Targeting Hypoxia-inducible Factor-1α With Tf–PEI–shRNA Complex via Transferrin Receptor–mediated Endocytosis Inhibits Melanoma Growth

Abstract

Malignant melanoma (MM) is a major public health problem. The development of effective, systemic therapies for MM is highly desired. We showed here that the transferrin receptor (TfR) was a suitable surface marker for targeting of gene therapy in MM and that the hypoxia-inducible factor-1α (HIF-1α) was an attractive therapeutic molecular target in MM. We observed that inhibition of HIF-1α blocked cell proliferation and induced cell apoptosis in vitro. We then showed that a transferrin–polyethylenimine–HIF-1α–short-hairpin RNA (Tf–PEI–HIF-1α–shRNA) complex could target MM specifically and efficiently both in vivo and in vitro, exploiting the high expression of the TfR in MM. The systemic delivery of sequence-specific small-interfering RNA (siRNA) against HIF-1α by the Tf– PEI–HIF-1α–shRNA complex dramatically inhibited tumor growth in the A375 MM xenograft model. The underlying concept of transfecting a HIF-1α shRNA expression vector complexed with Tf–PEI to block HIF-1α holds promise as a clinical approach to gene therapy for MM. Malignant melanoma (MM) is a major public health problem. The development of effective, systemic therapies for MM is highly desired. We showed here that the transferrin receptor (TfR) was a suitable surface marker for targeting of gene therapy in MM and that the hypoxia-inducible factor-1α (HIF-1α) was an attractive therapeutic molecular target in MM. We observed that inhibition of HIF-1α blocked cell proliferation and induced cell apoptosis in vitro. We then showed that a transferrin–polyethylenimine–HIF-1α–short-hairpin RNA (Tf–PEI–HIF-1α–shRNA) complex could target MM specifically and efficiently both in vivo and in vitro, exploiting the high expression of the TfR in MM. The systemic delivery of sequence-specific small-interfering RNA (siRNA) against HIF-1α by the Tf– PEI–HIF-1α–shRNA complex dramatically inhibited tumor growth in the A375 MM xenograft model. The underlying concept of transfecting a HIF-1α shRNA expression vector complexed with Tf–PEI to block HIF-1α holds promise as a clinical approach to gene therapy for MM. IntroductionMalignant melanoma (MM) is responsible for 80% of skin cancer deaths1Scala S Ierano C Ottaiano A Franco R La Mura A Liguori G et al.CXC chemokine receptor 4 is expressed in uveal malignant melanoma and correlates with the epithelioid-mixed cell type.Cancer Immunol Immunother. 2007; 56: 1589-1595Crossref PubMed Scopus (34) Google Scholar and has become a major public health problem.2Markovic SN Erickson LA Rao RD Weenig RH Pockaj BA Bardia A et al.Malignant melanoma in the 21st century, part 1: epidemiology, risk factors, screening, prevention, and diagnosis.Mayo Clin Proc. 2007; 82: 364-380Abstract Full Text Full Text PDF PubMed Scopus (368) Google Scholar Effective therapy for MM remains an unmet goal, with most traditional therapies representing inadequate trade-offs among the several goals of specificity, efficacy, and toxicity.3Becker JC Kirkwood JM Agarwala SS Dummer R Schrama D Hauschild A Molecularly targeted therapy for melanoma: current reality and future options.Cancer. 2006; 107: 2317-2327Crossref PubMed Scopus (44) Google Scholar Modulation of aberrant signaling pathways in MM cells has the potential to provide more effective and potentially nontoxic therapy for MM.3Becker JC Kirkwood JM Agarwala SS Dummer R Schrama D Hauschild A Molecularly targeted therapy for melanoma: current reality and future options.Cancer. 2006; 107: 2317-2327Crossref PubMed Scopus (44) Google Scholar In our previous studies, we suppressed the synthesis of vascular endothelial growth factor using small-interfering RNA (siRNA) and evaluated its therapeutic significance in a MM xenograft model.4Tao J Tu YT Huang CZ Feng AP Wu Q Lian YJ et al.Inhibiting the growth of malignant melanoma by blocking the expression of vascular endothelial growth factor using an RNA interference approach.Br J Dermatol. 