Abstract
Aim: To evaluate if the lentiviral accessory protein Nef can down-regulate the C-X-C chemokine receptor type 4 (CXCR4) in tumor cells and affect tumor cell proliferation, migration and angiogenesis. Materials and Methods: HeLa-ACC cells, which according to genotype analysis are virtually identical to the cervical cancer-derived HeLa cell line, were transfected with Nef from SIVmac239 and expression levels of cell surface CXCR4 were monitored by flow cytometry. Real-time proliferation and migration of cells was measured with the xCELLigence system or with the in vitro scratch assay. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis. Results: Cell surface down-regulation of CXCR4 was observed in HeLa-ACC cells after Nef transfection, as well as in the monkey kidney-derived COS-7 cell line after co-transfection of CXCR4 and Nef. Proliferation, as well as migration, of Nef-transfected HeLa-ACC cells appeared to be significantly reduced. In vitro tube formation was markedly lowered after Nef transfection, and CXCR4 knockdown with siRNA. Conclusion: SIV-Nef could serve as an interesting tool to study the biological behavior of CXCR4-expressing tumor cells and could be helpful in the discovery of new therapeutic approaches for the treatment of CXCR4-positive tumors.
The C-X-C chemokine receptor type 4 (CXCR4), also known as fusin or CD184, is a specific receptor for the CXC chemokine stromal cell-derived factor-1α (SDF-1α). CXCR4 has a short, acidic extracellular N-terminal end, as well as seven helical transmembrane domains with three intracellular hydrophilic loops (1). As typically observed for G-protein-coupled receptors, the intracellular C-terminus of CXCR4 interacts with G proteins and thereby enables transmembrane cell signaling after SDF-1α binding. CXCR4/SDF-1α activates numerous intracellular signaling pathways, influencing cell adhesion, chemotaxis, migration, proliferation and apoptosis (2-5).
CXCR4 is also known as a co-receptor for human and simian immunodeficiency viruses (HIV/SIV) during viral entry (6-8) and, together with its ligand SDF-1α, plays a central role in hematopoietic stem cell homing and bone marrow release. Knockout of CXCR4 or SDF-1α in transgenic mice results in embryonic lethality and underscores its critical role in the development of the nervous, cardiovascular and hematopoietic systems (9, 10). CXCR4 is overexpressed in a wide variety of human cancer types, such as lung, pancreatic, breast, prostate and kidney cancer (11-16). It is not surprising that more and more evidence indicate CXCR4/SDF-1α as playing an important role in tumor development, since it not only promotes cancer cell proliferation and migration, but also enhances metastasis and tumor angiogenesis (17, 18).
Negative factor (Nef) is an accessory protein expressed by primate lentiviruses, such as HIV-1 and -2 and SIV. Nef is also a key factor for viral infection and replication and can down-modulate the cell surface receptors CD4, CCR5 and CXCR4 that are required for HIV/SIV entry aiming to prevent a lethal viral superinfection (19). It can also down-regulate major histocompatibility (MHC) class I molecules due to retention in the Golgi apparatus, which promotes immune-escape of infected cells. Nef proteins from different viral strains exhibit large differences in their ability to down-regulate CXCR4. For example, Hrecka et al. (20) observed a strong down-regulating effect of Nef from SIVmac239 and HIV-2 on cell surface CXCR4 expression and SDF-1α-dependent lymphocyte migration. In contrast, HIV-1 Nef did not exhibit a comparable effect. However, Venzke et al. did not observe such differences between HIV and SIV Nef (19).
Short tandem repeat analysis.
Since down-regulation of CXCR4 by Nef was demonstrated in Jurkat T-cells, it is of interest to evaluate if Nef also affects expression of this receptor in CXCR4-positive cells derived from solid tumors, and subsequently influence tumor-promoting features, such as proliferation, migration and angiogenesis.
