Study On Mitochondria Dysfunction In Laryngeal Squamous Carcinoma Cell Lines And MtDNA Mutation In Laryngeal Carcinoma Tissue

Laryngeal carcinoma is one of cancer with high morbidity in Jilin province, especially superglottic laryngeal carcinoma is the most common type, which was examined with advantage stage generally. Laryngeal carcinoma in I stage has 90% 5-year survival rate,whereas in IV stage has less than 50% 5-year survival rate.Somatic mutation of mitochondria DNA was found in various tumors and was considered a biomarker of early detection. Carcinogenesis of larynx is related to long term smoking history. Carcinogen in tobacco include ROS may cause oxidative injury of mtDNA, which is more severe than that of nuclear DNA. mtDNA has an inefficient repair system and lack of protective histone. Mutation rate of mtDNA is 10 to 1000 higher than that of nDNA.Many evidence has showed mtDNA mutation is associated with various human tumor. It is still unclear that the mechanism of high mutation rate of mitochondia and if dysfunction of mitchondria will lead to carcinogenesis. Impaired apoptosis is a central characteristic of neoplastic and malignant transformation. In addition, chemotherapy and radiation therapy act primarily by inducing apoptosis, therefore defects in the apoptotic pathway can contribute to treatment resistance. Understanding the mechanisms by which tumor cells resist apoptosis at the molecular level will provide deeper insight about carcinogenesis, and may lead to new therapeutic approaches based on modulation of apoptotic sensitivity.We studied as follows:1. Mitochondrial membrane potential decreases after treatment with valinomycin Three laryngeal cell lines(JHU-011, 012, and 019) were given byHead and Neck research center, Johns Hopkins university. HL-60 is a control leukemia cell line(HL- 60) were treated with valinomycin and harvested at 15 mins, 30 mins, 1hour, 2hours, 4 hours, 1days, 2days, 3days. Valinomycin is K+ ionophore which can uncouple oxidative phosphonation and collapse mitochondria membrane potential(MMP). Damaged MMP was the earliest event of apoptosis. Once MMP collapse, is irreversible in apoptosis pathway. Untreated cells were used as controls. We used JC-1 to monitor mitochondrial membrane potential (ΔΨm) in laryngeal carcinoma cell lines and leukemia cell line. When cells were treated by valinomycin, the MMP of three head neck cell lines decreased gradually, but stabilized after 1 day of treatment. After 24 hours, the MMP of 011, 012 and 019 was 0.16, 0.28 and 0.05, respectively. The response of control HL60 cells was more rapid and complete, with significantly decreased MMPs after only 10 minutes treatment. The MMP nadir was 0.003 after only 24 hours of treatment. The effect of valinomycin was not related to the dose used. only 24 hours of treatment.2. laryngeal cell lines are resistant to mitochondrial depolarization induced apoptosis and Impaired cytochrome c release in HNSCC cell linesValinomycin treat that 4 kinds of cell by the same way。The MMP of laryngeal cancer cell lines and leukemia cell line were decreased at different levels after treatment with the mitochondrial depolarizing reagent. We therefore wanted to investigate the apoptotic status of valinomycin treated cells to detect any corresponding changes after treatment. All cells was treated and harvested by 4hours,1 day, 2day,3 days and 4days。Apoptosis is characterized by a variety of morphological features such as loss of membrane asymmetry and attachment, condensation of the cytoplasm and nucleus, and internucleosomal cleavage of DNA. One of the earliest indications of apoptosis is the translocation of the membrane phospholipid phosphatidylserine (PS) from the inner to the outer leaflet of the plasma membrane. Once exposed to the extracellular environment, binding sites on PS become available for Annexin V, which a phospholipid binding protein with a high affinity for PS. As such, Annexin V can be conjugated a fluorochrome such as FITC and used for flow cytometric identification of cells in the early stages of apoptosis. Because PS translocation also occurs during necrosis, Annexin V is not an absolute marker of apoptosis. Therefore, we used Annexin V-FITC in conjunction with 7-amino-actinomysin (7-AAD), which binds to nucleic acids, as a marker of later stages of apoptosis or necrosis. All cells were treated at 4hours, 1day, 2days, 3days, 4 days, collected after treatment, stained with Annexin V-FITC and 7AAD and analyzed by flow cytometry. As displayed in figure 3-4, cell death of three head and neck cancer lines changed only minimally even after 4 days of treatment with valinomycin. The overall rate of cell death three head and neck cell lines at any treatment time point was close to 5%, similar to that of the untreated controls. As to HL-60, cell death decreased dramatically after 1 day's treatment, and after 4 days, 87.8% necrotic rate。Since MMP of all laryngeal carcinoma cell lines had significantly decreased after treatment with valinomycin at 24 hours, ordinarily the mitochondrial transition pore would open and release mediators of the apoptotic cascade, including cytochrome c, which is stored in the intermembrane space between the outer and inter mitochondrial membrane. The detection of cytochrome c release into the cytosol is an early event in the activation of the intrinsic apoptotic pathway, therefore, we assayed for cytosolic release of cytochrome c to identify the compartment in which the apoptotic pathway was abrogated in laryngeal cells. We collected the cells after treatment by valinomycin at 12 hours, 1 day, 36 hours, 2days, 60 hours and 3days for the HL-60 control cell line, and 1 day, 2 days, 3 days, and 4 days for the laryngeal carcinoma cell lines. Cytosolic proteins were extracted as described above, and the cytochrome c content of this cellular compartment was determined by western blot. There was no cytochrome c detected in all three laryngeal carcinoma cell lines, in contrast to the strong cytochrome c band that was found after 24 hours of valinomycin treatment in the HL-60 cell line. These findings further support the argument for an intrinsic mitochondrial defect that impairs/abrogates depolarization induced mitochondrial membrane permeabilization pore opening and apoptosis in laryngeal cell lines.3.High mutation rate of mtDNA D-loop in laryngeal cancer tissue22 primary laryngeal carcinoma tissue and matched peripheral normal tissue and complete normal laryngeal pharynx and larynx tissue were selected for DNA direct sequencing. We found 68% laryngeal cancer were detected D-loop mutation,whereas 32% mutation were detected in matched tissue. mtDNA mutation are all basen substitution and insertion. Some mutations were polymorphism, some were found in many patients which imply they are potential biomarker of laryngeal carcinoma.We found that laryngeal cancer cell lines were resistant to its toxic effect compared with a control leukemia cell line. This suggests that mitochondrial dysfunction of cancer is a potential mechanism of carcinogenesis and development of metastasis of this type of cancer.we have demonstrated that laryngeal cell lines are defective in mitochondrial membrane depolarization induced apoptosis. This data's novel in that they relate the state of mitochondrial polarization to this defect. The dissipation of MMP induced by valinomycin treatment in laryngeal caner cell line is not a signal to open the MPT pore and induce apoptosis in these cell lines. This may explain that limited and reversibly opened MPT pore were not sufficient to open mitochondria inner membrane and release cytochrome c into the cytosol.The main function of mitochondria is generate energy by OXPHS, and produce ROS which may lead to oxidative damage of mtDNA. Dysfunction of mytochonria can promote ROS increase.aggravage mtDNA mutation. mtDNA mutation in laryngeal cancer will aggravate mitochondria defect and make cell resistance apoptosis. Due to cell's high copy number and high mutation rate, mtDNA Mutation were often detected in early stage in various tumor, The examine of mtDNA mutation is more sensititive and feasible than that of nDNA. The detection of mtDNA mutation may be the most potential biomarker for early diagnosis of laryngeal cancer.