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Chondrosarcoma is a difficult musculoskeletal tumor to treat. Surgical treatment leads to severe disability, with high rates of local recurrence and life threat. No adjuvant therapy is effective in differentiated chondrosarcomas. Bisphosphonates (BPs) are a class of molecules which is effective in malignant bone diseases. The aim of the present study was to determine the effects of zoledronic acid (ZOL) on chondrosarcoma tumor progression. ZOL was tested in vivo (s.c. 100 μg/kg, twice a week) in a rat chondrosarcoma model and in vitro (10−7–10−4 M) on cells derived from this model. Two types of animal models were assessed, the first simulated development after intralesional curettage, the second nonoperative development of the tumor. Cell proliferation, caspase-1, -3 activities and cell cycle analysis were studied. The results revealed that ZOL slows down primary tumor development, tumor progression after intralesional curretage and increases overall survival. ZOL inhibits cell proliferation and increases cell death, with no significant variation of caspase-1 and -3 activities and cell cycle profiles. The present study demonstrates for the first time that in addition to surgery, the therapy of chondrosarcoma with BPs might be beneficial. Because of these first results, new therapeutic approaches of chondrosarcoma must be considered, mainly for low grade chondrosarcoma when disabling operation is planned and when only intralesional resection can be undertaken. © 2006 Wiley-Liss, Inc.
Chondrosarcoma, identified by Lichtenstein and Jaffe as a malignant bone tumor clearly distinct from osteosarcoma, is currently defined as malignant cartilage tumor arising de novo or within a preexisting benign cartilage tumor.1 Human chondrosarcomas, which represent about 25% of all bone sarcoma,2, 3 are classified as low, intermediate or high grade on the basis of histological and cytological features.4 Overall local recurrence and 10-year survival are 24–33% and 46–70%, respectively.5, 7 Risk factors for the former include inadequate surgical margins5, 8 and tumor size, and for the later include local recurrence, extracompartmental spread and high histological grade.5 No effective adjuvant treatment (radiation therapy, chemotherapy) is available.6
As evidenced for bone metastases, a vicious cycle between osteoclasts, bone stromal cells/osteoblasts and cancer cells has been hypothesized during the progression of primary bone tumors.9 Accordingly, suppression of osteoclasts would be a primary approach to inhibit local cancer growth. Among the potential drugs available, bisphosphonates (BPs) are an important class of molecules for the treatment of bone diseases with different molecular mechanisms of action. Nitrogen-containing BPs act by inhibiting the recruitment, proliferation and differentiation of preosteoclasts, or by impeding the resorptive activity of mature osteoclasts.10, 11, 12, 13 They also shorten the life span of osteoclasts by inducing their apoptosis.14 Previous studies revealed that BPs have the ability to reduce the osteolytic bone resorption associated with multiple myeloma and breast cancer15, 16 and also show efficacy in cancer metastases to bone due to prostate cancer and other solid tumors, demonstrating that this BPs can reduce skeletal morbidity in both osteolytic and osteoblastic diseases.17, 18 A clear direct antitumor activity on breast cancer cells has been demonstrated in vitro.19 In primary malignant bone tumor, inhibition of human osteosarcoma cell growth by pamidronate and clodronate20, 21 and zoledronic acid (ZOL)22 have been reported in vitro. These results are consistent with our in vitro and in vivo studies demonstrated efficacy of ZOL on osteosarcoma tumor progression and metastatic spreading.23, 24
Hence, the purpose of the present study was to determine the efficacy of ZOL on chondrosarcoma in vitro and in vivo, in term of local tumor growth, animal survival after and before intralesional curettage using a rat transplantable model of chondrosarcoma.