Materials and Methods
Short tandem repeat (STR) analysis and cell culture. The cell line initially designated as ACC3 (21) that was used in our study was shown to exhibit high CXCR4 expression levels (22). Recently it was reported (23, 24) that this cell line was cross-contaminated with the cervical cancer-derived HeLa cell line in different laboratories world wide. In order to verify the identity of the cell line used in our study, DNA was isolated from ACC3 cells using the Blood and Cell Culture DNA Mini Kit from Qiagen (Hilden, Germany) and sent for STR analysis (German Biological Resource Centre Human and Animal Cell Lines, DSMZ, Braunschweig, Germany). The data revealed that the ACC3 cell line was most closely related to HeLa cells as previously reported by Phuchareon et al. (23) (Table I). To distinguish the cells used in our studies from regular HeLa cells, although according to the STR analysis no significant difference should be expected, we designated the cells as HeLa-ACC during the course of our study. Since HeLa cells exhibit high cell surface expression levels of CXCR4 and have often been used in research to evaluate the role of CXCR4 during the infection with HIV (25, 26), HeLa-ACC cells should also be suitable to evaluate the effect of SIVmac239-Nef on tumor cell proliferation, migration and angiogenesis.
COS-7 (27) and HeLa-ACC cells were cultured in Dulbecco's modified Eagle's medium (DMEM) in the presence of 10% fetal calf serum, 1% L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin. Human umbilical vein endothelial cells (HUVECs; C-003-5C; Life Technologies Corporation, Carlsbad, CA, USA) were cultured in EGM-2 medium supplemented with growth factors (Lonza Group Ltd., Basel, Switzerland). The culture medium was replaced every three days and the cells were passaged after reaching 80% confluence.
Construction of recombinant plasmids and transfection into cell lines. Full-length wild-type SIVmac239 Nef DNA (GenBank: M33262.1) and its functionally deficient mutant Nef-M8 (Y28A-Y39A-DE184/185AA-LM194/195AA-DD204/205AA) were amplified by polymerase chain reaction (PCR) using previously published Nef constructs as a template (28). Full-length CXCR4 (NCBI Reference Sequence: NM_003467.2) DNA was obtained by RT-PCR (Transcriptor First Strand cDNA Synthesis Kit; Roche Applied Science, Basel, Switzerland. REDTaq™ ReadyMix™ PCR reaction Mix; Sigma-Aldrich Corp. St. Louis, MO, USA) from total RNA (RNeasy Mini Kit; Qiagen) from HUVECs, which are known to highly express CXCR4. PCR was performed for 40 cycles (95°C, 30 s; 56°C, 1 min; 72°C, 1 min) with primers containing an EcoRI (Nef: 5’-AAACTTAAGCTTGCCACCATGGGTGGAG CTATTT-3’; CXCR4: 5’-AAACTTAAGCTTGCCACCATGTCCA TTCCT-3’) or HindIII (Nef: 5’-TCTGCAGAATTCTCAGCGAG TTTCCTTC-3’; CXCR4: 5’-GGCTAGGAATTCCATCTGTGTTAG-3’) restriction site. PCR products were gel-purified (QIAquick Gel Extraction Kit; Qiagen), double-digested with HindIII and EcoRI (New England Biolabs GmbH, Frankfurt, Germany) and subcloned into the pcDNA3.1(+) mammalian expression vector (Invitrogen Corporation, Carlsbad, CA, USA). The recombinant constructs, pcDNA3.1(+)-SIVmac239Nef-WT (Nef-WT), pcDNA3.1(+)-SIVmac239Nef-M8 (Nef-M8) and pcDNA3.1(+)-CXCR4 were confirmed by sequence analysis (4base lab GmbH, Reutlingen, Germany). Purified plasmid DNA (QIAprep Spin Midiprep Kit; Qiagen), CXCR4 siRNA and scrambled RNA (Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) were used to transfect (Lipofectamin™2000, Lipofectamin™RNAiMAX; Invitrogen Corporation. TranspassTM HUVEC Transfection Reagent; New England Biolabs GmbH, Ipswich, MA, USA) COS-7, HeLa-ACC and HUVECs according to the manufacturers' protocol.
