Abstract
Purpose
Combination treatment using the chemotherapy drug doxorubicin and the anti-resorptive agent zoledronic acid has shown to be very effective in inducing apoptosis in breast cancer cells, and also to eradicate breast tumour growth in vivo. Here, we investigated whether apoptotic cell death is increased when zoledronic acid and doxorubicin are given in sequence or in combination in prostate cancer cells in vitro.
Methods
PC3, DU145 and LNCaP prostate cancer cells were treated with zoledronic acid or doxorubicin alone, in sequence or in combination, and apoptosis was measured by evaluation of nuclear morphology following staining with Hoechst and PI. The involvement of the mevalonate pathway in the induction of apoptosis was assessed through the addition of the mevalonate pathway intermediate geranylgeraniol.
Results
Both agents induced PC3 cell death, with 5 μM zoledronic acid inducing 1.73% apoptosis and 50 nM doxorubicin 3.60% apoptosis following 24 h of exposure. In contrast, sequential exposure (doxorubicin followed by zoledronic acid) caused 8.87% apoptosis. Doxorubicin followed by zoledronic acid induced 4.77% apoptosis in LNCaP cells, compared to 1.53% caused by zol alone, 2.23% by dox alone and 2.5% following the reverse sequence (P < 0.001 in all cases). In DU145 cells doxorubicin followed by zoledronic acid induced 5.73% apoptosis, compared to 1.8% following zol alone, 2.93% by dox alone, and 3.20% following the reverse sequence (P < 0.001 in all cases).
Conclusions
This is the first detailed study to show that an increased anti-tumour effect is generated when doxorubicin and zoledronic acid are given in sequence in both hormone-sensitive and insensitive prostate cancer cells in vitro. Our results suggest that combined treatment with these agents is superior to single agent therapy, and should be explored in a tumour model of prostate cancer.
Similar content being viewed by others
Abbreviations
- BPs:
-
Bisphosphonates
- FPP:
-
Farnesyl diphosphate
- GGPP:
-
Geranylgeranyl diphosphate
- N-BPs:
-
Nitrogen-containing bisphosphonates
References
Ye L, Kynaston HG, Jiang WG (2007) Bone metastasis in prostate cancer: molecular and cellular mechanisms. Int J Mol Med 20(1):103–111
Saad F, Karakiewicz P, Perrotte P (2005) The role of bisphosphonates in hormone-refractory prostate cancer. World J Urol 23(1):14–18
Ottewell PD, Mönkkönen H, Jones M, Lefley DV, Coleman RE, Holen I (2008) Antitumor effects of doxorubicin followed by zoledronic acid in a mouse model of breast cancer. J Natl Cancer Inst 100:1167–1178
Ottewell PD, Deux B, Mönkkönen H, Coleman RE, Clezardin P, Holen I (2008) Differential effect of doxorubicin and zoledronic acid on intraosseous versus extraosseous breast tumor growth in vivo. Clin Cancer Res 14:4658–4666
Rogers MJ (2003) New insights into the molecular mechanisms of action of bisphosphonates. Curr Pharm Des 9(32):2643–2658
Van Beek E, Pieterman E, Cohen L, Lowik C, Papapoulos S (1999) Farnesyl pyrophosphate synthase is the molecular target of nitrogen-bisphosphonates. Biochem Biophys Res Commun 264:108–111
Luckman SP, Hughes DE, Coxon FP, Graham R, Russell G, Rogers MJ (1998) Nitrogen-containing bisphosphonates inhibit the mevalonate pathway and prevent post-translational prenylation of GTP-binding proteins, including Ras. J Bone Miner Res 13(4):581–589
Stresing V, Daubiné F, Benzaid I, Mönkkönen H, Clézardin P (2007) Bisphosphonates in cancer therapy. Cancer Lett 257(1):16–35
Senartne SG, Pirianov G, Mansi JL, Arnett TR, Colston KW (2000) Bisphosphonates induce apoptosis in human breast cancer cell lines. Br J Cancer 82(8):1459–1468
Coxon JP, Oades GM, Kirby RS, Colston KW (2004) Zoledronic acid induces apoptosis and inhibits adhesion to mineralised matrix in prostate cancer cells via inhibition of protein prenylation. BJU Int 94:164–170
Shipman CM, Rogers MJ, Apperley JF, Russell RG, Croucher PI (1997) Bisphosphonates induce apoptosis in human myeloma cell lines: a novel anti-tumour activity. Br J Haematol 98:665–672
