The Role Of NDRG2in Cancer Hyperthermia And The Signal Transduction Pathway Of It

The NDRG2(N-myc down-stream regulated gene2) was firstly discovered in1999. Itis a new gene, which is possibly related to cell proliferation and stress, but the exactlybiological function has not been completely elucidated. Previous findings have shown thatit take part in some stress reaction, for insistence: hypoxia, radiation, chemotherapy. Heatshock is a common stress reaction, and has come a new tumor therapy-hyperthermia.Hyperthermia is a type of cancer treatment except for surgery and chemotherapy, andresearches have shown that high temperatures can damage and kill cancer cells, usuallywith minimal injury to normal tissues. Hyperthermia is paid more attention to because ofits safe and little side effect. The physic factor cannot explain the curative effect ofhyperthermia, the heat shock can change signal transduction pathway and induce cellapoptosis. The undefined mechanism of hyperthermia brings blind to the application ofhyperthermia. Nuclear translocation of NDRG2under hypoxia stress and heat stress takesus to presume that NDRG2play a role in the process of heat stress. In order to prove above hypothesis, we treated two hepatoma cell lines (HepG2andHHCC) and two gastric cancer cell lines (MKN45and MKN28) with heat shock (42℃for1h).By real-time PCR and Western Blot, we found that NDRG2gene could be obviousdown regulated both at mRNA and protein level with recovery time going. All these datashowed that there existed a certain relationship between NDRG2and heat stress.We detected the apoptotic cell rate of the four cell lines and found that apoptosisincreased with the decrease of the expression of NDRG2. This result indicated thatNDRG2can active some signal transduction and induce apoptosis after heat stress. c-junis a kind of immediate early genes which are activated transiently and rapidly in responseto a wide variety of cellular stimuli. They represent a standing response mechanism that isactivated at the transcription level in the first round of response to stimuli, before any newproteins are synthesized. The research presented that NDRG2suppresses cell proliferationthrough down-regulation of AP-1activity in human colon carcinoma cells. Our team madea promoter scan for the promoter elements of NDRG2, and found a jun transcription factorbinding site. C-jun is widely distributed in cells and is the most active in Jun proteinfamily. C-jun can be active by many stressor, including heat stress. So we suppose thatc-jun and NDRG2has great relationship during the process of hyperthermia. NDRG2were over expressed and c-jun were silenced in HepG2cells by using adenovirus orsiRNA, cell apoptosis were detected by flow cell technology. The results showed that theexpression of c-jun protein increased with the expression of pro-apoptotic Bax increasedand the expression of anti-apoptotic Bcl-2decreased. These data showed heat stressinduce cell apoptosis by Bax/Bcl-2pathway, and NDRG2and c-jun were involved in it.To make clear the relationship between c-jun and NDRG2, C-jun was geneticallydown-regulated in HepG2cells by RNAi technique and we detected the protein level ofNDRG2of HepG2cells. NDRG2was unregulated by interfering the expression of c-jun.So there is a negative relationship between c-jun and NDRG2.It is interesting that we found that NDRG2total protein level was not changed with theincreasing apoptosis cell rate after heat stress with in6h. The result of Western Blotshowed the expression of pNDRG2（Ser332）and pNDRG2（Thr348） increased from0hand3h after heat shock. Kinases and phosphatases play important roles in heat shockresponse of cells. In general, both inhibitory and activating effects of heat shock onvarious kinases and phosphatases and the phosphorylation of proteins have been described. The identification of new mechanisms or factors able to induce apoptosis in gastric cellsmight therefore be a useful approach to treat advanced gastric cancer. In present, it hasbeen found that PI3K, AKT and GSK3, AKT are regulated by various stresses, also byheat shock. Recent researches have proved that NDRG2can be phosphorylated by AKTand GSK. AKT inhibitor could inhibit the phosphorylation of NDRG2induced by heatstress and induce apoptosis in gastric cancer cells.In summery，the hypothesis that NDRG2and c-jun take part in heat stress was firstproposed and proved. there is a negative relationship between c-jun and NDRG2, and bothof them induced apoptosis through Bax/Bcl-2pathway after hyperthermia. NDRG2issubstrate of protein kinase AKT. Heat stress induced apoptosis in MKN45and MKN28cells, accompanied with the variety of AKT-NDRG2pathway.