- Email: [email protected]
Noscapine inhibits tumor growth in TMZ-resistant gliomas
Cancer Letters 312 (2011) 245–252
Contents lists available at SciVerse ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Noscapine inhibits tumor growth in TMZ-resistant gliomas Niyati Jhaveri a,1, Heeyeon Cho b,1, Shering Torres a, Weijun Wang b, Axel H. Schönthal c, Nicos A. Petasis d, Stan G. Louie e, Florence M. Hofman a,⇑, Thomas C. Chen a,b,⇑ a
Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States Department of Neurosurgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States c Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States d Department of Chemistry, College of Letters, Arts & Sciences, University of Southern California, Los Angeles, CA, United States e Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, School of Pharmacy, University of Southern California, Los Angeles, CA, United States b
a r t i c l e
i n f o
Article history: Received 19 July 2011 Received in revised form 12 August 2011 Accepted 15 August 2011
Keywords: Glioma Noscapine Temozolomide-resistance Invasion Brain tumors
a b s t r a c t Noscapine, a common oral antitussive agent, has been shown to have potent antitumor activity in a variety of cancers. Treatment of glioblastoma multiforme (GBM) with temozolomide (TMZ), its current standard of care, is problematic because the tumor generally recurs and is then resistant to this drug. We therefore investigated the effects of noscapine on human TMZ-resistant GBM tumors. We found that noscapine significantly decreased TMZ-resistant glioma cell growth and invasion. Using the intracranial xenograft model, we showed that noscapine increased survival of animals with TMZ-resistant gliomas. Thus noscapine can provide an alternative therapeutic approach for the treatment of TMZresistant gliomas. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glioblastoma multiforme (GBM) is the most common and malignant of all gliomas, with an overall survival rate of less than 3.3% 5 years after diagnosis [1]. The median survival time of GBM patients with the currently best available therapy, consisting of surgery, radiation, and chemotherapy, is approximately 14.6 months [2]. This dismal prognosis is due, in part, to the highly invasive and infiltrative nature of this tumor, which makes complete eradication with surgical resection difficult. These tumors inevitably recur either locally, often within 2 cm of the
⇑ Corresponding authors. Address: Department of Pathology, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90033, United States. Tel.: +1 323 442 1153; fax: +1 323 442 3049 (Florence M. Hofman); Department of Neurosurgery, University of Southern Califorina, 2011 Zonal Avenue, CA90033, United States. Tel.: +1 323 442 3918; fax: +1 323 442 3049 (T.C. Chen). E-mail addresses: [email protected] (F.M. Hofman), tchen68670@aol. com (T.C. Chen). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.08.015
original tumor, or at distant sites within the brain. Furthermore, recurrent tumors are usually resistant to standard chemotherapy [3,4]. Although treatment of these recurrent lesions with a second surgery followed by chemotherapy may increase the symptom-free interval, the 5-year survival still remains low. Morphologically, GBM is highly vascular, characterized by extensive endothelial cell proliferation, particularly at the tumor edge [5]. Therefore, therapies that target tumor cell survival, invasion, and chemosensitization are relevant strategies for the treatment of this tumor. Temozolomide (TMZ) is currently the standard of care for the treatment of GBM [4]. This DNA alkylating agent causes DNA damage during cell replication, thereby destroying proliferating tumor cells. Clinical use of this drug has been shown to be effective in delaying tumor progression and leading to significant prolongation of survival [6]. However, GBM does recur, and when it does, it displays resistance to further TMZ treatment [7]. Thus resistance to TMZ has become a major obstacle to GBM therapy, and new treatments targeting TMZ-resistant tumors are sorely needed. Noscapine, a plant-derived benzylisoquinoline alkaloid, has been extensively used as a cough suppressant for
246
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
several decades without significant toxic side effects [8]. The potential of noscapine to inhibit cancer cell growth in vitro was discovered a half century ago [9,10], however it took 40 years to follow up on the initial promising findings [11]. The anti-tumor properties ascribed to noscapine have been primarily attributed to its ability to interfere with microtubule function, which leads to metaphase arrest of proliferating cells [11]. Noscapine functions by binding to microtubules and disrupting the microtubule dynamics without causing mass accumulation of tubulin [12,13]. Noscapine has been shown to have potent in vitro anti-tumor activity in a variety of tumor types, including glioma, non-small cell lung cancer, multiple myeloma, melanoma, ovarian and colon cancer [13–18]. In vivo studies using the xenograft murine model for human non-small cell lung cancer, T cell lymphoma, prostate and breast cancer [11,15,19,20] have demonstrated that noscapine has anti-cancer properties, which prompted a clinical investigation in patients with relapsed or refractory multiple myeloma [NCT00912899]. Overall, noscapine is an orally bioavailable agent with an excellent tolerability profile, making it an attractive agent for use as an anticancer agent. Dose escalating studies have shown that noscapine has minimal toxicity and does not interfere with the immune response [19]. Unlike other opium-derived products, noscapine does not have any analgesic, sedative, or euphoric properties and is not addictive. Recent publications have indicated the anti-tumor potency of noscapine in pre-clinical models of GBM. For example, noscapine has demonstrated activity in a mouse model of glioma in vivo [21]. The mechanism of noscapine activity in killing human glioma cells was attributed to the activation of the c-jun N-terminal kinase (JNK) signaling pathway and inactivation of the extracellular signal-regulated kinase (ERK) signaling pathway [18]. Noscapine also decreased hypoxia inducible factor (HIF)-1a expression and subsequently vascular endothelial cell growth factor (VEGF) secretion by U87 and T98G glioma cells exposed to hypoxia [22], indicating that this drug could be a potential anti-angiogenic agent as well. An important consideration for GBM therapy is the blood brain barrier. Drugs treating GBM must penetrate this barrier, and there is substantial evidence that noscapine is able to do so [21]. Using an in vitro blood brain barrier model, Landen et al. [21] demonstrated that noscapine (molecular mass: 413.4) transverses this barrier. Furthermore, in their intracranial model using rat C6 glioma cells oral administration of noscapine was detected in the brain parenchyma [21]. Thus noscapine is a potentially powerful agent for the treatment of central nervous system tumors. In the current study, we evaluated the use of noscapine for the treatment of TMZ-resistant gliomas, a major obstacle to successful GBM therapy. We found that noscapine significantly inhibited the proliferation of TMZ-resistant human glioma cells, and decreased migration and invasion of these cells in vitro. Furthermore, noscapine decreased the rate of migration of endothelial cells. In the intracranial rodent xenograft tumor model, noscapine significantly enhanced survival of animals bearing TMZ-resistant tumors. Thus noscapine demonstrated potent anti-tumor properties towards TMZ-resistant brain tumors.
2. Materials and methods 2.1. Cells and reagents The following human glioma cell lines were used: U87, U251, LN229, A172. These cell lines were cultured in 10% fetal calf serum (FCS) in DMEM supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin in a humidified incubator at 37 °C and 5% CO2. The TMZresistant glioma cells were prepared by treating the tumor cell lines with increasing doses of TMZ ranging from 10 to 100 lM over a period of 2–3 months. Human tissues were obtained in accordance with USC Institutional Review Board guidelines. Control, non-tumor human endothelial cells (BEC) were isolated from human brain tissues obtained following epileptic surgeries or trauma. Human tumor-associated brain endothelial cells (TuBEC) were obtained from human glioma tissue following resection. The human endothelial cells were isolated from these tissues, purified and characterized as described previously [23]. Endothelial cells were cultured in RPMI containing 10% FCS supplemented with Endogro (Millipore, Temecula, CA), HEPES buffer (Life Technologies, Inc., Carlsbad, CA), essential amino acids (Life Technologies, Inc.), heparin (Sigma Aldrich, St. Louis, MO), sodium bicarbonate (Life Technologies, Inc.), penicillin and streptomycin, and used only until passage 6. The following reagents were used: noscapine hydrochloride (Sigma Aldrich), TMZ (Schering Plough, Whitehouse Station, NJ), recombinant human IL-8 (R&D Systems, Minneapolis, MN). 2.2. MTT assay Tumor cells (200 cells/well) or endothelial cells (5 103 cells/well) were seeded in 96-well plates. After 24 h, noscapine was added to the cells at different concentrations; after 48 h, the medium containing the drug was replaced with fresh medium (no drug) and incubated for another 3 days. The MTT assay was performed according to the manufacturer’s protocol (Sigma Aldrich). Absorbance was measured using a microtiter plate reader (Dynatech MR4000). Percent viability was calculated relative to untreated control. All experiments were performed thrice. 2.3. Colony forming assay (CFA) Glioma cells were seeded into 6-well plates at 200 cells per well and allowed to adhere overnight. Subsequently, the cells were treated with noscapine and/or TMZ for 48 h; the medium was then removed and fresh medium (no drugs) added. Cells were incubated for another 12–14 days. At the termination of the assay, the colonies were visualized by staining with 1% methylene blue in methanol for 4 h and quantified. Groups were plated in triplicate. 2.4. Migration assay The migration assay was previously described [24]. Briefly, glioma cells or endothelial cells, at 1 104 cells/
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
well or 5 104 cells/well respectively, were seeded in the upper chamber of gelatin-coated Transwell plates with polyethylene terephthalate filters (8 lm pore) (BD Biocoat, Bedford, MA), and allowed to adhere. Noscapine was then added to the lower chamber and the chambers were incubated for 6 h. Subsequently the cells on the underside of the filter were stained, and counted at 40 magnification. Ten fields were counted per chamber. Groups were plated in duplicate, and experiments were repeated three times. Conditioned medium (CM) was prepared by growing immortalized mouse fibroblast NIH3T3 cells to 80% confluence. Culture supernatant was collected after 48 h, filtered through a 0.4 lm filter and stored at 20 °C. For migration studies, the CM, at 30% of total volume, was added to the lower chambers. IL-8 was added to the lower chamber as a chemoattractant. For hypoxic conditions, cell cultures were placed in the anaerobic gas generating pouch system (BD GASPak EZ) for 6 h.