Western blot and antibodies. Forty-eight hours after transfection, cells were harvested and lysed as described earlier (29). Thirty-five micrograms of total protein was separated in a 10% polyacrylamide gel by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to a nitrocellulose membrane. Membranes were blocked in 3% milk/phosphate buffered saline (PBS) (1 h; room temperature (RT)) and incubated (overnight, 4°C) with primary antibodies directed against SIV-Nef and human CXCR4 (SIV-Nef, sc-65911; Santa Cruz Biotechnology Inc.. CXCR4 antibody, ab2074; Abcam PLC, Cambridge, UK). Membranes were washed three times in 3% milk/PBS and incubated with horseradish peroxidase (HRP)-linked anti-rabbit or anti-mouse secondary antibodies (Santa Cruz Biotechnology Inc.) for 1 h at RT. After washing away excessive antibody, bands were visualized with the enhanced chemiluminescence (ECL) method on x-ray film.
Flow cytometry. HeLa-ACC and COS-7 cells were washed in PBS/0.5% bovine serum albumine (BSA) and incubated with a phycoerythrin (PE)-conjugated CXCR4 antibody (PE Mouse Anti-Human CD184; BD Biosciences, San Jose, CA, USA). IgG2α was used as an isotype control. Cell surface fluorescence was monitored by flow cytometry (BD LSR II; BD Biosciences) and data analysis was performed using FlowJo software (Tree Star, Inc., Ashland, OR, USA).
Immunocytochemistry. HeLa-ACC cells that were grown on coverslips were transfected with Nef or control vector and incubated for 48 h. Cells were washed in PBS, fixed for 30 min in cold (−20°C) methanol and incubated in staining buffer (3%BSA/0.3%NP40/PBS) for 30 min. Then anti-CXCR4 antibodies were added and incubation was continued for two more hours. Subsequently, cells were washed in staining buffer and secondary Texas Red (TR)-conjugated goat anti-mouse IgG (for CXCR4) was added for another hour at RT. Subcellular localization was evaluated by confocal laser scanning microscopy (Fluoview; Olympus Deutschland GmbH, Hamburg, Germany).
In vitro scratch assay. HeLa-ACC cells transfected with Nef, Nef-M8 or vector only were seeded into 6-well plates and cell growth was allowed to continue until confluence was reached. The cell monolayer was scratched with a 10 μl pipette tip and dislodged cells were washed away with PBS. Cell incubation was continued under standard conditions and the degree of cell migration into the scraped area was documented every 24 h (30).
Cell proliferation and migration. Real-time cell proliferation was monitored with the xCELLigence system (Roche, Mannheim, Germany). Cells were suspended in 150 μl DMEM and added into a 96-well microtiter plate specifically designed to measure cellular impedance (E-Plate; Roche). The measured impedance, which is dependent on the level of confluence, is expressed as an arbitrary unit called the Cell Index. The Cell Index at each time point is defined as (Rn-Rb)/(15Ω), where Rn is the cell-electrode impedance of the well when it contains cells and Rb is the background impedance of the well containing medium alone. A total of 5×103 HeLa-ACC cells transfected with Nef, Nef-M8, vector only, CXCR4-specific siRNA, or scrambled RNA were seeded on the E-plate. The proliferation of the cells was monitored every 15 min. SDF-1α was added to a final concentration of 100 ng/ml 24 h after cell seeding and proliferation was monitored continuously for 72 more hours. The cell monolayer was scratched once the impedance reached the maximum in the same way as described for the in vitro scratch assay and the Cell Index was monitored as described above.
