Clinical Cancer Research The Science of Cancer Health Disparities
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wakchoure, S.
Right arrow Articles by Selander, K. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wakchoure, S.
Right arrow Articles by Selander, K. S.
Clinical Cancer Research Vol. 12, 2862-2868, May 1, 2006
© 2006 American Association for Cancer Research

Cancer Therapy: Preclinical

Bisphosphonates Inhibit the Growth of Mesothelioma Cells In vitro and In vivo

Savita Wakchoure1, Melinda A. Merrell1, Wayne Aldrich1, Telisha Millender-Swain1, Kevin W. Harris1,2, Pierre Triozzi1 and Katri S. Selander1

Authors' Affiliations: 1 Department of Medicine, Division of Hematology-Oncology, University of Alabama at Birmingham and 2 Birmingham Veteran's Administration Medical Center, Birmingham, Alabama

Requests for reprints: Katri Selander, Department of Medicine, Division of Hematology-Oncology, University of Alabama at Birmingham, WTI T558, 1824 6th Avenue South, Birmingham, AL 35294-3300. Phone: 205-975-5973; Fax: 205-975-5650; E-mail: Katri.Selander{at}ccc.uab.edu.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
Purpose: Bisphosphonates (such as risedronate and zoledronate) are widely used inhibitors of bone resorption. Despite their in vitro antiproliferative effects in various cancer cells, bisphosphonates have not exhibited significant antitumor efficacy in animal models of visceral cancer, which may be due to their poor bioavailability. The diagnostic use of radioactive bisphosphonates has revealed the accumulation of bisphosphonates in mesothelioma, which prompted us to test the antitumor efficacy of bisphosphonates in this disease.

Experimental Design and Results: Treatment with either risedronate or zoledronate (2 x 10–4 to 2 x 10–6 mol/L) inhibited the growth of AB12 and AC29 mouse mesothelioma cells and induced the accumulation of unprenylated Rap1A in these cells. Both these in vitro effects were reversed by geranygeraniol, an end product of the mevalonate pathway that these bisphosphonates inhibit. Both bisphosphonates also induced the phosphorylation of the p38 mitogen-activated protein kinase in AB12 and AC29 cells. The inhibition of p38 augmented bisphosphonate-induced growth inhibition in these cells. Bisphosphonate-induced p38 phosphorylation was not reversible by geranylgeraniol. Risedronate (15 mg/kg) and zoledronate (0.5 mg/kg) inhibited the growth of s.c. tumors and increased the median survival of mice with i.p. mesothelioma tumors in vivo.

Discussion: In conclusion, risedronate and zoledronate inhibit the mevalonate pathway and induce p38 activation in mesothelioma cells in vitro. The effects on the mevalonate pathway dominate because the net result is growth inhibition. Both bisphosphonates also inhibit mesothelioma tumor growth in vivo and prolong the survival of mesothelioma-bearing mice. These results support further study of bisphosphonates in the management of mesothelioma.

Mesothelioma, an asbestos-related neoplasm of the pleural and peritoneal space, occurs in ~10,000 patients worldwide (1). Due to the long latency period for tumor development and the widespread use of asbestos for many years, the incidence is expected to rise until the year 2020 (2). The biological behavior is distinct from other solid tumors in that mesothelioma tends to grow in a sheet-like fashion, covering the surface of the pleura or peritoneum. It shows little tendency to invade, especially early in the course of the disease (3). Mesothelioma typically recurs even after the most aggressive attempts at surgical resection and is poorly responsive to radiotherapy and chemotherapy. Multimodality approaches have had a relatively small effect on the majority of the patients and have been associated with toxicity. The survival of patients with mesothelioma ranges between 4 and 12 months (4, 5). Clearly, new treatment modalities are needed.

Bisphosphonates are synthetic analogues of the naturally occurring pyrophosphate. Depending on their molecular structure, these drugs can be divided into pyrophosphate-resembling (p-bisphosphonates, such as clodronate) and nitrogen-containing bisphosphonates (n-bisphosphonates, such as risedronate and zoledronate; ref. 6). At the cellular level, the different bisphosphonates have different mechanisms of action; n-bisphosphonates inhibit the mevalonate pathway, whereas the effects of p-bisphosphonates are mediated via intracellular ATP-like analogues. The main effect of all bisphosphonates is their ability to inhibit osteoclast-mediated bone resorption. These drugs are therefore widely clinically used in the treatment of metabolic bone diseases that are due to increased bone resorption, such as osteoporosis (7). Bisphosphonates also inhibit the osteolytic complications of bone metastases of solid tumors and multiple myeloma (8). Data from animal models suggest that in addition to osteoclast inhibition at the site of bone metastasis, these drugs may also inhibit cancer cell proliferation in bone (9, 10). In particular, the newer n-bisphosphonates have also been suggested to actually inhibit the cancer spread to bones in animal models (11). Although these drugs significantly inhibit the growth of various cancer cells in vitro, they have not, however, proven effective as single agents in preventing tumor growth at visceral sites in various animal models of cancer (9–16). This may be due to their poor bioavailability to the tumors; bisphosphonates are poorly absorbed from the gastrointestinal tract and when given i.v., they are quickly cleared from the circulation and adsorbed to bone matrix hydroxyapatite, where they are retained for prolonged periods (17). It is indeed thought that this bone-seeking propensity of the otherwise very hydrophilic drugs makes them available for the cancer cells residing in the bone microenvironment.