247
the cells on the underside of the filters were stained and quantified as described above. 2.6. In vivo experiments All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at USC. U251 TMZ-resistant glioma (2 105) cells were implanted intracranially into 4–6 week old female athymic/nude mice according to a previously published method [23]. Seven days after implantation, treatment began in the following groups: vehicle control (n = 5); noscapine (n = 6); TMZ (n = 4); and noscapine + TMZ (n = 4). The drugs were administered by gavage, at the following doses: noscapine at 225 mg/kg twice daily (approximately 6–8 h apart); and TMZ at 5 mg/kg in a cycle of 7 days treatment followed by no TMZ treatment for 7 days. Survival was documented. 2.7. Cell cycle analysis
2.5. Invasion assay Glioma cells were treated with 10 lg/ml mitomycin C for 2 h, and then seeded on the top chamber of the Matrigel-coated Boyden chamber membrane (BD Biocoat Invasion Assay Kit, BD Bioscience) at 5 104 cells/well. The CM, derived from human glioma U251 cells, was added to the lower chamber to comprise 30% of the media volume; and the cells were incubated for 18 h. Subsequently,
Glioma cells were seeded at a density of 5 105 cells in a 10 cm plate. After 24 h, cells were treated with medium alone (control) or noscapine at 30 lM (based on the IC50 for noscapine) for an additional 24 h. Medium containing floating cells was collected. Attached cells were trypsinized and combined with the floating cells. The mixture was centrifuged and the cell pellet was washed with PBS, followed by fixation with 70% ethanol for 15 min. After removing
Fig. 1. Effects of drugs on colony formation and cell cycle in glioma cell lines. Three different TMZ-sensitive and TMZ-resistant glioma cell lines (TMZr) were treated with TMZ (A) or noscapine (B) at increasing concentrations. After 48 h, culture medium was replaced with fresh medium containing no drugs and cells were incubated for another 12–14 days. Colonies were visualized using methylene blue. Individual colonies were counted and IC50 was determined. U251 TMZ-sensitive (C) and U251 TMZ-resistant glioma cells (D) were treated with medium alone (left) or noscapine (right) at 30 lM for 24 h and cell cycle was analyzed by flow cytometry.
248
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
the ethanol, cells were incubated with 100 lL of a solution containing 50 lg/ml propidium iodide, 0.1 mg/ml RNase A and 0.05% Triton X-100 for 40 min at 37 °C. Cells were washed again with PBS and propidium iodide staining was analyzed by flow cytometry. Ten thousand cells per sample were analyzed. The experiments were repeated twice with similar results.
was used. An alpha of 0.05 was set a priori as a threshold for statistical significance. Statistical significance for survival was evaluated using the Logrank test. 3. Results 3.1. Noscapine inhibits growth of TMZ-resistant glioma cells Resistance to TMZ treatment is a major problem in glioma therapy, therefore identifying drugs that can decrease or inhibit the growth of TMZ-resistant tumors is paramount. To determine whether noscapine has an effect on TMZ-resistant glioma cells, three cell lines were established that were shown to be resistant to TMZ. To test whether these cells were indeed TMZ-resistant, the long-term CFA assay was used. Glioma
2.8. Statistical analysis Statistical significance was evaluated using the Student’s two-tailed t-test and where appropriate ANOVA
Table 1 IC50 of different glioma cell lines for noscapine. Cell lines
A172
LN229
U251
A172/TMZr
LN229/TMZr
U251/TMZr
Noscapine IC50 (lM)
20
70
40
60
75
30
Fig. 2. Effects of combination treatment of TMZ and noscapine on colony formation in glioma cell lines. Glioma cells lines were treated with noscapine (Nos) and TMZ alone or in combination for 48 h. Medium was changed to fresh medium and colonies were counted after 12–14 days. The following drug concentrations were used for the different cell lines: (A) U251 (Nos 30 lM, TMZ 20 lM), LN229 (Nos 50 lM, TMZ 5 lM), A172 (Nos 20 lM, TMZ 5 lM), or (B) U251/TMZr (Nos 20 lM, TMZ 100 lM), LN229/TMZr (Nos 40 lM, TMZ 100 lM) and A172/TMZr (Nos 20 lM, TMZ 100 lM). The doses used were based on drug IC50 values. Significance was designated by p < 0.05.
Fig. 3. Effects of noscapine on glioma cell migration. U251 cells were seeded in the upper chamber of a Transwell plate. Conditioned medium (CM) or noscapine (Nos) were added to the lower chamber as indicated. CM was derived from mouse fibroblast NIH3T3 cell line grown to 80% confluence. Plates were incubated for 6 h under normoxic conditions (5% CO2), and the number of migrated cells was quantified (A). The experiments were repeated under hypoxic conditions (B). Data are expressed as number of cells per field under high magnification; signifies p < 0.05.
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252 cell lines treated with TMZ were shown to be sensitive to TMZ, demonstrating an IC50 of 10 lM for LN229 and A172, and 35 lM for U251 (Fig. 1A); this is in sharp contrast to the minimal effect of TMZ on the TMZ-resistant populations (Fig. 1A) in doses reaching 1000 lM (data
249
not shown). Noscapine by itself has been shown to inhibit cell proliferation in glioma cells [18]. Our results showed that noscapine did indeed cause a significant decrease in the number of colonies in the drug-sensitive glioma cell lines, with an IC50 of 70 lM for LN229, 20 lM for A172,
Fig. 4. Effects of noscapine on glioma cell invasion. U251 TMZ-sensitive (A) and U251 TMZ-resistant (B) cells were treated with 10 lg/ml mitomycin C for 2 h to inhibit proliferation. Subsequently, cells were seeded in the upper chamber of a Matrigel-coated Transwell plate. Conditioned medium (CM) and/or noscapine (Nos) were added to the lower chamber as indicated. CM was derived from U251 glioma cells grown to 80% confluence. After an18 h incubation, the number of cells present on the underside of the filter was quantified. Data are expressed as numbers of cells per field under high magnification; signifies p < 0.0001 between the CM and the CM + Nos groups.
Fig. 5. Effects of noscapine on endothelial cell viability and migration. (A) For the cell survival assay, brain endothelial cells (BEC), tumor-associated brain endothelial cells (TuBEC) and U251 cells were treated with increasing concentrations of noscapine. After 48 h, the medium was replaced with fresh medium without drug, and cells were incubated for another 72 h. Subsequently the MTT assay was performed. Percent cell viability was calculated. U251 cells served as the positive control. (B) For the migration, BEC were added to the upper chambers of Transwell plates; noscapine (20 lM) and/or IL-8 (200 ng/ml) were added to the lower chambers. After 6 h, the cells that had migrated through the pores were quantified. Data are expressed as number of cells in ten highpower fields using 400 X magnification; signifies p < 0.0001. (C) For migration of TuBEC, these cells were placed in the upper chamber of Transwell plates; noscapine was added to the lower chamber as indicated. After 6 h the migrated cells were quantified; signifies p < 0.0001.