Evaluation of phospho-extracellular signal-regulated kinases (ERK), phospho-signal transducer and activator of transcription (STAT3) and caspase-3 in HeLa-ACC cells. Twenty-four hours after transfection of Nef or vector-only control, HeLa-ACC cells were treated with SDF-1α (100 ng/ml). Cells were lysed at time points 0, 5, 10 and 30 min. p-STAT3 (Santa Cruz Biotechnology Inc.), phosphorylated ERK1/2 (Sigma, Saint Louis, MO, USA) and caspase-3 (Santa Cruz Biotechnology Inc.) were evaluated by western blot analysis. Beta-actin (Sigma) was used as control. Fourty-eight hours after transfection with Nef, Nef-M8 or vector only, HeLa-ACC cells were lysed and protein expression was evaluated by western blot analysis.
In vitro tube formation assay. In order to evaluate the effect of SIVmac239-Nef on angiogenesis, endothelial tube formation was evaluated using an in vitro angiogenesis kit (Cell Biolabs, Inc. San Diego, CA, USA). A total of 2×104 HUVECs transfected with Nef, Nef-M8, vector only, CXCR4 siRNA, or scrambled RNA were seeded into a 96-well plate precoated with 50 μl extracellular matrix (ECM) gel and incubated at 37°C for 6 h. Endothelial tube formation was documented by light microscopy.
Statistical analysis. Nonparametric tests were used for data analysis. The Mann-Whitney test was implemented to evaluate the statistical significance of the proliferation rates between SIV-Nef, -Nef-M8 and vector only-transfected HeLa-ACC cells. A probability less than 0.05 (p<0.05) was considered statistically significant.
Results
Down-regulation of CXCR4 surface expression after co-transfection of SIVmac239-Nef and CXCR4 in COS-7 cells. Mammalian expression vectors containing the genes from Nef, Nef-M8 or CXCR4 were generated as described above. COS-7 cells were deployed to assess the expression levels of Nef, Nef-M8 and CXCR4 and to evaluate the influence of Nef on CXCR4 in this well-established cell system. Only little amounts of endogenous CXCR4 protein were detected in COS-7 cells (Figure 1A).
COS-7 cells were co-transfected with CXCR4 and Nef, Nef-M8 or pcDNA3.1(+) (vector only control) (Figure 1A). Flow cytometry analysis revealed a reduction in CXCR4 surface fluorescence of COS-7 cells after co-transfection of Nef but not of Nef-M8 or vector only, pointing to a Nef-dependent down-regulation of the receptor from the cell surface in this cell line (Figure 1B).
SIVmac239-Nef down-regulates expression of endogenous CXCR4 in tumor cells. Tumor cells, such as HeLa-ACC, overexpress CXCR4. Immunocytochemical analyses identified CXCR4 in the cytoplasm, as well as at the plasma membrane (Figure 2A). Substantial endogenous expression of CXCR4 was also documented in western blots of vector only (control)-transfected HeLa-ACC cells (Figure 2B). HeLa-ACC cells were further transfected with Nef or Nef-M8 and the expression of Nef was determined by western blot analysis. There was no visible change in total CXCR4 protein expression levels after transfection with Nef (Figure 2B). However, Flow cytometry analysis revealed a reduction of cell surface CXCR4 in HeLa-ACC cells after transfection with Nef-WT, similarly as previously observed in COS-7 cells co-transfected with Nef and CXCR4 (Figure 2C). No significant procaspase-3 cleavage was observed after Nef transfection (Figure 2B).
