Enhanced ERbeta immunoexpression and apoptosis in the germ cells of cimetidine-treated rats

<p>Abstract</p> <p>Background</p> <p>Cimetidine, refereed as antiandrogenic drug, causes hormonal changes in male patients such as increased testosterone and FSH levels. In the rat testis, structural alterations in the seminiferous tubules have been related to germ cell loss and Sertoli cell death by apoptosis. Regarding the important role of Sertoli cells in the conversion of testosterone into estrogen, via aromatase, the immunoexpression of estrogen receptors-beta (ERbeta) was evaluated in the germ cells of untreated and treated rats with cimetidine. A relationship between ERbeta immunoreactivity and apoptosis was also investigated in the germ cells of damaged tubules.</p> <p>Methods</p> <p>Immunohistochemistry for detection of ERbeta and TUNEL method were performed in testicular sections of adult male rats treated with 50 mg/Kg of cimetidine (CmG) or saline solution (CG) for 52 days.</p> <p>Results</p> <p>In CG, a cytoplasmic immunoexpression for ERbeta was observed in spermatogonia, primary spermatocytes and spermatids. An evident ERbeta immunoreactivity was always observed in the flagellum and residual bodies of late spermatids. In CmG, the cytoplasm or cytoplasm and nuclei of germ cells of the damaged tubules by cimetidine showed enhanced ERbeta immunostaining. TUNEL-labeling was usually observed in the same germ cell types exhibiting enhanced ERbeta immunoreactivity.</p> <p>Conclusion</p> <p>The presence of ERbeta immunolabeling in the flagellum and residual bodies of spermatids reinforces the role of estrogen in spermiogenesis. The overexpression of ERbeta in the germ cells of CmG could be related to a possible interference of cimetidine on tubular androgenization and/or on the intratubular aromatase due to Sertoli cell damage. The parallelism between ERbeta overexpression and apoptosis indicates a participation of ERbeta on germ cell death.</p

Targeting HSP90 for cancer therapy

Heat-shock proteins (HSPs) are molecular chaperones that regulate protein folding to ensure correct conformation and translocation and to avoid protein aggregation. Heat-shock proteins are increased in many solid tumours and haematological malignancies. Many oncogenic proteins responsible for the transformation of cells to cancerous forms are client proteins of HSP90. Targeting HSP90 with chemical inhibitors would degrade these oncogenic proteins, and thus serve as useful anticancer agents. This review provides an overview of the HSP chaperone machinery and the structure and function of HSP90. We also highlight the key oncogenic proteins that are regulated by HSP90 and describe how inhibition of HSP90 could alter the activity of multiple signalling proteins, receptors and transcriptional factors implicated in carcinogenesis

Decreased Hsp90 expression in infiltrative lobular carcinoma: an immunohistochemical study

Background: Elevated Hsp90 expression has been documented in breast ductal carcinomas, whereas decreased Hsp90 expression has been reported in precursor lobular lesions. This study aims to assess Hsp90 expression in infiltrative lobular carcinomas of the breast. Methods: Tissue specimens were taken from 32 patients with infiltrative lobular carcinoma. Immunohistochemical assessment of Hsp90 was performed both in the lesion and the adjacent normal breast ducts and lobules; the latter serving as control. Concerning Hsp90 assessment: i) the percentage of positive cells and ii) the intensity were separately analyzed. Subsequently, the Allred score was adopted and calculated. The intensity was treated as a

Withanolides-Induced Breast Cancer Cell Death Is Correlated with Their Ability to Inhibit Heat Protein 90

Withanolides are a large group of steroidal lactones found in Solanaceae plants that exhibit potential anticancer activities. We have previously demonstrated that a withanolide, tubocapsenolide A, induced cycle arrest and apoptosis in human breast cancer cells, which was associated with the inhibition of heat shock protein 90 (Hsp90). To investigate whether other withanolides are also capable of inhibiting Hsp90 and to analyze the structure-activity relationships, nine withanolides with different structural properties were tested in human breast cancer cells MDA-MB-231 and MCF-7 in the present study. Our data show that the 2,3-unsaturated double bond-containing withanolides inhibited Hsp90 function, as evidenced by selective depletion of Hsp90 client proteins and induction of Hsp70. The inhibitory effect of the withanolides on Hsp90 chaperone activity was further confirmed using in vivo heat shock luciferase activity recovery assays. Importantly, Hsp90 inhibition by the withanolides was correlated with their ability to induce cancer cell death. In addition, the withanolides reduced constitutive NF-κB activation by depleting IκB kinase complex (IKK) through inhibition of Hsp90. In estrogen receptor (ER)-positive MCF-7 cells, the withanolides also reduced the expression of ER, and this may be partly due to Hsp90 inhibition. Taken together, our results suggest that Hsp90 inhibition is a general feature of cytotoxic withanolides and plays an important role in their anticancer activity

A highly invasive human glioblastoma pre-clinical model for testing therapeutics

Animal models greatly facilitate understanding of cancer and importantly, serve pre-clinically for evaluating potential anti-cancer therapies. We developed an invasive orthotopic human glioblastoma multiforme (GBM) mouse model that enables real-time tumor ultrasound imaging and pre-clinical evaluation of anti-neoplastic drugs such as 17-(allylamino)-17-demethoxy geldanamycin (17AAG). Clinically, GBM metastasis rarely happen, but unexpectedly most human GBM tumor cell lines intrinsically possess metastatic potential. We used an experimental lung metastasis assay (ELM) to enrich for metastatic cells and three of four commonly used GBM lines were highly metastatic after repeated ELM selection (M2). These GBM-M2 lines grew more aggressively orthotopically and all showed dramatic multifold increases in IL6, IL8, MCP-1 and GM-CSF expression, cytokines and factors that are associated with GBM and poor prognosis. DBM2 cells, which were derived from the DBTRG-05MG cell line were used to test the efficacy of 17AAG for treatment of intracranial tumors. The DMB2 orthotopic xenografts form highly invasive tumors with areas of central necrosis, vascular hyperplasia and intracranial dissemination. In addition, the orthotopic tumors caused osteolysis and the skull opening correlated to the tumor size, permitting the use of real-time ultrasound imaging to evaluate antitumor drug activity. We show that 17AAG significantly inhibits DBM2 tumor growth with significant drug responses in subcutaneous, lung and orthotopic tumor locations. This model has multiple unique features for investigating the pathobiology of intracranial tumor growth and for monitoring systemic and intracranial responses to antitumor agents

