ANTICANCER RESEARCH 26: 1753-1758 (2006)

Tetrathiomolybdate Blocks bFGF- but not VEGF-induced Incipient In Vitro

FATEMA MAMOU1, KERSTIN S. MAY1, MATTHEW J. SCHIPPER1, NAVKIRANJIT GILL1, MUHAMMED S.T. KARIAPPER3, BINDU M. NAIR3, GEORGE BREWER2, DANIEL NORMOLLE1 and MOHAMED K. KHAN3

1Department of Radiation Oncology, Comprehensive Cancer and Geriatrics Center and 2Department of Human Genetics, University of Michigan Medical Center, Ann Arbor, Michigan; 3Roswell Park Cancer Institute, Department of Radiation Medicine, and Department of Cell Stress Biology, Buffalo, New York, U.S.A.

Abstract. Background: Angiogenesis is a multi-step process usually produces multiple pro-angiogenic molecules. A which involves endothelial cell sprouting from existing blood strategy that affects multiple activators of angiogenesis may vessels, followed by migration, proliferation, alignment and be highly applicable for the treatment of human tumors and tube formation. Tetrathiomolybdate (TM) is a multi-hit anti- could potentially suppress tumor angiogenesis more angiogenic agent with actions against multiple angiogenic aggressively than single target strategies. pathways. These inhibitory effects of TM are attributed to its Copper (Cu) is a required cofactor for the function of potent copper level-reducing property. Copper is needed for many pro-angiogenic mediators including bFGF (8-10), activation of various angiogenic pathways at the transcriptional VEGF and (11, 12). Several animal studies have and levels. Materials and Methods: The direct effects demonstrated that Cu is required for angiogenesis (13, 14), of TM on angiogenesis of endothelial cells were examined and that Cu depletion inhibits tumor growth in vivo (14-17). using an in vitro sprout-forming system. Results: It was shown Introducing a mild Cu deficiency in animals also that depletion of copper by TM selectively repressed bFGF- significantly reduces angiogenesis (18). In addition, Cu induced, but not VEGF-induced sprout formation (an early induces migration of endothelial cells, a crucial early event angiogenic step). Conclusion: This model permitted the in angiogenesis (19, 20). These data show that the molecular separation of VEGF- and bFGF- induced early angiogenesis mechanisms by which Cu promotes angiogenesis include: (i) in vitro, and indicated the existence of mechanistic differences binding to angiogenic growth factors, thereby increasing between bFGF- and VEGF- induced early angiogenic events. their affinity for endothelial cells, (ii) controlling secretion/mobilization of active angiogenic cytokines (as Angiogenesis, a process by which new microvasculature is demonstrated with bFGF and IL-1), or (iii) inducing the formed from existing microvasculature, occurs during expression of angiogenic growth factors like VEGF. Hence, physiologic conditions such as wound healing and therapy designed to deplete Cu levels may be a successful menstruation, and in pathologic conditions such as cancer, multi-step anti-angiogenic strategy that denies blood supply where tumors induce formation of new microvasculature in to tumors, thereby inhibiting tumor growth. order to grow beyond the critical mass (1-3). Numerous Tetrathiomolybdate (TM) is the most potent Cu-binding molecules stimulate angiogenesis (1, 4) or inhibit it (5, 6), agent known, and has been clinically used for over a decade in the balance between the two determining whether the treatment of the Cu storage disorder called Wilson's disease angiogenesis is "turned on" or "off" (7). A single tumor (21). TM forms a stable tripartite complex with Cu and protein, placing patients into a negative Cu balance immediately. Cu depletion with TM impedes development of mammary tumors in Her2-neu transgenic mice (22). The safety and antitumor Correspondence to: Mohamed K. Khan, Department of Radiation effects of TM therapy alone in patients with solid tumors have Medicine, Roswell Park Cancer Institute, Elm and Carlton Streets, been evaluated in a phase I clinical trial (23), where TM was Buffalo, NY 14263, U.S.A. Tel: (716) 845-1180, Fax: (716) 845- shown to impair angiogenesis, leaving other Cu-dependent 8254, e-mail: [email protected] cellular processes largely intact, thus preventing significant Key Words: Incipient angiogenesis, VEGF, bFGF, tetrathiomolybdate, clinical toxicity. Additionally, TM improved local control when collagen sprouting assay. combined with radiation therapy (RT) in pre-clinical models

0250-7005/2006 $2.00+.40 1753 ANTICANCER RESEARCH 26: 1753-1758 (2006)