2005; 153: 715-724Crossref PubMed Scopus (34) Google Scholar We found there were problems with this approach. Vascular endothelial growth factor is one of the downstream target genes of hypoxia-inducible factor-1α (HIF-1α), which is turned on in the presence of low oxygen levels, and HIF-1α is itself important in tumor neoangiogenesis.5Kamat CD Green DE Curilla S Warnke L Hamilton JW Sturup S et al.Role of HIF signaling on tumorigenesis in response to chronic low-dose arsenic administration.Toxicol Sci. 2005; 86: 248-257Crossref PubMed Scopus (63) Google Scholar The important role of HIF-1α in angiogenesis, glucose utilization, and tumor-cell survival and the fact that loss of HIF-1α activity inhibits growth of tumor xenografts in mice make HIF-1α a potentially attractive tumor-specific target.6Michaylira CZ Nakagawa H Hypoxic microenvironment as a cradle for melanoma development and progression.Cancer Biol Ther. 2006; 5: 476-479Crossref PubMed Scopus (39) Google Scholar In this study, our goal was to suppress the synthesis of HIF-1α in several human MM cell lines using short-hairpin RNA (shRNA) and to evaluate the effect of this suppression in a MM xenograft model. The utility of synthetic siRNAs for such applications is limited by both low-to-moderate transfection efficiency and by the short-term persistence of transient gene expression.7Masiero M Nardo G Indraccolo S Favaro E RNA interference: implications for cancer treatment.Mol Aspects Med. 2007; 28: 143-166Crossref PubMed Scopus (63) Google Scholar To overcome these limitations, we used a mammalian expression vector that directed the synthesis of shRNA to suppress the targeted messenger RNA (mRNA). Delivery of shRNA expression vectors into specific sites in vivo is a further obstacle for RNA interference–based therapy.8Vorhies JS Nemunaitis J Nonviral delivery vehicles for use in short hairpin RNA-based cancer therapies.Expert Rev Anticancer Ther. 2007; 7: 373-382Crossref PubMed Scopus (33) Google Scholar To achieve cell-specific delivery of shRNAs, one approach is to identify targeting ligands, antibodies, or aptamers for surface receptors to achieve site-specific gene delivery via receptor-mediated endocytosis.9Kim SI Shin D Choi TH Lee JC Cheon GJ Kim KY et al.Systemic and specific delivery of small interfering RNAs to the liver mediated by apolipoprotein A-I.Mol Ther. 2007; 15: 1145-1152Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar Most normal cells other than immature erythroid cells are low in transferrin receptor (TfR) expression, but actively proliferating tumor cells exhibit high levels of TfR.10Cheng Y Zak O Aisen P Harrison SC Walz T Structure of the human transferrin receptor-transferrin complex.Cell. 2004; 116: 565-576Abstract Full Text Full Text PDF PubMed Scopus (391) Google Scholar This suggests that targeting mediated by TfR might provide a method for delivery of anticancer agents into tumor cells in vivo as well as in vitro. In previous studies, our laboratory successfully constructed a human–mouse chimeric antibody and an intracellular antibody against TfR; they both displayed a tumor-specific distribution and had a strong antitumor effect. These studies showed that using TfR as a surface marker for tumor targeting was a plausible strategy.11Qing Y Shuo W Zhihua W Huifen Z Ping L Lijiang L et al.The in vitro antitumor effect and in vivo tumor-specificity distribution of human-mouse chimeric antibody against transferrin receptor.Cancer Immunol Immunother. 2006; 55: 1111-1121Crossref PubMed Scopus (34) Google Scholar,12Peng JL Wu S Zhao XP Wang M Li WH Shen X et al.Downregulation of transferrin receptor surface expression by intracellular antibody.Biochem Biophys Res Commun. 