A. Ugur Ural

The bisphosphonate zoledronic acid induces cytotoxicity in human myeloma cell lines with enhancing effects of dexamethasone and thalidomide.

Bisphosphonates have recently been introduced in the therapeutic armamentarium for long-term treatment of patients with multiple myeloma. These pyrophosphate analogs not only reduce the occurrence of skeletal events but also provide clinical benefit to patients and improve the survival of some of them. The existence of these capabilities raises the possibility that these compounds may have a direct antiproliferative effect on tumor cells. To investigate whether these drugs exert a direct antitumor effect, we exposed human myeloma cell lines ARH-77 and RPMI-8226 to increasing concentrations of zoledronic acid (ZOL) in vitro. A concentration- but not time-dependent cytotoxic effect was detected with drug treatment of ARH-77 and RPMI-8226 cell lines (30% and 60% at 48 hours and 38% and 62% at 72 hours, respectively, for 50µM of ZOL). Cytotoxicity was not due to ZOL-induced chelation of extracellular calcium as shown by control experiments with the calcium chelator ethylene glycol-bis(β-aminoethylether)-N,N,N’,N’-tetraacetic acid. Addition of the competitive inhibitor of the nitric oxide synthase Nω-nitro-L-arginine methyl ester did not modulate ZOL-induced cytotoxicity. However, a decrease in the number of apoptotic cells was detected when protein kinase C was inhibited by addition of staurosporine to ZOL-containing cultures. Cytotoxicity also was increased by addition of dexamethasone (Dex) and thalidomide (Thal) to ARH-77 and RPMI-8226 cultures. We demonstrated that exposing myeloma cell lines ARH-77 and RPMI-8226 to ZOL inhibits cell growth in a dose-dependent but not a time-dependent manner and that combination of Dex and Thal with ZOL induces apoptotic cell death, providing a rationale for potential applications in vivo.

Association of plasma adiponectin concentrations with chronic lymphocytic leukemia and myeloproliferative diseases.

Adiponectin, an adipocyte-secreted hormone, is an important negative regulator in the immune system and hematopoiesis. In this study, we investigated the association of adiponectin levels with chronic lymphocytic leukemia (CLL) and myeloproliferative diseases (MPDs). We measured adiponectin levels in 19 patients with CLL and 30 patients with MPD (chronic myelogenous leukemia, 15; polycythemia vera, 9; myelofibrosis, 4; essential thrombocythemia, 2). The data were compared with results from a control group of healthy volunteers who were matched according to age, sex, and body mass index. The adiponectin levels in patients with CLL were lower than in the controls (4.71 ± 1.33 μg/mL versus 16.61 ± 3.91 μg/mL; P < .001). They were also significantly lower in patients with MPD than in the controls (8.95 ± 2.64 μg/mL versus 17.16 ± 4.77 μg/mL; P < .001). In addition, we compared the adiponectin levels of MPD patients who were treated with interferon (IFN) to the levels of patients who were not treated with IFN. Adiponectin levels were significantly higher in IFN-treated patients (11.03 ± 1.39 μg/mL versus 6.87 ± 1.79 μg/mL; P < .001). These results suggest that lymphopoiesis and myelopoiesis negatively influence adiponectin levels. Adiponectin may be related to inflammatory cytokine release. IFN therapy appears to have a positive influence on adiponectin secretion by suppressing inflammatory cytokines. Future studies are needed to prove causality and to provide insight about this hormone’s mechanism of action and its potential role regarding the etiology and progression of CLL and MPD.

In vitro synergistic cytoreductive effects of zoledronic acid and radiation on breast cancer cells

IntroductionBisphosphonates are mostly used in the treatment of bone metastases. They have been shown to act synergistically with other chemotherapeutic agents. It is not known, however, whether similar synergistic effects exist with radiation on breast cancer cells.MethodsHuman MCF-7 breast cancer cells were treated with up to 100 μM zoledronic acid, were irradiated with up to 800 cGy or were exposed to combinations of both treatments to determine the antiproliferative effects of zoledronic acid and radiation.ResultsZoledronic acid and radiation caused a dose-dependent and time-dependent decrease in cell viability (approximate 50% growth inhibition values were 48 μM and 20 μM for 24 hours and 72 hours, respectively, for zoledronic acid and 500 cGy for radiation). A synergistic cytotoxic effect of the combination of zoledronic acid and radiation was confirmed by isobologram analysis.ConclusionThese data constitute the first in vitro evidence for synergistic effects between zoledronic acid and radiation. This combination therapy might thus be expected to be more effective than either treatment alone in patients with metastatic breast carcinoma.

