Pepro Tech
Originally published online as doi:10.1189/jlb.0907632 on May 5, 2008

Published online before print May 5, 2008
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
jlb.0907632v1
84/2/561    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koldehoff, M.
Right arrow Articles by Elmaagacli, A. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koldehoff, M.
Right arrow Articles by Elmaagacli, A. H.
(Journal of Leukocyte Biology. 2008;84:561-576.)
© 2008 by Society for Leukocyte Biology

Small-molecule inhibition of proteasome and silencing by vascular endothelial cell growth factor-specific siRNA induce additive antitumor activity in multiple myeloma

Michael Koldehoff1, Dietrich W. Beelen and Ahmet H. Elmaagacli

Department of Bone Marrow Transplantation, University Hospital of Duisburg-Essen, Essen, Germany

1 Correspondence: Department of Bone Marrow Transplantation, University Hospital of Duisburg-Essen, Hufelandstr. 55, 45122 Essen, Germany. E-mail: michael.koldehoff{at}uni-duisburg-essen.de

ABSTRACT

Angiogenesis plays an important role in the pathogenesis and progression in multiple myeloma (MM), and MM cells secrete vascular endothelial growth factor (VEGF), which further promotes proliferation of the tumor cells. Therefore, we evaluated the anti-myeloma effect of VEGF small interfering RNA (siRNA) silencing in MM cells and whether it can be augmented by the additional application of bortezomib directed against the 26S proteasome. After transfection with VEGF siRNA, we observed a reduction of VEGF expression in all studied cell lines: OPM-2, RPMI-8226, INA-6, Jurkat, Raji, and Karpas-299, as well as in cells of MM and lymphoma patients. VEGF siRNA significantly induced apoptosis and inhibited proliferation in OPM-2 cells (P<0.0001), RPMI-8226 (P<0.0001), and INA-6 (P<0.01) versus controls. Cotreatment with VEGF siRNA and bortezomib in MM cells resulted in an exaggerated inhibition of proliferation and induction of apoptosis compared with VEGF siRNA or bortezomib alone (P<0.001). In addition, the combination of VEGF siRNA and bortezomib significantly (P<0.01) reversed multidrug resistance gene 1-dependent resistance of MM cells. Our data suggest that small-molecule inhibition of proteasome and silencing by VEGF-specific siRNA may be associated with an additive antitumor activity and might be a suitable target for new, therapeutic strategies using RNA interference in MM.

Key Words: bortezomib • VEGF • small interfering RNA

INTRODUCTION

Multiple myeloma (MM) is a clonal B cell malignancy characterized by the accumulation of malignant plasma cells within the bone marrow (BM). Binding of MM cells to BM stromal cells promotes tumor cell growth, survival, and drug resistance by MM cells and BM microenvironment contact and triggering of cytokine secretion [1 ]. MM cells secrete vascular endothelial growth factor (VEGF), which further promotes cytokine production, especially IL-6 in BM stromal cells, as well as migration and proliferation of the tumor cells. Thus, VEGF is an autocrine growth factor and a trigger of IL-6-mediated paracrine MM cell growth. Recent reports have highlighted the major role of VEGF in MM pathogenesis, demonstrating that VEGF also increases microvessel density in the BM [2 3 ]. The VEGF family includes different forms of structurally related dimeric glycoproteins belonging to the platelet-derived growth factor superfamily of growth factors. Moreover, VEGF increases bone resorbtion by osteoclasts and inhibits maturation of dendritic cells [4 5 ]. Taken together, these reports have promoted preclinical evidence that has confirmed the promise of VEGF-targeting therapies [6 ]. The most successful approach to date to therapeutically target VEGF is the use of a humanized mAb against VEGF, bevacizumab (Avastin®), which is approved for the use in metastatic colorectal cancer, and metastatic renal-cell cancer and in combination with chemotherapy for various solid cancers [7 ].

The ubiquitin-proteasome pathway is the major extralysosomal machinery for protein degradation via ubiquitylation [8 ]. It is involved in cell homeostasis, regulating key functions of the cell such as cell cycle and apoptosis. Owing to their function, proteasomes also play an important role in other biological processes, including development of inflammation or resistance to cytostatics drugs. Inhibition of the proteasome by specific proteasome inhibitors has been shown to efficiently kill tumor cells in various types of leukemia and other malignant disorders. Bortezomib, a specific and selective inhibitor of the 26S proteasome with high response rates in patients with relapsed and refractory MM, has been approved for this indication [9 10 ]. Recently, several studies showed that the combination of proteasome inhibitors with other drugs may markedly increase cytotoxicity in different tumor cells [11 12 ], including hematological malignancies [13 14 ].

RNA interference (RNAi) represents an evolutionarily conserved cellular mechanism that mediates sequence-specific, post-transcriptional gene silencing initiated by dsRNA [15 16 ]. Small interfering RNAs (siRNAs) are the mediators of mRNA degradation in the process of RNAi. Synthetic siRNAs are able to mediate the cleavage of the target RNA, as shown by Elbashir et al. [17 18 ]. Recently, many groups demonstrated the functional and therapeutic intervention of RNAi against molecularly defined hematological targets in different hematopoietic cells and leukemic cell lines [19 20 21 22 ].

In the present study, we evaluated the anti-myeloma effect of VEGF siRNA silencing in MM cells and normal CD34-positive cells by studying the growth inhibition or proliferation rate, the induction of apoptosis, and the expression of VEGF protein levels or the VEGF mRNA by real-time RT-PCR. We also studied the influence of VEGF siRNA transfection on the autocrine or paracrine signaling and evaluated whether the anti-myeloma effect induced by VEGF siRNA in MM cells can be augmented by the additional application of bortezomib directed against the 26S proteasome. Finally, we analyzed the expression of the multidrug resistance gene 1 (MDR1) and its 170 kDa protein product, P-glycoprotein (P-gp) and the effect exerted by bortezomib in combination with VEGF siRNA.

MATERIALS AND METHODS

Cell culture
We used the following cell lines: OPM-2, human MM (IgG {lambda}) with hypertriploid/hypotetraploid karyotype; RPMI-8226, human MM with a flat-moded hypotriploid karyotype with 7.5% polyploidy; JURKAT, human T cell leukemia with a flat-moded hypotriploid karyotype with 7.8% polyploidy; RAJI, human Burkitt lymphoma with a flat-moded hypotriploid karyotype with 12% polyploidy; KARPAS-299, human T cell lymphoma with a hypodiploid karyotyp with 14% polyploidy. All cell lines were purchased from DSMZ (Braunschweig, Germany). The cells were grown in RPMI-1640 medium (Invitrogen, Heidelberg, Germany) supplemented with 10% FBS. The IL-6-dependent human MM cell line INA-6 was a generous gift from Martin Gramatzki (University Medical Center, Kiel, Germany). Cells were maintained in RPMI-1640 medium with Glutamax-1, supplemented with 10% FBS, antibiotics (all from Biochrom AG, Berlin, Germany), and 50 µM 2-β-ME in the presence of 1.0 ng/ml IL-6. Cells were maintained in a humidified 37°C incubator with 5% CO2. Recombinant human IL-6 was obtained from Sigma-Aldrich Chemie (Munich, Germany).

Isolation of patient cells
MM cells or leukemic cells were obtained from unmanipulated BM samples or by positive selection using the magnetic field of a MACS separator (Miltenyi Biotec, Bergisch Gladbach, Germany). CD34+ cells were obtained from healthy volunteers. BM stromal cells (BMSC) were obtained from long-term cultures of BM cells (4–8 weeks) in RPMI-1640 medium supplemented with 20% FBS. All patients and volunteers had given their prior informed consent. Patient characteristics, including grade of BM blast infiltration, are given in Table 1 .


View this table:
[in this window]
[in a new window]

  		Table 1. Patient’s Characteristics

Drug dosing and administration
Cells of the above cell lines or MM cells from patients were treated in vitro with bortezomib (Velcade®, Ortho Biotec, Germany), alone and in combination with siRNA (Dharmacon MWG Biotech, Ebersberg, Germany). The concentration of bortezomib varied in different experiments from 1 to 500 nM. The optimal doses of bortezomib and siRNA were determined in dose titration experiments. Bevacizumab (Avastin®, Roche Pharma, Grenzach-Wyhlen, Germany), a humanized anti-VEGF neutralizing mAb, was used at a concentration of 3.5 nM (500 ng/ml).

RNA isolation and real-time RT-PCR
RNA was isolated using the Rneasy mini kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. Real-time RT-PCR was performed as published previously for GAPDH real-time RT-PCR [23 ]. We used for VEGF-RT-PCR the following primers and hybridization probes: primers 5'-CCC TgC CCC CTT CAA TAT TC-3' and 5'-Agg AAA GTg Agg TTA CgT gCg-3', hybridization probes 5'-6FAM-TgA CAA AgA ggg AAC ggC TCT CAg gC-TAMRA-PH. For MDR1-RT-PCR, we used the following primers and hybridization probes: primers 5'-ggA AgC CAA TgC CTA TgA CTT TA-3' and 5'-gAA CCA CTg CTT CgC TTT CTg-3', hybridization probes 5'-6FAM-TgA AAC TgC CTC ATA AAT TTg ACA CCC Tgg-TAMRA. For IFN-{alpha}-RT-PCR, we used the following primers and hybridization probes: primers 5'-AgT gAT gAg CAA gCA gTA A-3' and 5'-Agg TTA ggA AAT ggC CAg-3', hybridization probes 5'-CCA TAg TgA CAC TgA AAT ggA TTg gTT ATA TA-Fl and 5'-LC-Red 640-gCT TAA gAA ATA gCC TCC CCA AAg TCT-PH. For IL-6R-RT-PCR, we used the following primers and hybridization probes: primers 5'-TTg CCA TTg TTC TgA ggT TC-3' and 5'-ATg CTT gTC TTg CCT TCC TT-3', hybridization probes 5'-6FAM-AgC CCg CAg CTT CCA CgT CTT CTT-TAMRA. Quantification of RNA transcript expression was normalized by determining the ratio between expression levels of targets and GAPDH.

