HSP70 Regulating The Effects Of Rapamycin On The Growth Inhibition Of Human Nasopharyngeal Carcinoma Cells

NPC (Nasopharyngeal Carcinoma, NPC) is one of common malignant tumors in south of China, the incidence rate ranges from 10/1 million people to 50/1 million people.In regard to the mortality rate of malignant tumors in the human body, nasopharyngeal carcinoma is 8th and it ranks third in Guangdong and Guangxi Province. Nowadays, the major therapeutic modality of NPC is radiotherapy. In the condition of the patients who are insensitive to radiotherapy as well as suffering from relapse of nasopharyngeal carcinoma or advanced stage of cancer,chemotherapy should be considered. But the anti-cancer ability of conventional radiotherapy and chemotherapy is regarded to be unsatisfactory specificity.Not only the killing-index is low,but also side effects can't be easily neglected.Molecular targeted therapy which act as an emerging hot spot of cancer research, not only is involed in the recent progress of molecular mechanism, but also it could greatly improve the efficacy of the treatment of nasopharyngeal carcinoma. Molecular targeted therapy is a new biological treatment model which aim at many aspects which may induce cellular carcinogenesis, such as cell signal transduction pathways, proto-oncogenes and tumor suppressor genes, cytokines and their receptors, anti-tumor angiogenesis, suicide genes, and so on at the molecular level. In order to reverse vicious biological behavior, molecular targeted therapy inhibits tumor growth, and even makes patient enjoy tumor-free life. As a novel molecular targeted therapy drugs Rapamycin and its derivatives have achieved varying degrees of efficacy, in different kinds of solid tumors and rapamycin has been approved to be carried on a Phase III clinical trials in renal cell carcinoma and carcinoid tumors.Upon so much encouraging achievement, we are confident that rapamycin will make an important role in the treatment of nasopharyngeal carcinoma. If this drug can be successfully used in the treatment of NPC, it will make a good result that can not only benefit patients and improve survival, but also generate considerable economic and social benefits.The purpose of this study is exploring the growth inhibition and its preliminary mechanism of rapamycin in different biological characteristics of nasopharyngeal carcinoma cell lines. On the basis of that, we silence the expression of HSP70 in nasopharyngeal carcinoma in order to find whether it impacts cell growth inhibition of cancer cell lines via rapamycin and try to make a probe into its preliminary molecular mechanism. The contents of the study consist of three parts:MethodsConfirming the best concentration and timepoint when growth inhibition of three nasopharyngeal carcinoma cell lines reaches maximum via mTOR inhibitor rapamycin according to MTS assay.Resutls1. Rapamycin can inhibit different biological characteristics of nasopharyngeal carcinoma cells, which results from the kinase activity inhibition of mTOR;2. Inhibition rate emerges in a dose and time-dependent manner, and there is no significance between different kinds of cell lines.Inhibition rate ranges from 40% to 50%, and the highest rate is 53.9%.3. In terms of inhibition rate, 100nM rapamycin for 48hrs may direct it onto the platform regardless of the kinds of cell lines.Methods1. Using Western Blotting method to detect the expression of critical protein markers (p-Akt Ser473, Akt, p-mTOR Ser2448, mTOR) in PI3K/Akt/mTOR signaling pathway of three cell lines which are exposed to different concentrations of rapamycin.2. Using Western Blotting method to detect the expression of critical protein markers (p-Akt Ser473, Akt, p-mTOR Ser2448, mTOR) in PI3K/Akt/mTOR signaling pathway of three cell lines which are exposed to 100nM rapamycin for 0hr,1hr,24hrs and 48hrs.Results:1. The expression of p-mTOR Ser2448 acts in time and dose-dependent manner in CNE-1 and 6-1 OB cell lines, but this phenomenon doesn't exist in 5-8F cell line.2. The expression of p-Akt Ser473 acts in time and dose-dependent manner in 6-1 OB and 5-8F cell lines, but whose changing seems to be "up and down"in CNE-1.3. The level of p-Akt Ser473 rises in three nasopharyngeal carcinoma cell lines when they are expose to rapamycin for 1hr.As the time went on, all the level of p-Akt Ser473 is lower than 1hr, but higher than Ohr except 6-1 OB when we prolong the drug-exposing time.4. The expression of mTOR,Akt and HSP70 does not change significantly whatever the concentration and exposing time of the drug is.Methods1. Using RT-PCR and Western Blotting methods to confirm the timepoint of the satisfactory inhibition rate of HSP70 in cell line when they are exposed to HSP70 siRNA.2. With MTS assay, we compare the growth inhibition rate of Control siRNA group,HSP70 siRNA group,Control siRNA+100nM Rapa group and HSP70 siRNA+100nM Rapa group.3. We observe morphological change of cells in different group with optical microscope.4. Upon Western Blotting, we make detection of key protein markers (p-Akt Ser473,Akt,p-mTOR Ser2448,mTOR) in PI3K/ Akt/mTOR signaling pathway in the three cell lines belongs to different group.Results1. RT-PCR shows that the expression of HSP70 may reach a relative low platform at the gene level after 48hrs upon the treatment of siRNA; Western Blotting shows that the expression of HSP70 may reach a relative low platform at the protein level after 72hrs upon the treatment of siRNA;2. There is highest growth inhibition rate of cancer cell in HSP70 siRNA+100nM Rapa group; 3. Compared with Control siRNA, Control siRNA+100nM Rapa group is similar with the treatment of rapamycin alone for 48hrs, which elevates the expression of intracellular p-Akt Ser473. The level of p-Akt Ser473 in cell which is treated with HSP70 siRNA and 100nM Rapa for 48hrs significantly decreases.4. Compared with the group treated with HSP70 siRNA alone, the expression of p-mTOR Ser2448 decreases in CNE-1 and 6-1OB cell lines which suffering from Control siRNA+100nM Rapa for 48hrs OR HSP70 siRNA+100nM Rapa for 48hrs, but 5-8F's does not.ConclusionsWith MTS assay,Western Blotting,transient transfection of liposome and reverse transcription-polymerase chain reaction, we observe that rapamycin's inhibitory effects on nasopharyngeal carcinoma cell lines with different biological characteristics and uncover the preliminary mechanism of growth inhibition by detecting the key protein markers in PI3K/Akt/mTOR signaling pathway. In the next step, we make an insight into the rapamycin's effect on the cells whose HSP70 is downregulated. Finally, we discuss that the preliminary mechanism of growth inhibition of nasopharyngeal carcinoma cell lines by combination HSP70 siRNA with rapamycin and conclude the following viewpoints:1. In vitro, rapamycin could inhibit the growth of nasopharyngeal carcinoma cell lines to some extent regardless of the cell type;2. The expression of p-mTOR Ser2448 and the kinase activity of mTOR are downregulated by rapamycin, which is the reason why the growth of cells is suppressed.3. Rapamycin inducing the elevating level of p-Akt Ser473 is the main reason why the growth inhibition rate of NPC cell lines is moderate. 4. Rapamycin induces the level of p-mTOR Ser2448 downregulation which does not mirror the actual inhibition rate of cell.5. Combination HSP70 siRNA and rapamycin may elevate the drug's inhibitory effects on the growth of NPC cells.6. HSP70 siRNA elevate the rapamycin's inhibitory effect on NPC cell by silencing the expression of HSP70 and downregulating the phosphory-lation of Akt Ser473.All results above provide precious data for combination HSP70 siRNA and rapamycin targeted therapy against NPC.