250
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
and 40 lM for U251 (Fig. 1B). However, the TMZ-resistant population also proved to be sensitive to noscapine (Fig. 1B). These data are summarized in Table 1. Furthermore, we showed that the effect of noscapine on the TMZ-resistant glioma cells was due to a growth arrest in cell proliferation in the G2/M phase (Fig. 1C). We then addressed the question whether noscapine could sensitize the glioma cells to TMZ. The cells were treated with doses of noscapine and TMZ at IC 80 so that additive effects could be observed. The results show that the combination of noscapine and TMZ exhibited a significant additive effect for the TMZ-sensitive cells, compared to each drug alone (Fig. 2A). However, in the TMZ-resistant glioma cells, the combination of noscapine and TMZ did not exhibit substantial anti-proliferative effects and was similar to the effect of noscapine alone (Fig. 2B). These data show that while noscapine inhibits glioma cell proliferation in the TMZ-resistant population, this agent has little effect in sensitizing these cells to TMZ in the combination treatment. 3.2. Noscapine decreases glioma cell migration and invasion Since noscapine was shown to be a microtubule targeting drug, this agent was evaluated for activity in cell migration. To test this, U251 glioma cells were exposed to conditioned media (CM) derived from NIH3T3 cells; CM was added to the cultures because the CM provides an environment which more closely corresponds to the environment in the tumor. Cells were then incubated in the absence or presence of noscapine for 6 h. With no CM stimulation, there was minimal migration of glioma cells. The results show that the non-cytotoxic dose of noscapine (20 lM) significantly decreased the migration of these glioma cells by >2-fold (Fig. 3A). Since tumor cells are often in a hypoxic environment, this migration assay was also performed under hypoxic as well as normoxic conditions. The results show that noscapine at 20 lM blocked cell migration of glioma cells in both hypoxic and normoxic environments (Fig. 3B). The invasive behavior of glioma cells presents a major clinical problem, especially with regard to tumor recurrence. We therefore examined whether noscapine decreases the invasive capacity of both the TMZ-sensitive and TMZ-resistant glioma cells in an 18 h invasion assay. Glioma cells were treated with mitomycin C to block cell proliferation, and with CM to stimulate migration. We noted that at baseline, the TMZ-resistant glioma cell lines exhibited approximately a 5-fold greater invasive capacity as compared to the TMZ-sensitive cells (Fig. 4). TMZ-sensitive cells treated with CM and exposed to noscapine showed significantly less invasion than control glioma cells (p < 0.0001) (Fig. 4A). TMZ-resistant glioma cells, tested in parallel, demonstrated that noscapine significantly (p < 0.0001) decreased tumor cell invasion (Fig. 4B). These data demonstrate that noscapine inhibits the invasive capacity of the TMZ-resistant glioma cells.
3.3. Noscapine decreases endothelial cell migration The vasculature is a critical component of tumor growth. To analyze the effects of noscapine on endothelial cell survival and proliferation, we performed a long-term MTT assay. Normal human brain endothelial cells (BEC) and human glioma-associated endothelial cells (TuBEC) were treated with increasing concentrations of noscapine (20–100 lM) for 48 h and then cultured for 72 h without the drug. U251 glioma cells served as the positive control. While noscapine decreased the proliferation of U251 as shown previously, endothelial cells did not show this effect (Fig. 5A). Even at the highest dose of noscapine (100 lM), there was little inhibition of TuBEC or BEC growth. These results show that noscapine at doses which inhibit proliferation of glioma cells, the drug had little effect on normal or tumor-associated brain endothelial cells. Since noscapine affected tumor cell migration, we tested whether this agent also had an effect on endothelial cell migration. Normal brain endothelial cells were seeded in the upper chamber and noscapine was added to the lower chamber. The potent endothelial cell chemokine, IL-8, was used as a chemoattractant. After 6 h incubation, normal brain endothelial cells treated with noscapine (20 lM) demonstrated a significant decrease in endothelial cell migration (p < 0.0001) (Fig. 5B). Thus noscapine blocked the migration of IL-8-activated endothelial cells. This experiment was repeated three times with different endothelial cell samples, giving similar results. Tumor-associated brain endothelial cells treated with noscapine showed significantly decreased (p < 0.0001) migration (Fig. 5C); this effect was independent of exogenous IL-8 (data not shown). These results indicate that noscapine acts as an anti-angiogenic agent on activated normal and tumor-associated endothelial cells.
3.4. Noscapine increases the survival of animals with orthotopic xenograft of TMZ-resistant GBM Based on the information that noscapine decreased TMZ-resistant glioma cell proliferation and acted as an anti-angiogenic agent, we tested this drug in the intracranial in vivo xenograft model. U251 TMZ-resistant glioma cells were injected intracranially into athymic/nude mice; and 7 days post-injection the mice were treated with noscapine (225 mg/ kg twice per day), TMZ (5 mg/kg) or both drugs. The survival results show that noscapine alone and noscapine in combination with TMZ significantly increased survival (p < 0.05) as compared to vehicle control and TMZ treatment alone (Fig. 6A). There was no significant difference in survival between the noscapine and noscapine plus TMZ groups. TMZ alone had no significant effect on the survival of the mice with TMZ-resistant tumors as compared to vehicle-treated control animals. In a parallel experiment, microvessel density of the tumors was evaluated at the time of sacrifice; the results show no apparent differences in microvessel density in noscapine-treated tumors as compared to vehicle treated tumors (data not shown). The data demonstrate that in this orthotopic xenograft in vivo model of TMZ-resistant tumors, treatment with noscapine increases animal survival.
4. Discussion
Fig. 6. Effects of noscapine in vivo. (A) TMZ-resistant U251 glioma cells (R) were implanted intracranially into nude mice. After 7 days, treatment was initiated. Mice were randomly divided into treatment groups: vehicle control (Con) (n = 5); noscapine (Nos) (n = 6); TMZ (n = 4); and noscapine + TMZ (N + T) (n = 4). The drugs were administered by gavage at the following doses: noscapine at 225 mg/kg twice daily (6–8 h apart); and TMZ at 5 mg/kg in a cycle of 7 days treatment followed by no TMZ treatment for 7 days. Survival was used as an endpoint; signifies statistical significance (p < 0.05).
The most challenging obstacle in the treatment of gliomas is tumor recurrence, because these recurring tumors are usually TMZ-resistant [3,4]. To date, there are only a few options for recurrent gliomas, and even these therapies have limited success [25]. We therefore focused this study on TMZ-resistant gliomas. Any therapy that either alone or in combination with other agents could cause decreased tumor growth or destruction of TMZ-resistant glioma cells would be a major contribution to our therapeutic arsenal. The studies presented here show that noscapine has this potential. Noscapine binds to microtubules, thereby interfering with cell mitosis resulting in decreased cell proliferation. Indeed, we show here that noscapine decreased the proliferation rate of TMZ-resistant human glioma cells in vitro. Similar to what was reported for TMZ-sensitive glioma
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
cells [18], the TMZ-resistant glioma cells exhibit an arrest in the G2/M phase of cell cycle. Such an arrest may lead to mitotic catastrophe and subsequent cytotoxicity [26]. Treatment with noscapine did indeed decrease the cell numbers observed in three different human glioma cell lines, U251, LN229, and A172, and their respective TMZresistant lines. These cell lines have different genetic characteristics; U251 and A172 express mutations in PTEN and p53, while LN229 expresses wild-type PTEN and mutant p53. These findings suggest that sensitivity to noscapine is independent of PTEN mutation, and is likely to be a common characteristic of glioma cells. In further studies, we tested whether TMZ, in combination with noscapine, had an additive effect on glioma cell proliferation. Our results show that in vitro, the TMZ-sensitive glioma cells can be further sensitized to TMZ with the addition of noscapine, while the TMZ-resistant cells were not affected by this combination. We next explored the capacity of noscapine to affect tumor cell invasion, a critical function of recurrent gliomas. The data showed that TMZ-resistant glioma cells consistently exhibited a higher baseline of invading cells as compared to the sensitive glioma cells, suggesting that a characteristic of TMZ-resistance is the capacity to migrate and invade more effectively than TMZ-sensitive cells. Our results demonstrated that noscapine alone significantly reduced this invasion process both in the sensitive and resistant glioma populations. The tumor vasculature is critical for tumor growth [27], and therapeutic targeting of the blood vessels with different anti-angiogenic agents has been shown to result in tumor shrinkage [28]. We therefore examined the effects of noscapine on normal and tumor-associated brain endothelial cells. Our results show that noscapine does not affect endothelial cell proliferation at doses potent for malignant tumor cells. However, noscapine did influence endothelial cell migration, a critical requirement of the angiogenic process. Others have shown that noscapine altered tubule formation and caused decreased microvessel density in tumors [29]. Our previously published studies demonstrated that TMZ does not have significant effects on endothelial cell proliferation or migration [23]. Microvessel density of tumors harvested from noscapine-treated tumors demonstrated no significant differences as compared to tumor treated with vehicle (data not shown). These data suggest that although noscapine decreased migration of endothelial cells in vitro, these effects of noscapine on the tumor vasculature may be observed earlier in the tumor growth process and may not be detectable at the later stage of disease. Thus noscapine does not appear to affect the tumor vasculature, and the results observed in vivo are related to noscapine activity directly on the tumor cells. The orthotopic in vivo xenograft TMZ-resistant tumor model demonstrated that noscapine decreased tumor growth. The decrease in tumor growth may be the result of decreased invasion of the tumor cells. Previous published work by Newcomb et al. [29] had shown a similar synergy with noscapine in combination with radiation, causing delays in murine glioma growth in vivo. Landen et al. [21] showed that noscapine inhibited rat C6 tumor cell growth in vivo by blocking tumor cell proliferation. In
251
our experiments using human TMZ-resistant glioma cells, noscapine appears to have a similar function. We show that inhibition of tumor invasion may play a role in decreased tumor growth. In summary, this study presents novel data on the function of noscapine on human TMZ-resistant glioma cells and human brain endothelial cells, demonstrating that in vitro, this agent decreased tumor cell growth and invasion. Furthermore, we show that noscapine increased survival in the orthotopic in vivo xenograft model of TMZ-resistant glioma. These studies demonstrate that noscapine is an efficient and non-toxic agent that can be used for the treatment of recurrent TMZ-resistant gliomas.