Transfection of siRNA
siRNA was purchased from Dharmacon MWG Biotech. siRNA nucleotides corresponded to the target sites AAA TGT GAA TGC AGA CCA AAG AA of the human VEGF-A gene region (GenBank accession no. NM_001033756). Sense and antisense were AUG UGA AUG CAG ACC AAA GAA dTdT and UUC UUU GGU CUG CAU UCA CAU dTdT, respectively. In vitro transfections with different concentrations of siRNA (22–350 pM) were performed in 24-well plates using TransMessenger [transfection reagent (1x105cells/well), Qiagen] following the manufacturer’s protocol and as described previously [20 24 ]. Transfection with siRNA was performed at least fivefold in each experiment, which was repeated at least twice. As controls, we used a fluorescence-labeled, control, nonsilencing siRNA from Qiagen or a mismatched human VEGF siRNA (until 525 pM) from Dharmacon MWG Biotech to prove transfection. To apply the drug bortezomib after siRNA transfection, 100 µl medium was removed, thoroughly mixed with a stock solution of the respective compound or compounds so as to yield a final concentration of 1–500 nM for bortezomib, and then returned to the respective well.

Growth inhibition assay
Cells were cultured in 96-well plates at a concentration of 5000 cells/well and left to recover. Viable cells were quantitatively estimated by a colorimetric assay using MTT (10 µl 5 mg/ml solution, Sigma-Aldrich Chemie), which was added to each well of the titration plate and incubated for 4 h at 37°C. The cells were then solibilized by the addition of DMSO (40 µl/well) and incubated for 60 min at 37°C. The absorbance of each well was determined in an ELISA plate reader using an activation wavelength of 570 nm and a reference wavelength of 630 nm. The percentage of viable cells was determined by comparison with untreated control cells.

Terminal transferase dUTP nick-end labeling assay
Apoptotic cells were identified using the In Situ Cell Death Detection Kit from Roche Diagnostics (Mannheim, Germany) following the manufacturer’s instructions. Apoptotic cells (dark-brown staining) were counted under a microscope. The apoptotic index was defined by the percentage of brown cells among the total number of cells for each sample. Five fields with 100 cells each were counted randomly for each sample. A minimum of 15 single analyses was performed on three samples.

Cell proliferation assay
Cell proliferation was determined by BrdU incorporation. Twenty-four hours after siRNA transfection, 2 x 105 cells were split into four-well chamber slides and incubated with culture medium containing BrdU for 4 h. BrdU staining was performed using the Roche kit following the manufacturer’s instructions. Analyses of proliferation was defined by the percentage of brown-stained cells among the total number of cells for each sample and performed as described for the apoptotic cells [20 ].

ELISA
After treatment with drugs or siRNA transfection, the supernatants were collected and analyzed by VEGF-ELISAs (Quantikine human VEGF immunoassay, R&D Systems, Wiesbaden, Germany), following the manufacturer’s instructions. Transfection with siRNA was performed at least fivefold in each experiment.

MACS
MM cells or plasma cells were positively selected with anti-CD19 (preselection) and anti-CD138 antibodies, or CD34+ cells were positively selected with an anti-CD34 antibody conjugated to iron-dextran microbeads using the MiniMACS device (Miltenyi Biotec), according to the manufacturer’s instructions.

Cell-cycle analysis
Cells were fixed in ice-cold ethanol (70% v/v) and stained with a solution of propidium iodide [PI; 20 µg/ml PI, 100 µg/ml RNase, 0.1% Triton X-100, and in 870 µg/ml trisodium citrate (Sigma-Aldrich Chemie)]. The DNA content was determined using a FACScan flow cytometer (Coulter, Krefeld, Germany). Cell-cycle distribution was analyzed using MultiCycle for Windows (Phoenix Flow System, San Diego, CA, USA). Red fluorescence (585±42 nm) was evaluated on a linear scale, and pulse-width analysis was used to exclude cell doublets and aggregates from the analysis. Cells with DNA content between 2N and 4N were designated as being in the G1-, S-, or G2/M-phase of the cell cycle. The number of cells in each compartment of the cell cycle was expressed as a percentage of the total number of cells present. In sequential combination experiments, cells were pretreated with VEGF siRNA or mismatched VEGF siRNA for 24 h, washed twice with PBS to remove the siRNA or without VEGF siRNA, and analyzed for cell cycles.

CFSE staining
After washing with PBS, the target cell suspensions were resuspended at 1 x 106 cells/ml and labeled with 1.25 µM CFSE (Invitrogen), according to the manufacturer’s instructions.

Flow cytometry
Briefly, 1 x 106 cells were labeled with antibody for multicolor flow cytometry using CFSE, FITC, PE, PerCP, or biotin (along with streptavidin-PerCP)-conjugated mAb directed against CD45, CD14, CD33, CD34, CD38, CD19, CD138, CD243 (P-gp), and 7-amino actinomycin (7-AAD). All antibodies were obtained from Coulter. Nonspecific binding was corrected with isotype-matched controls. Flow cytometric data were acquired using a four-color Epics XL AF 14075 flow cytometer with Expo 32 ADC software (Coulter).

Statistics
Variations in data between the different groups were tested by a two-tailed unpaired t-test or a Mann-Whitney U-test using the SPSS 12 program (SPSS Inc., Chicago, IL, USA).

RESULTS

Efficiency of transfection
Using fluorescence-marked nonsilencing siRNA (Qiagen), we evaluated the transfection rate in all cell lines and in patients’ cells. Twenty-four hours after transfection, the number of fluorescently marked cells was evaluated using a fluorescence microscope. We counted a minimum of five samples with 2 x 100 cells per samples. With this analysis method, the mean transfection rate was 67% (range: 63–72%) in OPM-2, 80% (range: 75–85%) in RPMI-8226, 41% (range: 37–45%) in INA-6, 79% (range: 73–84%) in JURKAT, 50% (range: 47–52%) in RAJI, 52% (range: 49–55%) in KARPAS-299, 56% (range: 47–64%) in CD34+ enriched cells, and 46% (range: 36–52%) in patients’ tumor cells, respectively.

Inhibition of cell growth in MM cell lines and MM cells of MM patients
To assess whether VEGF siRNA treatment of OPM-2 and RPMI-8226 affects the viability of these cells, cells were transfected with various doses (22–350 pM) of VEGF siRNA for 24 h, harvested, and analyzed for cell viability by MTT assay. As shown in Figure 1A and 1B , VEGF siRNA significantly decreased the viability of OPM-2 cells (IC50=94 pM) and of RPMI-6228 cells (IC50=88 pM). To exclude the possibility that this event was a nonspecific, adverse effect of VEGF siRNA treatment, we performed a similar experiment using another mismatched VEGF siRNA. OPM-2 or RPMI-8226 was treated with this mismatched VEGF siRNA up to a maximal dose of 525 pM for 24 h, harvested, and analyzed for cell viability. We found that neither OPM-2 nor RPMI-8226 was sensitive to the mismatched VEGF siRNA (Fig. 1A and 1B) . These siRNA titration experiments determined that 175 pM VEGF siRNA is the significant silencing dose (P<0.0001) for all other cell line experiments and for cells from MM patients.


Figure 1
View larger version (13K):
[in this window]
[in a new window]

  		Figure 1. (A and B) Inhibition of cell growth shown in MM cell lines. Growth inhibition was measured by MTT. Mean and SD are given. The percentage of viable cells was determined by comparison with untreated control cells. (A: OPM-2 cell line; B: RPMI-8226 cell line; VEGF siRNA=VEGF-specific siRNA; VEGF MM siRNA=VEGF-mismatched siRNA). siRNA titration experiments determined that 175 pM VEGF siRNA was the significant silencing dose (P<0.0001).

VEGF expression measured by ELISA and by real-time RT-PCR
In the first series of experiments, we performed MTT analysis to determine whether VEGF siRNA treatment could inhibit cell growth as measured by the MTT assay. As shown in Figure 2A , VEGF siRNA transfection reduced the cell growth in all malignant cell lines OPM-2, RPMI-8226, INA-6, Karpas, Jurkat, and Raji compared with controls (controls were set up to 100%). After transfection with VEGF siRNA in the MM cell lines OPM-2, RPMI-8226, and INA-6, a highly significant reduction of more than 45% of cell growth was found. Only a moderate reduction of cell growth after VEGF siRNA transfection was detected in the other cell lines Karpas, Jurkat, and Raji compared with controls. To judge the biological significance of these findings, we subsequently studied the effects of VEGF siRNA transfection on the VEGF expression, which was quantified by VEGF-ELISA or by VEGF real-time RT-PCR (quotient of VEGF/GAPDH). Spontaneous VEGF secretion (after 24–48 h culture supernatants) ranged in the cell lines from 524 pg/ml (INA-6 cells plus IL-6) to 144 pg/ml (Karpas cells). We found a marked reduction of VEGF expression in all MM cell lines, which was quantified by VEGF-ELISA. With this analysis method, a highly significant reduction of more than 75% of VEGF protein levels was observed after siRNA transfection in the MM cell lines OPM-2 (P<0.001), RPMI-8226, and INA-6 (both P<0.0001) compared with untreated controls as shown in Figure 2B . After transfection of Jurkat, Raji, and Karpas cells with VEGF siRNA, a mild reduction of VEGF protein level was found, and only in Jurkat cells, which spontaneously express high VEGF protein levels, did we detect a significant reduction of the VEGF protein level after VEGF siRNA transfection (P<0.05). Next, we performed experiments to verify the VEGF protein results by VEGF real-time RT-PCR, which was quantified after normalization to the corresponding expression of the housekeeping gene GAPDH. With this analysis method, a 50–67% reduction of VEGF mRNA levels was observed after siRNA transfection of OPM-2, RPMI-8226, and INA-6 cells compared with controls (controls were set up to 100%), as shown in Figure 2C . VEGF siRNA transfection of Jurkat, Raji, and Karpas cells resulted again in only a mild reduction of VEGF mRNA levels. Next, using the MM cell line OPM-2, we studied the time courses of VEGF siRNA transfection to investigate the knock-down efficiency of VEGF expression, and the efficiency of VEGF siRNA transfection in treated OPM-2 cells showed a reduction of 75.5% VEGF protein levels after 24 h compared with the untreated OPM-2 cells (P<0.001). The VEGF protein levels were reduced from 396 ± 22 pg/ml to 97 ± 52 pg/ml in the disrupting OPM-2 cells 24 h after the start of transfection and to 218 ± 20 pg/ml 48 h after the VEGF siRNA transfection (P<0.05). After this time, a single transient application of VEGF siRNA could not further inhibit VEGF production, and VEGF protein levels rose again to the spontaneous level (427±28 pg/ml as shown in Fig. 2D ).