The uptake of the bone scan agent 99Tcm diphosphonate, which is structurally similar to the actual bisphosphonate drugs, in malignant pleural effusions and, rarely, in nonmalignant pleural effusions is well established. Although the exact mechanism of uptake in these conditions remains unclear, passive transudation has been implicated (18). There are also several reports of uptake of 99Tcm diphosphonate by cancers localized at the soft tissue sites (19–21). Because mesothelioma has been associated with such an accumulation of 99Tcm diphosphonate, we hypothesized that bisphosphonates may also exhibit direct antitumor activity against this tumor type in vivo (22, 23). We show here that the new nitrogen-containing bisphosphonates, risedronate and zoledronate, effectively inhibit the proliferation of mesothelioma cells in vitro and the growth of mesothelioma tumors in vivo. Furthermore, we show that administration of these drugs after tumor formation can significantly extend the survival of tumor-bearing mice in experimental models of mesothelioma.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Bisphosphonates. Risedronate (a gift from Leiras OY, Turku, Finland) was dissolved in PBS and the stock solution was set to pH 7.4 with NaOH. Zoledronate (Novartis, Geneva, Switzerland) was obtained from the pharmacy and diluted into the cell culture medium. For animal studies, both bisphosphonates were diluted into sterile 0.9% saline.

Cell culture. The mouse mesothelioma cell lines AB12 and AC29 were provided by Dr. Steven Albelda (University of Pennsylvania, Philadelphia, PA). These cell lines were originally generated by Dr. Bruce Robinson at the Queen Elizabeth II Medical Center (Nedlands, Perth, Western Australia) by i.p. implantation of asbestos fibers in BALB/c and CBA/J mice, respectively, and have been well characterized (24). AB12 and AC29 cells were cultured and maintained in complete medium consisting of high-glucose DMEM (Mediatech, Washington, DC) supplemented with 10% heat-inactivated FCS, 100 units/mL penicillin, 100 µg/mL streptomycin, and 2 mmol/L glutamine (Sigma, St. Louis, MO). All cell cultures were done in incubators in a 37°C atmosphere of 5% CO2/95% air.

In vitro growth assay. Mesothelioma cells were plated in 96-well plates in normal culture medium, and treated for the indicated periods of time with various concentrations of zoledronate, risedronate, or PBS with or without the p38 inhibitor SB202190 or the inactive control compound SB207420 (both at the final concentration of 10–5 mol/L; Calbiochem, San Diego, CA), 25 µmol/L geranylgeraniol (cold, all trans, American Radiolabeled Chemicals, St. Louis, MO), or the same volume of ethanol as a vehicle control. DNA synthesis was measured as an indication of cell proliferation, using nonisotopic bromodeoxyuridine (BrdU) incorporation immunoassays (Exalpha Biologicals, Watertown, MA), according to the manufacturer's instructions. Briefly, 103 cells were plated onto 96-well plates in 100 µL of normal culture medium. The cells were then treated with the indicated agents for various times. BrdU was added to the wells for the final 24 hours and incorporated BrdU was detected with sequential additions of monoclonal mouse anti-BrdU antibody and horseradish peroxidase–conjugated anti-mouse antibody. After addition of the substrate for horseradish peroxidase, the intensity of the colored reaction product, which is proportional to the amount of BrdU incorporated into the cells, was read with a spectrophotometer at 450 nmol/L.

Western blotting. AB12 and AC29 cells were plated on six-well plates in normal culture medium until near confluency. The cells were then rinsed with sterile PBS and cultured for a further 24 hours in serum-free culture medium, in the presence or absence of 2 x 10–4 to 10–5 mol/L risedronate, zoledronate, or PBS control, with or without 25 µmol/L of geranylgeraniol, or the same volume of ethanol as a vehicle control. Culture medium was discarded and the cells were harvested in lysis buffer [20 mmol/L Tris (pH 7.4), 150 mmol/L NaCl, 1 mmol/L EDTA, 1 mmol/L EGTA, 1% Triton, 2.5 mmol/L sodium PPi, 1 mmol/L ß-glycerolphosphate, 1 mmol/L Na3VO4, 1 µg/mL leupeptin; Cell Signaling, Beverly, MA] and clarified by centrifugation. After boiling the supernatants in reducing SDS sample buffer, equal amounts of protein (~50 µg) were loaded per lane and the samples were electrophoresed on 10% polyacrylamide SDS gel and transferred to a nitrocellulose membrane. Unprenylated Rap1A was detected with the antibody SC-1482 and total Rap1 (both prenylated and unprenylated forms of both Rap1A and Rap1B) was detected with the antibody SC-65 (Santa Cruz Biotechnology, Santa Cruz, CA) according to the manufacturer's recommendations (25, 26). The phosphorylation status of p38 was studied with anti-phospho-p38 and anti-total p38 antibodies (Cell Signaling), as recommended by the manufacturer (27). The protein bands were visualized by chemiluminescence using SuperSignal West Pico enhanced chemiluminescence kit (Pierce, Rockford, IL).