Bisphosphonate treatment and radiotherapy in metastatic breast cancer

Patients with advanced breast cancer frequently develop metastasis to bone. Bone metastasis results in intractable pain and high risk of pathologic fractures due to osteolysis. The treatment of breast cancer patients with bone metastases requires a multidisciplinary approach. Radiotherapy is an established treatment for metastatic bone pain. It may be delivered either as a localized low dose treatment for localized bone pain or systemically for more widespread symptoms. Bisphosphonates have been shown to reduce morbidity and bone pain from bone metastases when given to patients with metastatic bone disease. In vivo studies indicate that early bisphosphonates administration in combination with radiotherapy improves remineralization and restabilization of osteolytic bone metastases in animal tumor models. This review focused on a brief discussion about biology of bone metastases, the effects of radiotherapy and bisphosphonate therapy, and possible mechanisms of combination therapy in metastatic breast cancer patients.

Aberrant expressions of leptin and adiponectin receptor isoforms in chronic myeloid leukemia patients

BACKGROUND Leptin and adiponectin receptors mediate the role of leptin in stimulating the growth of leukemic cells and the protective function of adiponectin undertaken in several malignancies such as leukemia. In this study, we investigated the involvement of the expression of leptin and adiponectin receptors in chronic myeloid leukemia (CML) pathogenesis. METHODS The expression of leptin receptor isoforms, OB-Rt, OB-Ra, and OB-Rb, and the expression of adiponectin receptors, AdipoR1 and AdipoR2, were measured as mRNA levels in two CML cell lines (K562 and Meg-01) and 20 CML patients and 24 healthy controls by using RT-PCR. RESULTS OB-Rt and OB-Ra isoforms expression of the leptin receptors were found to be significantly lower in Meg-01 cell lines than K562 cells. All leptin receptors were downregulated in CML patients and more particularly OB-Rb level was found to be undetectably low in normal PBMC as well as in CML patients. AdipoR1 expression level was higher in Meg-01 than in K562, whereas AdipoR2 level was found to be unchanged in both cell lines. Interestingly, while AdipoR1 expression increased in CML patients, AdipoR2 decreased. Moreover, imatinib therapy did not affect both leptin and adiponectin isoform expressions. CONCLUSION While the decrease in leptin receptor levels in CML patients was confirmed, the increase in AdipoR1 levels and relevant decrease in AdipoR2 levels depicted their possible involvement in CML pathogenesis. This suggests different functions of adiponectin receptors in CML development.

Treatment with Cerivastatin in Primary Mixed Hyperlipidemia Induces Changes in Platelet Aggregation and Coagulation System Components

Platelet activation, impairment of fibrinolysis, activation of the coagulation pathway, and dyslipidemia are important factors in the pathogenesis and progression of ischemic heart disease, and patients generally need to use an antiplatelet agent. Lipid-lowering cerivastatin, a novel 3-hydroxy-3-methylglutaryl coenzyme A reductase inhibitor, was administered to 20 patients with primary mixed hyperlipidemia for the assessment of the effect of cerivastatin on lipid levels, plasma fibrinogen concentration, factor VII, VIII, and X levels, plasminogen and antiplasmin concentrations, platelet count, and aggregation (adenosine diphosphate [ADP], collagen, and epinephrine induced). Assessments were made immediately after 2 months of a standard lipid-lowering diet, 4 weeks of placebo administration, and 4 weeks of cerivastatin treatment. Cerivastatin achieved significant reductions in triglyceride, total cholesterol, and low-density lipoprotein cholesterol levels. The significant improvement of the lipid profile was associated with platelet aggregation reduction in vitro stimulated by ADP, collagen, and epinephrine (P > .05,P = .05,P > .005, respectively). Significantly lower levels of factor VII and fibrinogen were observed (P = .001,P > .0001) immediately after cerivastatin treatment. No significant differences were detected in factor VIII level, plasminogen and antiplasmin concentrations, and platelet count after cerivastatin treatment. It was concluded that cerivastatin in mixed hyperlipidemia can exert beneficial changes on specific hemostatic variables and platelet aggregation in addition to its positive effects on plasma lipid values.