Figure 2
View larger version (41K):
[in this window]
[in a new window]

  		Figure 2. (A) Cell growth rate was measured 24 h after transfection with VEGF siRNA in different leukemic cell lines by the MTT assay. After transfection with VEGF siRNA, viable cell growth decreased significantly (P<0.005 in OPM-2, P<0.001 in RPMI-8226, and P<0.05 in INA-6) versus controls. (B) Effect of VEGF siRNA on the VEGF protein levels of different leukemic cell lines was measured by VEGF ELISA. After transfection with VEGF siRNA, the VEGF protein in pg/ml decreased significantly (P<0.001 in OPM-2, P<0.0001 in RPMI-8226, P<0.0001 in INA-6, and P<0.05 in Jurkat) versus untreated controls. (C) Effect of VEGF siRNA on the VEGF mRNA measurement of different leukemic cell lines. VEGF gene expression was measured by real-time RT-PCR and normalized to GAPDH expression. Control was set to 100%. Differences among OPM-2 (50%), RPMI-8226 (33%), and INA-6 (42%) versus control were significant (P<0.005 in OPM-2, P<0.001 in RPMI-8226, P<0.05 in INA-6, and P<0.05 in Jurkat). (D) The time-dependent effects of one transient application of 175 pM VEGF siRNA are shown by measured VEGF protein level with VEGF ELISA in OPM-2. We found an optimal knockdown of VEGF protein after 24–48 h of VEGF siRNA transfection compared with untreated controls. Mean and SD are given.

To verify the VEGF expression results, we assessed VEGF siRNA transfection in primary cells from patients with MM, lymphoplasmacytic lymphoma, and follicular cell lymphoma. We found a reduction of the VEGF mRNA amount in malignant cells with high BM infiltration derived from MM, lymphoplasmacytic lymphoma, and follicular cell lymphoma patients compared with untreated controls. The amount of VEGF mRNA was reduced significantly to 71% (mean) after 24 h VEGF siRNA transfection and to 61% (mean) after 48 h VEGF siRNA transfection in MM patients. In the non-MM patients (lymphoplasmacytic lymphoma or follicular cell lymphoma), we found only a minor reduction of the VEGF mRNA level after VEGF siRNA transfection, as shown in Table 2 A and 2B . Taken together, these data show that VEGF siRNA inhibits cell growth and VEGF expression in MM cell lines and in cells from MM patients.


View this table:
[in this window]
[in a new window]

  		Table 2A. Effects after 24 h of VEGF siRNA Transfection on VEGF mRNA Measurement in Leukemic Cells of Six Different Patients


View this table:
[in this window]
[in a new window]

  		Table 2B. Effects after 48 h of VEGF siRNA Transfection on VEGF mRNA Measurement in Leukemic Cells of Six Different Patients

Inhibition of proliferation and induction of apoptosis in MM cell lines and primary MM cells
Twenty-four hours after transfection with VEGF siRNA, we observed an induction of apoptosis in all studied MM cell lines. We found that 9.8% ± 1.0 of the OPM-2 cells, 9.2% ± 0.9 of the RPMI-8226 cells, and 7.9% + 0.6 of the IL-6-dependent INA-6 cells underwent apoptosis after transfection with 175 pM VEGF siRNA versus only 4.0% ± 1.1 of OPM-2 cells, 4.3% ± 0.8 of RPMI-8226 cells, and 3.4% ± 0.9 of INA-6 cells after transfection with up to 350 pM mismatched VEGF siRNA and spontaneously only 3.1% ± 0.9 (OPM-2), 2.8% ± 1.1 (RPMI-8226), and 2.5% ± 0.8 (INA-6) in each control group without siRNA, which was statistically significant (P<0.0001 for OPM-2, RPMI-8226; P<0.01 for INA-6), as shown in Figure 3A .


Figure 3
View larger version (33K):
[in this window]
[in a new window]

  		Figure 3. (A) Apoptosis rate was measured by terminal transferase dUTP nick-end labeling assay 24 h after transfection with VEGF siRNA in MM cell lines. After transfection with VEGF siRNA, the induced apoptosis rate of each MM cell line increased significantly (P<0.0001 in OPM-2, RPMI-8226; P<0.01 in INA-6) versus controls. (B) Proliferation rate measured by BrdU incorporation 24 h after transfection with VEGF siRNA in MM cell lines. After transfection with VEGF siRNA, the BrdU proliferation rate of each MM cell line decreased significantly (P<0.001 in OPM-2, RPMI-8226; P<0.01 in INA-6) versus controls. (C) The time-dependent effects of apoptosis in OPM-2 cells show a maximal induction of apoptosis rate (P<0.0001) after 24–48 h of VEGF siRNA transfection compared with untreated controls. (D) The time-dependent effects of proliferation in OPM-2 cells show a maximal reduction of BrdU proliferation rate (P<0.0001) after 48 h of VEGF siRNA transfection compared with untreated controls. Mean and SD are given.

Concordantly with the induction of apoptosis, the BrdU incorporation rate of all cell lines decreased. Proliferation was highly inhibited by VEGF siRNA in OPM-2, RPMI-8226, and INA-6 cells. The proliferation rate decreased spontaneously from 80.6% ± 4.4 or mismatched VEGF siRNA, 73.8% ± 7.2 to 36.4% ± 1.8 in OPM-2 cells (P<0.001) and spontaneously from 76.4% ± 5.2 or mismatched VEGF siRNA, 79.1% ± 3.2 to 42.4% ± 2.9 in RPMI-8226 cells (P<0.001) after transfection with 175 pM VEGF siRNA. In the IL-6-dependent INA-6 cells, the proliferation rate also decreased spontaneously from 77.2% ± 7.3 or mismateched VEGF siRNA, 82.7% ± 8.3 to 35.7% ± 7.1 after transfection with VEGF siRNA (P<0.01), as shown in Figure 3B .

To confirm that apoptosis or cell proliferation is dependent on the timing of VEGF siRNA silencing, we next investigated the time course of VEGF siRNA-induced effects in OPM-2 cells or RPMI-8226 cells. A strong stimulation of apoptosis to 10.9% ± 1.2 (OPM-2) and to 9.9% ± 1.5 (RPMI-8226) compared with the rate of spontaneous apoptosis in both cell lines was observed after 48 h of VEGF siRNA transfection, which was statistically significant (P<0.0001 for OPM-2 and RPMI-8226). After 72 h of VEGF siRNA transfection, no further stimulation of apoptosis was measured in OPM-2 or RPMI-8226; in fact, apoptosis decreased in both cell lines to 6.7% ± 0.9 (OPM-2) and to 5.5% ± 1.1 (RPMI-8226; Fig. 3C , and data not shown). We further observed a strong inhibition of BrdU incorporation to 16.0% ± 1.4 in OPM-2 cells (P<0.0001) and to 20.4% ± 1.9 in RPMI-8226 cells (P<0.0001) compared with the spontaneous proliferation rate after 48 h of VEGF siRNA transfection. Seventy-two hours after VEGF siRNA transfection, proliferation of OPM-2 cells could not be inhibited further, and BrdU incorporation increased to 56.2% ± 3.2 compared with the spontaneous rate (Fig. 3D , and data not shown). Transfection with 175 pM VEGF siRNA also induced an increased rate of apoptosis (P<0.01) and inhibited proliferation in cells from two MM patients with heavy BM infiltration (P<0.01) compared with controls, as shown in Table 3A .


View this table:
[in this window]
[in a new window]

  		Table 3A. Effects of VEGF siRNA on the Induction of Apoptosis and the Inhibition of Proliferation in Patients with MM

VEGF siRNA and its effect on induced apoptosis and proliferation in CD34+ cells
Proliferation in CD34+ enriched cells did not change after transfection with VEGF siRNA. We found similar proliferation rates of CD34+ cells after transfection with VEGF siRNA (29.6%±1.4) and mismatched VEGF siRNA (31.8%±1.5) and in nonmanipulated CD34+ cells (32.1%±2.8). The apoptosis rate (12.1%±1.6) in CD34+ enriched cells did not change after transfection with VEGF siRNA and mismatched VEGF siRNA (12.5%±1.4) and from nonmanipulated cells (12.8%±1.1), as shown in Figure 4A . To determine whether VEGF has direct effects on CD34+ enriched cells, we examined the autocrine production of VEGF protein by ELISA in these CD34+ cells. We found by ELISA only a mild, spontaneous VEGF protein expression of 58.3 ± 5.7 pg/ml and only a marginal reduction to 52.3 ± 5.0 pg/ml VEGF protein 24 h after VEGF siRNA transfection, as shown in Figure 4B . Taken together, these data show that CD34+ enriched cells synthesize and secrete only moderate amounts of VEGF and show no significant response to VEGF siRNA transfection.


Figure 4
View larger version (13K):
[in this window]
[in a new window]

  		Figure 4. (A) Proliferation rate and apoptosis rate 24 h after transfection with VEGF siRNA in CD34+ enriched cells. The proliferation rate (right) and the apoptosis rate (left) did not change after transfection with VEGF siRNA compared with controls (not significant). (B) Effect of VEGF siRNA on the VEGF protein levels in CD34+ enriched cells was measured by VEGF ELISA. The spontaneous VEGR protein expression in pg/ml CD34+ enriched cells was mild. After transfection with VEGF siRNA, the VEGF protein decreased only marginally as compared with untreated controls. Mean and SD are given.

Additive effects of VEGF siRNA and bortezomib in induction of apoptosis and inhibition of proliferation
As expected, we saw that transfection with VEGF siRNA alone or bortezomib alone inhibited the proliferation rate of OPM-2 cells compared with controls, as shown in Figure 5A (P<0.001 for 175 pM VEGF siRNA, and P<0.05 for 1 nM bortezomib vs. control). Transfection with VEGF siRNA combined with bortezomib resulted in an exaggerated decrease of proliferation from 75.5% ± 8.9 (controls) to 10.3% ± 4.1 compared with the inhibitory effect of VEGF siRNA or bortezomib (29.0%±5.4 after transfection with VEGF siRNA or 32.1±1.9 after 5 nM bortezomib treatment), as shown in Figure 5A (P<0.001 for 175 pM siRNA+5 nM bortezomib vs. siRNA or bortezomib alone). Concordantly, we observed an additive effect on induction of apoptosis by VEGF siRNA combined with bortezomib in OPM-2 cells. The rate of induced apoptosis increased from 10.2% ± 2.1 (controls) to 29.5% ± 1.6, whereas the use of VEGF siRNA alone or 1–5 nM bortezomib alone was again less effective (VEGF siRNA alone, 22.7%±1.7; 1 nM bortezomib alone, 16.0%±0.9), as shown in Figure 5B (P<0.01 for 175 pM siRNA+5 nM bortezomib vs. siRNA or bortezomib alone). An additive effect of VEGF siRNA and bortezomib (5 nM) on the induction of apoptosis and inhibition of proliferation was also seen in the MM cells of two male patients with heavy BM infiltration, as shown in Table 3B .