2007; 354: 864-871Crossref PubMed Scopus (23) Google Scholar Tf supplies iron to the cell via an interaction with TfR whereby Tf and TfR are internalized together, Tf releases its iron, and TfR is then recycled back to the surface. By virtue of this unique intracellular behavior, molecules conjugated to Tf may be efficiently delivered into cells with the TfR functioning through multiple internalization cycles.13Sato Y Yamauchi N Takahashi M Sasaki K Fukaura J Neda H et al.In vivo gene delivery to tumor cells by transferrin-streptavidin-DNA conjugate.Faseb J. 2000; 14: 2108-2118Crossref PubMed Scopus (43) Google ScholarIn the present study, we employed a transferrin–polyethylenimine (Tf–PEI) delivery system to carry HIF-1α shRNAs to distant tumors with the aim of silencing HIF-1α in MM tissue.ResultsHIF-1α expression in MMWe initially determined HIF-1α expression in patients with MM by histochemistry. Among the 77 MM lesions examined, HIF-1α expression was positive in 56/77 (72.7%). Among the 10 pigmented nevi lesions, HIF-1α expression was positive in 2/10(20%). Compared to the pigmented nevi lesions, MM lesions showed a significantly elevated (P = 0.008) frequency of HIF-1α expression (Figure 1a). We then evaluated HIF-1α expression by western blotting in 20 fresh MM tumors from patients and in 15 normal pigmented nevi lesions. The MM tumors showed a higher frequency of expression of HIF-1α than did the normal nevi (P = 0.004) (Figure 1b). Next, we observed abundant expression of HIF-1α protein in cells of the A375, A875, and KZ28 melanoma cell lines under hypoxic culture conditions, as determined by western blotting (Figure 1c). The strong upregulation of HIF-1α in MM, as found in this study, suggested that HIF-1α might be an important factor for MM growth and could be an attractive therapeutic molecular target for MM.Silencing HIF-1α in MM cell lines in vitro by HIF-1α shRNAsTo study the capacity of HIF-1α shRNA to downregulate HIF-1α, we transfected three shRNA constructs—shRNA1, shRNA2, and shRNA3—individually into cells of the A375, A875, and KZ28 melanoma cell lines. SYBR Green real-time reverse transcriptase PCR (RT-PCR) was used to measure downregulated levels of HIF-1α mRNA in all three transfected MM cell lines under hypoxic condition at time points of 6, 12, 24, and 48 hours after transfection. HIF-1α shRNAs showed variable efficacy in decreasing HIF-1α mRNA levels in the three cell lines relative to the control-scrambled shRNA and the mock group. ShRNA1 decreased HIF-1α mRNA by 74, 69, and 70% in the three cell lines, respectively, at 24 hours after transfection. ShRNA3 decreased HIF-1α mRNA in the three cell lines by 32, 24, and 22%, respectively, at the same time point. ShRNA2 resulted in a slightly decreased but no significant alteration of HIF-1α mRNA expression in any of the three cell lines. Downregulation of HIF-1α was not observed in the three cell lines transfected with control-scrambled shRNA or with mock transfection, and the mRNA level of β-actin remained unchanged after transfection in all three cell lines. Figure 2a illustrates the levels of HIF-1α mRNA in the A375 cells after knockdown with the HIF-1α shRNAs at the different time points. Among the HIF-1α shRNAs we tested, shRNA1 possessed the strongest inhibitory effect against HIF-1α and was effective in all three cell lines. For further experiments, we selected the shRNA1-A375 stable clone.Figure 2Hypoxia-inducible factor-1α (HIF-1α) short-hairpin RNAs (shRNAs) silencing of HIF-1α in malignant melanoma (MM) cell lines in vitro and its effects. (a) HIF-1α shRNAs downregulated HIF-1α expression in A375 cells. Comparison of HIF-1α messenger RNA (mRNA) levels of different HIF-1α shRNA-treated A375 cells revealed by real-time PCR at different time points. The expression level of HIF-1α was compared to the level of gene expression found in nontransfected negative controls, arbitrarily assigned the value 1. Bars represent the fold reduction in gene expression over the expression level in the nontransfected A375 cells, and shRNA1, shRNA2, and shRNA3 decreased HIF-1α mRNA by 74, 10, and 32%, respectively, in the A375 cells in the 24 hours after transfection. (b) The effects of shRNA1-mediated silencing of HIF-1α gene expression on the proliferation of A375 cells. Viable cells were determined by the MTT assay. The shRNA1-A375 stable clone, the negative control–scrambled shRNA-A375 stable clone, and untreated A375 cells (5 × 103) were plated on 96-well plates and evaluated by the MTT assay at 48 hours. Each bar represents the mean value of three identical wells from an experiment representative of three trials with different cell cultures. For all experiments combined, the shRNA1-A375 stable