Circulating endothelial progenitor cells in castration resistant prostate cancer: a randomized, controlled, biomarker study.

BACKGROUND: Endothelial progenitor cells (CEPs) and circulating endothelial cells (CECs) are potential biomarkers of response to anti-angiogenic treatment regimens. In the current study, we investigated the effect of docetaxel and sunitinib on CEP/CEC kinetics and clinical response in castration resistant prostate cancer (CRPC) patients. PATIENTS AND METHODS: Chemonaive patients with CRPC were enrolled in this study to receive either sunitinib (37.5 mg/d), in combination with docetaxel (75 mg/m2) or docetaxel alone. CEP and CEC kinetics were analyzed for every cycle. The primary objective was to compare CEP/CEC pharmacodynamics between both treatment arms. We also investigated if CEC/CEP spikes, induced by MTD docetaxel, are suppressed by sunitinib in patients treated with docetaxel/sunitinib relative to docetaxel monotherapy. RESULTS: A total of 27 patients were enrolled. We observed a significant increase of CEP/CEC (total/viable) counts over time within each cycle (coefficients 0.29233, 0.22092 and 0.26089, respectively; p<0.001). However, no differences between the treatment groups, in terms of CEP and CEC kinetics, were detected. In the docetaxel monotherapy arm 4 (30%) patients responded to therapy with a 50% PSA decline, while 9 (64%) patients showed a PSA decline in the combination group (n.s.). The median PFS in the docetaxel monotherapy group was 3.1 months (2.6-3.6 months, 95% CI) and 6.2 months (4.9-7.4 months, 95% CI; p = 0.062) in the combination arm. Sunitinib/docetaxel was reasonably well tolerated and toxicity manageable. CONCLUSION: In summary, no significant differences in CEC and CEP kinetics between the treatment arms were observed, although a highly significant increase of CEPs/CECs within each cycle over time was detected. These results mirror the challenge we have to face when employing anti-angiogenic strategies in CRPC. Additional preclinical research is needed to elucidate the underlying molecular mechanisms. However, docetaxel/sunitinib therapy resulted in a better response in terms of PSA decline and a trend towards improved PFS. TRIAL REGISTERY: clinicaltrialsregister.eu EudraCT 2007-003705-27.

Phenotyping of human melanoma cells reveals a unique composition of receptor targets and a subpopulation co-expressing ErbB4, EPO-R and NGF-R.

Malignant melanoma is a life-threatening skin cancer increasingly diagnosed in the western world. In advanced disease the prognosis is grave. Growth and metastasis formation in melanomas are regulated by a network of cytokines, cytokine-receptors, and adhesion molecules. However, little is known about surface antigens and target expression profiles in human melanomas. We examined the cell surface antigen profile of human skin melanoma cells by multicolor flow cytometry, and compared their phenotype with 4 melanoma cell lines (A375, 607B, Mel-Juso, SK-Mel28). Melanoma cells were defined as CD45-/CD31- cells co-expressing one or more melanoma-related antigens (CD63, CD146, CD166). In most patients, melanoma cells exhibited ErbB3/Her3, CD44/Pgp-1, ICAM-1/CD54 and IGF-1-R/CD221, but did not express CD20, ErbB2/Her2, KIT/CD117, AC133/CD133 or MDR-1/CD243. Melanoma cell lines were found to display a similar phenotype. In most patients, a distinct subpopulation of melanoma cells (4-40%) expressed the erythropoietin receptor (EPO-R) and ErbB4 together with PD-1 and NGF-R/CD271. Both the EPO-R+ and EPO-R- subpopulations produced melanoma lesions in NOD/SCID IL-2Rgamma(null) (NSG) mice in first and secondary recipients. Normal skin melanocytes did not express ErbB4 or EPO-R, but expressed a functional KIT receptor (CD117) as well as NGF-R, ErbB3/Her3, IGF-1-R and CD44. In conclusion, melanoma cells display a unique composition of surface target antigens and cytokine receptors. Malignant transformation of melanomas is accompanied by loss of KIT and acquisition of EPO-R and ErbB4, both of which are co-expressed with NGF-R and PD-1 in distinct subfractions of melanoma cells. However, expression of EPO-R/ErbB4/PD-1 is not indicative of a selective melanoma-initiating potential.

