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Targeting of apoptotic pathways in cancer cells by zoledronic acid
S102
ABSTRACTS / Bone 42 (2008) S17–S110
(P = .036). Histologic analysis revealed marginal differences in number of bone metastases between T + ZOL and vehicle (P = .058) and between combination treatment and vehicle. There was a trend toward differences in survival between groups (P = .151), and survival was significantly longer for the T + ZOL group vs vehicle (P = .022). Compared with vehicle and ZOL alone, cancerous cells in the bone of mice treated with T + ZOL expressed higher levels of Bax, and lower levels of Bcl-2 and Bcl-XL. ZOL produced a trend toward reduced NTX levels vs vehicle, and T + ZOL produced a profound reduction in NTX vs vehicle (P = .022). Conclusions: The results of this study suggest that ZOL + T is an effective combination that can reduce the incidence of bone metastasis from lung cancer and prolong survival in a mouse model of non-small cell lung cancer with a high potential for metastasis to bone.
significantly from each other in their BP-induced IPP and ATP analog accumulation: human estrogen-dependent (MCF7) and estrogen-independent (MDAMB436) breast cancer cell lines, human myeloma RPMI826 and human follicular lymphoma cell line (HF28RA). Determinations of apoptotic features upon zoledronic acid treatment were analyzed using flow cytometry assay for phosphatidylserine exposure, cell cycle analysis, mitochondrial potential, caspase activation and late apoptosis/ necrosis. Specific ApppI inhibitions with geranyl geraniol and statins (lovastatin) were perormed in order to elucidate its apoptosis involvement. Consequent mass spectrometry analyzes have been performed. ApppI/IPP accumulation seems to correlate with apoptosis but obviously multiple pathways are involved in zoledronate induced apoptosis in cancer cells.
doi:10.1016/j.bone.2007.12.195
[1] Mönkkönen, et al. Br J Pharmacol (2006) 147, 437–445. [2] Mönkkönen, et al. Bone (2006) 38 (suppl 1), S58.
186 Targeting of apoptotic pathways in cancer cells by zoledronic acid Laura Mitrofan-Oprea a, Marjo Jauhiainen b, Jukka Pelkonen c, Jukka Mönkkönen a a Department of Pharmaceutics, Faculty of Pharmacy, Kuopio, Finland b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuopio, Finland c Department of Clinical Microbiology, Faculty of Medicine, Kuopio, Finland
doi:10.1016/j.bone.2007.12.196
Biphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumour bone metastases. However, it is unclear whether this reflects an indirect effect via inhibition of bone resorption or a direct anti-tumour effect. Additionally, a better understanding of diverse modes of cell death induced by this class of drugs will provide further insight of the molecular events that account for the active bi-directional response between bone and associated tumor cells. Nitrogen-containing biphosphonates (N-BPs) act by inhibiting farnesyl pyrophosphate synthase (FPPs), one of the key enzymes of the intracellular mevalonate pathway. The pathway is blocked and accumulation of a pathway intermediate, isopentenyl pyrophosphate (IPP) consequently occurs. IPP becomes conjugates to AMP to form a novel ATP analogue (ApppI). The only characteristic of ApppI to promote apoptosis is so far linked to its potential to inhibit the mitochondrial adenine nucleotide translocase (ANT) [1] in a cell free system. However its significance in apoptosis is unclear even if the ability of ZOL or other biphosphonates to induce apoptosis in different cell types have been widely investigated. The purpose of the present study was to see whether ApppI has a direct involvement in cellular death. There are marked differences in zoledronic acid induced ApppI formation between different cancer cell lines as have been shown recently [2]. On this base we selected four cancer cell lines that differ
References
187 Zoledronic acid-induced IPP accumulation in cancer cells strongly correlates with gammadelta T-Cell mediated cancer cell death Hannu Monkkonen a, Ismahene Benzaid a, Verena Stresing a, Jonathan Green b, Jukka Monkkonen c, Jean L. Touraine d, Philippe Clezardin a a Faculty of Medicin, INSERM, Research Unit 664, Lyon, France b Novartis Pharma AG, Basle, Switzerland c Department of Pharmaceutics, University of Kuopio, Kuopio, Finland d Laboratory of Immunology and Transplantation, Hospital Edouard Herriot Pavillon P, Lyon, France Nitrogen-containing bisphosphonates (N-BPs), such as zoledronic acid (ZOL), have the ability to modulate the immune system. The internalization of N-BPs by PBMC (peripheral blood mononuclear cells) or cancer cells causes the intracellular accumulation of isopentenyl pyrophosphate (IPP) which, in turn, activates the expansion of human Vγ9Vδ2 T-cells, the dominant subset of gammadelta T-cells. Vγ9Vδ2 T-cells then exhibit strong cytotoxic activity against N-BP-treated cancer cells. However, the ability of N-BP-treated cells to activate Vγ9Vδ2 T-cells varies a lot from one cancer cell line to another. In the current study, we investigated whether the potency of Vγ9Vδ2 T-cells to kill cancer cells was dependent on intracellular IPP levels in ZOL-treated cancer cells. Different human breast cancer cells lines (MDA-MB-231, MDA-MB-435, MDA-MB-435S, MDA-MB-BO2, Hs578T, MCF-7, and T47D) were treated with 25 μM ZOL for 1h. The cultured cells were extracted 6 and 18 h after treatment using acetonitrile and the molar amount of IPP was determined in cell extracts by mass spectrometry. PBMC were isolated from healthy donors.
