Anjana Gupta

The Hedgehog Signaling Molecule Gli2 Induces Parathyroid Hormone-Related Peptide Expression and Osteolysis in Metastatic Human Breast Cancer Cells

Parathyroid hormone-related peptide (PTHrP) is a major factor involved in tumor-induced osteolysis caused by breast cancers that have metastasized to bone. However, the molecular mechanisms that mediate PTHrP production by breast cancer cells are not entirely clear. We hypothesized that Gli2, a downstream transcriptional effector of the Hedgehog (Hh) signaling pathway, regulates PTHrP expression in metastatic breast cancer because the Hh pathway regulates physiologic PTHrP expression in the developing growth plate. Here, we show that Gli2 is expressed in several human cancer cell lines that cause osteolytic lesions in vivo and produce PTHrP (MDA-MB-231, RWGT2, and PC-3) but is not expressed in nonosteolytic cancer cell lines that do not secrete PTHrP (MCF-7, ZR-75, and T47D). Transient expression of Gli2 in MDA-MB-231 and MCF-7 breast cancer cells increased PTHrP promoter-luciferase activity dose dependently. Stable expression of Gli2 in MDA-MB-231 cells resulted in an increase in PTHrP protein in the conditioned medium. Alternatively, MDA-MB-231 cells stably transfected with Gli2-EnR, a repressor of Gli2 activity, exhibited a 72% to 93% decrease in PTHrP mRNA by quantitative real-time PCR when compared with control cells. To examine the effects of Gli2 on breast cancer-mediated osteolysis in vivo, athymic nude mice were inoculated with MDA-MB-231 cells stably expressing Gli2 or the empty vector. Following tumor cell inoculation via the left cardiac ventricle, Gli2-expressing tumors caused significantly more osteolysis. Together, these data suggest that PTHrP expression and osteolysis in vivo in human breast cancer cells is driven at least in part by Gli2.

Therapeutic antitumor efficacy of anti-CD137 agonistic monoclonal antibody in mouse models of myeloma.

Purpose: Eradication of post-treatment residual myeloma cells is needed to prevent relapses, and immunostimulatory monoclonal antibodies (mAb) such as anti-CD137, CTLA-4, CD40, etc., which enhance the immune response against malignancies, represent a means of achieving this purpose. This study explores anti-CD137 mAbs for multiple myeloma treatment in preclinical models of the disease because they safely augment tumor immunity and are in clinical trials for other cancers. Experimental Design: The antitumor effect of anti-CD137 mAb on mouse plasmacytomas derived from HOPC and NS0 cell lines was studied and compared with that of anti-CTLA-4, anti-CD40, and anti-ICAM-2 mAbs. The antitumor effect of anti-CD137 mAb was also examined in a mouse syngeneic disseminated myeloma (5TGM1) model, which more closely resembles human multiple myeloma. Depletions of specific cell populations and gene-targeted mice were used to unravel the requirements for tumor rejection. Results: Agonistic mAb against CD137 and blocking anti-CTLA-4 mAb showed activity against i.p. HOPC tumors, resulting in extended survival of mice that also became immune to rechallenge. Anti-CD137 mAbs induced complete eradications of established s.c. NS0-derived tumors that were dependent on IFN-γ, natural killer cells, and CD8+ T lymphocytes. Natural killer cells accumulated in tumor draining lymph nodes and showed increased IFN-γ production. Antitumor efficacy of anti-CD137 mAb was preserved in CD28-deficient mice despite the fact that CD28 signaling increases the expression of CD137 on CD8+ T cells. Importantly, anti-CD137 mAb treatment significantly decreased systemic tumor burden in the disseminated 5TGM1 model. Conclusions: The immune-mediated antitumor activity of anti-CD137 mAb in mouse models holds promise for myeloma treatment in humans.

Detection of myeloma in skeleton of mice by whole-body optical fluorescence imaging

Development of new therapies for myeloma has been hindered by the lack of suitable preclinical animal models of the disease in which widespread tumor foci in the skeleton can be detected reliably. Traditional means of detecting skeletal tumor infiltration such as histopathology are cumbersome and labor-intensive and do not allow temporal monitoring of tumor progression or regression in response to therapy. To resolve this problem, we modified the Radl 5TGM1 model of myeloma bone disease such that fluorescent myeloma tumors can be optically imaged in situ. Here, we show that murine myeloma 5TGM1 tumor cells, engineered to express enhanced green fluorescent protein (eGFP; 5TGM1-eGFP cells), can be imaged in a temporal fashion using a fluorescence illuminator and a charge-coupled device camera in skeletons of live C57BL/KaLwRij mice. High-resolution, whole-body images of tumor-bearing mice revealed that myeloma cells homed almost exclusively to the skeleton, with multiple focal tumor foci in the axial skeleton, consistent with myeloma tumor distribution in humans. Finally, the tested antitumor treatment effect of Velcade (bortezomib), a proteasome inhibitor used clinically in myeloma, was readily detected by GFP imaging, suggesting the power of the technique in combination with the Radl 5TGM1-eGFP model for rapid preclinical assessment and sensitive monitoring of novel and potential therapeutics. Whole-body GFP imaging is practical, convenient, inexpensive, and rapid, and these advantages should enable a high throughput when evaluating in vivo efficacy of new potential antimyeloma therapeutics and assessing response to treatment. [Mol Cancer Ther 2007;6(6):1701–8]