Conflict of interest The authors declare no conflict of interest. References [1] S. Deorah, C.F. Lynch, Z.A. Sibenaller, T.C. Ryken, Trends in brain cancer incidence and survival in the United States: surveillance, epidemiology, and end results program, 1973 to 2001, Neurosurg. Focus 20 (4) (2006) E1. [2] R. Stupp, W.P. Mason, M.J. van den Bent, M. Weller, B. Fisher, M.J.B. Taphoorn, K. Belanger, A.A. Brandes, C. Marosi, U. Bogdahn, J. Curschmann, R.C. Janzer, S.K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe, G. Cairncross, E. Eisenhauer, R.O. Mirimanoff, Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma, New Engl. J. Med. 352 (10) (2005) 987–996. [3] R. Stupp, M.E. Hegi, M.J. van den Bent, W.P. Mason, M. Weller, R.O. Mirimanoff, J.G. Cairncross, Changing paradigms – an update on the multidisciplinary management of malignant glioma, Oncologist 11 (2) (2006) 165–180. [4] A.R. Dehdashti, M.E. Hegi, L. Regli, A. Pica, R. Stupp, New trends in the medical management of glioblastoma multiforme: the role of temozolomide chemotherapy, Neurosurg. Focus 20 (4) (2006) E6. [5] E.C. Holland, Glioblastoma multiforme: the terminator, Proc. Natl Acad. Sci. USA 97 (12) (2000) 6242–6244. [6] R. Stupp, P.Y. Dietrich, S. Ostermann Kraljevic, A. Pica, I. Maillard, P. Maeder, R. Meuli, R. Janzer, G. Pizzolato, R. Miralbell, F. Porchet, L. Regli, N. de Tribolet, R.O. Mirimanoff, S. Leyvraz, Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide, J. Clin. Oncol. 20 (5) (2002) 1375–1382. [7] C. Lu, A. Shervington, Chemoresistance in gliomas, Mol. Cell. Biochem. 312 (2008) 71–80. [8] M.S. Segal, M.M. Goldstein, E.O. Attinger, The use of noscapine (narcotine) as an antitussive agent, Dis. Chest 32 (3) (1957) 305–309. [9] H. Eagle, G. Foley, Report to CCNSC Screening Section, 1958. [10] H. Lettre, Synergists and antagonists of mitotic poisons, Ann. N. Y. Acad. Sci. 58 (1954) 1264. [11] K. Ye, Y. Ke, N. Keshava, J. Shanks, J.A. Kapp, R.R. Tekmal, J. Petros, H.C. Joshi, Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells, Proc. Natl Acad. Sci. USA 95 (4) (1998) 1601–1606. [12] J. Zhou, D. Panda, J.W. Landen, L. Wilson, H.C. Joshi, Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint, J. Biol. Chem. 277 (19) (2002) 17200–17208. [13] J.W. Landen, R. Lang, S.J. McMahon, N.M. Rusan, A.M. Yvon, A.W. Adams, M.D. Sorcinelli, R. Campbell, P. Bonaccorsi, J.C. Ansel, D.R. Archer, P. Wadsworth, C.A. Armstrong, H.C. Joshi, Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma, Cancer Res. 62 (14) (2002) 4109–4114. [14] J. Zhou, K. Gupta, J. Yao, K. Ye, D. Panda, P. Giannakakou, H.C. Joshi, Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2terminal kinase-mediated apoptosis in response to noscapine, J. Biol. Chem. 277 (42) (2002) 39777–39785. [15] T. Jackson, M.B. Chougule, N. Ichite, R.R. Patlolla, M. Singh, Antitumor activity of noscapine in human non-small cell lung cancer xenograft model, Cancer Chemother. Pharmacol. 63 (1) (2008) 117–126.
252
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
[16] N. Heidari, B. Goliaei, P.R. Moghaddam, N. Rahbar-Roshandel, M. Mahmoudian, Apoptotic pathway induced by noscapine in human myelogenous leukemic cells, Anticancer Drugs 18 (10) (2007) 1139– 1147. [17] R. Aneja, A.M. Ghaleb, J. Zhou, V.W. Yang, H.C. Joshi, P53 and p21 determine the sensitivity of noscapine-induced apoptosis in colon cancer cells, Cancer Res. 67 (8) (2007) 3862–3870. [18] E.W. Newcomb, Y. Lukyanov, I. Smirnova, T. Schnee, D. Zagzag, Noscapine induces apoptosis in human glioma cells by an apoptosisinducing factor-dependent pathway, Anticancer Drugs 19 (6) (2008) 553–563. [19] Y. Ke, K. Ye, H.E. Grossniklaus, D.R. Archer, H.C. Joshi, J.A. Kapp, Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses, Cancer Immunol. Immunother. 49 (4–5) (2000) 217–225. [20] I. Barken, J. Geller, M. Rogosnitzky, Noscapine inhibits human prostate cancer progression and metastasis in a mouse model, Anticancer Res. 28 (6A) (2008) 3701–3704. [21] J.W. Landen, V. Hau, M. Wang, T. Davis, B. Ciliax, B.H. Wainer, E.G. Van Meir, J.D. Glass, H.C. Joshi, D.R. Archer, Noscapine crosses the blood-brain barrier and inhibits glioblastoma growth, Clin Cancer Res 10 (15) (2004) 5187–5201. [22] E.W. Newcomb, Y. Lukyanov, T. Schnee, M.A. Ali, L. Lan, D. Zagzag, Noscapine inhibits hypoxia-mediated HIF-1alpha expression and angiogenesis in vitro: a novel function for an old drug, Int. J. Oncol. 28 (5) (2006) 1121–1130. [23] J.J. Virrey, E.B. Golden, W. Sivakumar, W. Wang, L. Pen, A.H. Schönthal, F.M. Hofman, T.C. Chen, Glioma-associated endothelial
[24]
[25]
[26]
[27] [28] [29]
cells are chemoresistant to temozolomide, J. Neurooncol. 95 (1) (2009) 13–22. C. Charalambous, L.B. Pen, Y.S. Su, J. Milan, T.C. Chen, F.M. Hofman, Interleukin-8 differentially regulates migration of tumor-associated and normal human brain endothelial cells, Cancer Res. 65 (22) (2005) 10347–10354. E. Franceschi, A. Tosoni, S. Bartolini, V. Mazzocchi, A. Fioravanti, A.A. Brandes, Treatment options for recurrent glioblastoma: pitfalls and future trends, Expert Rev. Anticancer Ther. 9 (5) (2009) 613– 619. G. Kroemer, L. Galluzzi, P. Vandenabeele, J. Abrams, E.S. Alnemri, E.H. Baehrecke, M.V. Blagosklonny, W.S. El-Deiry, P. Golstein, D.R. Green, M. Hengartner, R.A. Knight, S. Kumar, S.A. Lipton, W. Malorni, G. Nuñez, M.E. Peter, J. Tschopp, J. Yuan, M. Piacentini, B. Zhivotovsky, G. Melino, Classification of cell death: recommendations of the nomenclature committee on cell death 2009, Cell Death Differ. 16 (1) (2009) 3–11. J. Folkman, Tumor angiogenesis: therapeutic implications, New Engl. J. Med. 285 (21) (1971) 1182–1186. N. Ferrara, R.S. Kerbel, Angiogenesis as a therapeutic target, Nature 438 (7070) (2005) 967–974. E.W. Newcomb, Y. Lukyanov, M. Alonso-Basanta, M. Esencay, I. Smirnova, T. Schnee, Y. Shao, M.L. Devitt, D. Zagzag, W. McBride, S.C. Formenti, Antiangiogenic effects of noscapine enhance radioresponse for GL261 tumors, Int. J. Radiat. Oncol. Biol. Phys. 71 (5) (2008) 1477–1484.
