| BackgroundNasopharyngeal carcinoma(NPC) is one of common malignant tumor in southern China and southeast Asian countries.Etiology research has shown that NPC was associated with epstein-barr virus infection, genetic susceptibility, eating habits and some physical and chemical environment factors. The occurrence of NPC is a complex process which involved in many genes and muti-step,among them oncogene activation and tumor-suppressor gene inactivation is an important cause of tumorigenesis.NPC has many differentiation types,commonly been found in the pharyngeal recess,located in the inner sidewall of the inner corner of Pharyngeal port. In most countries, the incidence rate of NPC is low,less than1/100, OOO.However, in southern China, northern Africa and Alaska, the incidence rate is very high, particularly prevalent in Guangdong, China and the Inuit in Alaska. It has been reported that the incidence rate of NPC in Guangdong male and female was20to30/100000and15to20/100000respectively.NPC can occur at any age, peak age30to50years old, the minimum age of3, maximum90years.The main treatment of NPC is radiotherapy and chemotherapy in the clinic,with intensity modulated radiation therapy (IMRT) and image-guided radiotherapy (IGRT) technology widely used and improved, The curative effect of NPC compared with a decade ago has been improved to some extent, but5year overall survival (OS) was40-70%, and it has not yet achieved a satisfactory result.The main factor to curative effect is owning to the adverse reactions of radiotherapy and chemotherapy and multidrug resistance (MDR) phenomenon.MDR is the emergence of drug-resistant of an anticancer drug to tumor cells,at the same time producing cross-resistance to other anti-tumor drugs which have different mechanism and structure.Currently,it is very urgent to find a new generation of anticancer drugs which is efficient, radiosensitive and has litter adverse reactions.As a broad-spectrum anti-tumor molecule,Tea polyphenols (EGCG) can inhibit the proliferation of a variety of tumors,but its detailed biological mechanism is not been researched widely,especially the report about EGCG anti-NPC role is few.Although EGCG induces NPC HNE2to apoptosis by targeting mitochondrial signal transduction pathway, but the detailed biological mechanism of this process is still not clear.The EGCG gene methylation in the process of anti-NPC result of epigenetic changes and signaling pathways and many other issues still need to be further researched.Human telomerase reverse transcriptase(hTERT) is a determining factor to telomerase activation, the gene is associated with tumor formation and the degree of malignancy, and its expression is regulated by a variety of factors (23). C-myc not only is a gene can be translocation, a kind of adjustable gene regulation with variety of material, but also a kind of gene can make unlimited cell proliferation, and biochemical functions, promote cell division. C-myc involved in cell wither, associated with a wide variety of tumor development.The tumor suppressor gene RECK can inhibit matrix metalloproteinase (MMPs), thereby inhibiting tumor invasion and metastasis.Many studies have shown that the over-expression of MMPs is closely related to the degree of malignancy. By inhibiting the biological activity of the MMPs,RECK can hinder tumor metastasis. As a natural compound which is non-toxic, efficient and has dual role with both anti-tumor and immunomodulatory, EGCG is significantly different from the commonly used anti-tumor chemical synthetic drugs, having great significance in the development of new anticancer drugs,this is also the advantage that synthetic drugs don’t have.Therefore, as an efficient and low toxicity of anticancer drugs, EGCG is worthy further researching and developing, and has broad market prospects.PurposeIN this study,through the interaction of EGCG/PN with NPC cell lines or CSC in vitro culture,through a series of cell biology and molecular biology detection methods, the mechanism of EGCG/PN anti-NPC provides a theoretical basis for research and development of a new generation of anti-cancer targeted drug.EGCG/PN is expected to develop into a new type of anti-cancer drugs or health food, having important clinical and social significance to reduce patient suffering and economic burden and alleviate the side effects of chemical synthetic drugs.This project is involved in multidisciplinary field of research in medicine, oncology, cell biology, molecular biology, and has a positive role to promote the penetration of the integration between the various disciplines, and is in line with the trend of scientific development.MethodsWe use vitro cell culture, through CCK-8experiment, DNA gradient, apoptosis detection, methylation Western blot and other cell and molecular biology techniques and research methods to study the biological and radiosensitization mechanisms of EGCG/PN anti-NPC systematically.Innovations of the study The occurrence of tumor is a complex process which involved in many genes and muti-step.The project is based on the action of the EGCG, on the one hand,EGCG affects the key link of the NPC cell cycle, inhibits cancer cell proliferation,and induces programmed cell death, so as to achieving the purpose of anti-NPC cell proliferation; On the other hand, EGCG tries to regulate certain gene "off" and "open", namely the proto-oncogene inactivation and/or activation of the tumor suppressor gene and effectively controls the expression of tumor-related genes.The launching of this project will provide new ideas for new natural medicine EGCG in the prevention and treatment of NPC.Experimental program[1].The methods of cell culture, cryopreservation, recovery and morphological observationWe adopt conventional cell culture, cryopreservation and recovery methods to cultivate CNE2cells,the medium is DMEM,the morphological changes of the cells were observed and photographed under inverted microscope.