Synthesis and preclinical evaluation of the radiolabeled P-glycoprotein inhibitor [(11)C]MC113.

OBJECTIVES: With the aim to develop a PET tracer to visualize P-glycoprotein (Pgp) expression levels in different organs, the Pgp inhibitor MC113 was labeled with (11)C and evaluated using small-animal PET. METHODS: [(11)C]MC113 was synthesized by reaction of O-desmethyl MC113 with [(11)C]methyl triflate. Small-animal PET was performed with [(11)C]MC113 in FVB wild-type and Mdr1a/b((-/-)) mice (n=3 per group) and in a mouse model of high (EMT6Ar1.0) and low (EMT6) Pgp expressing tumor grafts (n=5). In the tumor model, PET scans were performed before and after administration of the reference Pgp inhibitor tariquidar (15mg/kg). RESULTS: Brain uptake of [(11)C]MC113, expressed as area under the time-activity curve from time 0 to 60min (AUC(0-60)), was moderately but not significantly increased in Mdr1a/b((-/-)) compared with wild-type mice (mean±SD AUC(0-60), Mdr1a/b((-/-)): 88±7min, wild-type: 62±6min, P=0.100, Mann Whitney test). In the tumor model, AUC(0-60) values were not significantly different between EMT6Ar1.0 and EMT6 tumors. Neither in brain nor in tumors was activity concentration significantly changed in response to tariquidar administration. Half-maximum effect concentrations (IC(50)) for inhibition of Pgp-mediated rhodamine 123 efflux from CCRFvcr1000 cells were 375±60nM for MC113 versus 8.5±2.5nM for tariquidar. CONCLUSION: [(11)C]MC113 showed higher brain uptake in mice than previously described Pgp PET tracers, suggesting that [(11)C]MC113 was only to a low extent effluxed by Pgp. However, [(11)C]MC113 was found unsuitable to visualize Pgp expression levels presumably due to insufficiently high Pgp binding affinity of MC113 in relation to Pgp densities in brain and tumors.

The PI3-kinase/mTOR-targeting drug NVP-BEZ235 inhibits growth and IgE-dependent activation of human mast cells and basophils.

The phosphoinositide 3-kinase (PI3-kinase) and the mammalian target of rapamycin (mTOR) are two major signaling molecules involved in growth and activation of mast cells (MC) and basophils (BA). We examined the effects of the dual PI3-kinase/mTOR blocker NVP-BEZ235 on growth of normal and neoplastic BA and MC as well as immunoglobulin E (IgE)-dependent cell activation. Growth of MC and BA were determined by measuring (3)H-thymidine uptake and apoptosis. Cell activation was determined in histamine release experiments and by measuring upregulation of CD63 and CD203c after challenging with IgE plus anti-IgE or allergen. We found that NVP-BEZ235 exerts profound inhibitory effects on growth of primary and cloned neoplastic MC. In the MC leukemia cell line HMC-1, NVP-BEZ235 showed similar IC(50) values in the HMC-1.1 subclone lacking KIT D816V (0.025 µM) and the HMC-1.2 subclone expressing KIT D816V (0.005 µM). Moreover, NVP-BEZ235 was found to exert strong growth-inhibitory effects on neoplastic MC in a xenotransplant-mouse model employing NMR1-Foxn1(nu) mice. NVP-BEZ235 also exerted inhibitory effects on cytokine-dependent differentiation of normal BA and MC, but did not induce growth inhibition or apoptosis in mature MC or normal bone marrow cells. Finally, NVP-BEZ235 was found to inhibit IgE-dependent histamine release in BA and MC (IC(50) 0.5-1 µM) as well as anti-IgE-induced upregulation of CD203c in BA and IgE-dependent upregulation of CD63 in MC. In summary, NVP-BEZ235 produces growth-inhibitory effects in immature neoplastic MC and inhibits IgE-dependent activation of mature BA and MC. Whether these potentially beneficial drug effects have clinical implications is currently under investigation.