van der Pluijm G, Vloedgraven H, van Beek E, van der Wee-Pals L, Lowik C, Papapoulos S (1996) Bisphosphonates inhibit the adhesion of breast cancer cells to bone matrices in vitro. J Clin Invest 98(3):698–705
Boissier S, Ferreras M, Peyruchaud O, Magnetto S, Ebetino FH, Colombel M, Delmas P, Delaisse JM, Clezardin P (2000) Bisphosphonates inhibit breast and prostate carcinoma cell invasion, an early event in the formation of bone metastases. Cancer Res 60:2949–2954
Virtanen SS, Vaananen HK, Harkonen PL, Lakkakorpi PT (2002) Alendronate inhibits invasion of PC3 prostate cancer cells by affecting the mevalonate pathway. Cancer Res 62:2708–2714
Fournier P, Boissier S, Filleur S, Guglielmi J, Cabon F, Colombel M, Clezardin P (2002) Bisphophonates inhibit angiogenesis in vitro and testosterone-stimulated vascular regrowth in the ventral prostate in castrated rats. Cancer Res 62:6238–6544
Croucher PI, De Hendrik R, Perry MJ, Hijzen A, Shipman CM, Lippitt J, Green J, van Marck E, van Camp B (2003) Zoledronic acid treatment of 5T2MM-bearing mice inhibits the development of myeloma bone disease: evidence for decreased osteolysis, tumour burden and angiogenesis, and increased survival. J Bone Miner Res 18(3):482–492
Giraudo E, Inoue M, Hanahan D (2004) An amino-bisphosphonate targets MMP-9 expressing macrophages and angiogenesis to impair cervical carcinogenesis. J Clin Invest 114:623–633
Green JR (2004) Bisphosphonates: preclinical review. Oncologist 9(Suppl 4):3–13
Santini D, Caraglia M, Vincenzi B, Holen I, Scarpa S, Budillon A, Tonini G (2006) Mechanisms of disease: preclinical reports of antineoplastic synergistic action of bisphosphonates. Nat Clin Pract Oncol 3(6):325–338
Neville-Webbe HL, Rostami-Hodjegan A, Evans CA, Coleman RE, Holen I (2005) Sequence and schedule-dependent enhancement of zoledronic acid induced apoptosis by doxorubicin in breast and prostate cancer cells. Int J Cancer 113:364–371
Ullen A, Lennartsson L, Harmenberg U, Hjelm-Eriksson M, Kalkner KM, Lennernas B (2005) Additive/synergistic anti-tumoral effects on prostate cancer cells in vitro following treatment with a combination of docetaxel and zoledronic acid. Acta Oncol 44(6):644–650
Tassone P, Forciniti S, Galea E, Morrone G, Turco MC, Martinelli V, Venuta S (2000) Growth inhibition and synergistic induction of apoptosis by zoledronate and dexamethasone in human myeloma cell lines. Leukaemia 14:841–844
Brubaker KD, Brown LG, Vessella RL, Corey E (2006) Administration of zoledronic acid enhances the effects of docetaxel on growth of prostate cancer in the bone environment. BMC Cancer 6:15
Kroger N, Achterrath W, Hegewisch-Becker S, Mross K, Zander AR (1999) Current options in treatment of anthracycline-resistant breast cancer. Cancer Treat Rev 25:279–291
Fornari FA Jr, Jarvis DW, Grant S, Orr MS, Randolph JK, White FK, Gewirtz DA (1996) Growth arrest and non-apoptotic cell death associated with the suppression of c-myc expression in MCF-7 breast tumor cells following acute exposure to doxorubicin. Biochem Pharmacol 51(7):931–940
Potter AJ, Gollahon KA, Palanca BJ, Harbert MJ, Choi YM, Moskovitz AH, Potter JD, Rabinovitch PS (2002) Flow cytometric analysis of the cell cycle phase specificity of DNA damage induced by radiation, hydrogen peroxide and doxorubicin. Carcinogenesis 23:389–401
Tewey KM, Rowe TC, Yang L, Halligan BD, Liu LF (1984) Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II. Science 226:466–468
Fornari FA, Randolph JK, Yalowich JC, Ritke MK, Gewirtz DA (1994) Interference by doxorubicin with DNA unwinding in MCF-7 breast tumour cells. Mol Pharmacol 45:649–656
Fornari FA Jr, Jarvis WD, Grant S, Orr MS, Randolph JK, White FK, Mumaw VR, Lovings ET, Freeman RH, Gewirtz DA (1994) Induction of differentiation and growth arrest associated with nascent (nonoligosomal) DNA fragmentation and reduced c-myc expression in MCF-7 human breast tumour cells after continuous exposure to a sublethal concentration of doxorubicin. Cell Growth Differ 5:723–733