[2].CCK-8experiment determine EGCG on the CNE2cell growth inhibition rateTaken the logarithmic growth phase CNE2cell lines (1.0×105/cell), packed in96-well plates for24h,then EGCG was added to make the final concentration as25,50,100,200,400μg/ml,each concentration had three parallel holes, saline was to as a blank control. Incubation was continued for48h, then add20μl WST-8solution (5mg/ml) and incubated for anther4h,200μl DMSO was added before the test, then fully shake. Enzyme immunoassay analyzer detect the value of the OD of each hole under570nm.[3]DNA ladder Electrophoresis detect the apoptotic fragments of DNAA.Different concentrations of EGCG processing CNE2cellsTaken the logarithmic growth phase CNE2cells,experimental group was added EGCG to make the final concentration as25,50,100,200,300,400μg/ml, control group added saline, cultured48h. Cells DNA was extracted then put it on1%agarose gel electrophoresis, automatically gel imaging system was used to observe and take a photo.B.Different duration of action of EGCG treat CNE2cellsIN the experimental group,we used EGCG(final concentration200μg/ml) to process CNE2cells6,12,24,36,48,72h, to the control group we joined saline. Cells DNA was extracted then put it on1%agarose gel electrophoresis, automatically gel imaging system was used to observe and take a photo. Observe whether there are characteristic DNA "ladder "strip.[4]Flow cytometry test cell apoptosis and cell cycleA.Different concentrations of EGCG treat CNE2line cellsTaken the logarithmic growth phase CNE2cells,experimental group was added EGCG to make the final concentration as25,50,100,200,300,400μg/ml, control group was added saline, cultured48h.Cells was fixed and digested,then added1.5ml PI-propidium iodide,fully shake,4C dark stained for30min. Again filtered with200mesh nylon net. At last,we used Row cytometry to test cell apoptosis and cell cycle.B.Different duration of action of EGCG treat CNE2cellsIN experimental group, we used EGCG(final concentration200μg/ml) to process CNE2cells6,12,24,36,48,72h, the control group was joined saline.The following steps above.[5] To detect the methylation of the hTERT and RECK1×106CNE2cells was treated by100μg/mL EGCG for0,1.5,3,6,12,24and36h, cells were collected, and cracked, the cells was extracted DNA. The hTERT and RECK methylation-specific primers were designed to detect the methylation level of these two genes by methylation-specific PCR (MCP), and compared with the control group.[6]To detect expression level of hTERT and C-Myc1×106CNE2cells was treated by100μg/mL EGCG for0,1.5,3,6,12,24and36h, cells were collected, and cracked.RNA and protein were extracted respectively. Specific expression levels of both mRNA and protein were detected by real-time PCR and Western, and compared with the control group.[7]Effect of CNE1/CNE2cell cycle and apoptosis for EGCG in combination with radiotherapy.To detecte CNE1/CNE2cloning rates and cell cycle change by EGCG and ray, compared with the control group.[8] Western blot method to detect Akt expression results.By EGCG and ray detection CNE1CNE2/Akt expression, compared with the control group.[9] EGCG adjuvant radiotherapy for nasopharyngeal carcinoma tumor radiosensitivity. This series of experiments including stem cell-like population of nasopharyngeal carcinoma cells culture and detecte its markers, CCK-8test、cell cycle、cloning rates, detecting expression of Akt, NF-κB, MnSOD (SOD2) by Q-PCR method and Westernblot tests.[10] Parthenolide (PN) in combination with radiotherapy for stem cell-like population of nasopharyngeal carcinoma cells. This series of experiments including confocal experiment, CCK-8test, cloning rates of stem cell-like population of nasopharyngeal carcinoma cells, detecting Akt, NF-κB, MnSOD (SOD2) expression by Q-PCR and Westernblot tests.ResultEGCG has a significant inhibitory effect on the proliferation of NPC CNE-2cell (P<0.05), and showed time and drug concentration-dependent manner. EGCG had significant time and concentration-dependent inhibitory effects on CNE-2cell proliferation。