A comparative small-animal PET evaluation of [11C]tariquidar, [11C]elacridar and (R)-[11C]verapamil for detection of P-glycoprotein-expressing murine breast cancer.

PURPOSE: One important mechanism for chemoresistance of tumours is overexpression of the adenosine triphosphate-binding cassette transporter P-glycoprotein (Pgp). Pgp reduces intracellular concentrations of chemotherapeutic drugs. The aim of this study was to compare the suitability of the radiolabelled Pgp inhibitors [(11)C]tariquidar and [(11)C]elacridar with the Pgp substrate radiotracer (R)-[(11)C]verapamil for discriminating tumours expressing low and high levels of Pgp using small-animal PET imaging in a murine breast cancer model. METHODS: Murine mammary carcinoma cells (EMT6) were continuously exposed to doxorubicin to generate a Pgp-overexpressing, doxorubicin-resistant cell line (EMT6AR1.0 cells). Both cell lines were subcutaneously injected into female athymic nude mice. One week after implantation, animals underwent PET scans with [(11)C]tariquidar (n = 7), [(11)C]elacridar (n = 6) and (R)-[(11)C]verapamil (n = 7), before and after administration of unlabelled tariquidar (15 mg/kg). Pgp expression in tumour grafts was evaluated by Western blotting. RESULTS: [(11)C]Tariquidar showed significantly higher retention in Pgp-overexpressing EMT6AR1.0 compared with EMT6 tumours: the mean ± SD areas under the time-activity curves in scan 1 from time 0 to 60 min (AUC(0-60)) were 38.8 ± 2.2 min and 25.0 ± 5.3 min (p = 0.016, Wilcoxon matched pairs test). [(11)C]Elacridar and (R)-[(11)C]verapamil were not able to discriminate Pgp expression in tumour models. Following administration of unlabelled tariquidar, both EMT6Ar1.0 and EMT6 tumours showed increases in uptake of [(11)C]tariquidar, [(11)C]elacridar and (R)-[(11)C]verapamil. CONCLUSION: Among the tested radiotracers, [(11)C]tariquidar performed best in discriminating tumours expressing high and low levels of Pgp. Therefore [(11)C]tariquidar merits further investigation as a PET tracer to assess Pgp expression levels in solid tumours.

Gastric cancer growth control by BEZ235 in vivo does not correlate with PI3K/mTOR target inhibition but with [18F]FLT uptake.

PURPOSE: In this study, we tested the antitumor activity of the dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor BEZ235 against gastric cancer in vitro and in vivo. EXPERIMENTAL DESIGN: Gastric cancer cell lines (N87, MKN45, and MKN28) were incubated with BEZ235 and assessed for cell viability, cell cycle, and PI3K/mTOR target inhibition. In vivo, athymic nude mice were inoculated with N87, MKN28, or MKN45 cells and treated daily with BEZ235. 3'-Deoxy-3'-[(18)F]fluorothymidine ([(18)F]FLT) uptake was measured via small animal positron emission tomography (PET). RESULTS: In vitro, BEZ235 dose dependently decreased the cell viability of gastric cancer cell lines. The antiproliferative activity of BEZ235 was linked to a G(1) cell-cycle arrest. In vivo, BEZ235 treatment resulted in PI3K/mTOR target inhibition as shown by dephosphorylation of AKT and S6 protein in all xenograft models. However, BEZ235 treatment only inhibited tumor growth of N87 xenografts, whereas no antitumor effect was observed in the MKN28 and MKN45 xenograft models. Sensitivity to BEZ235 in vivo correlated with downregulation of the proliferation marker thymidine kinase 1. Accordingly, [(18)F]FLT uptake was only significantly reduced in the BEZ235-sensitive N87 xenograft model as measured by PET. CONCLUSION: In conclusion, in vivo sensitivity of gastric cancer xenografts to BEZ235 did not correlate with in vitro antiproliferative activity or in vivo PI3K/mTOR target inhibition by BEZ235. In contrast, [(18)F]FLT uptake was linked to BEZ235 in vivo sensitivity. Noninvasive [(18)F]FLT PET imaging might qualify as a novel marker for optimizing future clinical testing of dual PI3K/mTOR inhibitors.

