Targeting Hypoxia Inducible Factor -1α With Tf-PEI-shRNA Complex Via Transferrin Receptor Mediated Endocytosis Inhibits Malignant Melanoma Growth

PartⅠExpressions of HIF-1αand its downstream genes VEGF and GLUT-1 in Malignant MelanomaObjectives To study expressions of HIF-1α,VEGF and GLUT-1 and their correlation to tumor clinical-pathology features in human malignant melanoma (MM) and MM cell lines. To analyze the possiblity of HIF-1αbeing an attractive therapeutic molecular target for MMMethods①Tissue sections from 77 MM patients were examined using immunohistochemical technique and morphological quantitative analysis for the protein expressions of HIF-1α,VEGF and GLUT-1 and their corelations to tumor clinical-pathology features in MM were analyzed. And tissue sections from 20 pigmented nevi patients were as controls.②MM cell lines under hypoxia condition were detected using immunocytochemistry ,western blot and ELISA for expressions of HIF-1α,VEGF and GLUT-1.And these cell lines under normoxia were as controls.Results①Positive HIF-1α,VEGF and GLUT-1 immunostaining were observed in 84.4% (65 cases),71.42% (55 cases) and 68.83%(53 cases) of 77 MM lesions respectively,whereas only one case of pigmented nevi expressed HIF-1αand two cases expressed VEGF and GLUT-1 ;②A positive correlation among HIF-1α,VEGF and GLUT-1 was observed in MM;③Expressions of HIF-1α,VEGF and GLUT-1 were positively correlated with malignant degree in MM;④Expressions of HIF-1α,VEGF and GLUT-1 in MM cell lines A375,A875 and KZ28 under hypoxia condition were higher than under normoxia⑤Expressions of secreted VEGF and membrane GLUT-1 in MM cell lines were steadily increased as hypoxia time was enlonged and were corelated to HIF-1αexpression. Conclusions①On the basis of the current data showing that malignant melanocytic tumors displayed strong HIF-1α,VEGF and GLUT-1 expressions, whereas benign melanocytic tumors showed almost no immunoreactivity for HIF-1α,VEGF and GLUT-1,they might be used as discriminating indicators to distinguish malignant melanocytic tumors from benign melanocytic tumors;②The strong upregulation of HIF-1αin MM found in this study suggested that HIF-1αmight be an important factor to MM growth and could be an attractive therapeutic molecular target for MM.PartⅡConstruction and Identification of HIF-1α-shRNAsObjectives Short interfering RNA (siRNA) eukaryotic expression vector for HIF-1αwas constructed and transfected into malignant melanoma cells. To explore its effect of interference for HIF-1αin malignant melanoma cells in vitro and observe its effect to cell apoptosis and proliferation.Methods①Three HIF-1α-shRNAs targeting human HIF-1αmRNA common sequence was synthesized and it was inserted into Bam HI-Hind III linearized Pgenesile-1 vector. The sequence of HIF-1α-shRNA plasmid was analyzed by DNA sequencer and restrict endonuclease cutting .②To screen the best silencing effect HIF-1αshRNA,the alteration of HIF-1αmRNA and protein was checked by real time Rt-PCR and western blot after HIF-1α-shRNAs were transfected in MM cell lines.And the level of secrected VEGF was checked by ELISA and membrance GLUT-1 was analyzed by FCM.③The monoclone A375 cells with stable expression of best effecting shRNA1 were obtained by G418 selection and were identified with checking GFP expression by FCM and observing under Fluorescence microscope④To observe cell apoptosis and proliferation ,shRNA1-A375 stable clone was cultured under hypoxia condition.Results①It was verified that the sequence of constructed recombinant plasmids were correct by DNA sequencing and restrict endonuclease cutting.②HIF-1αshRNAs showed variable efficacy in decreasing HIF-1αmRNA in the three cell lines, relative to the control scrambled shRNA and the mock group. ShRNA1 decreased HIF-1αmRNA by 74%, 69% and 70% in the three cell lines at time point of 24h after transfection, respectively. ShRNA3 decreased HIF-1αmRNA in the three cell lines by 32%, 24% and 22% at the same time point, respectively. Unexpectedly, shRNA2 resulted in a slightly decrease but no significant alteration of HIF-1αmRNA expression in the three cell lines. Among the HIF-1αshRNAs we tested , shRNA1 possessed the strongest inhibitory effect against HIF-1αand had paralleled effect on silencing HIF-1αin the three cell lines.And Silencing HIF-1αby HIF-1αshRNAs could down-regulated secreted VEGF and membrance GLUT-1 in MM cell lines.