SIVmac239-Nef reduces proliferation and migration of tumor cells. The xCELLigence system was deployed to measure real-time cell proliferation. Nef, Nef-M8, vector only, CXCR4 siRNA and scrambled RNA transfected HeLa-ACC cells were seeded into specialized 96-well culture plates and cellular impedance was measured continuously as described in the Materials and Methods. Control cells treated with SDF-1α exhibited an enhanced proliferation rate compared to untreated cells (Figure 3A). The proliferation rate of the control was taken as baseline (1.0) and the proliferation ratio of Nef and Nef-M8 to the vector control (relative proliferation rate) was plotted to highlight potential effects of Nef (Figure 3B). Nef-transfected HeLa-ACC cells exhibited a markedly reduced proliferation rate in both untreated and SDF-1α-treated groups, whereas Nef-M8-transfected cells exhibited a similar proliferation rate to the one of the vector only-transfected cells, pointing to a specific effect of the viral protein on HeLa-ACC cell proliferation. CXCR4 siRNA-transfected HeLa-ACC cells exhibited a pronounced inhibition of the proliferation rate compared to those transfected with scrambled RNA (as baseline 1.0) (Figure 3C) as similarly observed for the wild-type Nef (Figure 3B).
SIVmac239-Nef down-regulates cell surface expression of the C-X-C chemokine receptor type 4 (CXCR4) in COS-7 cells. A: Western blot analysis demonstrates co-expression of CXCR4 and wild-type Nef (Nef-WT) or Nef-M8 in COS-7 cells after co-transfection of the respective two constructs. Cells transfected with CXCR4 and with empty vector (control) express CXCR4 but exhibit no Nef signal. No significant change is noted in the total CXCR4 protein level after transfection with Nef. Actin was used as an internal control. B: Flow cytometry analysis demonstrates a reduction of CXCR4 from the cell surface in cells co-transfected with CXCR4 and Nef compared to cells co-transfected with CXCR4 and Nef-M8 or with empty vector (control).
Nef from SIVmac239 down-regulates endogenous CXCR4 in HeLa-ACC cells. A: Immunocytochemical analysis of HeLa-ACC cells. HeLa-ACC cells stain positively for endogenous CXCR4, which is found at the plasma membrane, as well as in the cytoplasm and nucleus. B: Western blot analysis reveals that all cells, transfected and untransfected, are positive for endogenous CXCR4, whereas only the Nef-transfected cells express the viral protein. No cleaved procaspase-3 was found in Nef-transfected cells. Actin was used as an internal control. C: Flow cytometry analysis demonstrates cell surface down-regulation of CXCR4 in wild-type Nef (Nef-WT)-transfected HeLa-ACC cells.
An in vitro scratch assay was performed to evaluate the influence of Nef on cellular migration. Cell numbers were normalized for better comparison. After scratching the cell layer, the subsequent rise in impedance was the lowest in the Nef-transfected HeLa-ACC cells compared with those transfected with Nef-M8 or vector only, pointing to Nef as an inhibitor of HeLa-ACC cell migration (Figure 4A). The short time required for impedance to rise again after scratching the cell layer indicates that migration rather than proliferation is responsible for this effect. Furthermore, microscopical analysis of HeLa-ACC cells revealed a difference in the rate of cells moving into the cell-free region (Figure 4B). Nef-transfected cells displayed the lowest migratory ability compared with those transfected with Nef-M8 or vector control, further pointing to an inhibitory effect of Nef on cell migration.
Nef from SIVmac239 reduces proliferation of HeLa-ACC cells. A: HeLa-ACC cells (vector control) exhibits enhanced proliferation after addition of 100 ng/ml stromal cell derived factor-1α (SDF-1α). B: Proliferation rate of wild-type Nef (Nef-WT) and Nef-M8-transfected HeLa-ACC cells relative to the vector-only transfected control cells, normalized to 1.0, reveals a significant (p=0.02 at 48 h) inhibition of proliferation in the Nef-WT-transfected cells. This effect was even more pronounced in the SDF-1α-treated cells (p=0.01 at 48 h). C: CXCR4 siRNA-transfected HeLa-ACC cells exhibited a reduced proliferation rate comparable to the one of wild-type Nef-transfected cells. (baseline=scrambled RNA).