Sphingosine kinase 1 is a relevant molecular target in gastric cancer.

Sphingosine kinase 1 (Sphk1), a lipid kinase implicated in cell transformation and tumor growth, is overexpressed in gastric cancer and is linked with a poor prognosis. The biological relevance of Sphk1 expression in gastric cancer is unclear. Here, we studied the functional significance of Sphk1 as a novel molecular target for gastric cancer by using an antisense oligonucleotide approach in vitro and in vivo. Gastric cancer cell lines (MKN28 and N87) were treated with Sphk1 with locked nucleic acid-antisense oligonucleotides (LNA-ASO). Sphk1 target regulation, cell growth, and apoptosis were assessed for single-agent Sphk1 LNA-ASO and for combinations with doxorubicin. Athymic nude mice xenografted with gastric cancer cells were treated with Sphk1 LNA and assessed for tumor growth and Sphk1 target regulation, in vivo. In vitro, nanomolar concentrations of Sphk1 LNA-ASO induced an approximately two-fold reduction in Sphk1 mRNA in both the cell lines. This resulted in a 1.6-fold increase in apoptosis and inhibited the growth of gastric cancer cells by more than 50% (P < 0.05). The combination of Sphk1 LNA-ASO with doxorubicin resulted in significant chemosensitization. In vivo, Sphk1 LNA-ASO displayed neither mRNA target regulation in xenografts nor antitumor activity in two independent nude mouse xenograft models. In conclusion, the potent single-agent activity and the synergistic effect of Sphk1 LNA-ASO in combination with chemotherapy in vitro highlight Sphk1 as a biologically relevant molecular target for gastric cancer. Further studies are warranted to overcome the challenge of delivering Sphk1-targeting RNA-therapeutics to solid tumors in vivo.

Vertical inhibition of the mTORC1/mTORC2/PI3K pathway shows synergistic effects against melanoma in vitro and in vivo.

The phosphatidyl inositol 3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway has been shown to be involved in the development of melanoma. PI-103 is a kinase inhibitor blocking PI3K class IA and mTOR complex 1 and 2. Here, we studied the effect of targeting the PI3K/mTORC1/mTORC2 pathway by PI-103 and rapamycin in melanoma cells and in a melanoma mouse model. Dual targeting of PI3K and mTOR by PI-103 induced apoptosis and cell-cycle arrest, and inhibited viability of melanoma cells in vitro. Combined treatment with PI-103 and the prototypic mTORC1 inhibitor rapamycin led to the synergistic suppression of AKT and ribosomal S6 protein phosphorylation and to the induction of apoptosis. In vivo, PI-103 and rapamycin displayed only modest single-agent activity, but the combination significantly reduced the tumor growth compared with both single agents. These data show that blocking the PI3K/mTORC1/mTORC2 pathway using the combination of two distinct small-molecule inhibitors ("vertical inhibition") leads to superior efficacy against malignant melanoma in vitro and in vivo.

mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and improves anti-tumor activity against gastric cancer in vivo.

VEGF receptor blockage has been reported to increase serum VEGF. We hypothesized that mTOR inhibition by everolimus counteracts VEGF induction by sunitinib resulting in an improved anti-tumor activity of sunitinib. In vitro, sunitinib in combination with everolimus did not outperform the respective monotherapies. In vivo, monotherapies reduced tumor growth by 60%, whereas the combination of sunitinib and everolimus led to an almost complete tumor growth inhibition. This superior anti-tumor activity coincided with attenuation of VEGF peaks. In conclusion mTOR inhibition by everolimus counteracts VEGF induction by sunitinib and results in significant reduction of tumor burden and long-lasting tumor growth control.