(24) and this combination with RT is also being evaluated in a that had not adhered to the collagen. The unstimulated set of clinical trial at Roswell Park Cancer Institute, U.S.A. plates received 8 ml of EGMì-2MV. The bFGF-stimulated set In vitro data demonstrated that TM reduced the expression received 8 ml EGMì-2MV with 50 ng/ml of bFGF (Intergen, Purchase, NY, USA). The TM + bFGF-stimulated set received of bFGF, VEGF, IL-1, IL-6 and IL-8 in tumor cells (22). In one of three concentrations of TM (0.1 nM, 1 nM and 10 nM) in an in vitro matrigel assay, TM inhibited formation of rat 50 ng/ml bFGF - EGMì-2MV solution. The VEGF stimulated aortic tubules (25). The levels of bFGF, VEGF, IL-6 and set received 8 ml EGMì-2MV with 50 ng/ml of VEGF IL-8 protein were decreased in the serum of TM-treated (Intergen). The TM + VEGF-stimulated set received one of two cancer patients (26); this decrease can be partly explained by concentrations of TM (1 nM and 10 nM) in 50 ng/ml VEGF - the fact that depletion of Cu by TM also inhibits NFκB (27), EGMì-2MV solution. Cu was tested against 1 nM TM on a master regulator of many angiogenic factors. This could bFGF-stimulated endothelial cells, as 2 nM cupric sulfate (CuSO ñ5H O, Sigma) in 1 nM TM, 50 ng/ml bFGF EGMì- cause a decrease in the transcription of several pro-angiogenic 4 2 2MV medium. After addition of fresh media, the plates were factors and decreasing levels produced by tumor cells. It is incubated at 37ÆC. Beyond day 0, the cells received fresh media likely that TM also exerts direct effects on the endothelium. with the same stimulation on days 2 and 4. The experiment was To gain insight into the direct effects of Cu depletion by repeated 9 times to give 9 replicates. TM on human endothelial cells, an in vitro sprout formation assay was used, where human dermal microvascular Sprout counting and harvesting of cells. On day 2, early "sprouts" or endothelial cells (HDMECs) are placed on a collagen matrix, associations of 2-3 cells were counted in a total of 10-20 random fields more accurately representing the in vivo situation (28), and per plate, using a microscope at 200X magnification. Three plates from induced by bFGF or VEGF. Induced sprout formation each set were used for counting. By day 4, full sprouts were seen as more cells clearly associating with one another, with a linear collagen involves proliferation, matrix reorganization, and migration pattern visibly aligned with the cells. Counting was repeated on day 6. of endothelial cells away from the proliferative foci, and an The same 3 plates per set were used throughout the experiment to absence of full tubule formation, indicative of incipient (very count the sprouts. Two different investigators did the blinded counting early) angiogenesis. We were, therefore, able to examine the (F.M., K.S.M.), and the values were averaged. On days 2, 4 and 6, cells effects of Cu removal by TM on incipient angiogenesis of were harvested from 3 plates per set and counted to analyze cell human endothelial cells induced by bFGF and VEGF. amplification. Collagen from these plates was liquefied using 6 ml of 2.5 mg/ml of type IV collagenase (Sigma) in PBS warmed to 37ÆC and Materials and Methods the cells were spun down for 7 minutes at 700 rpm and 4ÆC, reconstituted in PBS and counted using a hemocytometer. Preparation of collagen plates. The effects of bFGF, VEGF and TM on endothelial cells were examined using HDMECs grown on Statistical analysis. Data from the 9 experiments were analyzed in 100-mm plates of bovine dermal collagen. The collagen was two regression models. Poisson regression (SAS PROC prepared 1 day prior to seeding of cells on these plates, with all GENMOD, SAS Institute, Cary, NC, USA) was used to analyze ingredients kept at 4ÆC. Sterile filtered 10X phosphate buffered the number of sprouts per field. The day, stimulant (VEGF or saline (PBS, Sigma, St. Louis, MO, USA) solution was added to bFGF), Cu and TM levels, and their interactions were included as Vitrogen 100 collagen (Cohesion Technologies, Palo Alto, CA, predictors. Cu was not given with VEGF or with TM at the 0.1 and USA) in a 1 to 5 ratio. After mixing, the solution was added to 3 10 nM levels, and thus these interactions could not be estimated or parts sterile deionized water and 1 part sterile 0.1 M sodium tested. All other interactions, as well as the main effects, were hydroxide and the pH adjusted to 7.4±0.2 by sterile 0.1 M estimated and tested for significance. Interactions that were not hydrochloric acid. Aliquots of 6.5 ml of the collagen solution were significant at the 5% level were dropped from the model. The final dispensed evenly onto each 100 x 15 mm polystyrene petri dish model contained interaction terms for VEGF*Day, Cu*TM, and (Fisherbrand, Fisher Scientific, Pittsburgh, PA, USA) and the Stimulant*TM. This last interaction allowed TM to have different plates were placed in a 37ÆC incubator overnight to gelatinize. effects on bFGF- and VEGF-stimulated cells. A mixed model (SAS PROC MIXED) was fit to the logarithm of the cell counts with the Seeding of the HDMECs. The gelatinization collagen was washed with same predictors. All effects were included as categorical variables. 5 ml of Hank’s Balanced Salt Solution (HBSS, Cellgro®, Herndon, In this model, no interactions were significant, so only the main VA, USA) and incubated with 5 ml of Microvascular Endothelial Cell effects were included in the final model. A random effect for Medium-2 (EGMì-2MV, Biowhittaker, Walkersville, MD, USA) at experiment was included in both models to account for variations 37ÆC, for several hours, to allow for collagen equilibration. Passage 9 between experiments. HDMECs were harvested at a density of 96,667 cells/ml in EGMì- 2MV. After removal of the media, 6 ml of the cell suspension was Results gently dispensed over each collagen plate to give a final cell density of 5 5.8x10 cells/plate. The plates were then incubated overnight at 37ÆC The results of the statistical analysis of the sprout counts are and 5% CO to allow the cells to adhere to the collagen. 2 depicted in Tables I and II. The ‘Estimate’ in Table I is the Cell stimulation. The next day (day 0), the collagen plates were ratio of the expected sprout count results for the two grouped into 3 different stimulation sets, each comprising 3 treatment groups listed. The ratio of the expected sprout plates. The plates were washed once with HBSS to remove cells count for a field given bFGF to the expected sprout count