clone was significantly less than for the negative control–scrambled shRNA-A375 stable clone and untreated A375 cells. (c) The effects of shRNA1-mediated silencing of HIF-1α gene expression on the apoptosis of A375 cells. Cells in the bottom left quadrant represent viable cells (low annexin V-Alexa and BoBo-1 staining); cells in the bottom right quadrant represent early apoptotic cells (high annexin V-Alexa staining but low BoBo-1 staining); and cells in the top right quadrant represent late apoptotic cells (high annexin V-Alexa and BoBo-1 staining). The percentage of cells in each quadrant is indicated in the right up quadrant of the panels. Shown are representative data from one of three independent experiments with samples in triplicate. Increases in the percentage of apoptotic cells in the shRNA1-A375 stable clone compared with the negative control–scrambled shRNA-A375 stable clone and untreated A375 cells were of statistical significance (P = 0.003).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Downregulation of HIF-1α expression has been found to prevent cell proliferation and induce cell apoptosis in vitro. We, therefore, examined whether the loss of HIF-1α by shRNA treatment could affect cell proliferation and apoptosis in vitro. First, as shown in Figure 2b, the shRNA1-A375 stable clone was significantly affected compared to the negative control–scrambled shRNA-A375 stable clone and untreated A375 cells. The number of viable cells of the shRNA1-A375 stable clone was reduced significantly (~30%) at 48 hours under hypoxic conditions. Second, as shown in Figure 2c, at 48 hours under hypoxic conditions the siRNA1-A375 stable clone cells showed a 38.55% rate of apoptosis. This value compared with an apoptosis rate of 6.16 and 4.52% in the negative control–scrambled shRNA-A375 stable clone and the untreated A375 cells, respectively. The difference was statistically significant (P = 0.003). These results indicated that the downregulation of HIF-1α could significantly prevent cell proliferation and enhance apoptosis in A375 exposed to hypoxic conditions.The experiments above confirmed that HIF-1α could be an effective target for anticancer therapy by using HIF-1α shRNAs in MM cell lines in vitro. However, delivery of shRNA expression vectors into specific sites in vivo is a major obstacle for RNA interference–based therapy. To achieve cell-specific delivery of shRNAs, we needed to identify whether TfR, which might be used to achieve site-specific gene delivery via receptor-mediated endocytosis, was highly expressed in MM.TfR expression in MMTo explore TfR expression in MM, we used immunohistochemistry to initially test TfR expression in MM lesions of patients. Among the 77 MM lesions, TfR expression was positive in 73/77 (94.8%). Among the 10 pigmented nevi lesions, TfR expression was positive in 1/10 (10%). The MM tissues demonstrated significantly higher TfR expression than did the control nevus tissues (P = 0.002) (Figure 3a). Flow cytometry was used to evaluate qualitatively the level of expression of TfR at the cell surface membrane in MM cell lines. The levels of expression of TfR in A375, A875, and KZ28 cells were compared to that of the A2780 cells; we observed expression by 97, 82, 92, and 2% of the cells of the A375, A875, KZ28, and A2780 lines, respectively (Figure 3b). These results suggested that high expression of TfR on the MM cell surface membrane could provide an appropriate target receptor for gene therapy in MM.Figure 3Transferrin receptor (TfR) expression in malignant melanoma (MM) tissues and cell lines. (a) TfR staining in the tissues of MM (A1 and A2) and normal nevus (A3 and A4). A strong immune reaction in MM and a weak immune reaction in normal nevus were typically observed by immunohistochemistry (A1 and A3 ×100 magnification; A2 and A4 ×200 magnification). (b) TfR expression in the MM cell lines A375, A875, and KZ28 and in the human ovarian cancer cell line A2780 by flow cytometry. The levels of the cell surface TfR in A375, A875, and KZ28 cells are shown in the right top (B1: A2780, B2: A375, B3: A875, B4: KZ28).