Reduction of human melanoma tumor growth in severe combined immunodeficient mice by passive transfer of antibodies induced by a high molecular weight melanoma-associated antigen mimotope vaccine.

PURPOSE: The high molecular weight melanoma-associated antigen (HMW-MAA) is an attractive target for immunotherapy of malignant melanoma. We have recently generated a vaccine based on the HMW-MAA mimotope 225D9.2+ that was able to induce anti-HMW-MAA antibodies with antitumor activity in vitro. Here, we investigated the antitumor activity of these antibodies in a human melanoma xenotransplant severe combined immunodeficient (SCID) mouse model. EXPERIMENTAL DESIGN: Tumors were established by injecting the human melanoma 518A2 cells into C.B.17 SCID/SCID mice. In tumor prevention experiments, 200 microg purified total IgG antibodies were injected intravenously the same day or on day 5 in therapeutic experiments. Antibody administration was repeated every fourth day and tumor volumes were measured. Antibody specificity and tumor infiltration by macrophages were investigated by immunohistochemistry. RESULTS: Within 35 days after cell inoculation, antibody treatment reduced tumor growth up to 40% in the therapeutic and up to 62% in the tumor prevention experiments compared with the control mice. In tumors of all groups, a similar distribution of the HMW-MAA and no differences in infiltration of macrophages were detected by immunohistochemistry. CONCLUSIONS: Here, we showed that antibodies induced by the 225D9.2+ mimotope effectively inhibited melanoma tumor growth. Additional mechanisms besides antibody-dependent cell cytotoxicity like disruption of interactions of melanoma cells mediated by extracellular matrix components seem to be involved in tumor growth inhibition. Based on our findings, we suggest that active immunization with this mimotope might be a promising strategy for treatment of melanoma.

Everolimus (RAD001) and anti-angiogenic cyclophosphamide show long-term control of gastric cancer growth in vivo.

Metronomic dosing of cytotoxic drugs such as cyclophosphamide has shown anti-angiogenic activity, most likely by inducing hypoxia in tumors. Hypoxia leads to activation of escape mechanisms allowing tumor cell survival. This potentially limits the activity of anti-angiogenic strategies. We hypothesized that mTORC1 inhibition by everolimus (RAD001) leads to suppression of HIF-1alpha and VEGF resulting in synergistic anti-tumor activity in combination with anti-angiogenically dosed cyclophosphamide. In vitro, effects of everolimus on mTORC1 signaling, proliferation, cell cycle, HIF-1alpha expression and VEGF secretion were evaluated in two gastric cancer cell lines. In vivo, anti-tumor activity of everolimus in combination with metronomic cyclophosphamide was studied in a NCI-N87 human gastric cancer SCID mouse xenograft model. Expression of Ki-67 and HIF-1alpha, activated caspase 3, microvascular density (MVD) and tumor necrotic area assessed. Everolimus decreased proliferation and attenuated production of HIF-1alpha as well as VEGF in gastric cancer cells in vitro. In vivo, everolimus significantly inhibited tumor growth. This anti-tumor activity was linked to a significant increase in tumor necrotic area (p < 0.02) and trends for decreased proliferation, increased apoptosis, decreased HIF-1alpha and lower tumor MVD (p = n.s.). The combination of everolimus and cyclophosphamide resulted in a striking and highly significant long-term tumor growth control compared to monotherapy (p < 0.001), which was associated with a sharp increase in central tumor necrosis (p < 0.001). In conclusion, the combination of everolimus and metronomic cyclophosphamide showed synergistic anti-tumor activity. Depriving cancer cells by everolimus of factors necessary for their survival under hypoxia induced by anti-angiogenic chemotherapy appears to be a promising approach for treatment of gastric cancer.

mTOR inhibition sensitizes gastric cancer to alkylating chemotherapy in vivo.