Contents lists available at SciVerse ScienceDirect
Cancer Letters journal homepage: www.elsevier.com/locate/canlet
Noscapine inhibits tumor growth in TMZ-resistant gliomas Niyati Jhaveri a,1, Heeyeon Cho b,1, Shering Torres a, Weijun Wang b, Axel H. Schönthal c, Nicos A. Petasis d, Stan G. Louie e, Florence M. Hofman a,⇑, Thomas C. Chen a,b,⇑ a
Department of Pathology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States Department of Neurosurgery, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States c Department of Molecular Microbiology and Immunology, Keck School of Medicine, University of Southern California, Los Angeles, CA, United States d Department of Chemistry, College of Letters, Arts & Sciences, University of Southern California, Los Angeles, CA, United States e Department of Clinical Pharmacy and Pharmaceutical Economics and Policy, School of Pharmacy, University of Southern California, Los Angeles, CA, United States b
a r t i c l e
i n f o
Article history: Received 19 July 2011 Received in revised form 12 August 2011 Accepted 15 August 2011
Keywords: Glioma Noscapine Temozolomide-resistance Invasion Brain tumors
a b s t r a c t Noscapine, a common oral antitussive agent, has been shown to have potent antitumor activity in a variety of cancers. Treatment of glioblastoma multiforme (GBM) with temozolomide (TMZ), its current standard of care, is problematic because the tumor generally recurs and is then resistant to this drug. We therefore investigated the effects of noscapine on human TMZ-resistant GBM tumors. We found that noscapine significantly decreased TMZ-resistant glioma cell growth and invasion. Using the intracranial xenograft model, we showed that noscapine increased survival of animals with TMZ-resistant gliomas. Thus noscapine can provide an alternative therapeutic approach for the treatment of TMZresistant gliomas. Ó 2011 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Glioblastoma multiforme (GBM) is the most common and malignant of all gliomas, with an overall survival rate of less than 3.3% 5 years after diagnosis [1]. The median survival time of GBM patients with the currently best available therapy, consisting of surgery, radiation, and chemotherapy, is approximately 14.6 months [2]. This dismal prognosis is due, in part, to the highly invasive and infiltrative nature of this tumor, which makes complete eradication with surgical resection difficult. These tumors inevitably recur either locally, often within 2 cm of the
⇑ Corresponding authors. Address: Department of Pathology, University of Southern California, 2011 Zonal Avenue, Los Angeles, CA 90033, United States. Tel.: +1 323 442 1153; fax: +1 323 442 3049 (Florence M. Hofman); Department of Neurosurgery, University of Southern Califorina, 2011 Zonal Avenue, CA90033, United States. Tel.: +1 323 442 3918; fax: +1 323 442 3049 (T.C. Chen). E-mail addresses: [email protected] (F.M. Hofman), tchen68670@aol. com (T.C. Chen). 1 These authors contributed equally to this work. 0304-3835/$ - see front matter Ó 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.canlet.2011.08.015
original tumor, or at distant sites within the brain. Furthermore, recurrent tumors are usually resistant to standard chemotherapy [3,4]. Although treatment of these recurrent lesions with a second surgery followed by chemotherapy may increase the symptom-free interval, the 5-year survival still remains low. Morphologically, GBM is highly vascular, characterized by extensive endothelial cell proliferation, particularly at the tumor edge [5]. Therefore, therapies that target tumor cell survival, invasion, and chemosensitization are relevant strategies for the treatment of this tumor. Temozolomide (TMZ) is currently the standard of care for the treatment of GBM [4]. This DNA alkylating agent causes DNA damage during cell replication, thereby destroying proliferating tumor cells. Clinical use of this drug has been shown to be effective in delaying tumor progression and leading to significant prolongation of survival [6]. However, GBM does recur, and when it does, it displays resistance to further TMZ treatment [7]. Thus resistance to TMZ has become a major obstacle to GBM therapy, and new treatments targeting TMZ-resistant tumors are sorely needed. Noscapine, a plant-derived benzylisoquinoline alkaloid, has been extensively used as a cough suppressant for
246
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
several decades without significant toxic side effects [8]. The potential of noscapine to inhibit cancer cell growth in vitro was discovered a half century ago [9,10], however it took 40 years to follow up on the initial promising findings [11]. The anti-tumor properties ascribed to noscapine have been primarily attributed to its ability to interfere with microtubule function, which leads to metaphase arrest of proliferating cells [11]. Noscapine functions by binding to microtubules and disrupting the microtubule dynamics without causing mass accumulation of tubulin [12,13]. Noscapine has been shown to have potent in vitro anti-tumor activity in a variety of tumor types, including glioma, non-small cell lung cancer, multiple myeloma, melanoma, ovarian and colon cancer [13–18]. In vivo studies using the xenograft murine model for human non-small cell lung cancer, T cell lymphoma, prostate and breast cancer [11,15,19,20] have demonstrated that noscapine has anti-cancer properties, which prompted a clinical investigation in patients with relapsed or refractory multiple myeloma [NCT00912899]. Overall, noscapine is an orally bioavailable agent with an excellent tolerability profile, making it an attractive agent for use as an anticancer agent. Dose escalating studies have shown that noscapine has minimal toxicity and does not interfere with the immune response [19]. Unlike other opium-derived products, noscapine does not have any analgesic, sedative, or euphoric properties and is not addictive. Recent publications have indicated the anti-tumor potency of noscapine in pre-clinical models of GBM. For example, noscapine has demonstrated activity in a mouse model of glioma in vivo [21]. The mechanism of noscapine activity in killing human glioma cells was attributed to the activation of the c-jun N-terminal kinase (JNK) signaling pathway and inactivation of the extracellular signal-regulated kinase (ERK) signaling pathway [18]. Noscapine also decreased hypoxia inducible factor (HIF)-1a expression and subsequently vascular endothelial cell growth factor (VEGF) secretion by U87 and T98G glioma cells exposed to hypoxia [22], indicating that this drug could be a potential anti-angiogenic agent as well. An important consideration for GBM therapy is the blood brain barrier. Drugs treating GBM must penetrate this barrier, and there is substantial evidence that noscapine is able to do so [21]. Using an in vitro blood brain barrier model, Landen et al. [21] demonstrated that noscapine (molecular mass: 413.4) transverses this barrier. Furthermore, in their intracranial model using rat C6 glioma cells oral administration of noscapine was detected in the brain parenchyma [21]. Thus noscapine is a potentially powerful agent for the treatment of central nervous system tumors. In the current study, we evaluated the use of noscapine for the treatment of TMZ-resistant gliomas, a major obstacle to successful GBM therapy. We found that noscapine significantly inhibited the proliferation of TMZ-resistant human glioma cells, and decreased migration and invasion of these cells in vitro. Furthermore, noscapine decreased the rate of migration of endothelial cells. In the intracranial rodent xenograft tumor model, noscapine significantly enhanced survival of animals bearing TMZ-resistant tumors. Thus noscapine demonstrated potent anti-tumor properties towards TMZ-resistant brain tumors.
2. Materials and methods 2.1. Cells and reagents The following human glioma cell lines were used: U87, U251, LN229, A172. These cell lines were cultured in 10% fetal calf serum (FCS) in DMEM supplemented with 100 U/ml penicillin and 0.1 mg/ml streptomycin in a humidified incubator at 37 °C and 5% CO2. The TMZresistant glioma cells were prepared by treating the tumor cell lines with increasing doses of TMZ ranging from 10 to 100 lM over a period of 2–3 months. Human tissues were obtained in accordance with USC Institutional Review Board guidelines. Control, non-tumor human endothelial cells (BEC) were isolated from human brain tissues obtained following epileptic surgeries or trauma. Human tumor-associated brain endothelial cells (TuBEC) were obtained from human glioma tissue following resection. The human endothelial cells were isolated from these tissues, purified and characterized as described previously [23]. Endothelial cells were cultured in RPMI containing 10% FCS supplemented with Endogro (Millipore, Temecula, CA), HEPES buffer (Life Technologies, Inc., Carlsbad, CA), essential amino acids (Life Technologies, Inc.), heparin (Sigma Aldrich, St. Louis, MO), sodium bicarbonate (Life Technologies, Inc.), penicillin and streptomycin, and used only until passage 6. The following reagents were used: noscapine hydrochloride (Sigma Aldrich), TMZ (Schering Plough, Whitehouse Station, NJ), recombinant human IL-8 (R&D Systems, Minneapolis, MN). 2.2. MTT assay Tumor cells (200 cells/well) or endothelial cells (5 103 cells/well) were seeded in 96-well plates. After 24 h, noscapine was added to the cells at different concentrations; after 48 h, the medium containing the drug was replaced with fresh medium (no drug) and incubated for another 3 days. The MTT assay was performed according to the manufacturer’s protocol (Sigma Aldrich). Absorbance was measured using a microtiter plate reader (Dynatech MR4000). Percent viability was calculated relative to untreated control. All experiments were performed thrice. 2.3. Colony forming assay (CFA) Glioma cells were seeded into 6-well plates at 200 cells per well and allowed to adhere overnight. Subsequently, the cells were treated with noscapine and/or TMZ for 48 h; the medium was then removed and fresh medium (no drugs) added. Cells were incubated for another 12–14 days. At the termination of the assay, the colonies were visualized by staining with 1% methylene blue in methanol for 4 h and quantified. Groups were plated in triplicate. 2.4. Migration assay The migration assay was previously described [24]. Briefly, glioma cells or endothelial cells, at 1 104 cells/
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
well or 5 104 cells/well respectively, were seeded in the upper chamber of gelatin-coated Transwell plates with polyethylene terephthalate filters (8 lm pore) (BD Biocoat, Bedford, MA), and allowed to adhere. Noscapine was then added to the lower chamber and the chambers were incubated for 6 h. Subsequently the cells on the underside of the filter were stained, and counted at 40 magnification. Ten fields were counted per chamber. Groups were plated in duplicate, and experiments were repeated three times. Conditioned medium (CM) was prepared by growing immortalized mouse fibroblast NIH3T3 cells to 80% confluence. Culture supernatant was collected after 48 h, filtered through a 0.4 lm filter and stored at 20 °C. For migration studies, the CM, at 30% of total volume, was added to the lower chambers. IL-8 was added to the lower chamber as a chemoattractant. For hypoxic conditions, cell cultures were placed in the anaerobic gas generating pouch system (BD GASPak EZ) for 6 h.