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- Material and methods
The present study focuses on the potential effect of ZOL on tumor progression and residual disease after intralesional curettage in a rat model of chondrosarcoma, and demonstrates for the first time that bisphosphonate has a potent beneficial effect in vivo on chondrosarcoma tumor progression. Most of the literature on chondrosarcoma has confirmed that adequate surgery is the mainstay of treatment for local control which itself is a risk factor for survival.5, 6, 27, 28 Despite different definitions of adequate margins, this goal can be achieved in only 46–76% of patients.5, 6, 7, 28 Adequate surgery means wide margins of normal tissue, that leads to severe disability, mainly when tumor occurs on spine or pelvis, when possible. On the other hand, inadequate surgery is an independent risk factor for local recurrence (as well as tumor size greater than 10 cm). Adjuvant treatment is of great interest to improve local control of this tumor when wide margin is not possible to obtain metastatic spreading and even to consider intralesional treatment of low grade chondrosarcoma. Radiation therapy is not effective in local control of the tumor7, 27, 29 but can be used in exceptional situations delaying local recurrence probably only in patients who have minimum residual microscopic disease.30 Chemotherapy is recommended in high risk chondrosarcoma and dedifferentiated chondrosarcoma,6, 31, 32, 33 but it is not administered according to a specific protocol and no controlled study is available. Cryosurgery after intralesional curettage is much promising in grade 1 chondrosarcoma, with at least similar results of marginal excision in term of oncological control and mostly better than those of wide excisions in terms of functional results.34, 35, 36
A vicious cycle has been described in osteolytic metastasis; tumor cells release osteolytic mediators and bone resorption release growth factors which enhance tumor cell growth and further release of osteolytic mediators.9 One can speculate that this vicious cycle may also occur in the case of the primary bone tumor. Thus, previous study with RCS pointed out the role of bone microenvironment on tumor aggressiveness, interactions between bone and tumor-induced bone remodelling and modification of the grade (grade II with foci of grade III, according to O'Neal and Ackerman grading) when tumor tissue is transplanted in close contact to the scarified bone.26 Inhibitors of bone resorption such as BPs may interfere with primary tumor development at the skeletal site. To our knowledge, such hypothesis has never been tested in vivo and only one oral communication has been recently reported with chondrosarcoma.37 RCS simulates the conditions of human chondrosarcoma development. It has been well characterized histologically, biochemically and structurally.38, 39 Kenan and Steiner39 showed that RCS is a well-differentiated malignant tumor, histologically similar to well-differentiated human chondrosarcoma. In the present study, this RCS tumor tissue was transplanted in close contact to femur after mechanical scarifications to insure interaction between tumor progression and bone remodelling as previously described.26 Human low grade chondrosarcomas induce 3–12% of metastasis appearing along time after diagnosis (mean 47 months).5, 6 As the present corresponds to a low grade differentiated chondrosarcoma (mostly grade II),26 it is unsurprising that any metastasis was observed during this short experimental course (maximal 5 weeks postimplantation) with animals dying from local development of the tumor. Such condition is seldom observed in clinical situation.
In vivo results confirmed in all experiments of the present study, the efficacy of ZOL on RCS progression. On the other hand, ZOL treatment postponed local recurrence in treated group, but failed to prevent tumor recurrence after intralesional curettage. Because of extensive spreading of the tumor in the soft tissue, intralesional curettage in all cases simulated microscopic residual disease. Such situation is frequent in clinical conditions, and efficient medical adjuvant treatment such as bisphosphonate would be of greatest interest in potential microscopic residual disease, in order to reduce margins and improve functional results. Unfortunately, this condition is difficult to mimick with RCS. Indeed, intraosseous implantation, which could be a better model for an extensive curettage, leads to inconstant and small tumors in our experience. ZOL doses used in the present study are justified as the clinical dose (4 mg i.v. every 3–4 weeks) is equivalent to ∼100 μg/kg of the research grade disodium salt used in this study. However, even if dosing frequency of twice a week is greater than in human, it could be justified by the aggressive nature of the chondrosarcoma.
Initially, it was thought that the specific inhibition of osteoclastic bone resorption is the only mechanism of action of BPs, by which they are effective in the treatment of cancer patients bearing bone metastasis. However, evidence is emerging from both preclinical and clinical studies to suggest that BPs also have direct antitumor properties that may contribute to their therapeutic efficacy in malignant bone disease.40 Potential direct antitumor effect of pamidronate and clodronate have been suggested in 2 in vitro studies showing inhibition of osteosarcoma cell growth proliferation.20, 21 Morever, ZOL induced in a dose- and time-dependant decrease in cell proliferation in osteosarcoma cell lines in vitro, and reduced tumor progression and metastatic lung spreading in vivo.23, 24 The present study reports a significant decrease of chondrosarcoma cell proliferation and an increase of cell death at 1 and 10 μM in vitro with an IC50 comparable to those observed on osteosarcoma and breast carcinoma cells.1, 19, 24 ZOL appears to be a more potent inhibitor of cell proliferation compared to the other N-BPs.40 Indeed, while several BPs exert antitumor activities, their effects seem to vary among the different compounds, in relation to their basic structure. The nitrogen-containing BPs such as ZOL have been suggested to be clinically superior to their first generation counterparts.41 Moreover, the peak plasma levels of ZOL appear to be around 1 μM as shown by Skerjanec et al.42 which is in agreement with the IC50 of ZOL measured on cancer cells, thus then strengthening an in vivo potential effect of ZOL even at a low concentration. ZOL has been reported to inhibit cell cycle progression and increase the proportion of cells arrested in S-phase in rat and human osteosarcoma,21, 23 that was not confirmed in the present study with RCS cells. This observation can be explained by the slow proliferation rate of the RCS cells as shown in the control condition (Fig. 3d). Moreover, ZOL-induced RCS cell death is not mediated by activation of caspase 1 and 3 as in osteosarcoma cells.21, 22 Thus, our data support a cytotoxic effects of ZOL which could look like to an anoikis mechanism as already described in osteosarcoma cells.21 However, the precise molecular mechanisms implicated in the ZOL-induced cell death need further investigation.
The present study demonstrated for the first time, that in addition to surgery, the therapy of chondrosarcoma with BPs might be beneficial. Such results open the way to the development of effective adjuvant treatment associated with surgical approach for treatment of chondrosarcoma.