Effect of cobalt-60 (γ radiation) on multidrug-resistant multiple myeloma cell lines

Emergence of resistance to chemotherapy and radiotherapy is a major obstacle for the successful treatment of MM (multiple myeloma). Prednisone, vincristine and melphalan are commonly used chemotherapeutic agents for the treatment of MM. In the current study, we examined the presence of possible cross‐resistance between these drugs and gamma (γ) radiation. Prednisone, vincristine and melphalan resistant RPMI‐8226 and U‐266 MM cells were generated by stepwise increasing concentrations of the drugs. The sensitive and resistant cells were exposed to 200‐ and 800 cGy γ radiation, and proliferation was examined by XTT {2,3‐bis(2‐methoxy‐4‐nitro‐5‐sulfophenyl)‐5‐[(phenylamino)carbonyl]‐2H‐tetrazolium hydroxide} assay. The results showed that Prednisone‐ and melphalan‐resistant RPMI‐8226 cells were also cross‐resistant to 200 and 800 cGy γ radiation application, while vincristine‐resistant cells did not show resistance. On the other hand, Prednisone‐, vincristine‐ and melphalan‐resistant U‐266 cells showed cross‐resistance to 200‐ and 800 cGy γ radiation application. These results demonstrated that MM cells resistant to anticancer agents respond to radiation in different levels. These findings may be important in the clinical applications of radiation therapy in the treatment of vincristine resistant MM.

Additive/synergistic anti-tumoral effects of the combination of docetaxel and zoledronic acid on prostate cancer cells: possible mechanisms?

We read the interesting article by Ullen et al. [1] on the additive/synergistic effects of zoledronic acid (ZOL) combined with docetaxel (DOC) on prostate cancer cells, published in the September issue of Acta Oncologica . We especially appreciate their approach to study the anti-tumoral effects of ZOL alone and in combination with DOC on prostate cancer cells. In addition to showing that ZOL possesses anti-tumoral effects in terms of proliferation inhibition and apoptosis induction, they also reported the potential of ZOL and DOC to exert super-additive anti-tumoral effects on two hormonerefractory prostate cancer cell lines. They found an additive effect for PC3 cells when combining DOC and ZOL at concentrations of 1 ng/ml and 25 mM, respectively, whereas a synergistic anti-tumoral effect on DU145 cells was observed with DOC and ZOL at concentrations of 0.01 ng/ml and 50 mM, respectively. However, there was no mention of novel chemosensitizing effects of bisphosphonates (BPs) or possible mechanisms of action. This study supports the findings of other studies which showed that BPs act synergistically with other chemotherapeutic agents [2,3], a notion that further supports the combined use of BPs as chemosensitizing agents. As is well known, BPs exhibit a high affinity for calcified matrices, such as hydroxyapatite in bone, and are used successfully as powerful inhibitors of increased bone resorption in several bone diseases including Paget’s disease of bone, osteoporosis, and tumor-associated bone diseases. [4]. Therefore, BPs are used to decrease skeletal-related complications for a number of tumors including breast, prostate and multiple myeloma, leading to improved quality of life [5,6]. Although ZOL clearly has cytotoxic effects on prostate cancer cells in vitro, these may be mediated by the compound’s ability to chelate calcium. Our previous study with EGTA, which also chelates calcium, demonstrated only a small effect on the reduction on cell number which remained significantly different from the greater effect observed following ZOL treatment [3]. Addition of EGTA to ZOL-containing cultures showed a significant decrease in cytotoxicity rather than an enhanced cytotoxic effect, which one would have expected if the mechanism was via calcium chelation. These data suggest that a decrease in extracellular calcium can actually protect cells from these drugs. The BP alendronate induces calcium leakiness in osteoclasts that leads to a rise in free intracellular calcium. Such a rise in calcium has been previously suggested to be an inhibitory signal for cells [7]. Furthermore, increases in calcium have been implicated as second messengers during the induction of apoptosis [8]. In our study, we demonstrated that ZOL induced antiproliferative and apoptotic effects on MM cell lines in vitro by activating protein kinase C, and these effects were augmented when dexamethasone and thalidomide were combined with ZOL [3]. In

Bisphosphonates may retrieve endothelial function in vascular diseases similar to statins' effects.