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tumor-targeted distribution of the Tf–PEI–shRNA complex in vitro and in vivo after administrationTumor-targeted distribution in vitro. TfR-dependent uptake of the Tf–PEI–shRNA would predict that (i) shRNA expression is reduced in competition experiments with free Tf or with shRNA complexes lacking Tf, (ii) the transfection efficiency is decreased in cells with a low level of expression of TfR, and (iii) transfection efficiency is enhanced in cells with a high level of expression of TfR. The shRNA vector Pgenesil-1 had a green fluorescent protein (GFP) label, so it was possible to quantify shRNA delivery from the level of GFP expression as measured by flow cytometry. To determine whether Tf–PEI was able to deliver shRNA specifically into cells expressing TfR, the shRNA was added to the A375 and A2780 cells either (i) alone, (ii) with the unmodified Tf, (iii) with PEI, or (iv) with the Tf–PEI complex. Lipofectamine2000 transfection was used as a positive control. As shown in Figure 4a, shRNA transfected with Lipofectamine2000 was comparably taken up by both the A2780 and the A375 cells (~70% of the cells). Neither the A2780 cells nor the A375 cells appreciably took up shRNA by itself or when it was mixed either with Tf lacking PEI or with unmodified PEI. When mixed with Tf–PEI, the A2780 cells still not take up shRNA. In contrast, when mixed with Tf–PEI, the shRNA was taken up by ~52% of the A375 cells. Although lower than the ~70% rate of transfection observed for the A375 cells when treated with Lipofectamine2000, Tf–PEI transfection provided selective delivery of shRNA into the MM cells with high TfR expression. Therefore, these results indicated that Tf–PEI could be a useful tool for delivery of shRNA into MM cells in vitro.Figure 4Analysis of tumor-targeted distribution of the Tf–PEI–shRNA complex after administration. (a) Tumor-targeted distribution in vitro. Approximately ~50% of the A375 cells took up Tf–PEI–shRNA, whereas the A2780 cells did not. There was no difference in uptake between the A2780 and A375 cells when short-hairpin RNA (shRNA) was transfected by Lipofectamine2000. Neither the A2780 nor A375 cells appreciably took up shRNA-GFP by themselves or when mixed with transferring (Tf) lacking polyethylenimine (PEI) or with unmodified PEI. (b) Tumor-targeted distribution in vivo. Green fluorescent protein (GFP) messenger RNA (mRNA) expression in tumor tissues and major organs of nude mice bearing A375 or A2780 tumors and injected intravenous with Tf–PEI–shRNA was determined by the SYBR Green real-time RT-PCR assay. The method of quantitation of GFP mRNA was described in Materials and Methods. (b1) GFP mRNA distribution in tissues and major organs of A375 tumor–bearing nude mice. (b2) GFP mRNA distribution in tissues and major organs of A2780 tumor–bearing nude mice. The highest amounts of GFP mRNA were detected in tumors and only small amounts of GFP mRNA were detected in major organs such as liver, lungs, heart, and kidneys in the A375 tumor–bearing mice at a time point of 24 hours after the single injection (P = 0.004). The GFP mRNA distribution in tumor tissues and major organs of A2780 tumor–bearing mice revealed no significant differences (P = 0.496).View Large Image Figure ViewerDownload Hi-res image Download (PPT)Tumor-targeted distribution in vivo. To examine the ability of Tf–PEI to deliver shRNA, specifically to cells expressing TfR at a high level in tumor tissue in vivo, Tf–PEI–shRNA was injected via standard low-pressure tail-vein injection into nude mice bearing A375 subcutaneous (s.c.)xenograft tumors and nude mice bearing A2780 s.c.xenograft tumors, as described in Materials and Methods. Because the shRNA vector Pgenesil-1 had a GFP label, we could identify the efficiency of shRNA transfer by GFP expression. Figure 4b shows the distribution of GFP mRNA in major organs and tumor tissues of A375 and A2780 tumor-bearing mice at time points of 1, 12, 24, and 48 hours after application of Tf–PEI–shRNA. The highest amounts of GFP mRNA were detected in tumor tissues and in major organs such as liver, lungs, heart, and kidneys. Only small amounts of GFP mRNA were detected in the