ABSTRACTS / Bone 42 (2008) S17–S110
(P = .036). Histologic analysis revealed marginal differences in number of bone metastases between T + ZOL and vehicle (P = .058) and between combination treatment and vehicle. There was a trend toward differences in survival between groups (P = .151), and survival was significantly longer for the T + ZOL group vs vehicle (P = .022). Compared with vehicle and ZOL alone, cancerous cells in the bone of mice treated with T + ZOL expressed higher levels of Bax, and lower levels of Bcl-2 and Bcl-XL. ZOL produced a trend toward reduced NTX levels vs vehicle, and T + ZOL produced a profound reduction in NTX vs vehicle (P = .022). Conclusions: The results of this study suggest that ZOL + T is an effective combination that can reduce the incidence of bone metastasis from lung cancer and prolong survival in a mouse model of non-small cell lung cancer with a high potential for metastasis to bone.
significantly from each other in their BP-induced IPP and ATP analog accumulation: human estrogen-dependent (MCF7) and estrogen-independent (MDAMB436) breast cancer cell lines, human myeloma RPMI826 and human follicular lymphoma cell line (HF28RA). Determinations of apoptotic features upon zoledronic acid treatment were analyzed using flow cytometry assay for phosphatidylserine exposure, cell cycle analysis, mitochondrial potential, caspase activation and late apoptosis/ necrosis. Specific ApppI inhibitions with geranyl geraniol and statins (lovastatin) were perormed in order to elucidate its apoptosis involvement. Consequent mass spectrometry analyzes have been performed. ApppI/IPP accumulation seems to correlate with apoptosis but obviously multiple pathways are involved in zoledronate induced apoptosis in cancer cells.
doi:10.1016/j.bone.2007.12.195
[1] Mönkkönen, et al. Br J Pharmacol (2006) 147, 437–445. [2] Mönkkönen, et al. Bone (2006) 38 (suppl 1), S58.
186 Targeting of apoptotic pathways in cancer cells by zoledronic acid Laura Mitrofan-Oprea a, Marjo Jauhiainen b, Jukka Pelkonen c, Jukka Mönkkönen a a Department of Pharmaceutics, Faculty of Pharmacy, Kuopio, Finland b Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Kuopio, Finland c Department of Clinical Microbiology, Faculty of Medicine, Kuopio, Finland
doi:10.1016/j.bone.2007.12.196
Biphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumour bone metastases. However, it is unclear whether this reflects an indirect effect via inhibition of bone resorption or a direct anti-tumour effect. Additionally, a better understanding of diverse modes of cell death induced by this class of drugs will provide further insight of the molecular events that account for the active bi-directional response between bone and associated tumor cells. Nitrogen-containing biphosphonates (N-BPs) act by inhibiting farnesyl pyrophosphate synthase (FPPs), one of the key enzymes of the intracellular mevalonate pathway. The pathway is blocked and accumulation of a pathway intermediate, isopentenyl pyrophosphate (IPP) consequently occurs. IPP becomes conjugates to AMP to form a novel ATP analogue (ApppI). The only characteristic of ApppI to promote apoptosis is so far linked to its potential to inhibit the mitochondrial adenine nucleotide translocase (ANT) [1] in a cell free system. However its significance in apoptosis is unclear even if the ability of ZOL or other biphosphonates to induce apoptosis in different cell types have been widely investigated. The purpose of the present study was to see whether ApppI has a direct involvement in cellular death. There are marked differences in zoledronic acid induced ApppI formation between different cancer cell lines as have been shown recently [2]. On this base we selected four cancer cell lines that differ
References
187 Zoledronic acid-induced IPP accumulation in cancer cells strongly correlates with gammadelta T-Cell mediated cancer cell death Hannu Monkkonen a, Ismahene Benzaid a, Verena Stresing a, Jonathan Green b, Jukka Monkkonen c, Jean L. Touraine d, Philippe Clezardin a a Faculty of Medicin, INSERM, Research Unit 664, Lyon, France b Novartis Pharma AG, Basle, Switzerland c Department of Pharmaceutics, University of Kuopio, Kuopio, Finland d Laboratory of Immunology and Transplantation, Hospital Edouard Herriot Pavillon P, Lyon, France Nitrogen-containing bisphosphonates (N-BPs), such as zoledronic acid (ZOL), have the ability to modulate the immune system. The internalization of N-BPs by PBMC (peripheral blood mononuclear cells) or cancer cells causes the intracellular accumulation of isopentenyl pyrophosphate (IPP) which, in turn, activates the expansion of human Vγ9Vδ2 T-cells, the dominant subset of gammadelta T-cells. Vγ9Vδ2 T-cells then exhibit strong cytotoxic activity against N-BP-treated cancer cells. However, the ability of N-BP-treated cells to activate Vγ9Vδ2 T-cells varies a lot from one cancer cell line to another. In the current study, we investigated whether the potency of Vγ9Vδ2 T-cells to kill cancer cells was dependent on intracellular IPP levels in ZOL-treated cancer cells. Different human breast cancer cells lines (MDA-MB-231, MDA-MB-435, MDA-MB-435S, MDA-MB-BO2, Hs578T, MCF-7, and T47D) were treated with 25 μM ZOL for 1h. The cultured cells were extracted 6 and 18 h after treatment using acetonitrile and the molar amount of IPP was determined in cell extracts by mass spectrometry. PBMC were isolated from healthy donors.