In vivo mesothelioma models. Female BALB/c mice, 4 to 8 weeks of age, were obtained from the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD) and were housed in the Pathogen-Free Rodent Shared Facility (Comprehensive Cancer Center, University of Alabama at Birmingham). All animal procedures were done in accordance with recommendations for the proper care and use of laboratory animals and were approved by the local IACUC. S.c. and i.p. mouse mesothelioma models were evaluated. In the s.c. model, 3 x 106 AB12 cells were first injected s.c. into cohorts of BALB/c mice. Treatments with i.p. bisphosphonates or vehicle were started when tumors became palpable on day 10 and continued every 6 days for a total of four treatments. Tumor size was measured bidimensionally with calipers every 2 to 3 days, and tumor volume was calculated by the formula (length x width2) / 2. Mice were euthanized before tumors reached the size of 2,000 mm3. In the i.p. model, AC29 or AB12 cells (5 x 105 / 0.5 mL) were injected i.p. into cohorts of 10 to 12 BALB/c mice using a 26-gauge needle. Treatment was initiated 6 days after tumor inoculation and the mice were followed for survival. In the i.p. model, risedronate (15 mg/kg), zoledronate (0.5 mg/kg), or PBS were given i.p. thrice a week for 2 weeks.

Statistical analysis. Kaplan-Meier survival curves were analyzed with the Mantel-Cox Log-rank test. Fisher exact test was used to examine differences in the proportion of tumors responding and proportion of mice surviving. Student's t test (two-tailed) was used to examine differences in growth assays and for the time to death/sacrifice. Results are expressed as mean ± SD. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Risedronate and zoledronate effects on unprenylated Rap1A accumulation and growth inhibition can be partially reversed by geranylgeraniol in mesothelioma cells. Nitrogen-containing bisphosphonates have previously been shown to inhibit the growth of various epithelial cancer cells in vitro via inhibiting the mevalonate pathway (28, 29). This inhibition results in the depletion of intracellular prenyl-groups, such as geranylgeraniol, which are needed for the posttranslational modification and activation of small GTP-binding proteins, such as Ras, Rho, Rac, and Rap (7). For example, treatment with n-bisphosphonates has been shown to result in the accumulation of unprenylated Rap1A in CaCo-2 and leukemia cells (25, 30). To investigate whether risedronate and zoledronate similarly inhibit the mevalonate pathway in mesothelioma cells, AB12 and AC29 cells were treated for 24 hours with PBS or with 2 x 10–4 to 2 x 10–6 mol/L risedronate or zoledronate, with 25 µmol/L geranylgeraniol, or the same volume of ethanol as a vehicle control. The cells were then lysed and prepared for Western blot analysis. Accumulation of unprenylated Rap1A was used as a surrogate marker for the inhibition of the mevalonate pathway (26). Zoledronate and risedronate induced a dose-dependent accumulation of unprenylated Rap1A in both cell lines. Risedronate-induced accumulation of unprenylated Rap1A was almost completely reversed by 25 µmol/L geranylgeraniol in both cells. Zoledronate-induced accumulation of unprenylated Rap1A was partially reversed in both cells. Stripping and reblotting the membranes with the anti-total Rap1 antibody clearly indicated that the findings were not due to uneven loading of the gels (Fig. 1 ). Higher concentrations of geranylgeraniol were also tested and found effective, but because they compromised cell viability, they were not routinely used. Geranylgeraniol also reversed the bisphosphonate-induced inhibition of DNA synthesis in both cells, but the extent of this reversal was dependent on the cell line and the bisphosphonates used (Fig. 2 ).


Figure 1
View larger version (32K):
[in this window]
[in a new window]
  		Fig. 1. n-Bisphosphonates induce the accumulation of unprenylated Rap1A in mesothelioma cells. Accumulation of unprenylated Rap1A was detected in AB12 (A) and in AC29 (B) cells after treatment for 24 hours with PBS or with the indicated concentrations of risedronate or zoledronate, with 25 µmol/L geranylgeraniol (GG), or the same volume of ethanol as a vehicle control. The cells were then lysed and prepared for Western blot analysis. The levels of unprenylated Rap1A (top) were used as a surrogate marker to detect the inhibition of the mevalonate pathway, using the antibody SC-1482. The blots, which represent replicate experiments, were stripped and total Rap1 was detected with the antibody SC-65, to show that the effects were not due to a loading error.

