Abstract
Malignant gliomas are the most common primary intrinsic brain tumors and are highly lethal. The widespread migration and invasion of neoplastic cells from the initial site of tumor formation into the surrounding brain render these lesions refractory to definitive surgical treatment. Stathmin, a microtubule destabilizing protein that mediates cell cycle progression, can also regulate directed cell movement. Nitrosoureas, traditionally viewed as DNA alkylating agents, can also covalently modify proteins such as stathmin. We therefore sought to establish a role for stathmin in malignant glioma cell motility, migration, and invasion and determine the effects of nitrosoureas on these cell movement related processes. Scratch-wound healing recovery, Boyden chamber migration, Matrigel invasion, and organotypic slice invasion assays were performed before and after the down regulation of cellular stathmin levels and in the absence and presence of sub-lethal nitrosourea (CCNU; [1-(2-chloroethyl)-3-cyclohexyl-l-nitrosourea]) concentrations. We demonstrate that decreases in stathmin expression lead to significant decreases in malignant glioma cell motility, migration, and invasion. CCNU, at a concentration of 10 μM, causes similar significant decreases, even in the absence of any effects on cell viability. The direct inhibition of stathmin by CCNU is likely a contributing factor. These findings suggest that the inhibition of stathmin expression and function may be useful in limiting the spread of malignant gliomas within the brain and that nitrosoureas may have therapeutic benefits in addition to their anti-proliferative effects.
Keywords: stathmin, malignant gliomas, nitrosoureas, migration, invasion, glioblastoma
Introduction
Although the initial treatment for many malignant gliomas is maximal surgical resection, the widespread migration and invasion of tumor cells into the surrounding brain tissue render a complete removal virtually impossible. Patients are therefore commonly treated with radiotherapy and chemotherapy in efforts to inhibit tumor cell proliferation and increase survival (1). The inhibition of cell migration and invasion is in principle likely to prevent the spread of tumors into eloquent brain regions, and in turn preserve neurologic function, but clinical attempts to specifically effect such inhibition have been limited (2).
Stathmin regulates dynamic instability, the growth and shrinkage of microtubules, by stimulating microtubule plus end catastrophes and sequestering alpha-beta tubulin dimers (3). Consequently, stathmin has direct effects on cellular processes such as migration, division, and growth cone guidance by influencing the association of microtubules with the actin cytoskeleton (4). Consistent with this, stathmin has been shown to influence sarcoma cell migration and invasion (5). Nitrosoureas such as CCNU have previously been shown to decompose under physiological conditions to chloroethyl carbonium ions and reactive organic isocyanates. The former alkylate DNA and cause interstrand cross-links while the latter carbamoylate proteins on N-terminal amino groups and the amino groups of lysine side chains (6). Stathmin, which has a high percentage of lysine residues (16%), readily undergoes CCNU-mediated carbamoylation with a subsequent inhibition in microtubule depolymerization activity (7). We therefore hypothesized that CCNU, via its inhibitory effect on stathmin, could have an inhibitory effect on tumor cell migration and invasion that is independent of its anti-proliferative activity.
To test this hypothesis, we first sought to establish a correlation between stathmin expression level and cell motility, migration and invasion. We then analyzed the effects of sub-lethal concentrations of CCNU and as well as temozolomide (TMZ) on these cell movement related processes. TMZ was examined because of its increasing use in the treatment of patients with malignant gliomas (8). Finally, to establish a direct association between CCNU and stathmin, we assessed the effects of CCNU on the ability of stathmin to prevent the polymerization of microtubules, a well characterized function of stathmin. The direct significance of our findings on malignant glioma therapies is discussed.
Materials and Methods
Cell Culture
The U251-STMNi and U251-LacZi cell lines were derived as previously described (7). Briefly, parental U251 cells (ATCC; Manassas, VA) were transfected with pERV3 (Stratagene; La Jolla, CA) using the Lipofectamine 2000 transfection reagent (Invitrogen, Carlsbad, CA). Stable transfectants, selected for on the basis of G418 (Invitrogen) resistance, were then transfected with pEind-RNAi, which contains the hygromycin B resistance gene and either the stathmin-specific short-hairpin RNA (shRNA) oligonucleotide sequence 5′-AGTTGTTGTTCTCTTCTATTGCCTTCTGATTGGTCAGAAGGCAATAGAAGAGAACAACAACT-3′ (U251-STMNi cells) or the LacZ-specific shRNA oligonucleotide sequence 5′-CTACACAAATCAGCGATTTCGAAAA ATCGCTGATT TGTGTAG-3′ (U251-LacZi cells) downstream of a modified muristerone A (murA)-responsive U6 promoter sequence. Cells resistant to both G418 and hygromycin B (Invitrogen) were subcloned by limiting dilution.