Figure 5
View larger version (38K):
[in this window]
[in a new window]

  		Figure 5. (A) Proliferation rate 24 h after transfection with VEGF siRNA, bortezomib, and cotreatment of VEGF siRNA with bortezomib in the OPM-2 cell line. Controls with a spontaneous BrdU proliferation rate of 75.5% (mean) were set to 100%. The proliferation rate decreased after transfection with VEGF siRNA or 1 nM bortezomib to 38.4% (P<0.001) or 70% (P<0.05), respectively. Bortezomib (5 nM) had the same efficiency as 175 pM VEGF siRNA on the inhibition of the proliferation rate. Cotreatment of VEGF siRNA with bortezomib had additional effects on the inhibition of the proliferation rate, as shown in the right column (P<0.0001) versus untreated control and versus 5 nM bortezomib or VEGF siRNA alone (P<0.001). (B) Apoptosis rate 24 h after transfection with VEGF siRNA, bortezomib, and cotreatment of VEGF siRNA with bortezomib in the OPM-2 cell line. Controls with a spontaneous apoptosis rate of 10.2% (mean) were set to 100%. The apoptosis rate increased after transfection with VEGF siRNA or 1 nM bortezomib to 2.2-fold (P<0.0001) or 1.6-fold (P<0.01), respectively. Treatment of the OPM-2 cells with 5 nM bortezomib increased the apoptosis rate to ~1.8-fold (P<0.001) compared with the spontaneous apoptosis rate. Cotreatment of VEGF siRNAs with bortezomib had additional effects on the apoptosis rate, as shown in the right column (P<0.0001) versus untreated control and versus VEGF siRNA alone (P<0.01). (C) Comparison of the additive effects of VEGF siRNA and bortezomib to the humanized anti-VEGF antibody bevacizumab in OPM-2. VEGF protein levels of OPM-2 cells were measured by VEGF ELISA in pg/ml. Bevacizumab (500 mg/ml) had the strongest capability to reduce the VEGF protein level (P<0.0001) and could not be amplified with a cotreatment of VEGF siRNA and bevacizumab, which reduces the VEGF protein level a little stronger than the combination of VEGF siRNA with bortezomib (P<0.01) in OPM-2 cells. (D) Comparison of the proliferation rate with regard to VEGF siRNA and bortezomib with the humanized anti-VEGF antibody bevacizumab in OPM-2. The BrdU proliferation rate was measured by BrdU incorporation. VEGF siRNA with bortezomib had the strongest capability to reduce the proliferation rate (P<0.0001) compared with VEGF siRNA alone, bevacizumab alone, or bevacizumab with VEGF siRNA, which with bortezomib reduces the BrdU proliferation rate significantly as compared with all other treatment options (P<0.001) in OPM-2 cells. Mean and SD are given.


View this table:
[in this window]
[in a new window]

  		Table 3B. Additive Effects of VEGF siRNA and Bortezomib on the Induction of Apoptosis and the Inhibition of Proliferation in Patients with MM

Comparison of the additive effects of VEGF siRNA and bortezomib on the humanized anti-VEGF neutralizing mAb bevacizumab in OPM-2
The successful, therapeutic approach of targeting VEGF with the humanized anti-VEGF neutralizing mAb bevacizumab led us to study the effects of this antibody on VEGF expression in OPM-2 cells by VEGF ELISA. We compared these results with the findings described above—the additive effects of VEGF siRNA and bortezomib. With this analysis method, a highly significant reduction of more than 97% of VEGF protein level was observed after treatment with 3.5 nM bevacizumab compared with the untreated controls (P<0.0001). Concordantly, the combination of VEGF siRNA transfection with 5 nM bortezomib also showed a highly significant reduction of more than 90% of the VEGF protein level (P<0.001). Comparing the effects of VEGF siRNA with 5 nM bortezomib with 3.5 nM bevacizumab, we found that bevacizumab could reduce VEGF protein levels more strongly to 11 ± 27 pg/ml compared with VEGF siRNA with bortezomib with 43 ± 20 pg/ml (P<0.01). The combination of bevacizumab with VEGF siRNA showed no further reduction of the VEGF protein level, as shown in Figure 5C . Concordantly, with the inhibition of the VEGF protein level, the BrdU incorporation rate also decreased, as shown in Figure 5D . Bevacizumab or VEGF siRNA resulted in inhibition of proliferation to 34.3% ± 6 or to 37.5% ± 7 compared with controls (controls were set up to 100%). The strongest reduction of proliferation was observed with the combination of VEGF siRNA with bortezomib to 10.8% ± 5 compared with bevacizumab or VEGF siRNA or bevacizumab with siRNA to 28.9% ± 8 (P<0.001). Taken together, these data show that bevacizumab could strongly inhibit VEGF protein levels but that the additive effects of VEGF siRNA and bortezomib had the strongest functional influence on proliferation of OPM-2 cells.

VEGF siRNA and its effects on INA-6 cells
In the IL-6-dependent MM INA-6 cell line, we next examined the effects of VEGF siRNA, bortezomib, or the combination of VEGF siRNA with bortezomib in the presence of human IL-6 or in the presence of human BMSC. Twenty-four hours after treatment with bortezomib or transfection with VEGF siRNA or with the combination of bortezomib and siRNA, we saw an inhibition in the cell growth by MTT analysis of INA-6 cells with human IL-6 compared with the controls as shown in Figure 6A . Transfection with VEGF siRNA combined with bortezomib resulted in an exaggerated decrease in cell growth from 75.9 ± 11.3 (controls) to 25.6 ± 6.1 in IL-6-depentent INA-6 cells with 1.0 ng/ml human IL-6 (P<0.003). Concordantly with the inhibition of the cell growth, the BrdU incorporation rate also decreased as shown in Figure 6B . Transfection with VEGF siRNA combined with bortezomib also resulted in a decreased inhibition of the proliferation rate from 77.2 ± 7.3 (controls) to 22.7 ± 4.9 in IL-6-depentent INA-6 cells with 1.0 ng/ml human IL-6 (P<0.001). When we performed the same experiments with the IL-6-dependent INA-6 cell line in the presence of BMSC, we again found an inhibition in cell growth and a decreased proliferation rate after transfection of these cells with VEGF siRNA and cotreatment with bortezomib. VEGF siRNA combined with bortezomib decreased the viability detected by MTT analysis of INA-6 cells from 64.8 ± 5.3 (controls) to 24.2 ± 6.1 in INA-6 cells with 1 x 104 BMSC (p<0.001). Concordantly with the inhibition of viable cells, the proliferation rate decreased from 51.8 ± 8.9 (controls) to 10.2 ± 5.5 in INA-6 cells with 1 x 104 BMSC (P<0.0001), as shown in Fig. 6A and 6B . In addition, we determined the effects of VEGF siRNA silencing on INA-6 cells by quantitative VEGF real-time PCR. With this analysis method, a moderate reduction of VEGF mRNA levels of more than 34% and 60% was observed after treatment with bortezomib (P<0.005) or VEGF siRNA (P<0.05 for IL-6 and P<0.001 for BMSC). Transfection with VEGF siRNA and cotreatment with bortezomib resulted again in a twofold reduction of VEGF mRNA compared with the transfection with VEGF siRNA alone or the treatment with bortezomib alone (P<0.0001), as shown in Figure 6C . To elucidate some of the possible functional mechanisms for the VEGF siRNA- and bortezomib-induced inhibition of MM cells, we loaded INA-6 cells with the cell-division tracker CFSE. Cells were grown in coculture with BMSC or in a medium in the presence or absence of VEGF siRNA or the combination of VEGF siRNA with bortezomib for 3 days before the number of cell divisions was analyzed by flow cytometry. The experiments revealed that the number of INA-6 cells that had undergone one or more cell divisions was moderately inhibited by VEGF siRNA (P<0.001). Again, VEGF siRNA and cotreatment of VEGF siRNA with bortezomib reduced the division of INA-6 cells grown with BMSC (P<0.0001) compared with the control INA-6 cells grown in a medium and without VEGF siRNA, as shown in Figure 6D . Taken together, these results indicate that VEGF siRNA and the cotreatment of VEGF siRNA with bortezomib overcome the growth and survival advantages conferred by IL-6 and BMSC (paracrine signaling) in INA-6 cells.


Figure 6
View larger version (35K):
[in this window]
[in a new window]

  		Figure 6. (A) Effect of VEGF siRNA and bortezomib in the IL-6-dependent cell line INA-6 grown in human IL-6 (1 ng/ml) medium or in human BMSC (1x104 cells/ml). Cell growth rate was measured by the MTT assay. After treatment of bortezomib or transfection with VEGF siRNA or the combination of bortezomib with VEGF siRNA, cell growth was decreased significantly in INA-6 cells. Cotreatment with VEGF siRNA and bortezomib had the strongest inhibition effects in INA-6 cells (P<0.005 for INA-6 grown in IL-6 supplement, and P<0.001 for INA-6 grown with BMSC cells). (B) Proliferation rate of INA-6 after transfection with VEGF siRNA and bortezomib. Proliferation rate was measured by BrdU incorporation in INA-6, grown with IL-6 medium or in human BMSC. After treatment of bortezomib or transfection with VEGF siRNA or the combination of bortezomib with VEGF siRNA, the proliferation rate decreased significantly in INA-6 cells. Concordantly with the effects of cell growth, cotreatment of VEGF siRNA with bortezomib had an additive inhibition effect in INA-6 cells (P<0.001 for INA-6 grown in IL-6 supplement, and P<0.0001 for INA-6 grown with BMSC cells). (C) Effect of VEGF siRNA and bortezomib on VEGF mRNA expression in INA-6 cells. VEGF gene expression was measured by real-time RT-PCR and normalized to GAPDH expression. Controls were set to 100%. After transfection of VEGF siRNA and cotreatment with bortezomib, the VEGF mRNA level decreased significantly in INA-6 cells to ~28.8% (grown with IL-6, P<0.0001) and 24.5% (grown with BMSC, P<0.0001), respectively. (D) Effect of VEGF siRNA and VEGF siRNA with bortezomib on division of CFSE-labeled INA-6 cells. CFSE-labeled cells (1x106 cells/ml labeled with 1.25 µM CFSE) were measured by flow cytometry. Controls were set to 100%. After cotreatment with VEGF siRNA and bortezomib, the number of cell divisions was reduced significantly in INA-6 cells (P<0.0001) grown with BMSC (1x104 cells/ml) as compared with controls. Mean and SD are given.