Influence of SIVmac239-Nef on SDF-1/CXCR4 signaling in tumor cells. To determine whether SIV-Nef has an effect on MAPK/ERK signaling, HeLa-ACC cells were treated with SDF-1α and lysed at the indicated time points as described in the Materials and Methods. Phospho-ERK1/2 and phospho-STAT3 were detected by western blot (Figure 4C). No effect on procaspase-3 cleavage was observed. SIV-Nef appeared to induce a more enhanced decline in phospho-ERK1/2 and phospho-STAT3 expression after treatment with SDF-1α than did the negative control (vector only), however, this effect was not pronounced.
Effect of Nef on migration, signaling and angiogenesis. A and B: Nef from SIVmac239 inhibits migration of HeLa-ACC cells in an in vitro scratch assay. A: The xCELLigence system reveals a quick increase in electric impedance after scratching the cell layer of Nef-M8- and vector-only but not wild-type Nef (Nef-WT)-transfected HeLa-ACC cells. B: Similarly, microscopical analysis shows reduced migration of Nef-WT-transfected HeLa-ACC cells into the scratched cell-free area. C: Stromal cell derived factor-1α (SDF-1α)-treatment of HeLa-ACC cells reduces phospho-signal-transducer and activator of transcription-3 (STAT3) and phospho-extracellular signal-regulated kinase 1/2 (ERK1/2) levels. This effect appears to be more pronounced in the Nef-WT-transfected cells since the phospho-protein levels dropped as early as 10 min after SDF-1α addition. D: Nef inhibits angiogenesis. Nef-WT-transfected human umbilical vein endothelial cells lost most of their in vitro tube formation ability compared with control cells. A similar effect was observed after CXCR4 knockdown with siRNA.
SIVmac239-Nef inhibits in vitro angiogenesis. HUVECs were transfected with Nef, Nef-M8, CXCR4 siRNA or scrambled RNA. In vitro tube formation abilities were compared using light microscopy after 6 hours (Figure 4D). Nef- and CXCR4 siRNA-transfected HUVECs exhibited reduced tube formation ability compared with Nef-M8- and scrambled RNA-transfected HUVECs, pointing to an inhibitory effect of SIVmac239-Nef on angiogenesis that is comparable to CXCR4 inhibition.
Discussion
Nef is a 27 kDa, N-terminal myristoylated HIV/SIV accessory protein. During infection with HIV/SIV, Nef down-regulates CD4, the major receptor for virus entry, as well as its two co-receptors CXCR4 and CCR5, to prevent lethal viral superinfection of the infected cell (19, 20, 28, 31-33). Nef was also found to down-regulate MHC class I molecules from the cell surface of infected cells, reduce MHC class II levels in antigen-presenting cells and CD8 levels in cytotoxic T-lymphocytes (33-37), thereby promoting immune escape of the virus (38, 39).
CXCR4 is known not only as a co-receptor for HIV/SIV entry, but is also involved in malignant transformation, proliferation, migration and metastasis, where it plays an important role in directing metastatic CXCR4+ cells to organs that express high SDF-1α levels (22, 40-43). Therefore, targeting the CXCR4 receptor, e.g. with CXCR4-inhibiting peptides such as ALX40-4C, is used as a therapeutic approach to prevent HIV/SIV infection, as well as tumor metastasis (44, 45).
In the present study, wild-type Nef from SIVmac239, which was previously shown to exhibit a potent down-regulating effect on CXCR4 in Jurkat T-cells, was used to transfect HeLa-ACC cells to study if this viral protein can affect CXCR4 levels and related tumor relevant parameters, such as proliferation and migration.
In HeLa-ACC cells, CXCR4 was found located at the plasma membrane, as well as in the cytoplasm and the nucleus (Figure 2A). This is in accordance with previous reports that found a correlation of cytoplasmic and nuclear CXCR4 expression with lymph node metastasis and reduced overall survival in breast cancer and non-small cell lung cancer (46, 47). One explanation for the observed high level of intracellular CXCR4 could be a concomitant expression of SDF-1α, since autocrine secretion of this factor could result in enhanced endocytosis of the receptor, with subsequent cytoplasmic and nuclear localization. Indeed, the co-expression of CXCR4 and SDF-1α was found to predict lymph node metastasis in colorectal cancer (48).
