| ObjectiveNasopharyngeal carcinoma (NPC) is a common head and neck cancer in Southern China and Southeast Asia, particular in Guangdong, Guangxi and Fujian provinces. NPC is a multi-factorial disease. Both genetic predisposition and epigenetic alterations are important for the initiation and progression of NPC. In addition, the pathogenesis of NPC is closely linked to Epstein-Barr virus infection. Tobacco and alcohol consumption are critical risk factors as well. However, the molecular mechanisms underlying NPC pathogenesis remain unclear. In addition, NPC tends to be early invasive and metastatic. Due to its originating form a hidden anatomical site, it is usually in the advanced clinical stage at the time of diagnosis. Hence, the prognosis for NPC is poor with 5-year survival rate of less than 70%. Although its prognosis has improved due to advances in diagnostic and surgical techniques,30% to 40% of patients will develop distant metastases within 4 years. Once metastasis occurs, the prognosis is very poor and so it is of great clinical values to further understand the molecular mechanisms of this cancer and find early diagnostic markers as well as novel therapeutic strategies.Renin angiotensin system (RAS) components has become an important marker of cancer. This system is an endogenous blood pressure, fluid balance and cell growth regulation system. The angiotensin produced in liver, by degrading rennin and form a 10 peptide, angiotensin 1 (Ang I). Then Ang I was catalysised by angiotensin-converting enzyme (ACE) and peptide enzyme to generate two biological active peptide octapeptide angiotensin 2 (Ang II) and 7 peptide angiotensin-(1-7)[Ang-(1-7)]. Angiotensin-(1-7) [Ang-(1-7)], either produced in the circulation or in tissues, mediates biological responses by activating MasR, a unique G-protein-coupled receptor, is a biologically active peptide hormone of the renin-angiotensin system with vasodilator, antiproliferative, and antithrombotic properties. It has been shown that Ang-(1-7) reduced the growth of human lung tumor xenografts with a concomitant decrease in vascular endothelial growth factor (VEGF) and reduced vessel density, as well as orthotopic human estrogen receptor positive or HER2 over-expressing breast tumor xenografts. A Phase I clinical trial demonstrated that Ang-(1-7) is a first-in-class antiangiogenic drug with activity for treating cancer that is linked to reduction of plasma placental growth factor (P1GF) levels. Recent studies showed that Ang-(1-7) attenuates metastatic prostate cancer and reduces osteoclastogenesis, and also reduces proliferation and angiogenesis of human prostate cancer xenografts with a decrease in angiogenic factors and an increase in sflt-1. These findings suggest that Ang-(1-7) reduces tumor size by attenuating proliferation and angiogenesis. Until now, no functional evidence of Ang-(1-7) in NPC has been documented.However, the short half time of Ang-(1-7) in animals, which is only 0.42 to 0.61 hour in patients, limits its therapeutic effect and increase treatment cost. Recombinant adeno-associated virus (rAAV),with its low pathogenicity and low immunogenicity can lead to long-term and stable expression of foreign genes (up to more than 10 years), is widely used in gene therapy research. Recently, the European countries have approved the first gene therapy drug Glybera, and several clinical trials have proved the effectiveness and safety of AAV. AAV8-mediated hFIX gene transfer by a single peripheral-vein infusion consistently leads to long-term expression of the FIX transgene at therapeutic levels, without acute or long-lasting toxicity in patients with severe hemophilia B. AAV targeting and efficiency are further improved by using point mutations or Shuffling technique.In this study, we investigated the potential involvement of Ang-(1-7) in NPC using lentiviral or AAV vectors expressing