247
the cells on the underside of the filters were stained and quantified as described above. 2.6. In vivo experiments All animal protocols were approved by the Institutional Animal Care and Use Committee (IACUC) at USC. U251 TMZ-resistant glioma (2 105) cells were implanted intracranially into 4–6 week old female athymic/nude mice according to a previously published method [23]. Seven days after implantation, treatment began in the following groups: vehicle control (n = 5); noscapine (n = 6); TMZ (n = 4); and noscapine + TMZ (n = 4). The drugs were administered by gavage, at the following doses: noscapine at 225 mg/kg twice daily (approximately 6–8 h apart); and TMZ at 5 mg/kg in a cycle of 7 days treatment followed by no TMZ treatment for 7 days. Survival was documented. 2.7. Cell cycle analysis
2.5. Invasion assay Glioma cells were treated with 10 lg/ml mitomycin C for 2 h, and then seeded on the top chamber of the Matrigel-coated Boyden chamber membrane (BD Biocoat Invasion Assay Kit, BD Bioscience) at 5 104 cells/well. The CM, derived from human glioma U251 cells, was added to the lower chamber to comprise 30% of the media volume; and the cells were incubated for 18 h. Subsequently,
Glioma cells were seeded at a density of 5 105 cells in a 10 cm plate. After 24 h, cells were treated with medium alone (control) or noscapine at 30 lM (based on the IC50 for noscapine) for an additional 24 h. Medium containing floating cells was collected. Attached cells were trypsinized and combined with the floating cells. The mixture was centrifuged and the cell pellet was washed with PBS, followed by fixation with 70% ethanol for 15 min. After removing
Fig. 1. Effects of drugs on colony formation and cell cycle in glioma cell lines. Three different TMZ-sensitive and TMZ-resistant glioma cell lines (TMZr) were treated with TMZ (A) or noscapine (B) at increasing concentrations. After 48 h, culture medium was replaced with fresh medium containing no drugs and cells were incubated for another 12–14 days. Colonies were visualized using methylene blue. Individual colonies were counted and IC50 was determined. U251 TMZ-sensitive (C) and U251 TMZ-resistant glioma cells (D) were treated with medium alone (left) or noscapine (right) at 30 lM for 24 h and cell cycle was analyzed by flow cytometry.
248
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
the ethanol, cells were incubated with 100 lL of a solution containing 50 lg/ml propidium iodide, 0.1 mg/ml RNase A and 0.05% Triton X-100 for 40 min at 37 °C. Cells were washed again with PBS and propidium iodide staining was analyzed by flow cytometry. Ten thousand cells per sample were analyzed. The experiments were repeated twice with similar results.
was used. An alpha of 0.05 was set a priori as a threshold for statistical significance. Statistical significance for survival was evaluated using the Logrank test. 3. Results 3.1. Noscapine inhibits growth of TMZ-resistant glioma cells Resistance to TMZ treatment is a major problem in glioma therapy, therefore identifying drugs that can decrease or inhibit the growth of TMZ-resistant tumors is paramount. To determine whether noscapine has an effect on TMZ-resistant glioma cells, three cell lines were established that were shown to be resistant to TMZ. To test whether these cells were indeed TMZ-resistant, the long-term CFA assay was used. Glioma
2.8. Statistical analysis Statistical significance was evaluated using the Student’s two-tailed t-test and where appropriate ANOVA
Table 1 IC50 of different glioma cell lines for noscapine. Cell lines
A172
LN229
U251
A172/TMZr
LN229/TMZr
U251/TMZr
Noscapine IC50 (lM)
20
70
40
60
75
30
Fig. 2. Effects of combination treatment of TMZ and noscapine on colony formation in glioma cell lines. Glioma cells lines were treated with noscapine (Nos) and TMZ alone or in combination for 48 h. Medium was changed to fresh medium and colonies were counted after 12–14 days. The following drug concentrations were used for the different cell lines: (A) U251 (Nos 30 lM, TMZ 20 lM), LN229 (Nos 50 lM, TMZ 5 lM), A172 (Nos 20 lM, TMZ 5 lM), or (B) U251/TMZr (Nos 20 lM, TMZ 100 lM), LN229/TMZr (Nos 40 lM, TMZ 100 lM) and A172/TMZr (Nos 20 lM, TMZ 100 lM). The doses used were based on drug IC50 values. Significance was designated by p < 0.05.
Fig. 3. Effects of noscapine on glioma cell migration. U251 cells were seeded in the upper chamber of a Transwell plate. Conditioned medium (CM) or noscapine (Nos) were added to the lower chamber as indicated. CM was derived from mouse fibroblast NIH3T3 cell line grown to 80% confluence. Plates were incubated for 6 h under normoxic conditions (5% CO2), and the number of migrated cells was quantified (A). The experiments were repeated under hypoxic conditions (B). Data are expressed as number of cells per field under high magnification; signifies p < 0.05.
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252 cell lines treated with TMZ were shown to be sensitive to TMZ, demonstrating an IC50 of 10 lM for LN229 and A172, and 35 lM for U251 (Fig. 1A); this is in sharp contrast to the minimal effect of TMZ on the TMZ-resistant populations (Fig. 1A) in doses reaching 1000 lM (data
249
not shown). Noscapine by itself has been shown to inhibit cell proliferation in glioma cells [18]. Our results showed that noscapine did indeed cause a significant decrease in the number of colonies in the drug-sensitive glioma cell lines, with an IC50 of 70 lM for LN229, 20 lM for A172,
Fig. 4. Effects of noscapine on glioma cell invasion. U251 TMZ-sensitive (A) and U251 TMZ-resistant (B) cells were treated with 10 lg/ml mitomycin C for 2 h to inhibit proliferation. Subsequently, cells were seeded in the upper chamber of a Matrigel-coated Transwell plate. Conditioned medium (CM) and/or noscapine (Nos) were added to the lower chamber as indicated. CM was derived from U251 glioma cells grown to 80% confluence. After an18 h incubation, the number of cells present on the underside of the filter was quantified. Data are expressed as numbers of cells per field under high magnification; signifies p < 0.0001 between the CM and the CM + Nos groups.
Fig. 5. Effects of noscapine on endothelial cell viability and migration. (A) For the cell survival assay, brain endothelial cells (BEC), tumor-associated brain endothelial cells (TuBEC) and U251 cells were treated with increasing concentrations of noscapine. After 48 h, the medium was replaced with fresh medium without drug, and cells were incubated for another 72 h. Subsequently the MTT assay was performed. Percent cell viability was calculated. U251 cells served as the positive control. (B) For the migration, BEC were added to the upper chambers of Transwell plates; noscapine (20 lM) and/or IL-8 (200 ng/ml) were added to the lower chambers. After 6 h, the cells that had migrated through the pores were quantified. Data are expressed as number of cells in ten highpower fields using 400 X magnification; signifies p < 0.0001. (C) For migration of TuBEC, these cells were placed in the upper chamber of Transwell plates; noscapine was added to the lower chamber as indicated. After 6 h the migrated cells were quantified; signifies p < 0.0001.