Cell viability assays
5 × 104 cells were seeded into 24 well plates (Corning Inc.; Corning, NY) and treated with DMSO control, 10 to 30 μM CCNU (HNZ Portlink; ON, Canada), or 10 to 30 μM TMZ (Drug Synthesis and Chemistry Branch, NCI; Frederick, MD). After 3 days, cells were trypsinized and counted with a Coulter Particle Counter (Coulter Electronics; Luton, UK). Experiments were performed in sextuplicate.
Immunoblotting Analysis
Cells cultured for 72 hours in the absence or presence of increasing concentrations of murA (Invitrogen), which enhances the activity of a modified U6 promoter and results in the transcription of downstream shRNA oligonucleotide sequences, were solubilized using 1% Nonidet P-40 in Tris buffered saline (Sigma). Whole cell extracts were resolved by SDS-PAGE and transferred to polyvinylidene difluoride membranes (Millipore; Billerica, MD), which were subsequently probed with primary rabbit anti-stathmin (Calbiochem; San Diego, CA; 1:10,000 dilution) and mouse anti-β-actin (Sigma; St. Louis, MO; 1:40,000 dilution) antibodies followed by secondary alkaline phosphatase conjugated goat anti-rabbit and goat anti-mouse IgG antibodies (Promega; Madison, WI; 1:7500 dilution). Membranes were developed using DDAO phosphate (Invitrogen) and scanned using an FLA-5100 laser based scanner (Fujifilm; Stamford, CT). Image processing including laser densitometric analysis of stathmin and β-actin expression levels was performed using MultiGauge version 3.0 imaging software (Fujifilm). Quantitative relative stathmin expression levels were determined by measuring the quantitative light absorbance units minus background (QL - BG) for each of the stathmin and β-actin bands. The ratio of stathmin(QL-BG) to β-actin(QL-BG) was determined for each sample and normalized to the stathmin(QL-BG) to β-actin(QL-BG) ratio for the non-murA treated sample, which was assigned the value of 100.
Scratch-wound healing recovery assays
Cells cultured in the absence or presence of 10 μM murA for 72 hours were allowed to reach confluence. A 20 μl pipette tip (ThermoFisher Scientific; Waltham, MA) was used to scratch and create a wound in the confluent monolayer. Detached cells were immediately removed by replacement of medium and where indicated, CCNU (10 μM), TMZ (10 μM) or both were added. Images were subsequently captured, and then again 24 hours later, using an AxioCam MRm camera, an Axiovert 200 microscope and AxioVision version 4.2 software (all Zeiss; Thornwood, NY). Experiments were performed in triplicate.
Cell Migration Assays
Cells cultured in the absence or presence of 10 μM murA for 72 hours were added to the upper compartments of 96-well Boyden chamber plates containing 8-μm pore size polycarbonate filters (Neuro Probe, Gaithersburg, MD). After 16 hours of culture in medium containing drug vehicle, CCNU (10 μM), TMZ (10 μM), or both CCNU and TMZ, the polycarbonate filters were removed, fixed with methanol for 10 min, and stained with modified Trypan Blue stain (0.2% in PBS) for 20 minutes to facilitate visualization and counting of cells. Cells on the upper side of the filter were removed using a rubber scraper and the migrated cells on the underside of the filter were counted with the aid of a microscope. Experiments were performed in sextuplicate.