Goldenberg GJ, Wang H, Blair GW (1986) Resistance to adriamycin: relationship of cytotoxicity to drug uptake and DNA single-and double-strand breakage in cloned cell lines of adriamycin-sensitive and-resistant P388 leukaemia. Cancer Res 46:2978–2983
Petrioli R, Fiaschi AI, Francini E, Pascucci A, Francini G (2008) The role of doxorubicin and epirubicin in the treatment of patients with metastatic hormone-refractory prostate cancer. Cancer Treat Rev 34(8):710–718
Mike S, Harrison C, Coles B, Staffurth J, Wilt TJ, Mason MD (2006) Chemotherapy for hormone-refractory prostate cancer. Cochrane Database Syst Rev 18(4):CD005247
Oades GM, Senaratne SG, Clarke IA, Kirby RS, Colston KW (2003) Nitrogen containing bisphosphonates induce apoptosis and inhibit the mevalonate pathway, impairing RAS membrane localization in prostate cancer cells. The J Urol 170:246–252
Brown JE, Cook RJ, Major P et al (2005) Bone turnover markers as predictors of skeletal complications in prostate cancer, lung cancer, and other solid tumors. J Natl Cancer Inst 97:59–69
Thudi NK, Martin CK, Nadella MV, Fernandez SA, Werbeck JL, Pinzone JJ, Rosol TJ (2008) Zoledronic acid decreased osteolysis but not bone metastasis in a nude mouse model of canine prostate cancer with mixed bone lesions. Prostate 68(10):1116–1125
Corey E, Brown LG, Quinn JE, Poot M, Roudier MP, Higano CS, Vessella RL (2003) Zoledronic acid exhibits inhibitory effects on osteoblastic and osteolytic metastases of prostate cancer. Clin Cancer Res 9(1):295–306
Chung LW, Baseman A, Assikis V, Zhau HE (2005) Molecular insights into prostate cancer progression: the missing link of tumor microenvironment. J Urol 173(1):10–20
Clines GA, Guise TA (2008) Molecular mechanisms and treatment of bone metastasis. Expert Rev Mol Med 6(10):e7
Sun-Jin K, Hisanori U, Sertac Y, Junqin H, Robert RL, Paul M, Dominic F, Isaiah J (2005) Fidler modulation of bone microenvironment with zoledronate enhances the therapeutic effects of STI571 and paclitaxel against experimental bone metastasis of human prostate cancer. Cancer Res 65:3707–3715
van Beek ER, Lowik CW, van Wijngaarden J, Ebetino FH, Papapoulos SE (2008) Synergistic effect of bisphosphonate and docetaxel on the growth of bone metastasis in an animal model of established metastatic bone disease. Breast Cancer Res Treat [Epub ahead of print]
Jagdev SP, Coleman RE, Shipman CM, Rostami HA, Croucher PI (2001) The bisphosphonate, zoledronic acid, induces apoptosis of breast cancer cells: evidence for synergy with Paclitaxel. Br J Cancer 84(8):1126–1134
Pandha H, Birchall L, Meyer B, Wilson N, Relph K, Anderson C, Harrington K (2006) Antitumor effects of aminobisphosphonates on renal cell carcinoma cell lines. J Urol 176:2255–2261
Matsumoto S, Kimura S, Segawa H, Kuroda J, Yuasa T, Sato K, Nogawa M, Tanaka F, Maekawa T, Wada H (2005) Efficacy of the third-generation bisphosphonate, zoledronic acid alone and combined with anti-cancer agents against small cell lung cancer cell lines. Lung Cancer 47:31–39
Budman DR, Calabro A (2006) Zoledronic acid (Zometa) enhances the cytotoxic effect of gemcitabine and fluvastatin: in vitro isobologram studies with conventional and nonconventional cytotoxic agents. Oncology 70(2):147–153
Acknowledgments
R.D. Clyburn was sponsored by bursary by Yorkshire Cancer Research, UK. P. Reid is funded by a grant from Weston Park Hospital Cancer Charity, Sheffield, UK.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Clyburn, R.D., Reid, P., Evans, C.A. et al. Increased anti-tumour effects of doxorubicin and zoledronic acid in prostate cancer cells in vitro: supporting the benefits of combination therapy. Cancer Chemother Pharmacol 65, 969–978 (2010). https://doi.org/10.1007/s00280-009-1106-6
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00280-009-1106-6