1754 Mamou et al: TM Blocks bFGF- but not VEGF-induced Incipient Angiogenesis In Vitro

Table I. Sprout count statistical analysis. This table depicts a numerical statistical analysis of the data represented in Figures 1-3.

Comparison Estimate Lower 95% CL Upper 95% CL T-statistics P-value

Vs.

Treatment arm Control arm a)* bFGF Unstimulated 1.546 1.340 1.783 7.04 0.0001 b) VEGF (day 2) Unstimulated (day 2) 1.197 0.833 1.719 1.14 0.2865 c) VEGF (day 4) Unstimulated (day 4) 1.748 1.273 2.401 1.06 0.0036 d) VEGF (day 6) Unstimulated (day 6) 1.640 1.206 2.211 3.82 0.0051 e) bFGF + 0.1 nM TM bFGF 0.711 0.570 0.888 –3.55 0.0075 f) bFGF + 1 nM TM bFGF 0.643 0.557 0.743 –7.04 0.0001 g) bFGF + 1 nM TM + Cu bFGF + 1 nM TM 1.485 1.203 1.833 4.33 0.0025 h) bFGF + 10 nM TM bFGF 0.719 0.610 0.848 –4.62 0.0017 i) VEGF + 1 nM TM VEGF 0.866 0.668 1.121 –1.29 0.2343 j) VEGF + 10 nM TM VEGF 0.860 0.663 1.114 –1.34 0.2157 k) bFGF + 0.1 nM TM Unstimulated 1.099 0.873 1.384 0.94 0.3733 l) bFGF + 1 nM TM Unstimulated 0.994 0.848 1.164 –0.09 0.9314 m) bFGF + 10 nM TM Unstimulated 1.112 0.932 1.327 1.38 0.2045 n) bFGF + 1 nM TM + Cu bFGF 0.955 0.783 1.165 –0.54 0.6069 o) VEGF + 1 nM TM (day 2) Unstimulated (day 2) 1.036 0.716 1.498 0.22 0.8322 p) VEGF + 1 nM TM (day 4) Unstimulated (day 4) 1.513 1.094 2.094 2.94 0.0187 q) VEGF + 1 nM TM (day 6) Unstimulated (day 6) 1.419 1.044 1.929 2.63 0.0301 r) VEGF + 10 nM TM (day 2) Unstimulated (day 2) 1.029 0.711 1.488 0.18 0.8645 s) VEGF + 10 nM TM (day 4) Unstimulated (day 4) 1.503 1.086 2.081 2.89 0.0202 t) VEGF + 10 nM TM (day6) Unstimulated (day 6) 1.410 1.037 1.916 2.58 0.0328

*The effect of bFGF was same over time.

Table II. Statistical analysis of the cell count data.