We arranged the four different time points(12hours,24hours,48hours,72hours) to different concentrations of EGCG (50ug/mL and100ug/mL and150ug/mL,200ug/mL) as the experimental group,0ug/mL EGCG as the control group.At the same point in time, along with EGCG concentration increased, the inhibition rate of CNE-2gradually increased (P<0.05), in the same concentration, with time,the inhibition rate of CNE-2gradually increased (P<0.05). The performance showed time and concentration lazy.In low concentration group (100ug/mL), cell cycle arrest presented a time-dependent,with the increasing concentration and the prolonging time, cell cycle arrest was not obvious, but the rate of apoptosis increased. The same concentration (100ug/mL), with the prolonged duration of action, the G0/G1phase gradually increased, the S phase fraction gradually declined(compared between groups P<0.002), the performance was a time-dependent manner.EGCG promote apoptosis of NPC CNE-2cells, with the prolonged duration,the apoptosis rate increased (P<0.001), EGCG induced apoptosis in CNE-2cells in proportion to the exposure time (P<0.001). it was a time-dependent manner.The higher EGCG concentrations (150ug/ml,200ug/ml), and a longer duration of action, more obvious apoptosis is.This phenomenon was time-dependent(various experimental groups were compared respectively, P (0.01). This suggests that EGCG induce apoptosis of NPC CNE-2cell lines, and it is time-dependent.EGCG lowered NPC CNE-2cell lines hTERT mRNA (P<0.001) and its protein expression, and both a positive correlation. EGCG downregulated the expression of hTERT mRNA and protein in CNE-2cells (P<0.001), which were positively correlated.This experiment were designed with the same time (48hours) at different concentrations (Oug/mL,50ug/mL,100ug/mL,150ug/mL,200ug/mL) to detect the effect of EGCG on NPC CNE-2cell lines hTERT protein and expression of c-Myc.The Oug/mL EGCG were designed as the control group, GAPDH were used as internal reference. We used WesternBlot method to detect the relative level of protein expression.Compared with the control group, with EGCG concentration dose, the expression of hTERT and c-Myc showed a downward trend,this phenomenon in the high-dose group (200ug/mL) is more obvious. The downward trend of hTERT and c-Myc is consistent.EGCG could inhibit CNE1/CNE2cloning, at the same dose of EGCG, under the action of CNE1/CNE2cloning rates reduced gradually along with the increasing doses of radiation, after6gy, the experimental group (R+EGCG) cloning rates decreased significantly in the pure irradiation group (P<0.05), with statistical significance.The G2/M phase percentage is higher, indicating a G2/M phase retardation, pure rays than simple drug group of G2/M phase retardation is more obvious, the coalition has synergy from experimental group (EGCG+R) compared with control group.EGCG inhibits Akt expression, and radiation can inhibit Akt expression too, a combination of both Akt protein expression after lower than simple radiation group. Groups of Akt protein relative difference was statistically significant, the gray analysis shows that EGCG can enhance the role of the radiation, but EGCG is synergy to radiation or sensitization effect, its mechanism is not clear.EGCG and ray can make nasopharyngeal tumor cloning rates drops, cloning of inhibition on stem cell-like population of nasopharyngeal carcinoma cell with ray was dose dependent (P<0.05). EGCG concentration at the same time, with the increase of radiation dose, stem cell-like population of nasopharyngeal carcinoma cell cloning rates was dropped. significantly, the experimental group than simple ray group decreased significantly (P<0.05), with statistical significance. EGCG can increase inhibitory effect with ray on stem cell-like population of nasopharyngeal carcinoma cell.NF-KB, Akt, SOD2expression of EGCG+radiotherapy group was not significantly lower than the pure radiotherapy group and drug group, there is no statistical significance (p<0.05)。 we may think so, EGCG adjuvant radiotherapy, inhibition on stem cell-like population on nasopharyngeal carcinoma cell of nasopharyngeal than pure radiation, they may be a kind of synergy. There are, of course, through other possible signaling pathways of radiotherapy sensitization mechanism.PN can downregulate β-Catenin Expression of CNE1/CNE2tumor spheres, suggesting that the sensitization of radiotherapy may also be associated with Wnt/β-catenin signal pathway.ConclusionEGCG inhibit proliferation of NPC CNE-2cell lines and promote apoptosis, its possible cause is that EGCG inhibit the transcription of hTERT mRNA by down-regulating the expression of C-Myc protein in NPC CNE-2cell lines, make telomerase activity declined lastly.EGCG also has inhibition proliferation of stem cell-like population of nasopharyngeal carcinoma cell and promote apoptosis. It can increase the radiation damage of stem cell-like population of nasopharyngeal carcinoma cell, its possible mechanism is reduce the expression of Akt, which in turn by inhibiting its downstream substrate phosphorylation and biological effects, play a promoting apoptosis, and other functions. But the radiosensitization of EGCG is not clear.PN inhibit proliferation of stem cell-like population of nasopharyngeal carcinoma cell and promote apoptosis.it can obviously increase the radiation damage of stem cell-like population of nasopharyngeal carcinoma cell, its mechanism is by cutting the NF-KB and its downstream factor MnSOD (SOD2) expression.Not only EGCG but also PN have stronger inhibition proliferation of stem cell-like population of nasopharyngeal carcinoma cell and promote apoptosis. As natural compounds, their mechanisms of resistance to nasopharyngeal carcinoma each are not identical, but can be developed into the auxiliary therapeutic agents in the treatment of nasopharyngeal carcinoma, and non-toxic or low toxicity. PN could be a treatment of nasopharyngeal carcinoma radiosensitization agent and multiple targets antitumor medicine. |
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