageal cancer,and as a valuable indicator of predicting prognosis metastatic potential,and thus proposing a new therapeutic strategy.PI3K/Akt signaling pathway associated with tumor has get much attention in recent years. PI3K/Akt signaling pathway results in malignant transformation,and is closely related with tumor cell migration, adhesion and extracellular matrix degradation. PI3K/Akt signaling pathway can inhibit apoptosis induced by a variety of stimulators and promote cell cycle progression, then promote cell survival and proliferation.At the same time it is also involved in angiogenesis, transferring cancer invasion signaling mediated by integrin,which plays an important role in tumor formation,proliferation and metastasis. As one of the major downstream pathways of EGFR signaling pathway, PI3K/Akt pathway is considered as the primary cancer survival pathway, or anti-apoptosis pathway.PI3K contacts with receptor via SH2 region of regulatory subunit, leading to activation of the heterogeneity of the catalytic subunit. With the activation of PI3K, phosphatidylinositol diphosphate -PI3K lipid substrates in the inner surface of plasma membrane change into the second signal phosphatidylinositol.Phosphatidylinositol triphosphate binds with protein with PH region, leading to changes in the conformation of these proteins. Phosphorylated PH region is found in most protein,and promotes their downstream molecules Akt protein kinase B phosphorylation,and maximizes the activation of Akt.As an important protein kinase in signal transduction pathway, Akt is the target protein of PI3K downstream,and it is the core of PI3K/Akt signal transduction pathway,and its continued activation is closely related with tumor development.Akt,which is activated by PI3K,can activate or inhibit its downstream target protein Bad, Caspase9, NF-κB, Forkhead, mTOR, Par-4, P21 etc by phosphorylation, and regulate cell proliferation, differentiation and apoptosis. As an important anti-apoptotic intracellular signaling pathway, PI3K/Akt activity is affected by many factors, including PTEN making PIPS change into PIP2 by dephosphorylation,and PIP2 is a major negative regulator of PI3K/Akt signaling pathway.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, and has no light dynamic effect in itself,thus the induced protoporphyrinⅨ(PpⅨ) can be employed as a photosensitizer in PDT through a series of enzymes in the body.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, We investigated some gene and protein changes of EGFR/PI3K/Akt pathway to understand the mechanism of ALA-PDT for esophageal cancer; We investigated the relative the cell clony formation rate difference between the control group and the PDT group to understand the proliferation of ALA-PDT for esophageal cancer.Metheds:Chapter 1 Influence of 5-aminolevulinic acid-mediated photodynamic therapy on proliferation of esophageal cancerUnderstanding the cell clony formation and assaying: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 6h in dark, and irradiated with a light dose of 30 J/cm2(200 mW/cm2,150 s) under the 630 nm laser system. Using the two groups of cells of logarithmic growth phase, plating in 6 well plate,and100 per hole,each hole 3 comple..Arming cells to spread evenly.2w later cell clony formation have been observd,and disposing medium. The cell can be conventional fixed, Giemsa stained, cleaned. Counting the number of the cell clony formation which of the cell number are greater than or equal to 50,averaging the number of the cell clony formation, divided by 100,and calculating the number of the cell clony formation. The cell clony formation rate(%)=the cell clony formation number/seeded cells number.Statistical analysis:All datas were analysised by SPSS 13.0 statistical softwire. The results of experiment were expressed by the way of mean±SD. The relative the cell clony formation rate difference between the control group and the PDT group were analysised using independent t test. P-Values were considered to be significant at<0.05Chapter 2 Influence of 5-aminolevulinic acid-mediated photodynamic therapy on EGFR signaling pathway of esophageal cancerThe gene expression changes of EGFR,PI3K and Akt 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 were incubated with ALA to the final concentration of 0.25mM for 6h 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 EGFR,PI3K,Akt andβ-actin on ABI 7500 PCR detector using QuantiTect SYBR Green kits.The protein expression changes of EGFR,PI3K,Akt and p-Akt: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 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 EGFR,PI3K,Akt and p-Akt.Statistical analysis:All datas were analysised by SPSS 13.0 statistical software. The results of experiment were expressed by the way of mean±SD. The relative gene and protein expression difference between the control group and the PDT group were analysised using independent t test. P-Values were considered to be significant at <0.05.Conclutions:Chapter 1 Influence of 5-aminolevulinic acid-mediated photodynamic therapy on proliferation of esophageal cancer2w later the cell clony formation number in the control group was 66,56,53/well, and the colony-forming efficiency (CFE) was 58.33±6.81%.The cell clony formation number in the PDT group was 31,29,27/well, and the colony-forming efficiency (CFE) was 29.00±2.00%.The colony-forming efficiency (CFE) after ALA-PDT reduced,and the difference contrast to the control group was significant (t=7.161, P=0.002)The results indicated ALA-PDT significantly inhibited the proliferation of esophageal cancer cells.Chapter 2 Influence of 5-aminolevulinic acid-mediated photodynamic therapy on EGFR signaling pathway of esophageal cancerl.The expression of EGFR,PI3K and Akt genes were all significant down regulation at 24 h after ALA-PDT in the Eca-109 cell (t=9.623, P=0.011; t=27.392, P=0.001; t=28.315, P=0.001).2.The expression of EGFR,PI3K and p-Akt proteins were significant down regulation at 24 h psot-PDT in the Eca-109 cell, proteins were significant down regulation at 24h psot-PDT in the Eca-109 cell(t=5.323, P=0.006; t=4.73, P=0.009; t=7.871, P=0.001). The expression of Akt protein was slightly down-regulation, but the difference contrast to the control group was not significant (t=0.109, P=0.918).The results indicated EGFR/P13K/Akt was one of the cell apoptosis pathway in the esophageal cancer cells of 5-aminolevulinic acid-mediated photodynamic therapy.