BACKGROUND: Gastric cancer is a highly chemoresistant tumor. Previous studies suggest that cancer cells can be sensitized to standard chemotherapy, and especially alkylating agents, by inhibition of mammalian target of rapamycin (mTOR) signaling. The work presented here shows that the mTOR inhibitor everolimus, in combination with cyclophosphamide, exhibits synergistic antitumor activity in gastric cancer xenografts. MATERIALS AND METHODS: Treatment with everolimus at the minimal effective dose was studied in combination with cyclophosphamide at maximum tolerated dose in a human gastric cancer severe combined immunodeficient (SCID) mouse xenograft model. Besides tumor size, biomarker expression for proliferation (Ki-67), hypoxia (HIF-12alpha), apoptosis (activated caspase 3), angiogenesis (microvascular density, MVD) and levels of circulating endothelial progenitors (CEPs) were assessed. RESULTS: Everolimus single agent treatment significantly inhibited tumor growth relative to control and cyclophosphamide treatment (T/C 19%, p<0.01). This antitumor activity was linked to a significant decrease in tumor cell proliferation (p<0.01) and a trend towards lower tumor MVD, HIF-1alpha expression and levels of CEPs. Notably, the combination of everolimus with cyclophosphamide resulted in synergistic anti-tumor activity (T/C 9%, p<0.01). This antitumor activity coincided with a statistically significant decrease in MVD (p<0.01). Whereas treatment with everolimus was well tolerated, cyclophosphamide at maximum tolerated dose (MTD) showed significant toxicity both as monotherapy and in combination with everolimus. CONCLUSION: The antiangiogenic activity of everolimus combined with a high dose of cyclophosphamide shows synergistic antitumor activity against gastric cancer in vivo. In potential future clinical trials, the toxicity of cyclophosphamide in combination regimens with everolimus deserves careful evaluation.

Mcl-1 is a relevant molecular target for antisense oligonucleotide strategies in gastric cancer cells.

Gastric cancer is the second most common cause of death from cancer worldwide and resistant to various chemotherapeutic regimens. In gastric cancer, the anti-apoptotic Mcl-1 protein is expressed in up to 75% of all cases and associated with poor prognosis. The biological relevance of Mcl-1 expression in gastric cancer is unclear. Thus, we investigated the functional significance and potential role of Mcl-1 as a molecular target in gastric cancer by using an antisense strategy. Gastric cancer cell lines (NCI-N87, MKN-28, MKN-45) were treated with Mcl-1 antisense oligonucleotides. Protein expression, cell growth and apoptosis were assessed for single-agent Mcl-1 AS oligonucleotide treatment and for combinations with docetaxel or cisplatin. Treatment by Mcl-1 AS oligonucleotides resulted in approximately 50% reduction in Mcl-1 protein levels in all gastric cancer cell lines examined. Surprisingly, dose-dependent Mcl-1 downregulation produced a significant increase in apoptosis and up to 60% decrease in cell growth. Moreover, combination of Mcl-1 AS oligonucleotide with docetaxel or cisplatin displayed synergistic anti-tumor activity. In conclusion, the impressive single-agent anti-tumor activity and the synergistic effect of Mcl-1 AS oligonucleotides in combination with chemotherapy might qualify Mcl-1 as a promising molecular target for AS oligonucleotide based treatment strategies for gastric cancer in the future.

Mcl-1 overexpression in hepatocellular carcinoma: a potential target for antisense therapy.