VEGF siRNA and differentiation of CD34+ cells and MM cell lines
We next evaluated whether silencing of the VEGF gene might affect differentiation of transfected cells. As differentiation-specific surface markers, we used CD19, CD38, CD45, CD138, and 7-AAD. We did not observe effects on differentiation by transfection with VEGF siRNA of CD34+ cells or in MM cell lines. The fraction of CD138/CD45+ cells decreased by 50%, and the number of 7-AAD/CD45+ cells increased by 28% (data not shown) after transfection with VEGF siRNA compared with unmanipulated cells.

Effect of VEGF siRNA on the cell cycle of MM cells
To evaluate the effect of VEGF inhibition by VEGF siRNA, we performed flow cytometric cell-cycle analyses on two MM cell lines (OPM-2 and RPMI-8226). In OPM-2, 175 pM VEGF siRNA led to a considerable cell shift to the G0/G1-phase (increased by 16.5%), whereas cell numbers in the S- and G2-phase were decreased by 5.9% and 48.9%. In RPMI-8226, we saw a similar result with a shift to the G0/G1-phase (increased by 20.8%). Cells in the S- and G2-phase were decreased by 12.2% and 47.7%, respectively. Concordantly, we observed an additive effect in cell shift by VEGF siRNA combined with 5 nM bortezomib in OPM-2 cells and in RPMI-8226 cells. The rate of cell shift to the G0/G1-phase increased by 39.3% (OPM-2) and 38.2% (RPMI-8226), whereas cell numbers in the S-phase were decreased by 28.3% (OPM-2) and 29.4% (RPMI-8226), and the cell numbers in the G2/M-phase were decreased by 27.7% (OPM-2) and 26.1% (RPMI-8226), respectively (P<0.05 for 175 pM siRNA+5 nM bortezomib vs. siRNA or bortezomib alone), as shown in Figure 7A and 7B . Exposure to VEGF siRNA or to the combination of VEGF siRNA with bortezomib caused an increase in MM cells arrested at the G0/G1-phase, starting at 1 h after exposure. Over longer exposure times, the number of cells at the G0/G1-phase increased gradually, and long-term exposure to VEGF siRNA + bortezomib triggered an increased arrest in the G2/M-phase (data not shown).


Figure 7
View larger version (18K):
[in this window]
[in a new window]

  		Figure 7. (A and B) Effect of VEGF siRNA and bortezomib on the cell cycle in MM cells. (A) OPM-2 cell line; (B) RPMI-8226 cell line. VEGF siRNA induces G0/G1-phase arrest in OPM-2 and RPMI-8226 cells, which were exposed to VEGF siRNA and bortezomib for 24 h. After exposure, OPM-2 and RPMI-8226 cells were harvested, fixed, and incubated with 20 µg/ml PI for 1 h. The profiles of DNA content (measured by a flow cytometer) are presented in bar standings for cell-cycle distributions and represent the mean and SD of three independent experiments.

IFN-{alpha} and IL-6R gene expression in MM cells
To rule out that our observations are triggering effects of siRNA mediated by cytokines such as IFN-{alpha}, we next identified signaling pathways mediating nonspecific or specific effects of siRNA. In this series of experiments, we performed RT-PCR analysis to determine if IL-6R or IFN-{alpha}-mediated activation occurred via stimulatory properties of siRNA transfection in MM cells. We found in OPM-2 and RPMI-8226 a marginally increased level of IFN-{alpha} gene expression after transfection with VEGF siRNA and cotreatment with VEGF siRNA + bortezomib [3.5–8% and 10–15% (mean) compared with controls (controls were set up to 100%)]. IL-6R gene expression was induced significantly (VEGF siRNA, 22.2% and 39.3% in both cell lines; VEGF siRNA and bortezomib, 36.1% and 57.2%, respectively) compared with controls, as shown in Figure 8A and 8B . In the IL-6-dependent MM INA-6 cell line, which was cultured in the presence of human IL-6 (1 ng/ml) or BMSC (1x104 cells/ml), we could not find an increased IL-6R gene expression after transfection with VEGF siRNA or cotreatment with VEGF siRNA and bortezomib (data not shown).


Figure 8
View larger version (20K):
[in this window]
[in a new window]

  		Figure 8. (A) Effect of VEGF siRNA and bortezomib on IFN gene expression in MM cell lines. IFN gene expression was measured by real-time RT-PCR and normalized to GAPDH expression. Mean and SD are given. Controls were set to 100%. (B) Effect of VEGF siRNA and bortezomib on IL-6R gene expression in MM cell lines. IL-6R was measured by real-time RT-PCR. Differences between OPM-2 (%) and RPMI-8226 (%) versus controls were significant (P<0.01).

Effects of VEGF siRNA and/or bortezomib on P-gp and MDR1 gene expression
It has been reported that the MDR1 gene and its product P-gp protein are major obstacles for the improvement of treatment of MM. Bortezomib could alter important signaling pathways and attenuate P-gp-mediated multidrug resistance in variant hematological malignancies, including MM. To evaluate the effect of VEGF siRNA silencing and bortezomib, we performed flow cytometric analyses of P-gp expression in OPM-2 cells. We found a highly significant reduction of more than 60% of P-gp expression to 39.9% ± 5.7 (P<0.01) after 50 nM bortezomib compared with untreated controls (controls were set up to 100%), as shown in Figure 9A . VEGF siRNA transfection could decrease P-gp expression to 47.3% ± 3.0 (P<0.02), and the combination of VEGF siRNA and bortezomib led to an additional reduction of P-gp expression to 20.9% ± 2.8 (P<0.01) compared with controls. Next, we verified the P-gp expression results by evaluating MDR1 gene expression by real-time PCR. With this sensitive method, we further evaluated multidrug resistance after transfection and cotreatment with bortezomib in OPM-2. Twenty-four hours after treatment with the combination of VEGF siRNA (175 pM) and bortezomib (5 nM; P<0.01), we found an inhibition of MDR1 gene expression to 41.9% ± 31.3 versus high doses of bortezomib (50 nM), which inhibited to 52.9 ± 23.3% (P<0.05) compared with controls, as shown in Figure 9B . Taken together, these data show that the additive effect of bortezomib and VEGF siRNA could strongly inhibit MDR1 gene expression or its product, P-gp protein expression, in OPM-2 cells.


Figure 9
View larger version (15K):
[in this window]
[in a new window]

  		Figure 9. (A) Effect of VEGF siRNA and bortezomib on CD243 (P-gp expression) in OPM-2 cells. P-gp expression was measured by flow cytometry. Mean and SD are presented. Control was set to 100%. After 50 nM bortezomib, VEGF siRNA, and VEGF siRNA with 5 nM bortezomib, the P-gp expression was reduced significantly as compared with controls (P<0.01 by 50 nM bortezomib, P<0.02 after VEGF siRNA, and P<0.01 after VEGF siRNA with 5 nM boretzomib). VEGF siRNA with bortezomib had an additive inhibition effect on P-gp expression (P<0.03), comparing VEGF siRNA alone. (B) Effect of VEGF siRNA and bortezomib on MDR1 gene expression in OPM-2 cells. MDR1 gene expression was measured by real-time RT-PCR and normalized to GAPDH expression. Concordantly with the P-gp expression, the MDR1 gene expression shows similar effects after VEGF siRNA with bortezomib as compared with controls (P<0.01).

DISCUSSION

MM remains largely incurable despite conventional and high-dose therapies, and novel molecular therapies are urgently required. Recent studies demonstrate that various growth factors including VEGF play an important role in MM pathogenesis. Targeting VEGF therefore represents a promising therapeutic strategy in MM. The discovery that siRNA could be delivered effectively into mammalian cells has raised the possibility that selective intervention in leukemic cell gene regulation might be feasible for the treatment of leukemia. RNAi is highly specific, as shown in genome-wide gene expression profiles obtained during the inhibition of GFP expression [25 ] and in the isoform-specific suppression of mouse VEGF-A mRNA by siRNAs [26 ]. Moreover, various reports about VEGF siRNA show that VEGF silencing was accompanied by the inhibition of lymphangiogenesis and the spontaneous metastasis, suggesting that efficient suppression of VEGF expression may be useful for the treatment of diseases such as cancer and age-related macular degeneration [27 28 29 ]. The aim of this study was to define the role of VEGF for MM and lymphoma cell survival and explore whether it could serve as a target for cancer treatment by VEGF siRNA, alone or in combination with proteasome inhibitors such as bortezomib. As expected, we found that VEGF siRNA inhibits the growth of human MM cells in a time- and dose-dependent manner, contingent on the efficiency of the transfection in the cell lines. The response to high siRNA doses reached a plateau, and the maximum growth reduction in the MM cell lines was two- to fourfold. In MM, VEGF is produced by MM cells and BMSC and may account, at least in part, for the increased angiogenesis observed in the BM of MM patients [3 ]. We found differential, spontaneous VEGF secretion in all cell lines and observed that VEGF siRNA strongly inhibited VEGF protein or VEGF gene expression in three MM cell lines (VEGF protein decreased by 76% in OPM-2 cells, by 79% in RPMI-8226 cells, and 75% in INA-6 cells) and only mildly inhibited VEGF expression in the various B cell or T cell lines. In primary cells of patients with MM or lymphoma, VEGF expression was markedly reduced after VEGF siRNA transfection versus controls. In the primary cells of patients with MM or lymphoma, VEGF expression was less distinctly reduced after VEGF siRNA transfection in a more time-dependent manner, with maximal VEGF reduction after 48 h. One difficulty in the application of siRNA-based technology is that siRNAs display a wide range of activities reducing target mRNA or protein expression. It should be emphasized that the efficiency of siRNA-mediated gene silencing is affected by a combination of factors (variation in transfection efficiency, siRNA sequences, properties of the target mRNA, and others). These differences might, at least in part, be a result of the variable effectiveness of transfection with VEGF siRNA in these cell lines, which varied in mean from 41% to 80%. Moreover, Hu et al. [30] recently suggested that low-abundant transcripts are less susceptible to siRNA-mediated degradation than medium- and high-abundant transcripts. Concordantly with the reduction of VEGF expression, we observed growth suppression as measured by MTT assays in the tested cell lines. Notably, the rate of induced apoptosis declined significantly in all examined MM cell lines after transfection with VEGF siRNA versus mismatched VEGF or nonmanipulated controls. Moreover, the inhibitory effect on proliferation strictly correlated with or even overshot the degree of induced apoptosis in the examined cell lines. Consistent with our findings, it was reported previously that a 2'-O,4'-C-ethylene-bridged nucleic acid antisense oligonucleotide (AON) gapmer with RNase H-mediated activity was virtually stable in rat plasma and exhibited more than 90% inhibition of VEGF mRNA production in human lung carcinoma A549 cells [31 ]. Recently, a phase I study of AON against VEGF demonstrated at the maximum tolerated dose reductions in plasma VEGF in five of six patients. Other clinical responses included complete remission in one patient with AIDS-Kaposi’s sarcoma, a mixed but dramatic response in one patient with cutaneous T cell lymphoma, and prolongation of progression-free survival in six patients (12%) with renal cell, bronchoalveolar, small cell lung, thyroid, ovarian carcinomas, and chondrosarcoma [32 ]. Although siRNA and AONs are directed against the same target mRNA, it must be noted that the mechanisms of mRNA cleavage are different. siRNAs are thought to be much more effective than AONs in the degradation of their target mRNAs [24 33 ].

