| Hepatocellular carcinoma (HCC) is one of the most frequent malignancies worldwide,accounting for85%to90%of primary liver cancers. However, HCC is difficult todiagnosis and has a rapid progress of the course due to lack of the typical clinicalmanifestations, classical symptoms in early stage, and mostly with serious liver cirrhosis.Surgery is the mainly treatment in early stage. The disease course is usually late whendiagnosed, so the patients have lost the optimal time of surgery. At the meanwhile, theHCC is easy to recurrence and distant metastasis even though the tumor has been removed.Invasion and metastasis are the most important biologic characteristics for malignantcarcinoma, whiel chiefly accounts for clinic death of cancer patients. Once it hasprogressed to the metastatic stage that is extremely difficult to treat and does not respondto non-operative treatment. Therefore, the inhibition of invasion and metastasis has been the key factor for the successful treatment of HCC.Hyperthermia, a minimally invasive treatment with few side effects, has recentlybeen used for cancer therapy. A number of clinical and animal experiments have shownthat HT is a tolerable and clinically practical supplementary therapy for patients withadvanced malignant tumor, recurrent tumor and metastatic disease. HT exerts therapeuticeffects not only by delaying tumor growth but also by inhibiting metastasis. Althoughsome progress has been made in terms of assessing the biological effect of HT, themolecular mechanism that mediates the anti-metastatic effect of HT has not beenelucidated.Human NDRG2gene was named as N-Myc downstream regulated gene2. It belongsto the NDRG family. NDRG2was first discovered and cloned from normal human braintissue by our laboratory in1999(Syld GenBank AF159092). It was indicated by someprevious researches that NDRG2appeared low expression in some tumors. The ectopicexpression of NDRG2suppresses the proliferation of tumor cells. In addition,accumulated evidence indicates that the absence of NDRG2expression in a variety ofcarcinomas contributes to increased tumor metastatic potential. All of these findingssuggest that NDRG2has tumor suppressor role. In addition, increasingly more effortshave aimed to determine the role of NDRG2under stress conditions.At present, there was no report about relationship between HT and NDRG2oninvasion of HCC cells, so we decided to observe the changes of NDRG2in HCC cellsafter HT. So our research mainly sought to clarify the biological role of NDRG2ininvasion of HCC under HT. Understanding the molecular mechanism involved in HT mayhave valuable implications for developing optimized HT therapies for some NDRG2deficient cancers.Methods and Results：1. To assess the effects of anti-migration and anti-invasive of HT at different temperaturein HCC cells.We examined anti-migration and anti-invasive effect of HT on HepG-2andHuh-7cells by subjecting them to water bath at different temperatures (37C,39C,41C,43C and45C) for30min. Cell wound healing assay and Matrigel invasionassay were performed to evaluate the effects of HT on migration and invasion of cells, respectively. The migration and invasion of cells were affected by a heat treatment.Compared with control group (37C), heat treated at45C significantly reduced thepercentage invaded cells in HepG-2and Huh-7, respectively.2. To assess the expresses of MMP-2/9and NDRG2of HCC cells at differenttemperature of HT.To further confirm the anti-invasion effect of HT, we examined the expression ofNDRG2、MMP-2and MMP-9in HepG-2and Huh-7cells by subjecting them to waterbath at different temperatures (37C,39C,41C,43C and45C) for30min. Theexpression of MMP-2and MMP-9was significantly reduced by HT at45C.Meanwhile, Western-blot showed that expression of NDRG2was increased by heatshock at45C in both HepG-2and Huh-7cells.3. To investigate the biological role of NDRG2in the invasion of HCC cells exposed toHT.(1) We constructed the lentivirus vectors of NDRG2overexpression orNDRG2-shRNA to produce the lentiviral particles, and then infected the HepG-2cells. With long term selection of blasticidin or puromycin respectively, weestablished the stable cell lines with NDRG2overexpression or NDRG2knockdown.