BACKGROUND/AIMS: Recently, the anti-apoptotic Mcl-1 protein has been reported as a resistance factor in various types of cancer. Here we investigated the presence of Mcl-1 protein in hepatocellular carcinoma (HCC) tissues and its potential role as a molecular drug target for HCC therapy. METHODS: HCC specimens of 149 patients were examined by immunohistochemistry for Mcl-1 expression. Antisense oligonucleotides (ASO) targeting Mcl-1 were evaluated as monotherapy and in combination with cisplatin in the HCC cell lines HepG2 and Snu398. Protein regulation, cell viability, and apoptosis were assessed by western blotting, cell counting, and FACS analysis. RESULTS: Mcl-1 protein is overexpressed in 51% of all cases irrespective of underlying disease. Targeting Mcl-1 by ASO specifically downregulated Mcl-1 protein expression and led to significant dose and time dependent single agent activity in HCC cells characterized by increased apoptosis and decreased cell viability. No significant target regulation or cell death was observed for control oligonucleotide treatment. Upon combination with cisplatin, Mcl-1 ASO revealed a significant chemosensitizing effect. CONCLUSIONS: Mcl-1 is overexpressed in half of HCC-tissues. ASO targeting Mcl-1 revealed a prominent single agent and chemosensitizing activity against HCC in vitro. Targeting Mcl-1 might qualify as a promising novel approach in HCC therapy.

CpG oligonucleotides elicit antitumor responses in a human melanoma NOD/SCID xenotransplantation model.

For patients with advanced malignant melanoma, the 5 y survival rate with current treatment modalities is low. There is an urgent need for more effective therapeutic concepts. One approach with great potential is to stimulate the body's own immune defense to reject cancer cells using CpG oligonucleotides. Distinct oligonucleotides containing nonmethylated cytidine residues in cytidine-guanosine dinucleotides with particular flanking bases (CpG motifs) are capable of eliciting powerful immune stimulation by mimicking infectious disease. We evaluated the in vivo antitumoral effects of CpG oligonucleotides against human malignant melanoma xenografts in NOD/SCID mice. CpG oligonucleotides administered in single peritumoral subcutaneous injections three times per week resulted in elevated plasma levels of interleukin-12 and significant inhibition of the growth of established tumor xenografts by 60% (p<0.016) compared to the saline control. In addition to this a significant invasion of macrophages into tumor xenografts and increased numbers of Langerhans-cell-derived dendritic cells in draining lymph nodes could be observed. Our findings demonstrate the antitumor activity of oligonucleotides containing immune-stimulatory CpG motifs in a xenotransplantation model with absent B, T cells and a lack of natural killer cell function.

Antitumor effect of G3139 Bcl-2 antisense oligonucleotide is independent of its immune stimulation by CpG motifs in SCID mice.

The Bcl-2 antisense oligonucleotide (AS-ODN) G3139 chemosensitizes human malignancies by downregulating the antiapoptotic protein Bcl-2. Because G3139 contains two potential immunostimulatory CpG motifs, we asked if immune stimulation contributes to the antitumor activity observed previously. 5'-Methylation of cytosines in CpG motifs abrogates immune stimulation by oligonucleotides. We, therefore, studied the antitumor and immunostimulatory potential of G3139 vs. an identical oligonucleotide, except for methylation of cytosines in the two CpG motifs (G4232). In a human melanoma SCID mouse xenotransplantation model, G3139 or G4232 was administered by continuous subcutaneous (s.c.) or bolus intraperitoneal (i.p.) infusion. Both G3139 and G4232 significantly reduced tumor growth by about one third relative to the saline-treated group. Furthermore, we noted a similar downregulation of Bcl-2 expression and increase in tumor cell apoptosis caused by G3139 and G4232 compared with saline controls. However, mice treated with G3139 had a pronounced increase in spleen weight and interleukin-12 (IL-12) plasma levels relative to mice treated with either G4232 or saline. Splenomegaly and elevated IL-12 plasma levels suggest that G3139 can be immunostimulatory. However, there is clear evidence that the antitumor effect of G3139 in this model appears to be a Bcl-2 antisense effect that is independent of immune stimulation, as G3139 and its immune-silent counterpart G4232 caused similar tumor suppression and apoptosis induction.