 

Figure 2
View larger version (23K):
[in this window]
[in a new window]
  		Fig. 2. Geranylgeraniol partially reverses the n-bisphosphonate-induced growth inhibition. AB12 (A) and AC29 (B) were cultured in the presence of indicated concentrations of risedronate or zoledronate, with 25 µmol/L geranylgeraniol (GG) or the same volume of ethanol as a vehicle control. DNA synthesis as an indicator of cell proliferation rate was measured with BrdU incorporation after 5 days of treatment. Columns, mean percentage of PBS control; bars, ±SD; **. P < 0.01; ***, P < 0.001 versus the corresponding inactive control compound (n = 5).

 
Inhibition of p38 augments n-bisphosphonate induced growth inhibition. We have previously shown that in addition to the inhibitory effects on the mevalonate pathway, in particular, the n-bisphosphonates also activate the p38 mitogen-activated protein kinase in breast cancer cells. This activation signals for resistance against bisphosphonate-induced growth inhibition because blocking of the p38 mitogen-activated protein kinase pathway augments the growth-inhibitory effects of bisphosphonates (27). To study whether a similar mechanism operates in mesothelioma cells, we first investigated the effects of risedronate or zoledronate on p38 phosphorylation in these cells. Using phospho-p38-specific and total p38 antibodies in Western blotting, we show here that both risedronate and zoledronate induce a dose-dependent increase of p38 phosphorylation in AB12 and AC29 cells. Unlike the accumulation of unprenylated Rap1A, this effect was, however, not reversible by excess geranylgeraniol (25 µmol/L). Increasing the geranylgeraniol dose did not affect the bisphosphonate-induced, increased phosphorylation status of p38 either (data not shown for higher geranylgeraniol concentrations; Fig. 3 ). AB12 and AC29 cells were then cultured with risedronate or zoledronate, with or without the specific p38 inhibitor SB202190 (10–5 mol/L), or with the same concentration of an inactive control compound SB202474. Inhibition of p38 augmented both risedronate- and zoledronate-induced growth inhibition in both cell lines, even though there were cell-specific differences between the bisphosphonate concentrations at which these effects were seen. In general, AC29 cells were more sensitive to the effects of p38 inhibition (Fig. 4 ).


Figure 3
View larger version (19K):
[in this window]
[in a new window]
  		Fig. 3. n-Bisphosphonates induce p38 phosphorylation. AB12 and AC29 cells were cultured for 24 hours in the presence of the indicated concentrations of risedronate, zoledronate, or PBS, with 25 µmol/L geranylgeraniol (GG) or ethanol as a vehicle control. Phosphorylation of p38 was detected in AB12 (A) and in AC29 (B) cell lysates in Western blots, using phospho-p38 (top) and after stripping of the same blot, total p38 (bottom) specific antibodies.

 

Figure 4
View larger version (11K):
[in this window]
[in a new window]
  		Fig. 4. Inhibition of p38 augments n-bisphosphonate-induced growth inhibition. AB12 and AC29 cells were cultured with the indicated concentrations of risedronate (A) or zoledronate (B), with the specific p38 inhibitor SB202190 (10–5 mol/L) or the same volume of an inactive control compound SB202474. DNA synthesis as an indicator of cell proliferation rate was measured with BrdU incorporation after 5 days of treatment. Columns, mean percentage of PBS control; bars, ±SD; ***, P < 0.001 versus the corresponding inactive control compound (n = 5).

 
Risedronate and zoledronate inhibit mesothelioma growth in vivo. The antitumor activity of n-bisphosphonates was tested in vivo in a s.c. tumor model using AB12 cells, which are syngeneic in BALB/C mice, because AB12 tumors are more aggressive than the AC29 cells and have been resistant to most cancer chemotherapeutics in vivo (31). Groups of 10 BALB/c mice were inoculated s.c. with AB12 cells. Ten days later, when the tumors were palpable and between 100 and 175 mm3 in size, the mice were treated with risedronate or zoledronate, using higher doses and more infrequent dosing schedules than previously applied in mouse tumor models. Inoculations with PBS served as a vehicle control (9, 32). Tumor volume was measured over time. Mice were sacrificed when tumors reached 2,000 mm3. Both risedronate (P < 0.02) and zoledronate (P < 0.003) inhibited s.c. tumor growth (Fig. 5A ). However, neither of these bisphosphonates-treated tumors completely regressed, and after initial suppression, most tumors grew at a rate comparable to control tumors (data not shown). The effects of risedronate and zoledronate on survival were examined in vivo in an i.p. tumor model. In particular, AB12 cells form diffuse tumors throughout the peritoneal cavity following i.p. injection, a pattern similar to the presentation of human peritoneal mesothelioma (31). Six days after i.p. inoculation of AB12 or AC29 cells, groups of 10 to 12 mice were treated by i.p. injection of either risedronate, zoledronate, or PBS. Administration of zoledronate led to a significant increase in median survival (43 days for zoledronate versus 26 days for PBS; P < 0.001). Median survival in the risedronate group was 30 days, which was not statistically significantly different from the PBS group. All PBS-treated mice died by day 35. In contrast, there were three long-term (>60 days) and two long-term (>85 days) survivors in the risedronate and zoledronate-treatment groups, respectively (Fig. 5B). A similar survival experiment was also done with mice bearing AC29 cells. Although the differences were not statistically significant (P = 0.08) after a total of six inoculations with the drug, the median survival of mice in the zoledronate-treatment group was 39 days, whereas in the control group, the median survival was 26.5 days (Fig. 5C).