Matrigel™ Invasion Assay
The polycarbonate filter of each Boyden chamber well was coated with Matrigel™ (BD Biosciences; Franklin Lakes, NJ). Cells cultured in the absence or presence of 10 μM murA for 72 hours were inoculated into the upper chambers and the chemoattractant rhSDF-1α/PBSF (pre-B cell growth stimulating factor, 100ng/ml; R&D Systems; Minneapolis, MN) was added to the bottom chambers. After 24 hours of culture in medium containing drug vehicle, CCNU (10 μM), TMZ (10 μM), or both CCNU and TMZ, the Matrigel™ coated polycarbonate filters were removed, fixed, and stained as above. Invaded cells on the underside of the filter were counted as above. Experiments were performed in sextuplicate.
Glioma invasion in organotypic brain slices
Brain slices were obtained from 10-day-old NIH Swiss mice and cultured as previously described (9). Glioma cells incubated with DiI (3H-Indolium, 2-[3-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-, perchlorate; 30μmg/ml; Invitrogen) were plated onto 6-well plates at a density of 5×104cells/10 μl. Upside-down overnight culture of the cell suspensions led to the formation of glioma spheroids approximately 0.5mm in diameter. Spheroids were picked and implanted into the corpus callosa of the brain slices with the aid of an inverted microscope equipped with a calibrated micromanipulator. Slices were maintained in culture media containing no drugs, CCNU (10 μM), murA (10 μM), or both CCNU and murA. Media, including drug where appropriate, were replenished every three days. Images were captured after four weeks and analyzed using the same equipment and software as for the scratch wound healing assays. Animal studies were carried out in accordance with NIH institutional guidelines for the care and use of animals. Experiments were performed in triplicate.
In vitro Tubulin Polymerization Assay
Tubulin polymerization assays were performed using purified rat tubulin as described (10, 11). Six-histidine-tagged recombinant stathmin (5μM) was produced as previously described (7), and when indicated, was pretreated for 6 hours with CCNU (10μM) followed by removal of free CCNU using a Zeba micro desalt column (Pierce, Rockford, IL). Experiments were performed in triplicate.
Statistical Analysis
Student’s t-tests were performed where appropriate using JMP 5.1 (SAS Institute, Inc.; Cary, NC). P < 0.05 was considered significant.
Results
Stathmin knockdown and CCNU inhibit scratch wound healing
In order to determine the effects of stathmin knockdown on cell motility, we first generated a U251 malignant glioma derived cell line in which murA enhances the transcription of a stathmin-specific shRNA and induces the knockdown of stathmin expression (U251-STMNi). We also generated a control cell line in which murA enhances the transcription of a LacZ-specific shRNA (U251-LacZi). The murA-induced knockdown of stathmin protein expression by U251-STMNi cells is dose dependent with a 47% decrease occurring in response to treatment with 10 μM for 72 hours (Fig. 1A). In contrast, treatment of U251-LacZi cells with murA concentrations as high as 20 μM do not have appreciable effects on stathmin expression (Fig. 1B). All further experiments involving the use of murA were carried out using a concentration of 10 μM.
Figure 1. Stathmin knockdown and drug sensitivity profiles of malignant glioma cell lines.
A and B, evaluation of stathmin expression as determined by immunoblotting in U251-STMNi (A) and U251-LacZi (B) cells following treatment with increasing concentrations of murA for 72 hours, which induces the transcription of stathmin-specific and LacZi-specific shRNAs, respectively. The former causes the knockdown of stathmin expression while the latter serves as a control for the non-specific effects of murA treatment as well as the transcription of non-specific shRNAs. Evaluation of β-actin expression was performed on the same blots and used as an internal control to monitor loading and transfer of gel lanes. On the right side of each blot are molecular mass markers in kiloDaltons. Laser densitometric analysis of protein bands was performed and the ratio of stathmin expression to β-actin expression (STMN:β-actin) was determined and normalized to a value of 100 for the non-murA treated condition. There is a quantitative decrease in stathmin expression in the U251-STMNi cells following treatment of cells with increasing concentrations of murA (A). There is no such effect of murA treatment on stathmin expression in U251-LacZi cells (B). C, Cells were cultured for 72 hours in the presence of DMSO vehicle control (0 μM) or 10 μM to 30 μM CCNU or TMZ and viable cells were counted. Cell counts were normalized to the number of vehicle treated cells and the mean relative percentages of live cells under each culture condition, indicated along the X axis, is plotted along the Y axis. Experiments were performed in sextuplicate and error bars represent the standard deviations of the means. An asterisk denotes drug concentrations in which the mean number of viable cells differs significantly from the mean number of viable cells present in the vehicle control condition (P < 0.05).