Surprisingly, we found that VEGF-specific siRNA did not induce apoptosis in normal CD34+ progenitor cells and decreased the proliferation rate by only 8%. This result might be a result of a much lower degree of VEGF expression in normal CD34+ cells or other nonmaligant hematopoietic stem cells (HSCs), where VEGF release is dependent on exposure to factors such as hematopoietic cytokines for a longer time period [34 35 ].

Furthermore, we found additive effects on the rate of induced apoptosis and the rate of inhibition of proliferation by VEGF siRNA transfection combined with bortezomib treatment in MM cell lines. The proliferation rate decreased by two- to threefold compared with the use of VEGF siRNA alone (P<0.001). Binding of VEGF to MM cells triggers VEGFR, especially VEGFR-1 tyrosine phosphorylation. Subsequently, several downstream signaling pathways are activated: a PI-3K/protein kinase C{alpha}-dependent cascade mediating MM cell migration on fibronectin, a MEK-ERK pathway mediating MM cell proliferation, and a pathway mediating MM cell survival via up-regulation of myeloid cell leukemia 1 (MCL-1) and surviving in a dose- and time-dependent manner [36 ]. Proteasome inhibitor bortezomib induces apoptosis in drug-resistant MM cells and inhibits binding of MM cells in the BM microenvironment, as well as production and secretion of cytokines that mediate MM cell growth and survival. Bortezomib plays a fundamental role in degrading key regulatory proteins that govern cell cycle, transcription factor activation, apoptosis, and cell trafficking [37 ]. Regulatory proteins degraded by this system include p53, cyclins, cyclin-dependent kinase inhibitors p27 and p21, and NF-{kappa}B, which is important for cell survival, and it is activated in response to cell stress, including cytotoxic agents, radiation, and DNA damage. NF-{kappa}B also regulates the expression of genes involved in apoptosis, such as Bcl2 and Bcl-xL, cell-cycle progression, inflammation, and angiogenesis, including IL-6, IL-8, and VEGF [38 ]. Moreover, a recent study demonstrated that bortezomib down-regulates caveolin-1 expression and inhibitis caveolin-1 tyrosine phosphorylation, which are required for VEGF-mediated MM cell migration on fibronectin, and blocks VEGF-induced tyrosine phosphorylation of caveolin-1 in HUVECs, thereby inhibiting ERK-dependent endothelial cell proliferation [39 ]. This could indicate that VEGF siRNA and bortezomib affect MM cells by additive, selective, antitumor activity via different pathways and targeting different genes. Leukemic cells with disease-specific fusion genes, such as the BCR-ABL-positive chronic myelogenous leukemia cells, may be optimal targets for this purpose. It might therefore be possible to increase the antileukemic effect of a single siRNA directed against leukemic-specific fusion genes, as, e.g., AML1-ETO, PML-RAR{alpha}, or CBFβ-MYH11, heavy polypeptide 11, by combination with additional proteasome inhibitors such as bortezomib. The use of several siRNAs to induce additive effects toward target cells has already been described in other systems [20 24 40 ]. To date, the most successful approach to therapeutically target VEGF expression, bevacizumab (Avastin®), which was recently approved by the U.S. Food and Drug Administration for the therapy of metastatic colorectal cancer, and specifically bevacizumab in combination with conventional chemotherapy, is a new treatment option [41 ]. Ongoing studies in MM are evaluating the efficacy of bevacizumab. We found that bevacizumab powerfully inhibited VEGF expression in MM cells (up to 95%) compared with the additive effect of VEGF siRNA combined with bortezomib (90%, P<0.01), although the proliferation rate of MM cells measured by BrdU incorporation was significantly stronger when inhibited by the VEGF siRNA in combination with bortezomib compared with bevacizumab alone (up to 90% for VEGF siRNA and bortezomib vs. 66% for bevacizumab, P<0.001). Combining bevacizumab with VEGF siRNA did not improve reduction of VEGF expression or proliferation of OPM-2 cells. The function of VEGF and its receptors is one component of the regulatory processes contributing to the pathogenesis of hematologic malignancies, including MM. External autocrine loops, where growth factors (e.g., VEGF) are secreted by tumor cells followed by activation of receptors on tumor cells and other cells, have been described in several hematologic malignancies, including MM. Internal autocrine loops, where the factor activates autonomous cell growth via an intracellular receptor without being secreted, have been described in acute leukemia [42 ]. Lee and co-workers [43] showed recently that in vivo autocrine VEGF signaling is required for endothelial cell survival under nonpathological conditions in a cell-autonomous manner. Cell-autonomous signaling triggers a response that does not fully overlap with the events initiated by paracrine activation. Thus, paracrine VEGF signaling is essential for the angiogenic cascade, proliferation, survival, permeability responses, and endothelial differentiation. In contrast, autocrine VEGF signaling only conveys survival signals. Interestingly, paracrine and autocrine activation is mediated by the main initiating receptor (VEGFR-2). These findings indicate that the functional significance of cell-autonomous signaling is broader than anticipated, and it impacts on fully differentiated, normal endothelial cells as part of a homeostatic program [43 ]. In contrast to the external autocrine loop, cell proliferation mediated by an internal VEGF autocrine loop is not cell density-dependent, and neutralizing antibodies do not prevent continued cell growth or differentiation. Importantly, this independence of factor secretion contrasts the regulatory role of VEGF during hematopoiesis versus angiogenesis [44 ]. In addition to the known paracrine/external autocrine/juxtacrine loops, an internal autocrine loop of VEGF similar to acute leukemia may contribute to growth factor and cell density-independent proliferation of MM cells.

Anderson and co-workers [45, 46] demonstrated that the BM microenvironment confers cell growth and drug resistance in MM cells. We therefore considered the functional sequelae of VEGF siRNA in the presence or absence of bortezomib in MM INA-6 cells grown in an IL-6-supplemented medium or in a BMSC milieu. We found that VEGF siRNA and VEGF siRNA combined with bortezomib inhibited IL-6-induced or BMSC-induced cell growth and proliferation rate in INA-6 cells. In terms of MM cell–host interaction, the expression and regulation of adhesion molecules mediating the binding of MM cells to extracellular matrix proteins and BMSC, as well as the resultant growth, survival, and drug-resistance advantages as a result of tumor cell-binding and induction of cytokines, have been delineated [36 39 ]. Of importance, it has been reported that IL-6 is incapable of protecting MM cells from bortezomib-induced cell death [47 ]. The induction of IL-6R signaling interacts in OPM-2 and RPMI-8226 cells only after transfection with VEGF siRNA or would be triggered by a combined treatment of VEGF siRNA and bortezomib. In the IL-6-dependent INA-6 cell line, we could not find an induction of IL-6R mRNA levels after treatment with VEGF siRNA or the combination of VEGF siRNA with bortezomib in the presence of human IL-6 or in the presence of human BMSC. This observation in OPM-2 and RPMI-8226 cells could be a bystander effect or a deregulation of trans-signaling in the molecular mechanisms of the IL-6/IL-6R pathway [48 49 50 ]. Differentiation-specific surface markers were not affected by VEGF siRNA transfection in any studied cell lines or in normal CD34+ progenitor cells. However, cell-cycle phase distribution was affected by VEGF siRNA transfection, suggesting that the above-described inhibition of proliferation and the apoptotic rate were based on cell-cycle arrest or on the induction of apoptosis. It transpired that the transfection with VEGF siRNA depends on effects in cell-cycle arrests in the G1-phase and on an increased apoptotic distribution with DNA fragmentation (sub-G0/G1-phase) [51 ]. By adding bortezomib to VEGF siRNA treatment, these effects increased about ~1.3-fold (P<0.05). These results strongly suggest that dual inhibition of silencing by VEGF siRNA with bortezomib additively enhances MM cytotoxicity and apoptosis. The complexity of VEGF actions is determined by a multitude of target cells. In addition, effects of VEGF on targets cells other than endothelial cells may contribute to the clinical manifestation of leukemias, lymphomas, and MM. Direct and indirect targeting of VEGF and its receptors (VEGFR inhibitors, GW654652, or others) is therefore a promising novel, therapeutic approach to improve patient outcome [6 ]. Furthermore, as VEGF plays a pivotal role in HSC differentiation, the potential role of VEGF and VEGFR in clonal leukemogenic development is also under investigation.