Figure 5
View larger version (12K):
[in this window]
[in a new window]
  		Fig. 5. Risedronate and zoledronate mediate antitumor activity in vivo. A, AB12 cells were inoculated s.c. into the flanks of mice. Ten days later, groups of 10 mice were treated s.c. with PBS, zoledronate (0.5 mg/kg), or risedronate (15 mg/kg) every 6 days for four injections. Points, mean tumor volume; bars, ±SE. B, AB12 cells were inoculated into the peritoneal cavities of mice. Six days later, groups of 12 mice were treated by i.p. injection of zoledronate (0.5 mg/kg), risedronate (15 mg/kg), or an equal volume of PBS thrice a week for 2 weeks. Data is expressed as the percentage of survival. C, AC29 cells were inoculated into the peritoneal cavities of mice (n = 10). Six days later, the mice were treated by i.p. injection of zoledronate (0.5 mg/kg) or with an equal volume of PBS thrice a week for 2 weeks. Data is expressed as the percentage of survival.

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The in vivo anticancer effects of bisphosphonates have been previously detected in models of bone metastases. In this setting, the growth-inhibitory effects of these drugs have been attributed to their ability to inhibit osteoclasts and thereby, to the inhibition of growth factor liberation from the bone matrix during bone resorption (9, 10, 16). We show here for the first time that both risedronate and zoledronate inhibit mesothelioma tumor growth in vitro and in vivo. Treatment of mesothelioma-bearing mice with these agents also promoted their survival. Thus, bisphosphonates might also have bone-independent, direct anticancer effects against certain tumors.

Our in vivo results with mesothelioma tumors established at soft tissue sites seem to be superior to those achieved with bisphosphonates in other tumor models. For example, bisphosphonates have not been shown to inhibit tumor growth in a mouse breast cancer model using s.c. injection of human MDA-MB-231 breast cancer cells (9, 10). Furthermore, in a mouse model of breast cancer metastasis, zoledronate increased the overall survival of the tumor-bearing mice by, at most, 4 days, whereas the median survival in our studies was increased by 17 days with zoledronate (32). Although given less frequently, the bisphosphonate doses used in our study were much higher than those used in the earlier studies. Therefore, despite the less frequent dosing, which mimics the use of i.v. bisphosphonates in oncology, the cumulative doses are high and possibly not directly translatable into clinical use (8).

Our findings are surprising in light of the poor bioavailability of bisphosphonates to visceral tumors. For example, serum concentrations of 1 to 3 µmol/L are maintained only for a few hours after a 4 mg dose of zoledronate (33). Bisphosphonates are rapidly adsorbed from the circulation to bone tissue hydroxyapatite, where they are retained for prolonged periods (17). The fact that mesothelioma tumors have been reported to accumulate Tc-99m hydroxymethylene diphosphonate in patients with mesothelioma suggests that these cells may possess a yet to be identified mechanism to accumulate bisphosphonates, which may also explain their in vivo sensitivity to these drugs. Such an extraosseous accumulation of the bone scanning agent has also been described for several other epithelial cancer cells, including small cell lung cancer (19, 22, 34). Interestingly, it was shown recently that treatment with zoledronate also significantly inhibited the growth of small cell lung cancer cells in the flanks of nude mice (35). The mechanism by which cancer cells accumulate Tc-99m hydroxymethylene diphosphonate is not understood, although tumor necrosis and calcification have been suggested to have a role in this process (20, 21). Recent studies conducted in macrophages suggest the presence of an active, yet uncharacterized uptake mechanism, for which n-bisphosphonates and p-bisphosphonates compete (26). Further studies are required to characterize whether a similar mechanism is also active in mesothelioma and other cancer cells.

n-Bisphosphonates have also recently been shown to inhibit angiogenesis in several models (36–39). n-Bisphosphonate treatment also resulted in decreased amounts of circulating mediators of angiogenesis in patients with breast cancer (40, 41). Therefore, another possible mechanism through which the n-bisphosphonates may have inhibited mesothelioma growth in our study is through inhibited angiogenesis.