Comparison Estimate Lower 95% CL Upper 95% CL T-statistics P-value

Vs.

Treatment arm Control arm a) bFGF Unstimulated 0.043 0.004 0.082 2.158 0.0317 b)* VEGF Unstimulated 0.111 0.111 0.177 3.331 0.0010 c)* 0.1 nM TM + bFGF (or VEGF) bFGF or VEGF –0.055 –0.110 –0.000 –1.970 0.0497 d)* 1 nM TM + bFGF (or VEGF) bFGF or VEGF –0.059 –0.095 –0.023 –3.226 0.0014 e)* 10 nM TM + bFGF (or VEGF) bFGF or VEGF –0.067 –0.113 –0.021 –2.894 0.0041

*TM has the same effect on both stimulants (bFGF and VEGF).

for a field not given bFGF was 1.546 (Table I, row a, column sending out projections of endothelial cells (sprouts). Our Estimate), indicating that bFGF increased the expected data showed that HDMEC sprouts in the in vitro collagen sprout count of an unstimulated field by 54%. An Estimate matrix system can be significantly increased above those of value of 1 indicates no differences between the two unstimulated HDMECs by using angiogenic factors bFGF treatment arms. The last 10 effects analyzed were combined or VEGF (Figures 1-3). bFGF induced a statistically effects (Table I, rows k-t) and the expected sprout count for significant increase in sprout formation on all days tested fields given both TM and bFGF was not significantly (p=0.0001) (Table I, row a). The VEGF-induced increase different from the unstimulated fields (Table I, rows k-m). in sprouts was insignificant on day 2 (p=0.2865), but During early angiogenesis, endothelial cells proliferate significant (p=0.0036 and 0.0051) by days 4 and 6, and breakdown nearby basement membranes and matrix, respectively (Table I, rows b-d).

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Figure 1. Levels of HDMEC sprout formation in bFGF-stimulated cells in Figure 2. Reversal of the inhibitory effect of TM by Cu. Addition of 1 nM an in vitro collagen matrix system. HDMECs were stimulated with bFGF TM to bFGF-stimulated HDMECs reduced sprout formation to the level alone, bFGF and one of three concentrations of TM (0.1 nM, 1 nM and 10 shown by unstimulated cells. This effect was reversed by the addition of nM), and media alone (unstimulated control), and the sprouts were counted. 2 nM Cu2+ to the 1 nM TM + bFGF-stimulated cells.

When TM was added to lower Cu levels, bFGF- stimulated HDMEC sprout formation was significantly reduced (Figure 1, Table I). TM decreased sprout counts in bFGF-induced HDMECs at all concentrations tested (p=0.0075, 0.0001, 0.0017 for TM=0.1, 1, 10 nM, respectively) (Table I, rows e, f, h). Further, when TM was added to bFGF, the resulting curves (Figure 2) were statistically insignificantly different from those of the unstimulated endothelial cells (Table I, rows k-m). As 1 nM TM showed the strongest inhibition of bFGF-treated endothelial cell sprout formation (Figure 1), this concentration of TM was used in experiments attempting to reverse this inhibition via Cu addition to the media. The inhibition of sprout formation by TM was reversed when excess Cu was added (Figure 2), compelling evidence that Cu depletion induced by TM caused the inhibition. Adding Cu to the TM + bFGF-treated cells had a statistically significant effect (p=0.0025) on sprout formation, when compared to TM + bFGF alone (Table I, row g). Adding Cu to TM + bFGF-stimulated cells did not show a Figure 3. Levels of HDMEC sprout formation in VEGF-stimulated cells statistically significant difference in sprout formation, when in an in vitro collagen matrix system. HDMECs were stimulated with compared to bFGF alone (p=0.6069) (Table I, row n). The VEGF alone, VEGF + 1 nm TM, VEGF + 10 nm TM and media alone (unstimulated control), and the sprouts were counted. mean number of sprouts appeared similar under both conditions (Figure 2). Finally, TM + bFGF-stimulated cells did not show a statistically significant difference in number of sprouts, when compared to unstimulated cells (Table I, Although the estimated effects of TM on endothelial cell rows k-m), showing that addition of TM nullified bFGF sprout formation induced by VEGF were less than 1 (Table I, induction. rows i-j), indicating reduced sprout count, these reductions