Given the cytotoxic effects of CCNU and TMZ, we sought to establish experimental conditions under which decreased cell motility rather than decreased cell viability would be the primary potential cause for decreased scratch wound healing. Treatment of cells with 10 μM CCNU does not have a significant effect on cell viability (p = 0.6520), however, treatment with 20 μM CCNU leads to a 28% decrease in cell number after 72 hours (P = 0.0100) (Fig. 1C). Similarly, treatment of cells with 10 μM TMZ does not have a significant effect on cell viability (P = 0.5935), however, treatment with 20 μM TMZ leads to a 17% decrease in cell number after 72 hours (P = 0.0411) (Fig. 1C). All further experiments involving CCNU and/or TMZ were therefore carried out using a concentration of 10 μM to minimize the potential confounding effect of decreased cell viability.
U251-STMNi cells treated with murA alone (Fig. 2A.3) demonstrate decreased wound healing compared to non-murA treated U251-STMNi cells (Fig. 2A.2). As controls for the effects of murA treatment and shRNA expression per se, U251-LacZi cells with (Fig. 2B.3) and without (Fig. 2B.2) murA pre-treatment were also assessed, and there appear to be no differences in wound healing. CCNU treatment alone inhibits wound healing by both U251-STMNi (Fig. 2A.4) and U251-LacZi (Fig. 2B.4) cells, but TMZ treatment alone does not affect either cell type (Figs. 2A.5 and 2B.5). The combination of stathmin knockdown and CCNU treatment appears to be additive in inhibiting U251-STMNi (Fig. 2A.6) but not U251-LacZi (Fig. 2B.6) cell wound healing. There is no further decrease in wound healing in either cell type due to the addition of TMZ to murA or CCNU (Figs. 2A.7, 2A.8, 2B.7, and 2B.8).
Figure 2. Scratch wound healing assay.
A and B, U251-STMNi (A) and U251-LacZi (B) cells grown to confluence were scratched to create a wound and then washed with medium to remove loosened cells. Drug vehicle (A.2, A.3, B.2 and B.3), 10 μM CCNU (A.4, A.6, A.8, B.4, B.6 and B.8), and/or 10 μM TMZ (A.5, A.7, A.8, B.5, B.7 and B.8) were added to cultures as indicated. Cultures were photographed subsequently and then again 24 hours later to assess the degree of wound healing. Cells cultured in the presence of 10 μM murA (A.3, A.6, A.7, B.3, B.6 and B.7) were done so for 72 hours prior to scratching and murA was not replaced after the removal of loosened cells. Panels marked with an asterisk (A.1 and B.1) are representative photographs of the respective cell lines immediately after scratching and washing. White scale bar equals 500 μm. Experiments were performed in triplicate and representative results are shown.
Stathmin knockdown and CCNU inhibit cell migration and invasion
To quantitate the effects seen in the scratch wound healing assays, Boyden chamber migration assays were performed (Fig. 3). Mur-A induced knockdown of stathmin expression in U251-STMNi cells causes a 32% decrease in migration (P = 0.0008) while CCNU treatment causes a 35% decrease (P < 0.0001). Treatment of U251-STMNi cells with the combination of murA and CCNU causes a 62% decrease in migration which is significantly greater than that seen following treatment with the former (P = 0.0002) or the latter (P = 0.0004) alone. In contrast, TMZ treatment by itself has no significant effect on migration (P = 0.1092) and does not enhance the effects of murA (P = 0.6701) or CCNU (P = 0.4027) either (Fig. 3A). CCNU treatment of U251-LacZi cells causes a 30% decrease in migration (P = 0.0008), but murA treatment of these cells has no significant effect when used alone (P = 0.5960) and does not enhance the effects of CCNU (P = 0.1297). TMZ alone also has no significant effects on the migration of U251-LacZi cells (P = 0.3839), and also does not enhance the effects of CCNU either (P = 0.1138) (Fig. 3B).
Figure 3. Boyden chamber migration assay.