③We screened and obtained A375 cells with stable expressions of HIF-1α-siRNA as A375 stable clone could observed under Fluorescence microscope and 98% A375 stable clone expressed GFP detected by FCM;④The down-regulation of HIF-1αcould significantly prevent cell proliferation and enhance apoptosis in A375 exposed to hypoxic conditions.Conclusions It indicated that hairpin siRNA eukaryotic expression vector for HIF-1αwould be successfully established,and it played a specific inhibitory role in three kinds of tumor cell lines and had parellel silencing effect and also down-regulated VEGF and GLUT-1. At the same time we obtained cells with the stable expression of HIF-1α-shRNA which could cause cell apoptosis and inhibit cell proliferation under hypoxia condition in A375 stable clone. This study laid experimental foundation for further research of the therapy of HIF-1α-shRNA vector in MM.Part III Expression of TfR in MMObjectives To study expressions of TfR in human malignant melanoma (MM) and MM cell lines and to analyze the possiblity of TfR being an attractive target receptor for MM gene therapy.Methods①Tissue sections from 77 MM patients were examined using immunohistochemical technique and morphological quantitative analysis for the protein expressions of TfR and its corelations to tumor clinical-pathology features in MM were analyzed. And tissue sections from 20 pigmented nevi patients were as controls.②MM cell lines under normoxia and hypoxia condition were detected using immunocytochemistry ,western blot and FCM for expressions of TfR.And A2780 cell lines which TfR expression was low as controls.③To analyze the mechanism of the dual phase change of expression of TfR under hypoxia in different time course by checking cell apoptosis and proliferation under hypoxia.Results①Positive TfR immunostaining were observed in 90.9% (70 cases) of 77 MM lesions,whereas only five case of pigmented nevi expressed TfR ;②Expressions of TfR were positively correlated with tumor graduation in MM;③Expressions of TfR in MM cell lines A375,A875 , KZ28 and A2780 under normoxia condition were 97%,89%,79% and 2%,respectively.④hypoxia has a dual effect on the expression of TfR in human Melanoma A375 cell line during different hypoxic time course which hypoxia down-regulated TfR expression under hypoxia at time points 12 and 24h and up-regulated TfR expression under hypoxia at time point (36,48 and 60h).⑤hypoxia has multiple opposing effects on tumor growth, with a variable net outcome at various time periods and hypoxia might down-regulate TfR expression by inducing apoptosis which decreases the iron requirements in A375 cells in early hypoxic time course, and up-regulates TfR expression by promoting proliferation in A375 cells in late hypoxic time course.Conclusions①On the basis of the current data showing that malignant melanocytic tumors displayed strong TfR expressions,whereas benign melanocytic tumors showed almost no immunoreactivity for TfR,they might be used as target receptor for gene therapy in MM;②Hypoxia has dual effect on the expression of TfR in human Melanoma A375 cell line. These findings may have important implications for more rational, individualized gene-based therapy using TfR as target receptor in melanoma. Part IV To study Tumor-targeted distribution after administration of Tf-PEI-shRNA complex in vitro and in vivoObjectives By detecting the ability of Tf-PEI to deliver shRNA specifically to TfR-high-expressed cells in vitro and TfR-high-expressed cells bearing tumor tissue in vivo, we expect to provide the experimental data and basis of theory for the clinical treatment of malignant melanoma with Tf-PEI-shRNA complex.Methods①t ransfection efficiency for each group was estimated as the mean value of GFP per 10,000 cells by FCM in vitro.