A375 tumor–bearing mice at 24 hours after the single injection. At the same time, the result revealed that the distribution of GFP mRNA expression in the tumor tissues and major organs of A2780 tumor–bearing mice had no significant differences. The results demonstrated that Tf–PEI could deliver shRNA with tumor-targeted specific distribution in the nude mice bearing A375 s.c.xenograft tumors.Taken together, these results showed the tumor-targeted, relatively specific distribution of Tf–PEI–shRNA complex in vitro and in vivo after administration.Observation of the growth rate of MM xenograft tumors after injection with Tf–PEI–HIF-1α–shRNA1 in vivoWe carried out the following experiment to evaluate the therapeutic potential of the Tf–PEI–shRNA1 complex in the A375 tumor xenograft. The method of administration of the “therapeutic” plasmid Tf–PEI–shRNA1 complex for the A375 and A2780 tumor xenografts was described in Materials and Methods. As shown in Figure 5a, at the time of killing, the volumes of the A375 group tumors in the control second and third treatment groups were 610 ± 145 mm3 and 655 ± 90 mm3, a 7.5-fold increase over the starting volume, whereas the A375 tumors injected with the Tf–PEI–HIF-1α–shRNA1 complex had a volume of 310 ± 90 mm3, resulting in a volume increase of only 3.9-fold. The delay in tumor growth was statistically significant (P = 0.0014) from day 5 after the beginning of therapy until the day the mice were killed. During days 10–15, after treatment, we observed the strongest effect of the Tf–PEI–shRNA1 complex; in this period, the growth rates of the second and third control groups were 64 ± 7.5 mm3/day and 68 ± 9.5 mm3/day, respectively, whereas in the treated group it was 8 ± 8.6 mm3/day (P = 0.002), demonstrating that the growth rate of the tumor of the two control groups was nearly eightfold greater compared with the treated group. As shown in Figure 5b, at the time point of killing, the A2780 tumors in the three groups had volumes of 710 ± 145 mm3, 755 ± 90 mm3, and 695 ± 120 mm3, all ~7.5-fold larger than the starting volume. A delay in tumor growth was not found in the therapeutic group. These preliminary data clearly showed that a single injection of the Tf–PEI–shRNA1 complex was able to slow down growth of the A375-derived xenografts and, most importantly, it had a lasting effect on tumor development, being effective for at least 25 days. We concluded that Tf–PEI was able to deliver shRNAs specifically to TfR-expressed MM cells to suppress MM growth even when administered systemically.Figure 5Observation of the growth rate of malignant melanoma (MM) xenograft tumors after injection with Tf–PEI–shRNA1 in vivo. Each point represents the mean volume ± SD. N = 12 for the shRNA1 and scrambled shRNA–injected groups (n = 12) and n = 6 for the untreated group. One arrow represents the day of the Tf–PEI–shRNA injection, and two arrows is the time of killing of the mice (**P < 0.001). (a) The therapeutic potential of the Tf–PEI–shRNA1 complex in the A375 tumor xenograft. At the time point of killing, for the A375 group, the tumors in the control second and third groups had volumes of 610 ± 145 mm3 and 655 ± 90 mm3, respectively, which were 7.5-fold larger than the starting volume, whereas the A375 tumors of the mice injected with the Tf–PEI–shRNA1 complex had a volume of 310 ± 90 mm3, resulting in a volume increase of only 3.9-fold. The tumor growth delay was statistically significant (P = 0.0014) from day 5 after the beginning of therapy until the day the mice were killed. (b) The effect of the Tf–PEI–shRNA1 complex in the A2780 tumor xenograft. At the time point of sacrificing, the tumors for the A2780 xenografts in the three groups had volumes of 710 ± 145, 755 ± 90, and 695 ± 120 mm3, respectively, which were 7.5-fold larger than the starting volume. A tumor growth delay was not found in the therapeutic group (P = 0.2460). Tf, transferring; PEI, polyethylenimine; shRNA, short-hairpin RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Detection in tumor sections of HIF-1α knockdown after systemic administration of the Tf–PEI–HIF-1α–shRNA complexNext, we needed to explore