n-Bisphosphonate-induced cellular effects, such as inhibition of osteoclast activity, have been suggested to be due to inhibition of the mevalonate pathway (42–45). This inhibition results in the lack of geranylgeraniol and thereby, in defective protein prenylation in cells. Inversely, excess geranylgeraniol has been shown to reverse all the inhibitory effects of n-bisphosphonates in osteoclasts (42, 44). Geranylgeraniol was also shown to partially reverse the antiproliferative and mineralization-promoting effects of zoledronate, but not of pamidronate, in human fetal osteoblasts (46). Geranylgeraniol also reversed the inhibitory effects of n-bisphosphonates on migration, in human ovarian and breast cancer cells (47, 48). n-Bisphosphonate-induced decrease in breast cancer viability was not, however, reversed by geranylgeraniol (46). Our results showed that geranylgeraniol also reversed, completely or partially, the n-bisphosphonate-induced accumulation of unprenylated Rap1A in the studied mesothelioma cells. The effects of risedronate on the accumulation of unprenylated Rap1A were more completely reversed by geranylgeraniol than those of zoledronate, which may represent potency differences between these two drugs (49). Furthermore, the inhibitory effects on proliferation were more completely reversed by geranylgeraniol than the inhibitory effects on accumulation of unprenylated Rap1A (for example with zoledronate in AC29 cells). This suggests that the function of the small GTPases controlling various aspects of cell behavior are differentially regulated by n-bisphosphonates. Similar findings have also been reported by other investigators recently (48, 50).

We further discovered that similar to the effects seen in breast cancer cells, these drugs induce the phosphorylation of the p38 mitogen-activated protein kinase in mesothelioma cells as well (27). Furthermore, similar to breast cancer cells, the n-bisphosphonate-induced p38 activation results in resistance against the growth-inhibitory effects of these drugs, because inhibition of p38 also augments bisphosphonate-induced growth inhibition in mesothelioma cells. Whether n-bisphosphonate-induced p38 phosphorylation promotes survival or increases cell cycling, remains to be studied. The effects on the mevalonate pathway dominate over the activation of the p38-mediated pathway, because the net result of the bisphosphonate-treatment is decreased growth.

In conclusion, our demonstration that n-bisphosphonates inhibit the growth of mesothelioma cells in vitro and in vivo, and prolong the survival of mesothelioma-bearing mice in vivo support further study of bisphosphonates in the management of mesothelioma.


   Acknowledgments
 
Dr. Yufeng Li is kindly acknowledged for the statistical analyses. Dr. Denise Shaw is acknowledged for critical review of the manuscript.


   Footnotes
 
Grant support: University of Alabama Comprehensive Cancer Center Mesothelioma Center Core grant (5P30CA13148).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Note: P. Triozzi and K.S. Selander contributed equally to this work.

Received 12/19/05; revised 2/ 6/06; accepted 2/23/06.