fusion proteins which secrete the heptapeptide. We first examined the effects of Ang-(1-7) on NPC cell growth and migration. Second, we investigated a potential role of Ang-(1-7) on NPC tumorigenesis in a murine model. Finally, we explored the underlying mechanism of Ang-(1-7) functions in NPC. Our study will provide a novel therapeutic target gene of nasopharyngeal carcinoma.Methods1. Identification of expression of MasR in NPCThe expression of MasR mRNA and protein in 5 NPC cell lines (CNE-1, CNE-2, HNE-1,5-8F, C666-1) and 30 NPC biopsy specimens was detected by real-time RT-PCR and Western blot respectively.2. Effects of Ang-(1-7) over-expression on biological behaviors of CNE1 cellsThe LV-Ang-(1-7) was driven by the human elongation factor la promoter (EF1α) containing a human prorenin signal peptide and the immunoglobulin fragment from the mouse IgG2b linked to a portion of the human prorenin prosegment. Thus, the transgene encodes a fusion protein that is capable of releasing the Ang-(1-7) peptide by taking advantage of the constitutive presence of the furin enzyme, which ultimately releases the peptide from the fusion protein. Both IgG2b and Ang-(1-7) can be detected intracellularly. The enhanced green fluorescent protein (eGFP) lentiviral vector LV-eGFP was used as a control. The lentivirus was transfected into CNE-1 and CNE-2 cells and then western blot and EIA were used to detect the efficiency of the Ang-(1-7) expression. The cell growth and migration were detected by cell counting, MTT, plate colony formation and wound scratch assay respectively. MasR antagonist A-779 was used to detect the effect of Ang-(1-7) on nasopharyngeal carcinoma cells when MasR was blocked.3. Effects of Ang-(1-7) over-expression on the growth of human nasopharyngeal tumor xenograftsAthymic mice were subjected to s.c. injections of human CNE-1 nasopharyngeal cancer cells (1.0×106) in Matrigel (1:1) into the lower flank to induce tumor growth. After the tumors reach about 0.4cmx0.3 cm, the mice were placed into three groups at random and the animals received tail vein injections of AAV8 (Y733F)-CBA-Ang-(1-7), AAV8 (Y733F)-CBA-eGFP (5×1011 v.g/mouse) or PBS. Tumor size was measured every 3 days. The mice were anesthetized on day 35 and euthanized by decapitation and tumors were dissected. Tumor volumes were calculated as follows:volume = (D×d2)/2, where D meant the longest diameter and d meant the shortest diameter. Both livers and tumors were isolated for Western blot and quantitative real-time RT-PCR analysis, or fixed in 10% buffered formalin and used for histologic and immunohistochemical analysis.4. The mechanism of Ang-(1-7) on the inhibitory of the growth of NPC cellsTo better understand the mechanism underlying the role of Ang-(1-7), VEGF, PIGF, HIF-1α and VEGF receptor Flt-1, Flk-1α and sFlt-1 was detected in the Ang-(1-7) overexpressed NPC cells and in in vivo study using real-time RT-PCR. MAPK signaling pathway was also detected by Western blot.Results1. Identification the expression of MasR in NPC cell lines and tissuesA panel of human NPC cell lines (CNE-1, CNE-2, HNE-1,5-8F, C666-1) and immortalized nasopharyngeal epithelia cells NP69 was first analyzed to quantitate the expression level of MasR mRNA. The results of one way ANOVA analysis showed that the expression level of MasR was significantly different from each other (F=27.608, P=0.000). Compared with NP69 cells, the expression level of MasR was significantly increased in all 5 NPC cell lines. MasR mRNA was highly expressed in CNE-2 (△△Ct=-3.608±0.717) with metastatic ability, low expressed in CNE-1 (△△Ct=-1.505±0.398) and HNE-1 (△△Ct=-1.543±0.270) with no metastatic ability, and moderately in C666-1 (AACt=-2.851±0.274) and 5-8F(△△Ct=-2.709±0.507).Western blot results showed that the expression level of MasR protein were consistent with the expression levels of mRNA.The expression level of MasR