250
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
and 40 lM for U251 (Fig. 1B). However, the TMZ-resistant population also proved to be sensitive to noscapine (Fig. 1B). These data are summarized in Table 1. Furthermore, we showed that the effect of noscapine on the TMZ-resistant glioma cells was due to a growth arrest in cell proliferation in the G2/M phase (Fig. 1C). We then addressed the question whether noscapine could sensitize the glioma cells to TMZ. The cells were treated with doses of noscapine and TMZ at IC 80 so that additive effects could be observed. The results show that the combination of noscapine and TMZ exhibited a significant additive effect for the TMZ-sensitive cells, compared to each drug alone (Fig. 2A). However, in the TMZ-resistant glioma cells, the combination of noscapine and TMZ did not exhibit substantial anti-proliferative effects and was similar to the effect of noscapine alone (Fig. 2B). These data show that while noscapine inhibits glioma cell proliferation in the TMZ-resistant population, this agent has little effect in sensitizing these cells to TMZ in the combination treatment. 3.2. Noscapine decreases glioma cell migration and invasion Since noscapine was shown to be a microtubule targeting drug, this agent was evaluated for activity in cell migration. To test this, U251 glioma cells were exposed to conditioned media (CM) derived from NIH3T3 cells; CM was added to the cultures because the CM provides an environment which more closely corresponds to the environment in the tumor. Cells were then incubated in the absence or presence of noscapine for 6 h. With no CM stimulation, there was minimal migration of glioma cells. The results show that the non-cytotoxic dose of noscapine (20 lM) significantly decreased the migration of these glioma cells by >2-fold (Fig. 3A). Since tumor cells are often in a hypoxic environment, this migration assay was also performed under hypoxic as well as normoxic conditions. The results show that noscapine at 20 lM blocked cell migration of glioma cells in both hypoxic and normoxic environments (Fig. 3B). The invasive behavior of glioma cells presents a major clinical problem, especially with regard to tumor recurrence. We therefore examined whether noscapine decreases the invasive capacity of both the TMZ-sensitive and TMZ-resistant glioma cells in an 18 h invasion assay. Glioma cells were treated with mitomycin C to block cell proliferation, and with CM to stimulate migration. We noted that at baseline, the TMZ-resistant glioma cell lines exhibited approximately a 5-fold greater invasive capacity as compared to the TMZ-sensitive cells (Fig. 4). TMZ-sensitive cells treated with CM and exposed to noscapine showed significantly less invasion than control glioma cells (p < 0.0001) (Fig. 4A). TMZ-resistant glioma cells, tested in parallel, demonstrated that noscapine significantly (p < 0.0001) decreased tumor cell invasion (Fig. 4B). These data demonstrate that noscapine inhibits the invasive capacity of the TMZ-resistant glioma cells.
3.3. Noscapine decreases endothelial cell migration The vasculature is a critical component of tumor growth. To analyze the effects of noscapine on endothelial cell survival and proliferation, we performed a long-term MTT assay. Normal human brain endothelial cells (BEC) and human glioma-associated endothelial cells (TuBEC) were treated with increasing concentrations of noscapine (20–100 lM) for 48 h and then cultured for 72 h without the drug. U251 glioma cells served as the positive control. While noscapine decreased the proliferation of U251 as shown previously, endothelial cells did not show this effect (Fig. 5A). Even at the highest dose of noscapine (100 lM), there was little inhibition of TuBEC or BEC growth. These results show that noscapine at doses which inhibit proliferation of glioma cells, the drug had little effect on normal or tumor-associated brain endothelial cells. Since noscapine affected tumor cell migration, we tested whether this agent also had an effect on endothelial cell migration. Normal brain endothelial cells were seeded in the upper chamber and noscapine was added to the lower chamber. The potent endothelial cell chemokine, IL-8, was used as a chemoattractant. After 6 h incubation, normal brain endothelial cells treated with noscapine (20 lM) demonstrated a significant decrease in endothelial cell migration (p < 0.0001) (Fig. 5B). Thus noscapine blocked the migration of IL-8-activated endothelial cells. This experiment was repeated three times with different endothelial cell samples, giving similar results. Tumor-associated brain endothelial cells treated with noscapine showed significantly decreased (p < 0.0001) migration (Fig. 5C); this effect was independent of exogenous IL-8 (data not shown). These results indicate that noscapine acts as an anti-angiogenic agent on activated normal and tumor-associated endothelial cells.
3.4. Noscapine increases the survival of animals with orthotopic xenograft of TMZ-resistant GBM Based on the information that noscapine decreased TMZ-resistant glioma cell proliferation and acted as an anti-angiogenic agent, we tested this drug in the intracranial in vivo xenograft model. U251 TMZ-resistant glioma cells were injected intracranially into athymic/nude mice; and 7 days post-injection the mice were treated with noscapine (225 mg/ kg twice per day), TMZ (5 mg/kg) or both drugs. The survival results show that noscapine alone and noscapine in combination with TMZ significantly increased survival (p < 0.05) as compared to vehicle control and TMZ treatment alone (Fig. 6A). There was no significant difference in survival between the noscapine and noscapine plus TMZ groups. TMZ alone had no significant effect on the survival of the mice with TMZ-resistant tumors as compared to vehicle-treated control animals. In a parallel experiment, microvessel density of the tumors was evaluated at the time of sacrifice; the results show no apparent differences in microvessel density in noscapine-treated tumors as compared to vehicle treated tumors (data not shown). The data demonstrate that in this orthotopic xenograft in vivo model of TMZ-resistant tumors, treatment with noscapine increases animal survival.
4. Discussion
Fig. 6. Effects of noscapine in vivo. (A) TMZ-resistant U251 glioma cells (R) were implanted intracranially into nude mice. After 7 days, treatment was initiated. Mice were randomly divided into treatment groups: vehicle control (Con) (n = 5); noscapine (Nos) (n = 6); TMZ (n = 4); and noscapine + TMZ (N + T) (n = 4). The drugs were administered by gavage at the following doses: noscapine at 225 mg/kg twice daily (6–8 h apart); and TMZ at 5 mg/kg in a cycle of 7 days treatment followed by no TMZ treatment for 7 days. Survival was used as an endpoint; signifies statistical significance (p < 0.05).
The most challenging obstacle in the treatment of gliomas is tumor recurrence, because these recurring tumors are usually TMZ-resistant [3,4]. To date, there are only a few options for recurrent gliomas, and even these therapies have limited success [25]. We therefore focused this study on TMZ-resistant gliomas. Any therapy that either alone or in combination with other agents could cause decreased tumor growth or destruction of TMZ-resistant glioma cells would be a major contribution to our therapeutic arsenal. The studies presented here show that noscapine has this potential. Noscapine binds to microtubules, thereby interfering with cell mitosis resulting in decreased cell proliferation. Indeed, we show here that noscapine decreased the proliferation rate of TMZ-resistant human glioma cells in vitro. Similar to what was reported for TMZ-sensitive glioma
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
cells [18], the TMZ-resistant glioma cells exhibit an arrest in the G2/M phase of cell cycle. Such an arrest may lead to mitotic catastrophe and subsequent cytotoxicity [26]. Treatment with noscapine did indeed decrease the cell numbers observed in three different human glioma cell lines, U251, LN229, and A172, and their respective TMZresistant lines. These cell lines have different genetic characteristics; U251 and A172 express mutations in PTEN and p53, while LN229 expresses wild-type PTEN and mutant p53. These findings suggest that sensitivity to noscapine is independent of PTEN mutation, and is likely to be a common characteristic of glioma cells. In further studies, we tested whether TMZ, in combination with noscapine, had an additive effect on glioma cell proliferation. Our results show that in vitro, the TMZ-sensitive glioma cells can be further sensitized to TMZ with the addition of noscapine, while the TMZ-resistant cells were not affected by this combination. We next explored the capacity of noscapine to affect tumor cell invasion, a critical function of recurrent gliomas. The data showed that TMZ-resistant glioma cells consistently exhibited a higher baseline of invading cells as compared to the sensitive glioma cells, suggesting that a characteristic of TMZ-resistance is the capacity to migrate and invade more effectively than TMZ-sensitive cells. Our results demonstrated that noscapine alone significantly reduced this invasion process both in the sensitive and resistant glioma populations. The tumor vasculature is critical for tumor growth [27], and therapeutic targeting of the blood vessels with different anti-angiogenic agents has been shown to result in tumor shrinkage [28]. We therefore examined the effects of noscapine on normal and tumor-associated brain endothelial cells. Our results show that noscapine does not affect endothelial cell proliferation at doses potent for malignant tumor cells. However, noscapine did influence endothelial cell migration, a critical requirement of the angiogenic process. Others have shown that noscapine altered tubule formation and caused decreased microvessel density in tumors [29]. Our previously published studies demonstrated that TMZ does not have significant effects on endothelial cell proliferation or migration [23]. Microvessel density of tumors harvested from noscapine-treated tumors demonstrated no significant differences as compared to tumor treated with vehicle (data not shown). These data suggest that although noscapine decreased migration of endothelial cells in vitro, these effects of noscapine on the tumor vasculature may be observed earlier in the tumor growth process and may not be detectable at the later stage of disease. Thus noscapine does not appear to affect the tumor vasculature, and the results observed in vivo are related to noscapine activity directly on the tumor cells. The orthotopic in vivo xenograft TMZ-resistant tumor model demonstrated that noscapine decreased tumor growth. The decrease in tumor growth may be the result of decreased invasion of the tumor cells. Previous published work by Newcomb et al. [29] had shown a similar synergy with noscapine in combination with radiation, causing delays in murine glioma growth in vivo. Landen et al. [21] showed that noscapine inhibited rat C6 tumor cell growth in vivo by blocking tumor cell proliferation. In
251
our experiments using human TMZ-resistant glioma cells, noscapine appears to have a similar function. We show that inhibition of tumor invasion may play a role in decreased tumor growth. In summary, this study presents novel data on the function of noscapine on human TMZ-resistant glioma cells and human brain endothelial cells, demonstrating that in vitro, this agent decreased tumor cell growth and invasion. Furthermore, we show that noscapine increased survival in the orthotopic in vivo xenograft model of TMZ-resistant glioma. These studies demonstrate that noscapine is an efficient and non-toxic agent that can be used for the treatment of recurrent TMZ-resistant gliomas.