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

  1. Sterman DH, Kaiser LR, Albelda SM. Advances in the treatment of malignant pleural mesothelioma. Chest 1999;116:504–20.[Abstract/Free Full Text]
  2. Peto J, Decarli A, La Vecchia C, Levi F, Negri E. The European mesothelioma epidemic. Br J Cancer 1999;79:666–72.[CrossRef][Medline]
  3. Craighead JE. Current pathogenetic concepts of diffuse malignant mesothelioma. Hum Pathol 1987;18:544–57.[Medline]
  4. Rusch VW. Pleurectomy/decortication and adjuvant therapy for malignant mesothelioma. Chest 1993;103:382–4S.
  5. Jaklitsch MT, Grondin SC, Sugarbaker DJ. Treatment of malignant mesothelioma. World J Surg 2001;25:210–7.[CrossRef][Medline]
  6. Rogers MJ, Watts DJ, Russell RG. Overview of bisphosphonates. Cancer 1997;80:1652–60.[CrossRef][Medline]
  7. Rogers MJ. New insights into the molecular mechanisms of action of bisphosphonates. Curr Pharm Des 2003;9:2643–58.[CrossRef][Medline]
  8. Conte P, Coleman R. Bisphosphonates in the treatment of skeletal metastases. Semin Oncol 2004;31:59–63.[Medline]
  9. Sasaki A, Boyce BF, Story B, et al. Bisphosphonate risedronate reduces metastatic human breast cancer burden in bone in nude mice. Cancer Res 1995;55:3551–7.[Abstract/Free Full Text]
  10. Hiraga T, Williams PJ, Mundy GR, Yoneda T. The bisphosphonate ibandronate promotes apoptosis in MDA-MB-231 human breast cancer cells in bone metastases. Cancer Res 2001;61:4418–24.[Abstract/Free Full Text]
  11. Neudert M, Fischer C, Krempien B, Bauss F, Seibel MJ. Site-specific human breast cancer (MDA-MB-231) metastases in nude rats: model characterisation and in vivo effects of ibandronate on tumour growth. Int J Cancer 2003;10:468–77.
  12. Senaratne SG, Pirianov G, Mansi JL, Arnett TR, Colston KW. Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer 2000;82:1459–68.[Medline]
  13. Shipman CM, Croucher PI, Russell RG, Helfrich MH, Rogers MJ. The bisphosphonate incadronate (YM175) causes apoptosis of human myeloma cells in vitro by inhibiting the mevalonate pathway. Cancer Res 1998;58:5294–7.[Abstract/Free Full Text]
  14. Shipman CM, Rogers MJ, Apperley JF, Graham R, Russell G, Croucher PI. Anti-tumour activity of bisphosphonates in human myeloma cells. Leuk Lymphoma 1998;32:129–38.[Medline]
  15. Forsea AM, Muller C, Riebeling C, Orfanos CE, Geilen CC. Nitrogen-containing bisphosphonates inhibit cell cycle progression in human melanoma cells. Br J Cancer 2004;9:803–10.
  16. Mundy GR, Yoneda T, Hiraga T. Preclinical studies with zoledronic acid and other bisphosphonates: impact on the bone microenvironment. Semin Oncol 2001;28:35–44.[Medline]
  17. Fleisch H. Bisphosphonates. Pharmacology and use in the treatment of tumour-induced hypercalcaemic and metastatic bone disease. Drugs 1991;42:919–44.[Medline]
  18. Sandler ED, Hattner RS, Parisi MT, Miller TR. Clinical utility of bone scan features of pleural effusion: sensitivity and specificity for malignancy based on pleural fluid cytopathology. J Nucl Med 1994;35:429–31.[Abstract/Free Full Text]
  19. Vorne M, Saukko T. Detection of malignant soft tissue tumors in bone imaging. Eur J Nucl Med 1984;9:180–4.[Medline]
  20. Kaida H, Ishibashi M, Baba K, Nishida H, Matsuoka K, Hayabuchi N. Extraosseous uptake of metastatic lymph nodes of ureteral cancer on 99Tcm hydroxymethylene diphosphonate bone scintigraphy. Br J Radiol 2004;77:869–70.[Free Full Text]
  21. Shih WJ, DeLand FH, Domstad PA, Johnston EH. Extraosseous localization of 99mTc-MDP in ganglioneuroblastoma. Eur J Nucl Med 1982;7:336–8.[Medline]
  22. Tanabe Y, Suto Y. Tc-99m MDP accumulation in primary malignant peritoneal mesothelioma. Clin Nucl Med 1998;23:180–1.[Medline]
  23. Swayne LC, Hediger RG, Wolff M. Bone scan detection of pelvic metastasis from pleural mesothelioma. Clin Nucl Med 1992;17:965–6.[Medline]
  24. Davis MR, Manning LS, Whitaker D, Garlepp MJ, Robinson BW. Establishment of a murine model of malignant mesothelioma. Int J Cancer 1992;52:881–6.[Medline]
  25. Suri S, Mönkkönen J, Taskinen M, et al. Nitrogen-containing bisphosphonates induce apoptosis of Caco-2 cells in vitro by inhibiting the mevalonate pathway: a model of bisphosphonate-induced gastrointestinal toxicity. Bone 2001;29:336–43.[Medline]
  26. Frith JC, Rogers MJ. Antagonistic effects of different classes of bisphosphonates in osteoclasts and macrophages in vitro. J Bone Miner Res 2003;18:204–12.[CrossRef][Medline]
  27. Merrell M, Suarez-Cuervo C, Harris KW, Väänänen HK, Selander KS. Bisphosphonate induced growth inhibition of breast cancer cells is augmented by p38 inhibition. Breast Cancer Res Treat 2003;81:231–41.[Medline]
  28. Oades GM, Senaratne SG, Clarke IA, Kirby RS, Colston KW. Nitrogen containing bisphosphonates induce apoptosis and inhibit the mevalonate pathway, impairing Ras membrane localization in prostate cancer cells. J Urol 2003;170:246–52.[CrossRef][Medline]
  29. Jagdev SP, Coleman RE, Shipman CM, Rostami HA, Croucher PI. The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with paclitaxel. Br J Cancer 2001;84:1126–34.[CrossRef][Medline]
  30. Segawa H, Kimura S, Kuroda J, et al. The anti-leukemic efficacy of the third generation bisphosphonate ONO5920/YM529. Leuk Res 2005;29:451–7.[Medline]