whether suppressing tumor growth when the Tf–PEI–HIF-1α–shRNA complex was administered systemically was related to knockdown of HIF-1α. As shown in Figure 6a, the results of the immunohistochemistry showed that, in the nude mice bearing A375 s.c.xenograft tumors, HIF-1α expression in the Tf–PEI–shRNA1 complex group were significantly lower than in the Tf–PEI–scrambled shRNA and A375 groups, and no significant difference in HIF-1α expression was found between the Tf–PEI–scrambled shRNA and A375 groups. In contrast, in the nude mice bearing A2780 s.c.xenograft tumors, HIF-1α expression in the three groups was not significantly different. The results by western blotting for HIF-1α expression (Figure 6b) were in agreement with those by immunohistochemistry (Figure 6a). Therefore, we concluded that systemic administration of the Tf–PEI–HIF-1α–shRNA complex in the nude mice bearing A375 s.c.xenograft tumors could knockdown HIF-1α, which slowed the rate of growth of the MM xenograft tumors.Figure 6Detection of hypoxia-inducible factor-1α (HIF-1α) knockdown in tumor sections after systemic Tf–PEI–HIF-1α–shRNA complex administration. (a) HIF-1α expression was determined by immunohistochemistry. HIF-1α expression in the Tf–PEI–shRNA1 complex group was significantly lower than in the Tf–PEI–HIF-1α-scrambled shRNA and control groups in the nude mice bearing A375 s.c.xenograft tumors. No significant differences in HIF-1α expression were found between the three groups in the nude mice bearing A2780 s.c.xenograft tumors (×200 magnification). (b) HIF-1α expression determined by western blotting. The results as determined by western blotting were in agreement with those determined by immunohistochemistry. Tf, transferring; PEI, polyethylenimine; shRNA, short-hairpin RNA.View Large Image Figure ViewerDownload Hi-res image Download (PPT)DiscussionAn ideal therapy for cancer would be one that selectively targets tumor cells, is nontoxic to normal cells, and could be systemically delivered, thereby reaching metastases as well as the primary tumor.14Kircheis R Schuller S Brunner S Ogris M Heider KH Zauner W et al.Polycation-based DNA complexes for tumor-targeted gene delivery in vivo.J Gene Med. 1999; 1: 111-120Crossref PubMed Google Scholar The receptor-mediated (such as TfR-mediated) gene delivery approach has a further attractive feature because it provides an opportunity to achieve cell-specific delivery of the targeted gene while enhancing the transfection efficiency.15Hu-Lieskovan S Heidel JD Bartlett DW Davis ME Triche TJ Sequence-specific knockdown of EWS-FLI1 by targeted, nonviral delivery of small interfering RNA inhibits tumor growth in a murine model of metastatic Ewing's sarcoma.Cancer Res. 2005; 65: 8984-8992Crossref PubMed Scopus (497) Google Scholar In previous studies, our lab successfully constructed a human–mouse chimeric antibody and an intracellular antibody against TfR and they both displayed a tumor-specific distribution and had a strong antitumor effect. It has further been shown that using TfR as a surface marker for targeting is a tumor therapeutic strategy.11Qing Y Shuo W Zhihua W Huifen Z Ping L Lijiang L et al.The in vitro antitumor effect and in vivo tumor-specificity distribution of human-mouse chimeric antibody against transferrin receptor.Cancer Immunol Immunother. 2006; 55: 1111-1121Crossref PubMed Scopus (34) Google Scholar,12Peng JL Wu S Zhao XP Wang M Li WH Shen X et al.Downregulation of transferrin receptor surface expression by intracellular antibody.Biochem Biophys Res Commun. 2007; 354: 864-871Crossref PubMed Scopus (23) Google Scholar The strategy of exploiting the Tf/TfR system for gene targeting is based on elevated levels of TfR present on the surface of tumor cells. In this study, we have shown that MM tissue as well as cell lines demonstrated abundant expression of TfR, especially on the cell surface membrane (Figure 3). These results suggested that TfR was a suitable targeting molecule for gene therapy in MM and that modification of shRNA formulations to contain Tf as a ligand for the TfR might lead to successful cell-specific delivery of siRNA complexes in MM. In this study, w