A and B, U251-STMNi (A) and U251-LacZi cells (B) that had migrated through Boyden chamber membranes during the course of a 16 hour incubation period were counted and normalized to the number of migrating, untreated U251-STMNi and U251-LacZi cells, respectively. Where indicated, cells were exposed to CCNU or TMZ at the start of the 16 hour assay period. Cells treated with murA were done so for 72 hours prior to the initiation of the assay. Various treatment conditions are indicated along the X axis and the normalized mean relative cell numbers are plotted along the Y axis. Experiments were performed in sextuplicate and error bars represent the standard deviations of the means. Asterisks denote drug treatment conditions in which the mean number of migrating cells differs significantly from the mean number of migrating cells in the vehicle control condition (P < 0.05).
The movement of cells through Matrigel™, an in vitro model of basement membrane invasion, was evaluated. In U251-STMNi cells, murA-induced stathmin knockdown inhibits invasion by 49% (P < 0.0001) while CCNU treatment does so by 50% (P < 0.0001). The combination of the two inhibits invasion by 70% (P < 0.0001), which is significantly greater than that seen in response to either stathmin knockdown (P = 0.0013) or CCNU treatment (P = 0.0014) alone (Fig 4A). CCNU treatment of U251-LacZi cells causes a 40% decrease in invasion (P = 0.0003), but murA treatment of these cells has no significant effects (P = 0.0756) (Fig. 4B). Again, TMZ has no effects by itself and does not enhance the effects of either murA treatment or CCNU treatment in either U251-STMNi or U251-LacZi cells (all P > 0.0821) (Figs. 4A and 4B).
Figure 4. Matrigel invasion assay.
A and B, U251-STMNi (A) and U251-LacZi cells (B) that had invaded through Matrigel-coated Boyden chamber membranes during the course of a 24 hour incubation period were counted and normalized to the number of invading, untreated U251-STMNi and U251-LacZi cells, respectively. Where indicated, cells were exposed to CCNU or TMZ at the start of the 24 hour assay period. Cells treated with murA were done so for 72 hours prior to the initiation of the assay. Various treatment conditions are indicated along the X axis and the normalized mean relative cell numbers are plotted along the Y axis. Experiments were performed in sextuplicate and error bars represent the standard deviations of the means. Asterisks denote drug treatment conditions in which the mean number of invading cells differs significantly from the mean number of invading cells in the vehicle control condition (P < 0.05).
We also assessed the movement of tumor cells within organotypic slice preparations, a model for the in vivo invasion of tumor cells into surrounding brain tissue. As expected, U251-STMNi cell invasion is decreased by murA, CCNU and the combination of murA and CCNU (Fig. 5A). U251-LacZi cell invasion is also decreased by CCNU, but murA treatment does not by itself appear to have any appreciable effects nor enhance the effects of CCNU in this cell type (Fig. 5B).
Figure 5. Organotypic slice invasion assay.
A and B, DiI (3H-Indolium, 2-[3-(1,3-dihydro-3,3-dimethyl-1-octadecyl-2H-indol-2-ylidene)-1-propenyl]-3,3-dimethyl-1-octadecyl-, perchlorate)-labeled U251-STMNi (A) and U251-LacZi (B) tumor cell spheroids were implanted into organotypic brain slice cultures and treated with 10 μM murA and/or 10 μM CCNU as indicated below each photomicrograph. Red DiI-labeled tumor cells were monitored for invasion into surrounding tissues. A, MurA-treated U251-STMNi cells exhibited less invasion into surrounding tissues when compared to untreated cells. CCNU treatment also inhibited invasion of U251-STMNi cells into surrounding tissues, and the effects of murA and CCNU appeared to additive. B, In contrast, murA treatment of U251-LacZi cells did not have any appreciable effects on the invasion of cells into surrounding tissues. CCNU treatment did however inhibit the invasion of U251-LacZi cells. MurA treatment did not augment the inhibitory effects of CCNU on U251-LacZi cell invasion. White arrows point out the extent of tumor cell invasion into surrounding tissues. Red scale bar equals 500 m. Experiments were performed in triplicate and representative results are shown.
CCNU directly inhibits stathmin function
Stathmin prevents microtubule polymerization by sequestering alpha-beta tubulin dimers (3). To determine if CCNU could inhibit this stathmin function, tubulin polymerization assays were performed. The steady state concentration of polymerized microtubules in a tubulin containing solution is decreased 63% by the addition of native stathmin. This stathmin-mediated decrease is abrogated by 59% if the stathmin is pre-treated with 10 μM CCNU (Fig. 6). CCNU by itself has no significant effects on tubulin polymerization (data not shown).