CCR1 blockade reduces tumor burden and osteolysis in vivo in a mouse model of myeloma bone disease

The chemokine CCL3/MIP-1α is a risk factor in the outcome of multiple myeloma (MM), particularly in the development of osteolytic bone disease. This chemokine, highly overexpressed by MM cells, can signal mainly through 2 receptors, CCR1 and CCR5, only 1 of which (CCR1) is responsive to CCL3 in human and mouse osteoclast precursors. CCR1 activation leads to the formation of osteolytic lesions and facilitates tumor growth. Here we show that formation of mature osteoclasts is blocked by the highly potent and selective CCR1 antagonist CCX721, an analog of the clinical compound CCX354. We also show that doses of CCX721 selected to completely inhibit CCR1 produce a profound decrease in tumor burden and osteolytic damage in the murine 5TGM1 model of MM bone disease. Similar effects were observed when the antagonist was used prophylactically or therapeutically, with comparable efficacy to that of zoledronic acid. 5TGM1 cells were shown to express minimal levels of CCR1 while secreting high levels of CCL3, suggesting that the therapeutic effects of CCX721 result from CCR1 inhibition on non-MM cells, most likely osteoclasts and osteoclast precursors. These results provide a strong rationale for further development of CCR1 antagonists for the treatment of MM and associated osteolytic bone disease.

Volumetric and compressibility studies for (L-arginine + D-maltose monohydrate + water) system in the temperature range of (298.15 to 308.15) K

ABSTRACT The density and speed of sound of L-arginine (0.025–0.2 mol kg−1) in aqueous + D-maltose (0–6 mass% of maltose in water) were obtained at temperatures of (298.15, 303.15 and 308.15) K. The apparent molar volume, limiting apparent molar volume, transfer volume, as well as apparent molar compressibility, limiting apparent molar compressibility, transfer compressibility, pair and triple interaction coefficients, partial molar expansibilities, coefficient of thermal expansion and also the hydration number, were calculated using the experimental density and speed of sound values. The results have been discussed in terms of solute–solute and solute–solvent interactions in these systems. Solute–solvent (hydrophilic–ionic group and hydrophilic–hydrophilic group) interactions were found to be dominating over solute–solute (hydrophobic–hydrophilic group) interactions in the solution, which increases with increase in maltose concentration.

Abstract 4216: Antitumor effect of Nexrutine, a phellodendron amurense bark extract, in multiple myeloma

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Multiple myeloma (MM), a clonal malignancy of plasma cells, is the second most common adult hematological malignancy with approximately 15,000 new cases diagnosed annually. Despite recent advances in its clinical management, morbidity and mortality associated with MM remain high and there is a need for continuing development of novel anti-myeloma agents. Nexrutine, a non-toxic herbal extract from the bark of Chinese tree Phellodendron amurense, has been shown to exert significant anti-tumor effects in animal models of prostate cancer and is currently being evaluated in phase I/II clinical trials in patients with prostate cancer. In this study, we investigated the potential anti-tumor effect of nexrutine in MM. Exposure to nexrutine (3-100µg/ml) reduced viability of murine 5TGM1 and human RPMI 8226 MM cells in a dose- and time-dependent manner, independent of cell cycle disruption. Loss of cell viability was due primarily to apoptosis as indicated by >2-fold increase in annexin V+, 7AAD- cells and increased DNA fragmentation detectable by 24 hours. Previous studies showed that down-regulation of short-lived anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1), in part by caspase-dependent cleavage of the full-length protein (Mcl-1L) to generate smaller (pro-apoptotic) Mcl-1 fragments, plays a major role in execution of apoptosis in MM cells. Nexrutine-induced apoptosis in MM cells was associated with reduced levels of Mcl-1L, caspase activation as indicated by a large increase in the active 17kDa cleavage product of procaspase 3 and an increase in pro-apoptotic Mcl-1 cleavage product. Nexrutine also inhibited the mammalian target of rapamycin (mTOR) and this was accompanied by accumulation of LC3-II, a marker of autophagosomes, in autophagy-competent MM cells. However, treatment of MM with cells with either pepstatin A and E64D that target lysosomal proteases involved in autophagy or with chloroquine, a lysosomotrophic autophagy inhibitor also resulted in cytotoxicity and these autophagy inhibitors did not rescue nexrutine-induced loss of viability in MM cells suggesting that autophagy plays a role constitutively in MM cell survival. Finally, we evaluated potential in vivo anti-tumor efficacy of nexrutine in the well-characterized preclinical model of MM in which 5TGM1 cells are inoculated intravenously in syngeneic naive female C57BL/KaLwRij mice. Administration of nexrutine (400µg/mouse p.o. by gavage 6 days/week for 5 weeks) to tumor-bearing mice reduced serum levels of the monoclonal IgG2bκ paraprotein, a surrogate for tumor progression and overall tumor burden. Collectively, our results indicate an anti-myeloma effect of nexrutine and support further extensive preclinical testing and early phase clinical trial of this non-toxic herbal extract in MM. Our data also indicate that a combination of nexrutine and one or more autophagy inhibitors would result in enhanced anti-tumor effect in MM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4216. doi:10.1158/1538-7445.AM2011-4216