(2) Matrigel invasion assay and Western-blot were performed to evaluate the effect ofHT on invasion of cells. In NDRG2overexpressing cells, HT at45C reduced theinvasive potential significantly, compared with HepG-2cells treated by HT alone.We next detected the expression of MMP-2and MMP-9. Western-blot revealed thatexpression of MMP-2and MMP-9was decreased in NDRG2overexpressing HepG-2cells and even lower after the cells were treated by HT.(3) We further examined the anti-invasion potential of NDRG2in HCC-implantedmice. NDRG2dificient HepG-2cells were injected into nude mice. H&E staining ofhistological sections from mouse xenograft model revealed that HT suppressed theinvasion ability of HepG-2cells significantly. Malignant tumors slightly invaded intonearby tissues in HT treated mouse model. In contrast，suppression of NDRG2facilitate invasion of tumor nodules and reversed anti-invasive effect of HT inHepG-2cells with significant destruction of the muscle layer. The expression of bothMMP-2and MMP-9was also detected in tissues sections，and a representativeimmunohistochemical staining was presented. Consistent with H&E staining results， we found that down-regulation of NDRG2alleviated the repression of MMP-2andMMP-9expression by HT.4. To elucidate the molecular mechanism of NDRG2mediated the anti-invasion effect ofHT.(1) We examined the expression profiles of HSP27, HSP70and HSP90in HepG2cells of control and overexpression of NDRG2at different time points after cellswere subjected to HT at45C for30min. There was no change in expression levelsof HSP27and HSP90after HT between these two groups. HSP70expression wasinduced at6h and kept at high levels through12h after HT. However, the expressionpattern of HSP70was the same between these two groups.(2) We investigated the effect of NDRG2on the downstream activation of the MAPKby detecting phosphorylated p38MAPK, ERK1/2and JNK which were induced byHT. The phosphorylation of ERK1/2increased up within2h, then declined to thebasal level rapidly4h after heat shock treatment, and even lower later. Moreover,overexpression of NDRG2abrogated the intrinsic and HT induced activation ofERK1/2pathway. HT was found to increase activation of p38MAPK in a timedependent manner, while the phosphorylation level of p38MAPK remainedunchanged in NDRG2overexpressing cells.(3) To further confirm the role of NDRG2on ERK1/2activation, lentivirus-mediatedshRNA was employed to downregulate endogenous NDRG2expression in HepG-2cells. NDRG2deficient cells were treated with the ERK1/2inhibitor, PD98059,p38MAPK inhibitor, SB203580or JNK inhibitor, SP600125, respectively. Silence ofNDRG2expression significantly increased the invasion ability of HT treated cells.Meanwhile, PD98059treatment could significantly enhance the anti-invasive effectof HT and decrease MMP-2/MMP-9expression in NDRG2deficient HepG-2cells.These findings indicated that ERK1/2activation could be selectively inhibited byNDRG2expression in HepG-2cells.5. To demonstrate the therapeutic effect of NDRG2combined with HT against HCC.(1) We examined the type of cell death induced by HT at different temperature (37C,43C and45C) for30min, using double staining with Annexin V and PI. HepG-2cells treated at45C showed significantly higher rate of cell apoptosis, compared withthose treated at43C. HT at45C caused massive increase in number of necrotic cells in comparison with cells treated at43C. Intriguingly, we found that combination ofNDRG2expression and HT at43C resulted in increased level of apoptosis andreduced necrosis, compared with treatment of HT at45C.(2) We examined anti-invasive effect of HT on HepG-2by subjecting them to waterbath at different temperatures (37C,43C and45C) for30min. Matrigel invasionassay were performed to evaluate the effects of HT on invasion of cells. The cellstreated at45C showed significantly lower percentage of invaded cells, comparedwith those treated at43C. The combined treatment of HT at43C and NDRG2expression decreased expression of MMP-2and MMP-9as well as invaded cellpercentage.Conclusion：1. HT at45C could inhibit migration and invasion of HCC cells.2. NDRG2involves in HT inhibiting HCC cell invasion ability, moreover theexpression of NDRG2and cell invasion ability are negative correlation.3. NDRG2mediates the anti-invasion effect of HT via inhibition of the ERK1/2signaling pathway.4. NDRG2synergizes with HT to inhibit invasiveness of HCC cells and decreasespontaneous necrosis. |
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