1756 Mamou et al: TM Blocks bFGF- but not VEGF-induced Incipient Angiogenesis In Vitro were statistically insignificant (p=0.2343, 0.2157). TM + the matrix in other experiments (29). Cu also induced the VEGF-treated endothelial cells were statistically significantly assembly of a multiprotein aggregate implicated in the different in sprout formation from unstimulated cells by days release of acidic fibroblast growth factor in response to 4 and 6, at TM concentrations of 1 nM and stress (29, 30). This, however, does not fully explain the 10 nM (Table I, rows p, q, s, t). In other words, VEGF effects seen in our study, as bFGF was present in the media induced sprout formation, but TM could not significantly (and did not need to be mobilized from the matrix). The decrease VEGF-induced sprout formation. blockage of bFGF-induced sprout formation indicates a Both bFGF and VEGF are known mitogens for direct blockage of pathways leading to incipient endothelial cells in culture and, therefore, increase angiogenesis (sprout formation). This is most consistent endothelial cell proliferation. The total number of cells were with a direct inhibition of bFGF protein function. determined using additional collagen plates seeded with the The fact that VEGF-induced sprout formation was not same number of endothelial cells at the same time as each affected by Cu depletion with TM could indicate two sprout count experiment (see materials and methods). possibilities: (i) VEGF-induced angiogenesis is resistant to These experiments demonstrated that both bFGF and the effects of Cu depletion with TM; this is not supported VEGF stimulation increased the total number of cells by other experiments where Cu depletion blocked tubule present on each plate when compared to unstimulated formation (25) and (ii) VEGF induced a series of molecular plates (Table II, rows a-b) and TM opposed this cell cascades that permitted incipient (early) angiogenesis to number effect. The effect of TM on cell number occur even in the absence of Cu, but later steps (tubule demonstrated a dose response, with increasing Cu reduction formation, matrix organization, proper attachment to the by TM causing lower cell numbers (Table II, rows c-f). The vascular side of the capillaries, etc.) require the presence of data show that both bFGF and VEGF caused the expected Cu. Our data are more consistent with the second cell number increases, and that TM was able to affect the possibility. mitogenic response of both factors, demonstrating that the In the future, we expect that the combination of this type TM was also functional. of reductional model system with microarray or proteomic techniques could permit analysis of the different complex Discussion steps in angiogenesis. In particular, this may permit the dissection of molecular steps between sprout formation (lines We were able to demonstrate that Cu depletion by TM of endothelial cells) and tubule formation (higher level blocked bFGF-induced early (incipient) angiogenesis in reorganization of vascular structures). As various non- HDMECs. This was measured by the blockage of sprout invasive molecular correlates of angiogenesis are discovered, formation (proliferation, collagen reorganization and this may have particular importance when trying to measure migration of cells) in an in vitro collagen matrix assay. the effects of TM in clinical situations. For example, one Interestingly, Cu depletion by TM did not cause blockage of could be fooled into thinking TM has no anti-angiogenic VEGF-induced HDMEC sprout formation. These results activity on a tumor with high VEGF activity, if the molecular demonstrated, for the first time, differences in Cu-regulated markers one is using detect incipient angiogenesis, thereby angiogenesis of bFGF and VEGF on human endothelial cells. missing crucial later anti-angiogenic activities. The in vitro Previous studies have indicated that Cu depletion by TM system utilized here, and the finding of disparate TM effects interferes with NFκB, thereby decreasing transcription of on bFGF- vs. VEGF-induced incipient angiogenesis, point to numerous pro-angiogenesis mediators from tumor cells and the need for further consideration of a temporal molecular creating a negative angiogenic balance overall. It was also analysis of the many different aspects of angiogenesis, and shown that Cu depletion by TM inhibited full tubule indicate the way for a molecular dissection of these pathways formation in a mixed factor matrigel assay (25). As TM via the use of inhibitors like TM. appears to affect multiple factors from tumors, separation of those candidates responsible for most of the effects Acknowledgements shown by TM was needed. The direct effects of TM-induced Cu depletion on human endothelial cells had not been We wish to thank Peter Polverini for teaching Dr. Khan’s laboratory previously examined in detail. We expected Cu depletion by how to use the collagen system, and Stephanie Linn for some TM to apply equally to both bFGF- and VEGF-induced preliminary modification of the collagen system. We also wish to angiogenesis. We were surprised to find that TM blocked thank Sofia Merajver for her moral and intellectual support during this and several TM studies. Kerstin May was supported for part of bFGF- but not VEGF-induced sprout formation. these studies by an undergraduate University of Michigan Summer The mechanism of bFGF blockade by TM was due to Cu Biomedical Fellowship through the UROP-Undergraduate depletion, as Cu addition restored sprout formation in the Research Opportunities Program at the University of Michigan, presence of TM. Cu has been shown to mobilize bFGF from U.S.A. The studies were funded by a start-up fund for Dr. Khan.

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