There are several mechanisms by which tumor cells develop resistance to cytotoxic agents. One mechanism is mediated by drug transporter proteins, such as P-gp or MDR1, and MDR-associated protein and lung resistance-related protein in MM cells, which represent the pharmacologic basis for decreased intracellular drug accumulation mediated by enhanced efflux [52 ]. Specifically, a predominant acquirement of MDR1-dependent resistance is found in the majority of patients with refractory lymphomas and MM, as well as in patients during relapse [53 ]. It should be stressed that our data of P-gp expression by flow cytometric analysis show a highly significant reduction of more than 60% of P-gp expression only after a high dose of 50 nM bortezomib (P<0.01) compared with untreated OPM-2 cells. VEGF siRNA transfection could also decrease P-gp expression up to 50% (P<0.02), and the combination of VEGF siRNA with a lower dose bortezomib (5 nM) resulted in an additional reduction of P-gp expression to 79% in OPM-2 cells (P<0.01). These data are consistent with our results regarding MDR-1 gene expression that demonstrated that the combination of VEGF siRNA and 5 nM bortezomib seems to be sufficiently effective to overcome MDR1 gene expression in the OPM-2 cells. In addition to impaired drug transport, aberrant activation of signal transduction proteins including NF-{kappa}B has been implicated in treatment failure in hematological malignancies, including MM. Recent in vitro data also demonstrated that bortezomib may change P-gp-mediated efflux in MM cells [54 ]. Furthermore, to complement pharmacological approaches, silencing of the MDR-1 gene via siRNA, resulting in reduced P-gp expression, represents an attractive means to revert drug resistance [55 ].

In conclusion, our study demonstrates that VEGF siRNA induces apoptosis and inhibition of proliferation in MM cells. Moreover, we found that the combination of VEGF siRNA and bortezomib results in an additive, antiproliferative, and exaggerated proapoptotic effect in MM cells. In addition, the combination of VEGF siRNA and bortezomib significantly reversed MDR1-dependent multidrug resistance of primary malignant cells in patients with advanced MM and various cell lines without general toxic effects. Therefore, the combination of VEGF siRNA with a selective inhibitor of the 26S proteasome can be considered a potential therapeutic strategy in various hematological malignancies, where MDR-1 overexpression and activities are a major impediment for a cure.

ACKNOWLEDGEMENTS

This work was supported in part by grants from the Deutsche José Carreras Leukämie-Stiftung e.V. DJCLS-R06/36v and the Deutsche Kulturstiftung Essen. M. K. designed, performed, and analyzed research and wrote the manuscript. D. W. B. and A. H. E. participated in the coordination of the study and funded the study. The authors declare no competing financial interests. The authors thank Melanie Kroll, Silke Gottwald, Christiane Schary, and Ines Riepenhoff for their excellent technical performance of the PCR analyses and siRNA experiments. The authors also thank Martina Franke and Ramona Dittloff for their flow-cytometric studies.

Received September 14, 2007; revised March 12, 2008; accepted March 31, 2008.

