Biology contributions
Synergistic cytotoxic effects of zoledronic acid and radiation in human prostate cancer and myeloma cell lines

https://doi.org/10.1016/j.ijrobp.2004.09.065Get rights and content

Purpose

The clinical use of the potent bisphosphonate zoledronic acid has increased recently, especially for the treatment of bone metastases. Synergistic effects with chemotherapeutic agents (e.g., doxorubicin, paclitaxel) have been shown. It is not known whether similar synergistic effects exist with radiation.

Methods and materials

IM-9 myeloma cells and C4-2 prostate cancer cells were treated with up to 200 μM concentrations of zoledronic acid, irradiated with single doses of up to 1,000 cGy, or exposed to combinations of both treatments. Cell viability was then determined via yellow dye 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide assay and the affected fractions analyzed using the median effect principal, a method developed and validated by Chou and Talalay.

Results

A statistically significant synergistic cytotoxic effect of the combination of zoledronic acid and radiation was documented. The extent of the effect was cell type–dependent, with the C4-2 cells showing a greater synergistic effect than the IM-9 cells.

Conclusions

The combined use of zoledronic acid and radiotherapy shows enhanced in vitro cytotoxicity for two human prostate and myeloma cancer cell lines over that expected for a simple additive effect from each treatment alone. A clinical trial is under way to test this combination therapy.

Introduction

Bisphosphonates (BP) are a family of pyrophosphate analogs characterized by a phosphorus-carbon-phosphorus backbone (Fig. 1). Their chemical structure is responsible for the BP affinity to bone tissue. In the early 1980s, their potential in the treatment of metastatic bone disease was recognized (1). In the last few years, a large number of clinical studies have confirmed that BP effectively inhibits the progression of bone metastases of breast cancer, prostate cancer, and multiple myeloma (2, 3, 4, 5, 6, 7). The different BP differ in potency in part because of the structure of their side chains, which can be grouped into the older and less active class of non-nitrogen-containing BP and the more recent, more potent nitrogen-containing BP (amino-BP) (3, 8).

Bisphosphonates inhibit both bone resorption and inflammatory processes. Although the reasons for these effects are still under investigation, it seems that BP bind with high affinity to bone minerals (hydroxyapatite crystals), especially in areas of bone destruction (9, 10). The accumulated high concentrations of BP are taken up by osteoclasts, specifically inhibit adenosine triphosphate (ATP)-dependent enzyme systems, induce apoptosis of osteoclasts by inhibiting mevalonic acid pathway (11), and thus disrupt the biochemical processes that lead to bone destruction (12).

One of the most potent nitrogen-containing BP is zoledronic acid (Fig. 1) with the chemical formula (1-hydroxy-2-imidazol-1-yl-phosphonoethyl) phosphonic acid monohydrate (Zometa; Novartis Pharmaceuticals Corp., East Hanover, NJ). If the potency of the first non-nitrogen-containing BP etidronate is set at 1, then the relative potency of zoledronic acid is 100,000 (8). Its use in the treatment of multiple myeloma and bone metastases of breast carcinoma was approved by the Food and Drug Administration in 2002 and it has now become an integral part of the treatment of multiple myeloma (13). Zometa is also approved in the United States and Europe for the treatment of bone metastases associated with breast, prostate, lung, and renal cancer, as well as other solid tumors (14). The long-term efficacy and safety of zoledronic acid has been shown in a clinical trial (15). Zoledronic acid is superior to pamidronate for the treatment of bone metastases in breast carcinoma patients with at least one osteolytic lesion (16). It delays the onset of skeletal-related events and progression of skeletal disease in patients with advanced renal cell carcinoma (17) and hormone-refractory metastatic prostate carcinoma (18).

A heavily investigated application of zoledronic acid is in the management of bone metastases from prostate carcinoma (5, 19, 20, 21).

Another well-established treatment modality for the local treatment of bone metastases is radiotherapy. Although the treatment effects of radiation for this application are primarily the result of the direct destruction of tumor cells, it has recently been shown in a pilot study by Hoskin et al. (22) that the effectiveness of radiation could partially be explained by the inhibition of osteoclastic activity. Scheven et al. (23) also showed that osteoclast-forming progenitors are rather radiosensitive. A combination therapy of radiation and BP might thus be expected to be even more effective than either treatment alone. To investigate possible combination effects, we examined in this study the cytotoxic effects of zoledronic acid and radiation on IM-9 myeloma cells and C4-2 prostate carcinoma cells, both alone and in combination.

Section snippets

Zoledronic acid solutions

Zoledronic acid was obtained as pure research compound in the form of the disodium salt from Novartis Pharma AG, Basel, Switzerland. A 10-mM stock solution of zoledronic acid was prepared and sterile filtrated through a 0.22-μm filter. Zoledronic acid dilutions of 10, 20, 50, 100, and 200 μM were prepared with RPMI-1640 medium (Lerner Media Laboratory, Cleveland Clinic Foundation, Cleveland, OH).

Cell lines

C4-2 cells are a subline of LNCaP, an androgen-dependent human prostate cancer cell line, and were

Results

Treatment of C4-2 and IM-9 cells with increasing concentrations of zoledronic acid caused a dose-dependent decrease in cell number (Fig. 1). The toxic effects were similar for both cell lines with an effective dose required to produce a specified effect in 50% of the population (ED50) of 82.0 μM for the C4-2 cells and of 58.6 μM for the IM-9 cells. Irradiation also caused a dose-dependent decrease in cell number (Fig. 2). The C4-2 prostate cancer cells with an ED50 of 1,314.6 cGy, however, were

Discussion

Our study substantiates the cytotoxic effects of zoledronic acid on both prostate cancer and lymphoma cells. These results agree with those reported by Oades et al. (32), except that their ED50 using three different prostate cell lines was about five times lower (10 μM) than the one determined in our study with C4-2 cells. Another article by Lee et al. reported cell growth inhibition levels closer to our results, between 10–50 μM (19). It is not clear why such differences are seen, although

Conclusion

Zoledronic acid combined with radiotherapy can induce cytotoxicity in a synergistic manner in prostate cancer and myeloma cells. This in vitro finding agrees with previous studies showing a synergistic effect of zoledronic acid with different chemotherapeutic drugs. The interaction of BP-induced apoptosis and radiation-induced cell death, however, is not clear yet and requires more fundamental studies. Transferred to the clinic, the combination of zoledronic acid and radiotherapy might allow a

Acknowledgments

We thank Novartis for supplying zoledronic acid for these experiments. Many thanks also to Marcela Oancea (Alex Almasan's laboratory, Cleveland Clinic Foundation) for providing us with IM-9 cells and to Skip Heston (Cleveland Clinic Foundation) for providing us with C4-2 cells.

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