Conflict of interest The authors declare no conflict of interest. References [1] S. Deorah, C.F. Lynch, Z.A. Sibenaller, T.C. Ryken, Trends in brain cancer incidence and survival in the United States: surveillance, epidemiology, and end results program, 1973 to 2001, Neurosurg. Focus 20 (4) (2006) E1. [2] R. Stupp, W.P. Mason, M.J. van den Bent, M. Weller, B. Fisher, M.J.B. Taphoorn, K. Belanger, A.A. Brandes, C. Marosi, U. Bogdahn, J. Curschmann, R.C. Janzer, S.K. Ludwin, T. Gorlia, A. Allgeier, D. Lacombe, G. Cairncross, E. Eisenhauer, R.O. Mirimanoff, Radiotherapy plus concomitant and adjuvant temozolomide for glioblastoma, New Engl. J. Med. 352 (10) (2005) 987–996. [3] R. Stupp, M.E. Hegi, M.J. van den Bent, W.P. Mason, M. Weller, R.O. Mirimanoff, J.G. Cairncross, Changing paradigms – an update on the multidisciplinary management of malignant glioma, Oncologist 11 (2) (2006) 165–180. [4] A.R. Dehdashti, M.E. Hegi, L. Regli, A. Pica, R. Stupp, New trends in the medical management of glioblastoma multiforme: the role of temozolomide chemotherapy, Neurosurg. Focus 20 (4) (2006) E6. [5] E.C. Holland, Glioblastoma multiforme: the terminator, Proc. Natl Acad. Sci. USA 97 (12) (2000) 6242–6244. [6] R. Stupp, P.Y. Dietrich, S. Ostermann Kraljevic, A. Pica, I. Maillard, P. Maeder, R. Meuli, R. Janzer, G. Pizzolato, R. Miralbell, F. Porchet, L. Regli, N. de Tribolet, R.O. Mirimanoff, S. Leyvraz, Promising survival for patients with newly diagnosed glioblastoma multiforme treated with concomitant radiation plus temozolomide followed by adjuvant temozolomide, J. Clin. Oncol. 20 (5) (2002) 1375–1382. [7] C. Lu, A. Shervington, Chemoresistance in gliomas, Mol. Cell. Biochem. 312 (2008) 71–80. [8] M.S. Segal, M.M. Goldstein, E.O. Attinger, The use of noscapine (narcotine) as an antitussive agent, Dis. Chest 32 (3) (1957) 305–309. [9] H. Eagle, G. Foley, Report to CCNSC Screening Section, 1958. [10] H. Lettre, Synergists and antagonists of mitotic poisons, Ann. N. Y. Acad. Sci. 58 (1954) 1264. [11] K. Ye, Y. Ke, N. Keshava, J. Shanks, J.A. Kapp, R.R. Tekmal, J. Petros, H.C. Joshi, Opium alkaloid noscapine is an antitumor agent that arrests metaphase and induces apoptosis in dividing cells, Proc. Natl Acad. Sci. USA 95 (4) (1998) 1601–1606. [12] J. Zhou, D. Panda, J.W. Landen, L. Wilson, H.C. Joshi, Minor alteration of microtubule dynamics causes loss of tension across kinetochore pairs and activates the spindle checkpoint, J. Biol. Chem. 277 (19) (2002) 17200–17208. [13] J.W. Landen, R. Lang, S.J. McMahon, N.M. Rusan, A.M. Yvon, A.W. Adams, M.D. Sorcinelli, R. Campbell, P. Bonaccorsi, J.C. Ansel, D.R. Archer, P. Wadsworth, C.A. Armstrong, H.C. Joshi, Noscapine alters microtubule dynamics in living cells and inhibits the progression of melanoma, Cancer Res. 62 (14) (2002) 4109–4114. [14] J. Zhou, K. Gupta, J. Yao, K. Ye, D. Panda, P. Giannakakou, H.C. Joshi, Paclitaxel-resistant human ovarian cancer cells undergo c-Jun NH2terminal kinase-mediated apoptosis in response to noscapine, J. Biol. Chem. 277 (42) (2002) 39777–39785. [15] T. Jackson, M.B. Chougule, N. Ichite, R.R. Patlolla, M. Singh, Antitumor activity of noscapine in human non-small cell lung cancer xenograft model, Cancer Chemother. Pharmacol. 63 (1) (2008) 117–126.
252
N. Jhaveri et al. / Cancer Letters 312 (2011) 245–252
[16] N. Heidari, B. Goliaei, P.R. Moghaddam, N. Rahbar-Roshandel, M. Mahmoudian, Apoptotic pathway induced by noscapine in human myelogenous leukemic cells, Anticancer Drugs 18 (10) (2007) 1139– 1147. [17] R. Aneja, A.M. Ghaleb, J. Zhou, V.W. Yang, H.C. Joshi, P53 and p21 determine the sensitivity of noscapine-induced apoptosis in colon cancer cells, Cancer Res. 67 (8) (2007) 3862–3870. [18] E.W. Newcomb, Y. Lukyanov, I. Smirnova, T. Schnee, D. Zagzag, Noscapine induces apoptosis in human glioma cells by an apoptosisinducing factor-dependent pathway, Anticancer Drugs 19 (6) (2008) 553–563. [19] Y. Ke, K. Ye, H.E. Grossniklaus, D.R. Archer, H.C. Joshi, J.A. Kapp, Noscapine inhibits tumor growth with little toxicity to normal tissues or inhibition of immune responses, Cancer Immunol. Immunother. 49 (4–5) (2000) 217–225. [20] I. Barken, J. Geller, M. Rogosnitzky, Noscapine inhibits human prostate cancer progression and metastasis in a mouse model, Anticancer Res. 28 (6A) (2008) 3701–3704. [21] J.W. Landen, V. Hau, M. Wang, T. Davis, B. Ciliax, B.H. Wainer, E.G. Van Meir, J.D. Glass, H.C. Joshi, D.R. Archer, Noscapine crosses the blood-brain barrier and inhibits glioblastoma growth, Clin Cancer Res 10 (15) (2004) 5187–5201. [22] E.W. Newcomb, Y. Lukyanov, T. Schnee, M.A. Ali, L. Lan, D. Zagzag, Noscapine inhibits hypoxia-mediated HIF-1alpha expression and angiogenesis in vitro: a novel function for an old drug, Int. J. Oncol. 28 (5) (2006) 1121–1130. [23] J.J. Virrey, E.B. Golden, W. Sivakumar, W. Wang, L. Pen, A.H. Schönthal, F.M. Hofman, T.C. Chen, Glioma-associated endothelial
[24]
[25]
[26]
[27] [28] [29]
cells are chemoresistant to temozolomide, J. Neurooncol. 95 (1) (2009) 13–22. C. Charalambous, L.B. Pen, Y.S. Su, J. Milan, T.C. Chen, F.M. Hofman, Interleukin-8 differentially regulates migration of tumor-associated and normal human brain endothelial cells, Cancer Res. 65 (22) (2005) 10347–10354. E. Franceschi, A. Tosoni, S. Bartolini, V. Mazzocchi, A. Fioravanti, A.A. Brandes, Treatment options for recurrent glioblastoma: pitfalls and future trends, Expert Rev. Anticancer Ther. 9 (5) (2009) 613– 619. G. Kroemer, L. Galluzzi, P. Vandenabeele, J. Abrams, E.S. Alnemri, E.H. Baehrecke, M.V. Blagosklonny, W.S. El-Deiry, P. Golstein, D.R. Green, M. Hengartner, R.A. Knight, S. Kumar, S.A. Lipton, W. Malorni, G. Nuñez, M.E. Peter, J. Tschopp, J. Yuan, M. Piacentini, B. Zhivotovsky, G. Melino, Classification of cell death: recommendations of the nomenclature committee on cell death 2009, Cell Death Differ. 16 (1) (2009) 3–11. J. Folkman, Tumor angiogenesis: therapeutic implications, New Engl. J. Med. 285 (21) (1971) 1182–1186. N. Ferrara, R.S. Kerbel, Angiogenesis as a therapeutic target, Nature 438 (7070) (2005) 967–974. E.W. Newcomb, Y. Lukyanov, M. Alonso-Basanta, M. Esencay, I. Smirnova, T. Schnee, Y. Shao, M.L. Devitt, D. Zagzag, W. McBride, S.C. Formenti, Antiangiogenic effects of noscapine enhance radioresponse for GL261 tumors, Int. J. Radiat. Oncol. Biol. Phys. 71 (5) (2008) 1477–1484.