The 4C5 Cell-Impermeable Anti-HSP90 Antibody with Anti-Cancer Activity, Is Composed of a Single Light Chain Dimer

MAb 4C5 is a cell impermeable, anti-HSP90 murine monoclonal antibody, originally produced using hybridoma technology. We have previously shown that mAb 4C5 specifically recognizes both the α- and to a lesser extent the β-isoform of HSP90. Additionally, in vitro and in vivo studies revealed that by selectively inhibiting the function of cell-surface HSP90, mAb 4C5 significantly impairs cancer cell invasion and metastasis. Here we describe the reconstitution of mAb 4C5 into a mouse-human chimera. More importantly we report that mAb 4C5 and consequently its chimeric counterpart are completely devoid of heavy chain and consist only of a functional kappa light chain dimer. The chimeric antibody is shown to retain the original antibody's specificity and functional properties. Thus it is capable of inhibiting the function of surface HSP90, leading to reduced cancer cell invasion in vitro. Finally, we present in vivo evidence showing that the chimeric 4C5 significantly inhibits the metastatic deposit formation of MDA-MB-453 cells into the lungs of SCID mice. These data suggest that a chimeric kappa light chain antibody could be potentially used as an anti-cancer agent, thereby introducing a novel type of antibody fragment, with reduced possible adverse immunogenic effects, into cancer therapeutics

Induction of Premature Senescence by Hsp90 Inhibition in Small Cell Lung Cancer

BACKGROUND: The molecular chaperone Hsp90 is a promising new target in cancer therapy and selective Hsp90 inhibitors are currently in clinical trials. Previously these inhibitors have been reported to induce either cell cycle arrest or cell death in cancer cells. Whether the cell cycle arrest is reversible or irreversible has not generally been assessed. Here we have examined in detail the cell cycle arrest and cell death responses of human small cell lung cancer cell lines to Hsp90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: In MTT assays, small cell lung cancer cells showed a biphasic response to the Hsp90 inhibitors geldanamycin and radicicol, with low concentrations causing proliferation arrest and high concentrations causing cell death. Assessment of Hsp90 intracellular activity using loss of client protein expression showed that geldanamycin concentrations that inhibited Hsp90 correlated closely with those causing proliferation arrest but not cell death. The proliferation arrest induced by low concentrations of geldanamycin was not reversed for a period of over thirty days following drug removal and showed features of senescence. Rare populations of variant small cell lung cancer cells could be isolated that had additional genetic alterations and no longer underwent irreversible proliferation arrest in response to Hsp90 inhibitors. CONCLUSIONS/SIGNIFICANCE: We conclude that: (1) Hsp90 inhibition primarily induces premature senescence, rather than cell death, in small cell lung cancer cells; (2) small cell lung cancer cells can bypass this senescence through further genetic alterations; (3) Hsp90 inhibitor-induced cell death in small cell lung cancer cells is due to inhibition of a target other than cytosolic Hsp90. These results have implications with regard to how these inhibitors will behave in clinical trials and for the design of future inhibitors in this class

Revisions to the International Neuroblastoma Response Criteria: A Consensus Statement From the National Cancer Institute Clinical Trials Planning Meeting

Purpose More than two decades ago, an international working group established the International Neuroblastoma Response Criteria (INRC) to assess treatment response in children with neuroblastoma. However, this system requires modification to incorporate modern imaging techniques and new methods for quantifying bone marrow disease that were not previously widely available. The National Cancer Institute sponsored a clinical trials planning meeting in 2012 to update and refine response criteria for patients with neuroblastoma. Methods Multidisciplinary investigators from 13 countries reviewed data from published trials performed through cooperative groups, consortia, and single institutions. Data from both prospective and retrospective trials were used to refine the INRC. Monthly international conference calls were held from 2011 to 2015, and consensus was reached through review by working group leadership and the National Cancer Institute Clinical Trials Planning Meeting leadership council. Results Overall response in the revised INRC will integrate tumor response in the primary tumor, soft tissue and bone metastases, and bone marrow. Primary and metastatic soft tissue sites will be assessed using Response Evaluation Criteria in Solid Tumors (RECIST) and iodine-123 (123I) –metaiodobenzylguanidine (MIBG) scans or [18F]fluorodeoxyglucose–positron emission tomography scans if the tumor is MIBG nonavid. 123I-MIBG scans, or [18F]fluorodeoxyglucose–positron emission tomography scans for MIBG-nonavid disease, replace technetium-99m diphosphonate bone scintigraphy for osteomedullary metastasis assessment. Bone marrow will be assessed by histology or immunohistochemistry and cytology or immunocytology. Bone marrow with ≤ 5% tumor involvement will be classified as minimal disease. Urinary catecholamine levels will not be included in response assessment. Overall response will be defined as complete response, partial response, minor response, stable disease, or progressive disease. Conclusion These revised criteria will provide a uniform assessment of disease response, improve the interpretability of clinical trial results, and facilitate collaborative trial designs