Transfection of HeLa-ACC cells with Nef resulted in down-regulation of CXCR4 from the cell surface, however, without markedly influencing the total CXCR4 protein expression. Investigating the real-time proliferation the and migration of Nef-transfected cells revealed that tumor cells transfected with the viral protein had significantly lower proliferation and migration rates. HUVECs transfected with Nef exhibited a reduction in angiogenesis in the tube formation assay. CXCR4 with SDF-1α influence numerous intracellular signaling pathways, such as the phosphatidylinositol 3-kinase (pI3K)/protein kinase B (PKB, AKT), cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA), mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) and diacylglycerol (DAG)/Ca2+/protein kinase C (PKC) pathways to regulate cell adhesion, chemotaxis, migration, proliferation and apoptosis. The MAPK/ERK pathway plays an important role in CXCR4/SDF-1α signaling. ERK can phosphorylate and activate downstream cellular proteins and further phosphorylate nuclear transcription factors, such as STAT3, leading to changes in gene expression and cell cycle progression. No signs of apoptosis, such as cleavage of procaspase-3, were observed in any of the Nef-transfected cells, indicating that the inhibitory effect of SIV-Nef exerted on HeLa-ACC cells is not necessarily related to the activation of programmed cell death (Figure 2B and 4C). However, Nef affects intracellular signals through complex mechanisms which are not fully understood. STAT3 can also be phosphorylated or activated from a crosstalk signaling pathway via the mammalian target of rapamycin and Janus kinases. ERK was induced and PI3K inhibited by HIV-Nef in Jurkat T-cells and primary peripheral CD4+ T-lymphocytes (49).
Wild-type but not mutated HIV-Nef was previously found to induce apoptosis in Jurkat T-cells (50). Subsequent mapping of HIV-Nef revealed two specific regions (motifs) that exert apoptosis, with motif 1 (M1) being the most powerful (51). Interestingly, in a recent study, this group used an HIV-Nef peptide (Nef-M1) representing the pro-apoptotic motif 1 to treat four colorectal cancer cell lines, as well as colorectal xenografts, in SCID mice. Here, they observed a potent antitumor effect and concluded that the interaction of the Nef-M1 peptide with CXCR4 is responsible for the observed effect (52). However, there are no obvious sequence similarities between these two HIV-Nef apoptosis-inducing motifs and SIVmac239-Nef.
Taken together, it was demonstrated that Nef from SIVmac239 can down-regulate expression of plasma membrane-associated CXCR4 in the CXCR4+ tumor cell line HeLa-ACC and that expression of the viral protein resulted in reduction of cell proliferation, migration and angiogenesis. SIV-Nef could therefore serve as an interesting experimental tool for the study of CXCR4-expressing tumors and could potentially help to pinpoint new therapeutic approaches for the treatment of these malignancies.
Acknowledgements
We thank Ms. Roswitha Peldszus and Ms. Maria Sadowski (Department of Otorhinolaryngology, UKGM GmbH) and Mr. Thorsten Volkmann (Department of Hematology, Oncology and Immunology, UKGM GmbH) for their excellent technical assistance. This study was supported by the Alfred und Ursula Kulemann Stiftung (Philipps University Marburg, Germany).
Footnotes
-
Note
A previously accepted submission of the data presented in this manuscript erroneously assuming use of a head and neck adenoid-cystic carcinoma cell line (ACC3), which indeed was virtually identical to the cervical cancer-derived cell line HeLa (see Table I of this article), was retracted from Oral Oncology since this journal covers head and neck cancer only (http://dx.doi.org/10.1016/j.oraloncology.2011.06.502).
- Received December 2011.
- Revision received January 30, 2012.
- Accepted February 1, 2012.
- Copyright© 2012 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved
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