mRNA in 30 NPC specimens and 23 chronic nasopharyngitis tissue samples were further detected using real-time RT-PCR. MasR positive rate was higher in 22 of 30(73.3%) carcinomatous tissues compared with 7 of 23 (30.4%) inflammatory tissues (P=0.000). Consistent with the data obtained from NPC cell lines, the average expression level of MasR was significantly higher in NPC specimens than in chronic nasopharyngitis tissues (P<0.01). Further analysis found that MasR expression were significantly related to tumor invasion (P=0.007), lymph node metastasis (P=0.020), and distant metastasis (P=0.020) and clinical stage (P =0.027).2. Effects of Ang-(1-7) over-expression on biological behaviors of NPC cellsThe Ang-(1-7) lentivirus were packaged and titered, and then transduced into CNE-1 and CNE-2 cells. Successful overexpression of Ang-(1-7) was confirmed by western blot and EIA method. Increased Ang-(1-7) showed a significantly decreased proliferation when compared with control cells as determined by cell counting assay and in vitro MTT assay. In addition, CNE-1 and CNE-2 cells overexpressing Ang-(1-7) cells had a decreased ability of colony formation. These results indicated that over-expression of Ang-(1-7) inhibited the proliferation of NPC cells.The results of in vitro migration assay showed that the CNE-1 cells transduced with Lenti-Ang-(1-7) had a significantly decreased migration when compared with the controls. The same results were also showed in CNE-2 cells. These results indicated that over-expression of Ang-(1-7) lead to downregulation of NPC cell migration. MasR antagonist A-779 was used to detect the effect of Ang-(1-7) on nasopharyngeal carcinoma cells when MasR was blocked. The results showed that A-779 can completely block Ang-(1-7)’ effect in proliferation and migration inhibitory.3. Effects of Ang-(1-7) over-expression on the growth of NPC xenograft1) The growth of NPC transplanted tumor CNE-1 cells were injected subcutaneously into the dorsal flank of nude mice to assess the effect of Ang-(1-7) on nasopharyngeal tumor growth. When xenograft tumors were about 0.4 cm×0.3 cm, mice were randomized for tail intravenous injection with AAV8 (Y733F)-CBA-Ang-(1-7), AAV8 (Y733F)-CBA-eGFP (5×1011 v.g/mice,200 μl) or PBS to reach a high level of Ang-(1-7) stable expression. Tumors in the control group mice continued to grow faster over the 30 day period, whereas administration of the AAV8 (Y733F)-CBA-Ang-(1-7) resulted in a significant reduction in tumor volume. Tumors from mice in the control group were 2.0-fold larger than tumors from AAV8 (Y733F)-CBA-Ang-(1-7)-treated mice. There were no differences in body weight or pathological abnormalities between the groups. The mice were euthanized at the end of the study and the tumors were dissected and weighed. The tumors from mice treated with AAV8 (Y733F)-CBA-Ang-(1-7) weighed about 60% less than the tumors from mice infused with AAV8 (Y733F)-CBA-eGFP and PBS, demonstrating that Ang-(1-7) reduces tumor growth.2) Ang-(1-7) expression mediated by AAV vectors in mice Livers from mice treated with AAV8 (Y733F)-CBA-Ang-(1-7), AAV8 (Y733F)-CBA-eGFP or saline were harvested on the end day of the experiment. Fluorescence detection, Western blot hybridization and immunohistochemical analysis were performed to detect foreign gene expression. The results showed that the eGFP fluorescence was only observed in livers from the eGFP group. Ang-(1-7) fusion protein containing mouse IgG Fc region was positive only in livers from the Ang-(1-7) group by immunohistochemistry and Western blot hybridization using horseradish peroxidase-conjugated anti-mouse IgG.3) The cell proliferation in nasopharyngeal tumor xenografts Tumor sections from mice infused with AAV8 (Y733F)-CBA-Ang-(1-7), AAV8 (Y733F)-CBA-eGFP or PBS underwent immunohistochemistry using antibody of the cell proliferation marker Ki67. The results showed