  31. Lanuti M, Rudginsky S, Force SD, et al. Cationic lipid:bacterial DNA complexes elicit adaptive cellular immunity in murine intraperitoneal tumor models. Cancer Res 2000;60:2955–63.[Abstract/Free Full Text]
  32. Hiraga T, Williams PJ, Ueda A, Tamura D, Yoneda T. Zoledronic acid inhibits visceral metastases in the 4T1/luc mouse breast cancer model. Clin Cancer Res 2004;10:4559–67.[Abstract/Free Full Text]
  33. Skerjanec A, Berenson J, Hsu C, et al. The pharmacokinetics and pharmacodynamics of zoledronic acid in cancer patients with varying degrees of renal function. J Clin Pharmacol 2003;43:154–62.[Abstract/Free Full Text]
  34. Tanabe M, Mizukawa K, Sugita K, Tamai T, Wakabayashi H, Morino Y. 99mTc-diphosphonate uptake in small cell carcinoma of the lung. Radioisotopes 1979;28:177–9.[Medline]
  35. Matsumoto S, Kimura S, Segawa H, et al. Efficacy of the third-generation bisphosphonate, zoledronic acid alone and combined with anti-cancer agents against small cell lung cancer cell lines. Lung Cancer 2005;47:31–9.[CrossRef][Medline]
  36. Hamma-Kourbali Y, Di Benedetto M, Ledoux D, et al. A novel non-containing-nitrogen bisphosphonate inhibits both in vitro and in vivo angiogenesis. Biochem Biophys Res Commun 2003;310:816–23.[CrossRef][Medline]
  37. Fournier P, Boissier S, Filleur S, et al. Bisphosphonates inhibit angiogenesis in vitro and testosterone-stimulated vascular regrowth in the ventral prostate in castrated rats. Cancer Res 2002;62:6538–44.[Abstract/Free Full Text]
  38. Bezzi M, Hasmim M, Bieler G, Dormond O, Ruegg C. Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt. J Biol Chem 2003;278:43603–14.[Abstract/Free Full Text]
  39. Giraudo E, Inoue M, Hanahan D. An amino-bisphosphonate targets MMP-9-expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest 2004;114:623–33.[CrossRef][Medline]
  40. Vincenzi B, Santini D, Dicuonzo G, et al. Zoledronic acid-related angiogenesis modifications and survival in advanced breast cancer patients. J Interferon Cytokine Res 2005;25:144–51.[Medline]
  41. Ferretti G, Fabi A, Carlini P, et al. Zoledronic-acid-induced circulating level modifications of angiogenic factors, metalloproteinases and proinflammatory cytokines in metastatic breast cancer patients. Oncology 2005;69:35–43.[CrossRef][Medline]
  42. Fisher JE, Rogers MJ, Halasy JM, et al. Alendronate mechanism of action: geranylgeraniol, an intermediate in the mevalonate pathway, prevents inhibition of osteoclast formation, bone resorption, and kinase activation in vitro. Proc Natl Acad Sci U S A 1999;96:133–8.[Abstract/Free Full Text]
  43. Fisher JE, Rodan GA, Reszka AA. In vivo effects of bisphosphonates on the osteoclast mevalonate pathway. Endocrinology 2000;141:4793–6.[Abstract/Free Full Text]
  44. van beek E, Löwik C, van der Pluijm G, Papapoulos S. The role of geranylgeranylation in bone resorption and its suppression by bisphosphonates in fetal bone explants in vitro: a clue to the mechanism of action of nitrogen-containing bisphosphonates. J Bone Miner Res 1999;14:722–9.[CrossRef][Medline]
  45. van Beek E, Pieterman E, Cohen L, Löwik C, Papapoulos S. Farnesyl pyrophosphate synthase is the molecular target of nitrogen-containing bisphosphonates. Biochem Biophys Res Commun 1999;264:108–11.[CrossRef][Medline]
  46. Reinholz GG, Getz B, Sanders ES, et al. Distinct mechanisms of bisphosphonate action between osteoblasts and breast cancer cells: identity of a potent new bisphosphonate analogue. Breast Cancer Res Treat 2002;71:257–68.[CrossRef][Medline]
  47. Sawada K, Morishige K, Tahara M, et al. Alendronate inhibits lysophosphatidic acid-induced migration of human ovarian cancer cells by attenuating the activation of rho. Cancer Res 2002;62:6015–20.[Abstract/Free Full Text]
  48. Denoyelle C, Hong L, Vannier JP, Soria J, Soria C. New insights into the actions of bisphosphonate zoledronic acid in breast cancer cells by dual RhoA-dependent and -independent effects. Br J Cancer 2003;88:1631–40.[CrossRef][Medline]
  49. Sanders JM, Gomez AO, Mao J, et al. 3-D QSAR investigations of the inhibition of Leishmania major farnesyl pyrophosphate synthase by bisphosphonates. J Med Chem 2003;46:5171–83.[CrossRef][Medline]
  50. Nogawa M, Yuasa T, Kimura S, et al. Zoledronic acid mediates Ras-independent growth inhibition of prostate cancer cells. Oncol Res 2005;15:1–9.[Medline]



This article has been cited by other articles:


Home page
 Am. J. Respir. Crit. Care Med.Home page
Y. C. G. Lee and S. Wilkosz
Malignant Pleural Effusions: Fixing the Leaky Faucet
Am. J. Respir. Crit. Care Med., July 1, 2008; 178(1): 3 - 5.
[Full Text] [PDF]

Home page
 Clin. Cancer Res.Home page
A. J. Roelofs, K. Thompson, S. Gordon, and M. J. Rogers
Molecular mechanisms of action of bisphosphonates: current status.
Clin. Cancer Res., October 15, 2006; 12(20): 6222s - 6230s.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Wakchoure, S.
Right arrow Articles by Selander, K. S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Wakchoure, S.
Right arrow Articles by Selander, K. S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Cancer Research Clinical Cancer Research
Cancer Epidemiology Biomarkers & Prevention Molecular Cancer Therapeutics
Molecular Cancer Research Cancer Prevention Research
Cancer Prevention Journals Portal Cancer Reviews Online
Annual Meeting Education Book Meeting Abstracts Online