REFERENCES

    1
  1. Dankbar, B., Padro, T., Leo, R., Feldmann, B., Kropff, M., Mesters, R. M., Serve, H., Berdel, W. E., Kienast, J. (2000) Vascular endothelial growth factor and interleukin-6 in paracrine tumor-stromal cell interaction in multiple myeloma Blood 95,2630-2636[Abstract/Free Full Text]
    2. 2
  2. Vacca, A., Ribatti, D., Presta, M., Minischetti, M., Iurlaro, M., Ria, R., Albini, A., Bussolino, F., Dammacco, F. (1999) Bone marrow neovascularization, plasma cell angiogenic potential, and matrix metalloproteinase-2 secretion parallel progression of human multiple myeloma Blood 93,3064-3073[Abstract/Free Full Text]
    3. 3
  3. Podar, K., Tai, Y. T., Davies, F. E., Lentzsch, S., Sattler, M., Hideshima, T., Lin, B. K., Gupta, D., Shima, Y., Chauhan, D., Mitsiades, C., Raje, N., Richardson, P., Anderson, K. C. (2001) Vascular endothelial growth factor triggers signaling cascades mediating multiple myeloma cell growth and migration Blood 98,428-435[Abstract/Free Full Text]
    4. 4
  4. Oyama, T., Ran, S., Ishida, T., Nadaf, S., Kerr, L., Carbone, D. P., Gabrilovich, D. I. (1998) Vascular endothelial growth factor affects dendritic cell maturation though the inhibition of nuclear factor-{kappa} B activation in hemopoietic progenitor cells J. Immunol. 160,1224-1232[Abstract/Free Full Text]
    5. 5
  5. Gabrilovich, D. I., Chen, H. L., Girgis, K. R., Cunningham, H. T., Meny, G. M., Nadaf, S., Kavanaugh, D., Carbone, D. P. (1996) Production of vascular endothelial growth factor by human tumors inhibits the functional maturation of dendritic cells Nat. Med. 2,1096-1103[CrossRef][Medline]
    6. 6
  6. Podar, K., Catley, L. P., Tai, Y. T., Shringarpure, R., Carvalho, P., Hayashi, T., Burger, R., Schlossman, R. L., Richardson, P. G., Pandite, L. N., Kumar, R., Hideshima, T., Chauhan, D., Anderson, K. C. (2004) GW654652, the pan-inhibitor of VEGF receptors, blocks the growth and migration of multiple myeloma cells in bone marrow microenvironment Blood 103,3474-3479[Abstract/Free Full Text]
    7. 7
  7. Ferrara, N. (2002) Role of vascular endothelial growth factor in physiologic and pathologic angiogenesis: therapeutic implications Semin. Oncol. 29,10-14[Medline]
    8. 8
  8. Fang, S., Weissmann, A. M. (2004) A field guide to ubiquitylation Cell. Mol. Life Sci. 61,1546-1561[Medline]
    9. 9
  9. Richardson, P. G., Barlogie, B., Berenson, J., Singhal, S., Jagannath, S., Irwin, D., Rajkumar, S. V., Srkalovic, G., Alsina, M., Alexanian, R., Siegel, D., Orlowski, R. Z., Kuter, D., Limentani, S. A., Lee, S., Hideshima, T., Esseltine, D. L., Kauffman, M., Adams, J., Schenkein, D. P., Anderson, K. C. (2003) A phase 2 study of bortezomib in relapsed, refractory myeloma N. Engl. J. Med. 348,2609-2617[Abstract/Free Full Text]
    10. 10
  10. Bross, P. F., Kane, R., Farrell, A. T., Abraham, S., Benson, K., Brower, M. E., Bradley, S., Gobburu, J. V., Goheer, A., Lee, S. L., Leighton, J., Liang, C. Y., Lostritto, R. T., McGuinn, W. D., Morse, D. E., Rahman, A., Rosario, L. A., Verbois, S. L., Williams, G., Wang, Y. C., Pazdur, R. (2004) Approval summary for bortezomib for injection in the treatment of multiple myeloma Clin. Cancer Res. 10,3954-3964[Free Full Text]
    11. 11
  11. Mimnaugh, E. G., Xu, W., Vos, M., Yuan, X., Isaacs, J. S., Bisht, K. S., Gius, D., Neckers, L. (2004) Simultaneous inhibition of hsp 90 and the proteasome promotes protein ubiquitination, causes endoplasmic reticulum-derived cytosolic vascuolization, enhances antitumor activity Mol. Cancer Ther. 3,551-566[Abstract/Free Full Text]
    12. 12
  12. Catley, L., Tai, Y. T., Chauhan, D., Anderson, K. C. (2005) Perspectives for combination therapy to overcome drug-resistant multiple myeloma Drug Resist. Updat. 8,205-218[CrossRef][Medline]
    13. 13
  13. Gatto, S., Scappini, B., Pham, L., Onida, F., Milella, M., Ball, G., Ricci, C., Divoky, V., Verstovsek, S., Kantarjian, H. M., Keating, M. J., Cortes-Franco, J. E., Beran, M. (2003) The proteasome inhibitor PS-341 inhibits growth and induces apoptosis in Bcr/Abl-positive cell lines sensitive and resistance to imatinib mesylate Haematologica 88,853-863[Abstract/Free Full Text]
    14. 14
  14. Chauhan, D., Li, G., Podar, K., Hideshima, T., Shringarpure, R., Catley, L., Mitsiades, C., Munshi, N., Tai, Y. T., Suh, N., Gribble, G. W., Honda, T., Schlossman, R., Richardson, P., Sporn, M. B., Anderson, K. C. (2004) The bortezomib/proteasome inhibitor PS-341 and triterpenoid CDDO-Im induce synergistic anti-multiple myeloma (MM) activity and overcome bortezomib resistance Blood 103,3158-3166[Abstract/Free Full Text]
    15. 15
  15. Fire, A., Xu, S., Montgomery, M. K., Kostas, S. A., Driver, S. E., Mello, C. C. (1998) Potent and specific genetic interference by double-stranded RNA in Caenorhabditis elegans Nature 391,806-811[CrossRef][Medline]
    16. 16
  16. Novina, C. D., Sharp, P. A. (2004) The RNAi revolution Nature 430,161-164[CrossRef][Medline]
    17. 17
  17. Elbashir, S. M., Harborth, J., Lendeckel, W., Yalcin, A., Weber, K., Tuschl, T. (2001) Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells Nature 411,494-498[CrossRef][Medline]
    18. 18
  18. Elbashir, S. M., Lendeckel, W., Tuschl, T. (2001) RNA interference is mediated by 21- and 22-nucleotide RNAs Genes Dev. 15,188-200[Abstract/Free Full Text]
    19. 19
  19. Wilda, M., Fuchs, U., Wössmann, W., Borkhardt, A. (2002) Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi) Oncogene 21,5716-5724[CrossRef][Medline]
    20. 20
  20. Elmaagacli, A. H., Koldehoff, M., Peceny, R., Klein-Hitpass, L., Ottinger, H., Beelen, D. W., Opalka, B. (2005) WT1 and BCR-ABL specific small interfering RNA have additive effects in the induction of apoptosis in leukemic cells Haematologica 90,326-334[Abstract/Free Full Text]
    21. 21
  21. Koldehoff, M., Steckel, N. K., Beelen, D. W., Elmaagacli, A. H. (2006) Synthetic small interfering RNAs reduce bcr-abl gene expression in leukemic cells of de novo Philadelphia(+) acute myeloid leukemia Clin. Exp. Med. 6,45-47[CrossRef][Medline]
    22. 22
  22. Takei, Y., Kadomatsu, K., Yuzawa, Y., Matsuo, S., Muramatsu, T. (2004) A small interfering RNA targeting vascular endothelial growth factor as cancer therapeutics Cancer Res. 64,3365-3370[Abstract/Free Full Text]
    23. 23
  23. Elmaagacli, A. H., Freist, A., Hahn, M., Opalka, B., Seeber, S., Schaefer, U. W., Beelen, D. W. (2001) Estimating the relapse in chronic myeloid leukemia patients after allogeneic stem cell transplantation by the amount of bcr-abl fusion transcripts detected using a new real-time polymerase chain reaction method Br. J. Haematol. 113,1072-1075[CrossRef][Medline]
    24. 24
  24. Koldehoff, M., Steckel, N. K., Beelen, D. W., Elmaagacli, A. H. (2007) Therapeutic application of small interfering RNA directed against bcr-abl transcripts to a patient with imatinib-resistant chronic myeloid leukemia Clin. Exp. Med. 7,47-55[CrossRef][Medline]
    25. 25
  25. Chi, J. T., Chang, H. Y., Wang, N. N., Chang, D. S., Dunphy, N., Brown, P. O. (2003) Genomewide view of gene silencing by small interfering RNAs Proc. Natl. Acad. Sci. USA 100,6343-6346[Abstract/Free Full Text]
    26. 26
  26. Zhang, L., Yang, N., Mohamed-Hadley, A., Rubin, S. C., Coukos, G. (2003) Vector-based RNAi, a novel tool for isoform-specific knock-down of VEGF and anti-angiogenesis gene therapy of cancer Biochem. Biophys. Res. Commun. 303,1169-1178[CrossRef][Medline]
    27. 27
  27. Filleur, S., Courtin, A., Ait-Si-Ali, S., Guglielmi, J., Merle, C., Harel-Bellan, A., Clézardin, P., Cabon, F. (2003) siRNA-mediated inhibition of vascular endothelial growth factor severely limits tumor resistance to antiangiogenic thrombospondin-1 and slows tumor vascularization and growth Cancer Res. 63,3919-3922[Abstract/Free Full Text]
    28. 28
  28. Guan, H., Zhou, Z., Wang, H., Jia, S. F., Liu, W., Kleinerman, E. S. (2005) A small interfering RNA targeting vascular endothelial growth factor inhibits Ewing’s sarcoma growth in a Xenograft mouse model Clin. Cancer Res. 11,2662-2669[Abstract/Free Full Text]
    29. 29
  29. Reich, S. J., Fosnot, J., Kuroki, A., Tang, W., Yang, X., Maguire, A. M., Bennett, J., Tolentino, M. J. (2003) Small interfering RNA (siRNA) targeting VEGF effectively inhibits ocular neovascularization in a mouse model Mol. Vis. 9,210-216[Medline]
    30. 30
  30. Hu, X., Hipolito, S., Lynn, R., Abraham, V., Ramos, S., Wong-Staal, F. (2004) Relative gene-silencing efficiencies of small interfering RNAs targeting sense and antisense transcripts from the same genetic locus Nucleic Acids Res. 32,4609-4617[Abstract/Free Full Text]
    31. 31
  31. Morita, K., Yamate, K., Kurakata, S., Abe, K., Watanabe, K., Koizumi, M., Imanishi, T. (2006) Inhibition of VEGF mRNA by 2'-O,4'-C-ethylene-bridged nucleic acids (ENA) antisense oligonucleotides and their influence on off-target gene expressions Nucleosides Nucleotides Nucleic Acids 25,503-521[CrossRef][Medline]
    32. 32
  32. Levine, A. M., Tulpule, A., Quinn, D. I., Gorospe, G., III, Smith, D. L., Hornor, L., Boswell, W. D., Espina, B. M., Groshen, S. G., Masood, R., Gill, P. S. (2006) Phase I study of antisense oligonucleotide against vascular endothelial growth factor: decrease in plasma vascular endothelial growth factor with potential clinical efficacy J. Clin. Oncol. 24,1712-1719[Abstract/Free Full Text]
    33. 33
  33. Kretschmer-Kazemi Far, R., Sczakiel, G. (2003) The activity of siRNA in mammalian cells is related to structural target accessibility: a comparison with antisense oligonucleotides Nucleic Acids Res. 31,4417-4424[Abstract/Free Full Text]
    34. 34
  34. Mohle, R., Rafii, S., Moore, M. A. (1998) The role of endothelium in the regulation of hematopoietic stem cell migration Stem Cells 16,159-165[Medline]
    35. 35
  35. Bautz, F., Rafii, S., Kanz, L., Mohle, R. (2000) Expression and secretion of vascular endothelial growth factor-A by cytokine-stimulated hematopoietic progenitor cells. Possible role in the hematopoietic microenvironment Exp. Hematol. 28,700-706[CrossRef][Medline]
    36. 36
  36. Le Gouill, S., Podar, K., Amiot, M., Hideshima, T., Chauhan, D., Ishitsuka, K., Kumar, S., Raje, N., Richardson, P. G., Harousseau, J. L., Anderson, K. C. (2004) VEGF induces MCL-1 upregulation and protects multiple myeloma cells against apoptosis Blood 104,2886-2892[Abstract/Free Full Text]
    37. 37
  37. Hideshima, T., Mitsiades, A., Akiyama, M., Hayashi, T., Chauhan, D., Richardson, P., Schlossman, R., Podar, K., Munshi, N. C., Mitsiades, N., Anderson, K. C. (2003) Molecular mechanisms mediating antimyeloma activity of proteasome inhibitor PS-341 Blood 101,1530-1534[Abstract/Free Full Text]
    38. 38
  38. Boccadoro, M., Morgan, G., Cavenagh, J. (2005) Preclinical evaluation of the proteasome inhibitor bortezomib in cancer therapy Cancer Cell Int. 5,18-26[CrossRef][Medline]
    39. 39
  39. Podar, K., Shringarpure, R., Tai, Y. T., Simoncini, M., Sattler, M., Ishitsuka, K., Richardson, P. G., Hideshima, T., Chauhan, D., Anderson, K. C. (2004) Caveolin-1 is required for vascular endothelial growth factor-triggered multiple myeloma cell migration and is targeted by bortezomib Cancer Res. 64,7500-7506[Abstract/Free Full Text]
    40. 40
  40. Wohlbold, L., Van Der Kuip, H., Miething, C., Vornlocher, H. P., Knabbe, C., Duyster, J., Aulitzky, W. E. (2003) Inhibition of bcr-abl gene expression by small interfering RNA sensitizes for imatinib mesylate (ST571) Blood 102,2236-2239[Abstract/Free Full Text]
    41. 41
  41. Hurwitz, H., Fehrenbacher, L., Novotny, W., Cartwright, T., Hainsworth, J., Heim, W., Berlin, J., Baron, A., Griffing, S., Holmgren, E., Ferrara, N., Fyfe, G., Rogers, B., Ross, R., Kabbinavar, F. (2004) Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer N. Engl. J. Med. 350,2335-2342[Abstract/Free Full Text]
    42. 42
  42. Santos, S. C., Dias, S. (2004) Internal and external autocrine VEGF/KDR loops regulate survival of subsets of acute leukemia through distinct signaling pathways Blood 103,3883-3889[Abstract/Free Full Text]
    43. 43
  43. Lee, S., Chen, T. T., Barber, C. L., Jordan, M. C., Murdock, J., Desai, S., Ferrara, N., Nagy, A., Roos, K. P., Iruela-Arispe, M. L. (2007) Autocrine VEGF signaling is required for vascular homeostasis Cell 130,691-703[CrossRef][Medline]
    44. 44
  44. Podar, K., Anderson, K. C. (2005) The pathophysiologic role of VEGF in hematologic malignancies: therapeutic implications Blood 105,1383-1395[Abstract/Free Full Text]
    45. 45
  45. Hideshima, T., Chauhan, D., Richardson, P., Anderson, K. C. (2005) Identification and validation of novel therapeutic targets for multiple myeloma J. Clin. Oncol. 23,6345-6350[Abstract/Free Full Text]
    46. 46
  46. Mitsiades, C. S., Mitsiades, N. S., Munshi, N. C., Richardson, P. G., Anderson, K. C. (2006) The role of the bone microenvironment in the pathophysiology and therapeutic management of multiple myeloma: interplay of growth factors, their receptors and stromal interactions Eur. J. Cancer 42,1564-1573[CrossRef][Medline]
    47. 47
  47. Hideshima, T., Richardson, P., Chauhan, D., Palombella, V. J., Elliott, P. J., Adams, J., Anderson, K. C. (2001) The proteasome inhibitor PS-341 inhibits growth, induces apoptosis, and overcomes drug resistance in human multiple myeloma cells Cancer Res. 61,3071-3076[Abstract/Free Full Text]
    48. 48
  48. Rose-John, S., Waetzig, G. H., Scheller, J., Grötzinger, J., Seegert, D. (2007) The IL-6/sIL-6R complex as a novel target for therapeutic approaches Expert Opin. Ther. Targets 11,613-624[CrossRef][Medline]
    49. 49
  49. Chalaris, A., Rabe, B., Paliga, K., Lange, H., Laskay, T., Fielding, C. A., Jones, S. A., Rose-John, S., Scheller, J. (2007) Apoptosis is a natural stimulus of IL6R shedding and contributes to the pro-inflammatory trans-signaling function of neutrophils Blood 110,1748-1755[Abstract/Free Full Text]
    50. 50
  50. Kallen, K-J. (2002) The role of transsignaling via the agonistic soluble IL-6 receptor in human diseases Biochim. Biophys. Acta 1592,323-343[Medline]
    51. 51
  51. Kumar, V., Varma, N., Varma, S., Vohra, H., Malhotra, P., Dutta, U., Sharma, S. C. (2004) Flow cytometric analysis of DNA indices, expression of p53 and multidrug resistance genes in multiple myeloma patients Anal. Quant. Cytol. Histol. 26,271-277[Medline]
    52. 52
  52. Schwarzenbach, H. (2002) Expression of MDR1/P-glycoprotein, the multidrug resistance protein MRP, and the lung-resistance protein LRP in multiple myeloma Med. Oncol. 19,87-104[CrossRef][Medline]
    53. 53
  53. Sonneveld, P. (2000) Multidrug resistance in haematological malignancies J. Intern. Med. 247,521-534[Medline]
    54. 54
  54. Rumpold, H., Salvador, C., Wolf, A. M., Tilg, H., Gastl, G., Wolf, D. (2007) Knockdown of PgP resensitizes leukemic cells to proteasome inhibitors Biochem. Biophys. Res. Commun. 361,549-554[CrossRef][Medline]
    55. 55
  55. Szakacs, G., Paterson, J. K., Ludwig, J. A., Booth-genthe, C., Gottesman, M. M. (2006) Targeting multidrug resistance in cancer Nat. Rev. Drug Discov. 5,219-234[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF) Free
Right arrow All Versions of this Article:
jlb.0907632v1
84/2/561    most recent
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Koldehoff, M.
Right arrow Articles by Elmaagacli, A. H.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Koldehoff, M.
Right arrow Articles by Elmaagacli, A. H.