Figure 6. Tubulin polymerization assay.
The polymerization of tubulin into microtubules was initiated by warming a tubulin containing solution from 0°C to 37°C. The polymerization reaction was carried out in the absence of stathmin (0), the presence of native unmodified stathmin (STMN), or the presence of 10 μM CCNU-pretreated stathmin (STMN + CCNU). The concentration of polymerized microtubules present in the solution was monitored over time (X axis) for up to 120 min by determining the optical density (O.D. - Y axis) at 340 nm in a temperature-controlled 96-well microplate spectrophotometer. Unmodified stathmin inhibits microtubule formation but modification of stathmin by 10 μM CCNU abrogates this inhibition. Experiments were performed in triplicate and representative results are shown.
Discussion
The infiltration of malignant gliomas into eloquent brain regions compromises neurologic function and is a major cause of morbidity. While most malignant glioma therapies are intended to be cytostatic or cytotoxic, therapies intended to inhibit tumor cell migration and invasion are also likely to be beneficial. With this in mind, we investigated the roles and interactions of stathmin and CCNU in the directed movements of malignant glioma cells. The results presented above demonstrate that both decreases in stathmin expression and treatment with CCNU can inhibit the migration and invasion of malignant glioma cells. The concentration of CCNU used throughout these studies (10 μM) was specifically selected because it has no significant effects on cell survival and thereby minimizes decreased cell viability per se as a potential confounding effect on cell motility related functions. Ten μM is also the approximate mean peak plasma concentration achieved in patients following oral administration of CCNU at a dose of 15 mg/kg (12).
A plausible mechanism by which CCNU can retard migration and invasion is through the direct inhibition of stathmin function as demonstrated in the tubulin polymerization assay. Consistent with this model, TMZ, an imidazotetrazine DNA alkylating agent with no direct effects on stathmin function (7), has no effects on cell migration or invasion. TMZ also does not act to enhance the inhibitory effects of stathmin knockdown or CCNU treatment on these cell movement related functions.
The median survival of malignant glioma patients treated with radiotherapy plus TMZ has been shown to be significantly greater than that of patients treated with radiotherapy alone (P < 0.001) (13). In contrast, the addition of nitrosoureas to radiotherapy has not been shown to provide a significant overall survival benefit (P = 0.108) (1). One implication of these two studies is that TMZ is more effective than nitrosoureas in inducing lethal DNA cross links in proliferating tumor cells. While this is likely the case, our results indicate that nitrosoureas are superior to TMZ in inhibiting tumor cell motility. A possible therapeutic strategy would then be radiotherapy plus the combination of TMZ and a nitrosourea, the former to control proliferation and the latter to control invasion.
In a recent phase II clinical trial of radiotherapy and TMZ plus CCNU, malignant glioma patients whose tumors had promoter methylation of the MGMT (O6-methylguanine-DNA methyltransferase) gene had a median progression free survival of 19 months (14). In a phase III clinical trial of patients also with MGMT promoter methylated tumors, the median progression free survival following radiotherapy and TMZ treatment was 10.3 months (13). The almost two fold increase in progression free survival with the addition of CCNU is consistent with a model in which progression is due to a variable combination of local and/or distant tumor recurrence defined as expanding and/or new areas, respectively, of contrast enhancement on magnetic resonance imaging. The latter requires the movement of tumor cells away from the original tumor site. Due to its inhibitory effects on migration and invasion, CCNU may delay this infiltration of tumor cells into surrounding brain tissues and consequently, the appearance of new areas of contrast enhancement. When eloquent brain regions are spared, there may also be a delay in the deterioration of neurologic function. A phase III clinical trial assessing the addition of nitrosoureas to radiotherapy and TMZ should therefore be considered given our experimental data and the promising results of the phase II clinical trial described above.
Acknowledgements
This research was supported by the Intramural Research Program of the NIH, NINDS and NICHD. We thank A. Sedlock for providing technical assistance, G. Park for critical review of the manuscript, and L. Wu and J. Isaac for advice on the organotypic slice cultures.
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