Sequence‐ and schedule‐dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells

Abstract

We investigated whether the combination of zoledronic acid and doxorubicin induced apoptosis of breast and prostate cancer cell lines, and if synergistic interaction was present. We investigated whether the levels of cell death altered depending on the sequence in which the drugs were administered and the possible mechanism of action responsible for the increased cell death following combined treatments. Breast and prostate cancer cells were treated with zoledronic acid alone, doxorubicin alone, or drugs in sequence (doxorubicin before, after, or with zoledronic acid), and the levels of apoptotic death were determined by evaluation of nuclear morphology. We found that clinically relevant concentrations of doxorubicin and zoledronic acid induced sequence-and schedule-dependent apoptosis of breast and prostate cancer cells. For maximal apoptosis, cells had to be pretreated for 24 hr with doxorubicin before immediate treatment with zoledronic acid for 1 hr. This observation is a characteristic feature of cell cycle phase-specific synergistic effect. Replacing zoledronic acid with the nonnitrogen-containing bisphosphonate clodronate did not induce increased apoptosis. Induction of apoptosis was mainly via inhibition of the mevalonate (MVA) pathway, as addition of the MVA pathway intermediary geranylgeraniol inhibited the induction of apoptosis by doxorubicin followed by zoledronic acid. In conclusion, combined treatment of breast and prostate cancer cell lines with clinically relevant doses of doxorubicin and zoledronic acid induces apoptosis in a synergistic fashion. These findings may have relevance for the clinical setting, particularly breast cancer patients receiving these drugs in the adjuvant setting.Key words: breast cancer; apoptosis; zoledronic acid; doxorubicin; synergistic interaction Zoledronic acid is a potent third-generation nitrogen-containing bisphosphonate and is the only bisphosphonate licensed to treat bone metastases from a variety of solid tumors and in multiple myeloma. In clinical practice, an increasing number of breast and prostate cancer patients are receiving zoledronic acid at earlier stages of their treatment in order to manage their metastatic bone disease.Zoledronic acid inhibits osteoclastic bone resorption, which occurs at an accelerated pace due to the presence of tumor cells in the bone microenvironment. 1 Nitrogen-containing bisphosphonates affect osteoclast action by inhibition of enzymes of the mevalonate pathway. 2 Target enzymes include farnesyl pyrophosphate synthase 3,4 and/or geranylgeranylpyrophosphate synthase, 5 leading to a lack of formation of farnesyl pyrophosphate and geranylgeranyl pyrophosphate. These isoprenoids are requisite for posttranslation lipid modification (i.e., farnesylation and geranylgeranylation) of signaling GTPases, such as Ras, Rho and Rac. 6 As these control a variety of important osteoclast cell functions, their loss ultimately leads to osteoclast apoptosis through caspase-3 enzyme activation. 7 In addition to their inhibitory effect on osteocl...

Combined effects of the bisphosphonate, zoledronic acid and the aromatase inhibitor letrozole on breast cancer cells in vitro: evidence of synergistic interaction

“…This includes a combination with paclitaxel (Jagdev et al, 2001;Neville-Webbe et al, 2006), tamoxifen (Jagdev et al, 2000) and doxorubicin (Neville-Webbe et al, 2005). In our earlier study with ZA and chemotherapy drugs, we have similarly found that sequential administration of drugs is of paramount importance for inducing maximal (potentially synergistic) levels of apoptosis.…”

“…before inhibition of the mevalonate pathway by ZA (Neville-Webbe et al, 2005) and subsequent apoptosis. In combination, ZA and letrozole have potential for synergistic induction of MCF7-Ca cells in vitro.…”

“…Inhibition of the mevalonate pathway by nitrogen-bisphosphonates is integral for inducing a variety of effects including apoptosis of osteoclasts (Amin et al, 1992), breast cancer cells (Jagdev et al, 2001), prostate cancer cells (Oades et al, 2003), myeloma cells (Shipman et al, 1998) and inhibition of tumour cell invasion (Denoyelle et al, 2003). Furthermore, inhibition of this pathway also contributes to the apoptosis induced when breast (and prostate) cancer cells are treated in combination with ZA and doxorubicin (Neville-Webbe et al, 2005), or when breast cancer cells are treated with ZA and paclitaxel (Neville-Webbe et al, 2006).…”

Anticancer mechanisms of doxorubicin and zoledronic acid in breast cancer tumor growth in bone

“…Administration of doxorubicin combined with zoledronic acid induces apoptosis and inhibits proliferation of breast cancer cells in vitro and in vivo, with maximum effects detected when the chemotherapeutic agent is given 24 hours before the bisphosphonate (24,32,33). Here, we have shown the molecular pathways by which these two drugs interact to increase apoptosis, reduce proliferation, and ultimately inhibit MDA-MB-436 breast tumor growth in bone.…”

“…Zoledronic acid has been shown to synergistically increase cancer cell death when combined with doxorubicin, paclitaxel, or cyclophosphamide in breast cancer cells (24)(25)(26); paclitaxel, etoposide, cisplatinum, and irinotecan in lung cancer cells (27); and dexamethasone in myeloma cells (28) in vitro. Combining zoledronic acid (250 μg/kg) with the oral fluoropyrimidine UFT (20 mg) daily decreased the number of bone metastases compared with single agents in the 4T1 mouse mammary tumor model (29).…”