②tumors and major organs were excised for GFP mRNA and protein expression by real time RT-PCR and western blot analysis.Results①t ransfected shRNA by Lipofectamine2000 was comparably taken up by ~ 70% of both A2780 and A375. Neither A2780 and A375 appreciably took up shRNA by themselves nor when mixed either with Tf lacking PEI or with unmodified PEI.When mixed with Tf-PEI, did A2780 still not take up shRNA. In contrast, when mixed with Tf-PEI, shRNA was comparably taken up by ~ 52% in A375. Although lower than Lipofectamine2000 ~ 70% transfection in A375, Tf-PEI transfection specific delivery of shRNA into TfR-high-expressed MM cell was verified.②the highest amounts of GFP mRNA and protein were detected in tumor tissues and in major organs such as liver, lungs, heart and kidneys only small amounts of GFP mRNA and protein were detected in the A375 bearing tumor mice at time point of 24h after the single injection. At the same time, the result revealed that the GFP mRNA and protein expression distribution in the tumor tissues and major organs of A2780 bearing tumor mice had no significant differences. The results demostrated that Tf-PEI could deliver shRNA with tumor-targeted specific distribution in the nude mice bearing A375 sc.xenograft tumors.Conclusions these results showed the tumor-targeted relatively specific distribution after administration of Tf-PEI-shRNA complex in vitro and in vivo. Part V Observation of the growth rate of MM xenograft tumor after injection with Tf-PEI- HIF-1αshRNA1 in vivo and exploration of potential mechanismsObjectives To observe growth inhibition of MM xenograft after receiving systemic admnistration of Tf-PEI- HIF-1αshRNA1 and to explore the potential mechanismsMethods①To evaluate therapeutic potential of Tf-PEI-shRNA1 complex in A375 tumor xenograft, the nude mice bearing A375 s.c.xenograft tumors and the nude mice bearing A2780 s.c.xenograft tumors were randomized and divided in three experimental groups, respectively: one group (n=12) was received direct injection of a total of 20μg of"therapeutic"plasmid Tf-PEI- shRNA1 complex conjugate in100μl volume through tail vein, whereas the second group (n=12) was injected with a total of 20μg of negative control Tf-PEI- scrambled shRNA conjugate in 100μl volume through the tail vein and the third group (n=12) was not received any treatment . Tumor diameter was measured at regular intervals with calipers, and tumor length (L) and width (W) were measured at the end of the experiment, and tumor volume was estimated by the formula (L×W2)/2.②At 25 days after injection, mice were sacrificed and tumors were dissected for further analysis.Expression of HIF-1α,VEGF and GLUT-1 and MVD in tumor tissues were detected by immunohistochemistry and western blot.③tumor glucolysis product Lactic Acid (LA) was detected by LA kit.④tumor cell apotosis was detected by TUNEL method.Results①Tumor growth delay was statistically significant (P=0.0014) from day 5 after the beginning of therapy until the day the mice were sacrificed. During day 10-15 after treatment we observed the strongest effect of Tf-PEI-shRNA1 complex; A2780 group tumors in the three groups had a volume of 710±145mm3 , 755±90mm3 and 695±120mm3, which were 7.5-fold larger than the starting volume. Tumor growth delay was not found in the"therapeutic"group.②the immunohistochemistry results shown that in the nude mice bearing A375 s.c.xenograft tumors, HIF-1α,VEGF and GLUT-1 expressions in Tf-PEI-shRNA1 complex group were significantly lower than in Tf-PEI-scrambled shRNA and A375 groups, and no significant difference in HIF-1αexpression was found between Tf-PEI-scrambled shRNA and A375 groups.③But expression of MVD in the three groups has no significant diference (P>0.05)④tumor glucolysis product Lactic Acid (LA) in Tf-PEI-shRNA1 complex group were significantly lower than in Tf-PEI-scrambled shRNA and A375 groups, and no significant difference in HIF-1αexpression was found between Tf-PEI-scrambled shRNA and A375 groups.⑤the number of apotosis tumor cell was larger in Tf-PEI-shRNA1 complex group that other two groups.Conclusions①Tf-PEI was able to deliver shRNAs specifically to TfR-expressed MM cells to suppress MM growth even when administered systemically.②systemic Tf-PEI-HIF-1α-shRNA complex administration in the nude mice bearing A375 sc.xenograft tumors could knock down HIF-1αwhich slowed the growth rate of MM xenograft tumor.③Inhibiting tumor glucolysis ,not tumor angiogenesis maybe involve to enhance tumor cell apotosis and to slow the growth rate of MM xenograft tumor.