To the Editor: We read the article by Koizumi et al. on the apoptotic effects and possible mechanisms of zoledronate (ZOL) in dexamethasone-resistant multiple myeloma cells (Dex-R) with great interest (1). They concluded that ZOL reduced the viability and induced apoptosis of Dex-R cells by inducing actin fiber rearrangement and unprenylating portions of the small G proteins. These results are consistent with our previous studies (2). The authors demostrated that inhibition of farnesylpyrophosphate (FPP) synthase by ZOL results in reduced production of isoprenoid intermediates like geranylgeranylpyrophosphate (GGPP) and FPP. They observed that the addition of geranylgeraniol (GGOH), which is essential for geranylgeranylation and for membrane attachment of small GTP-binding proteins such as Rho and Rap1A, inhibited the viability-suppressing, apoptotic and actin fiber-disrupting effects of ZOL. Authors’ these important and novel findings raise the possibility that pleitropic effects of bisphosphonates can be attributed to their effects on mevalonate pathway which is also important in the regulation of endothelial function (Fig. 1). Endothelial dysfunction is associated with vascular diseases may result from impairment of the synthesis, release, or activity of endothelium-derived vasorelaxing factors including nitric oxide (NO). Endothelium-derived NO inhibits several components of the atherogenic process including monocyte adhesion to the endothelial surface, platelet aggregation, vascular smooth muscle cell proliferation, and vasoconstriction. Therefore, the NO production within the endothelium is served as a target system in the treatment of endothelial dysfunction associated with the vascular diseases. The possible role of RhoA-Rho-kinase system in the regulation of endothelial NO was provided by the observation that RhoA can negatively regulate eNOS (endothelial NO synthase) mRNA expression in human cultured endothelial cells (3). Therefore, Rho-kinase might inhibit NO production within the endothelium, and thus excessive endothelial Rho-kinase activity might also contribute to endothelial dysfunction and atherosclerosis. In mevalonate pathway, inhibition of 3-hydroxy-3methylglutaryl coenzyme A (HMG-CoA) reductase by statins, widely used to lower elevated plasma cholesterol levels, not only reduces plasma cholesterol levels, but also decreases intracellular levels of isoprenoid intermediates. Therefore, the protective effects of statins on the endothelium can be attributed the rapid restoration of NO bioactivity by upregulation and activation of eNOS and the enhancement of NO release by inhibiting of

Immunosuppressive effects of multipotent mesenchymal stromal cells on graft-versus-host disease in rats following allogeneic bone marrow transplantation.

Objective: Graft-versus-host disease (GVHD) is a major obstacle to successful allogeneic bone marrow transplantation (allo-BMT). While multipotent mesenchymal stromal cells (MSCs) demonstrate alloresponse in vitro and in vivo, they also have clinical applications toward prevention or treatment of GVHD. The aim of this study was to investigate the ability of MSCs to prevent or treat GVHD in a rat BMT model. Materials and Methods: The GVHD model was established by transplantation of Sprague Dawley rats’ bone marrow and spleen cells into lethally irradiated (950 cGy) SDxWistar rat recipients. A total of 49 rats were randomly assigned to 4 study and 3 control groups administered different GVHD prophylactic regimens including MSCs. After transplantation, clinical GVHD scores and survival status were monitored. Results: All irradiated and untreated control mice with GVHD died. MSCs inhibited lethal GVHD as efficiently as the standard GVHD prophylactic regimen. The gross and histopathological findings of GVHD and the ratio of CD4/CD8 expression decreased. The subgroup given MSCs displayed higher in vivo proportions of CD25+ T cells and plasma interleukin-2 levels as compared to conventional GVHD treatment after allo-BMT. Conclusion: Our results suggest that clinical use of MSCs in both prophylaxis against and treatment of established GVHD is effective. This study supports the use of MSCs in the prophylaxis and treatment of GVHD after allo-BMT; however, large scale studies are needed. Conflict of interest:None declared.