Antioxidants as potential anti-osteolytic therapies for breast cancer bone metastasis

© 2013 Dr. Walter PfisterReactive oxygen species (ROS) are known to play a crucial role in different stages of breast cancer progression, in particular in the maturation and activation of bone-resorbing osteoclast cells. In support of this, several recent reports have shown that antioxidants suppress bone resorption in mouse models of osteoporosis. On this basis, antioxidants may have therapeutic potential in inhibiting breast cancer-induced osteolysis. While measurement of antioxidant activity in cell culture is relatively easy, the assessment of antioxidant activity and ROS levels in human and animal tissue is far more problematic. The constant challenge of antioxidant studies is a) to demonstrate treatment effect and b) providing evidence that the observed effect was indeed caused by antioxidant activity. To investigate the impact of antioxidant treatment on breast cancer primary tumour, metastasis to secondary organs and cancer-induced osteolysis, we employed the orally available antioxidant N-acetylcysteine (NAC) in appropriate pre-clinical mouse models, using grafted mouse (4T1.2) and human (MDA-MB-231, MDA-MB-468) cell lines. Fluorescent-tagged (mCherry) breast cancer cells were inoculated into the mammary fat pad of Balb/C wild type (syngeneic) or Balb/C nu/nu (xenograft) mice. Tumour growth rates for the three cell lines grafted were similar for Placebo (water)-treated mice as well as the doses of NAC examined (0.1 to 2 g/kg/day). Tumour architecture and ratio of necrotic to viable cell mass in the MDA-468-mCherry tumours was unaffected by NAC treatment. Metastatic foci in the lungs of 4T1.2-mCherry and MDA-468-mCherry inoculated mice formed within 30 and 100 days respectively. However, overall tumour burden was not reduced by NAC treatment in the 4T1.2-mCherry model and sub-optimal duration of tumour growth limited the detection of metastatic lung lesions in the MDA-468-mCherry and MDA-231-mCherry models. Contrary to recent reports in the literature, antioxidant treatment did not promote metastasis in our pre-clinical breast cancer models. NAC, when combined with the chemotherapeutic agent doxorubicin (DOX) had no impact on the efficacy of the anthracycline. Supporting in vitro studies demonstrated that NAC at sub-lethal concentration (10 mM) has no effect on DOX-induced inhibition of clonogenicity in MDA-231-mCherry cells, but the antioxidant has an additive, positive impact on the DOX-induced inhibition of clonogenicity when the same assessment is conducted with murine 4T1.2-mCherry cancer cells. Breast cancer-induced osteolysis was initiated by inoculation of 4T1.2-mCherry and MDA-231-mCherry into the tibial marrow of syngeneic and xenograft mice respectively. Treatment with NAC inhibited the onset of osteolysis in the xenograft model and showed mild suppression of osteolysis in the syngeneic mice. To assess impact of NAC treatment on anti-osteolytic therapy with the bisphosphonate Zometa, mice were inoculated with the 4T1.2-mCherry and MDA-231-mCherry breast cancer cell lines following combination treatment with the two agents. Zometa inhibited osteolysis in the MDA-231-mCherry model and supplementation with NAC appeared to increase the inhibition, although the added bone protection did not reach statistical significance. Zometa therapy reduced bone resorption in the 4T1.2-mCherry model, but this effect was only statistically significant when Zometa and 30 mM NAC treatment were combined. This finding suggests that supplementing bisphosphonate therapy with antioxidants like NAC may increase efficacy in anti-osteolysis therapies, including therapies against breast cancer-induced bone resorption. An important part of our studies was the evaluation of pharmacodynamic assays that directly or indirectly measure antioxidant activity in vivo. A modified prooxidant/antioxidant balance (PAB) assay proved useful to measure antioxidant capacity in the NAC drug formulation and in plasma from NAC-treated, non-tumour challenged mice. The dihydroethidium (DHE) assay was employed to detect superoxide formation in kidneys from NAC-treated and control mice. We were able to demonstrate that the fluorescent oxidation product 2-hydroxyethidium can be detected by other means other than high performance liquid chromatography (HPLC) and mass spectrometry, which supports the aim to establish reliable pharmacodynamic assays that can be employed by research and clinical laboratories without expensive instrumentation or specialist skills. The thiobarbituric acid reactive substances (TBARS) assay identified higher malondialdehyde (MDA) levels in tumour challenged mice compared to non-tumour-challenged mice, indicating breast cancer-induced oxidative stress in this model. Furthermore, the assay identified significantly lower MDA levels after treatment with the antioxidant Trolox, in comparison with other antioxidants, including NAC. An assessment of 8-OH-dG urine levels in the same mice revealed significantly higher antioxidant activity in the Olive leaf extract- and Curcumin-treated mice when compared with the control group and NAC-treated mice. This offers the possibility to assess antioxidant impact on breast cancer progression and in particular breast cancer-induced osteolysis with potentially stronger antioxidants