that both the staining intensity and the number of hyper-proliferative Ki-67 tumor cells were significantly decreased compared with both control groups, suggesting that Ang-(1-7) reduces cell proliferation in vivo.4. The mechanism of Ang-(1-7) on the inhibitory of cell growth1) Effect of Ang-(1-7) on VEGF, PIGF, HIF-la and VEGF receptors in nasopharyngeal tumor xenografts VEGF was reduced by 57% and 53% respectively in Lenti-Ang-(1-7) treated CNE-1or CNE-2 cells compared to control cells. Similarly, CNE-1or CNE-2 cells transduced with Lenti-Ang-(1-7) had respective ～60% and 70% reductions approximately in PIGF mRNA compared to control cells. HIF-la mRNA was also shown to be significantly decreased in transduced NPC cells compared to control cells. VEGF and PIGF bind to and activate two distinct VEGF receptors-VEGF receptor 1 or Flt-1, and VEGF receptor 2 or Flk-1-to initiate intracellular signaling cascades. Flt-1 and Flk-1 were significantly reduced in Lenti-Ang-(1-7) treated NPC cells compared to controls, suggesting that this heptapeptide may also attenuate VEGF and PIGF signaling by reducing the number of receptors available to these ligands. In contrast to the Ang (1-7)-mediated reduction in membrane associated VEGF receptors, sFlt-1 was significantly increased in Lenti-Ang-(1-7) treated CNE-1 or CNE-2 cells compared to control cells.Consistent with the in vitro results, VEGF mRNA and protein were significantly reduced in tumors from nude mice administered with AAV8 (Y733F)-CBA-Ang-(1-7) when compared to tumors from mice treated with AAV8 (Y733F)-CBA-eGFP or PBS. Similarly, PIGF and HIF-1α were also significantly reduced in tumors from nude mice administered with AAV8 (Y733F)-CBA-Ang-(1-7), compared to tumors from AAV8 (Y733F)-CBA-eGFP or PBS-treated animals. Flt-1 and Flk-1 were quantified by real-time RT-PCR in tumors from mice treated with AAV8 (Y733F)-CBA-Ang-(1-7), AAV8 (Y733F)-CBA-eGFP or PBS to determine the effect of the heptapeptide on VEGF receptor expression. Flt-1 and Flk-1 were significantly reduced in tumors from mice administered with Ang (1-7), compared to tumors from control mice, suggesting that the heptapeptide may also attenuate VEGF and PIGF signaling by reducing the number of available receptors. In contrast to the Ang(1-7)-mediated reduction in membrane associated VEGF receptors, sFlt-1 was significantly increased in tumor tissue following administration of the heptapeptide. These results suggest that VEGF, PIGF, HIF-1α and VEGF receptors were involved in Ang(1-7) inhibiting cell and tumor growth both in vivo and in vitro.2) Effect of Ang-(1-7) on p44/42 MAPK, JNK and p38MAPK signaling pathway MAPK signaling pathways are implicated in cell survival, growth and proliferation. Phosphorylated p44/42, p38 and JNK were measured in transduced NPC cells using phospho-specific antibodies to determine whether Ang-(1-7) inhibited NPC cell growth by attenuating MAP kinases. Western blot results showed that the p44/42 phosphorylation level in Lenti-Ang-(1-7) treated cells was decreased. The phosphorylation of p38, another MAPK pathway, showed a similar response pattern. However, the level of JNK phosphorylation was not changed in NPC cells treated with Lenti-Ang-(1-7). These results suggest that Ang-(1-7) reduces tumor growth, in part, by attenuating p44/42 and p38 MAP kinase activities.Conclusions1. The expression of MasR is upregulated in NPC and correlates statistically with the malignant status of NPC.2. Ang-(1-7) inhibited the growth and migration of NPC cells in vitro.3. Ang-(1-7) inhibited the growth of nasopharyngeal tumor xenografts.4. VEGF, PIGF, HIF-1α and VEGF receptors were involved in Ang(1-7)’s role in inhibiting cell and tumor growth both in vivo and in vitro, and Ang-(1-7) reduces tumor growth, in part, by attenuating p44/42 and p38 MAP kinase activities. |
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