Raman spectroscopy for accurately characterizing biomolecular changes in androgen-independent prostate cancer cells.
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ABSTRACT:
SUBMITTER: Corsetti S
PROVIDER: S-EPMC6538931 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:BackgroundCastration resistant prostate cancer (CRPC) develops as a consequence of hormone therapies used to deplete androgens in advanced prostate cancer patients. CRPC cells are able to grow in a low androgen environment and this is associated with anomalous activity of their endogenous androgen receptor (AR) despite the low systemic androgen levels in the patients. Therefore, the reactivated tumor cell androgen signaling pathway is thought to provide a target for control of CRPC. Previously, we reported that Hedgehog (Hh) signaling was conditionally activated by androgen deprivation in androgen sensitive prostate cancer cells and here we studied the potential for cross-talk between Hh and androgen signaling activities in androgen deprived and androgen independent (AI) prostate cancer cells.ResultsTreatment of a variety of androgen-deprived or AI prostate cancer cells with the Hh inhibitor, cyclopamine, resulted in dose-dependent modulation of the expression of genes that are regulated by androgen. The effect of cyclopamine on endogenous androgen-regulated gene expression in androgen deprived and AI prostate cancer cells was consistent with the suppressive effects of cyclopamine on the expression of a reporter gene (luciferase) from two different androgen-dependent promoters. Similarly, reduction of smoothened (Smo) expression with siRNA co-suppressed expression of androgen-inducible KLK2 and KLK3 in androgen deprived cells without affecting the expression of androgen receptor (AR) mRNA or protein. Cyclopamine also prevented the outgrowth of AI cells from androgen growth-dependent parental LNCaP cells and suppressed the growth of an overt AI-LNCaP variant whereas supplemental androgen (R1881) restored growth to the AI cells in the presence of cyclopamine. Conversely, overexpression of Gli1 or Gli2 in LNCaP cells enhanced AR-specific gene expression in the absence of androgen. Overexpressed Gli1/Gli2 also enabled parental LNCaP cells to grow in androgen depleted medium. AR protein co-immunoprecipitates with Gli2 protein from transfected 293T cell lysates.ConclusionsCollectively, our results indicate that Hh/Gli signaling supports androgen signaling and AI growth in prostate cancer cells in a low androgen environment. The finding that Gli2 co-immunoprecipitates with AR protein suggests that an interaction between these proteins might be the basis for Hedgehog/Gli support of androgen signaling under this condition.
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Project description:Although blockade of androgen receptor (AR) signaling represents the main treatment for advanced prostate cancer (PrCa), many patients progress to a lethal phenotype of "Castration-Resistant" prostate cancer (CR-PrCa). With the hypothesis that early PrCa may harbor a population of androgen-unresponsive cancer cells as precursors to CR-recurrent disease, we undertook the propagation of androgen-independent cells from PrCa-prostatectomy samples of early, localized (Stage-I) cases. A collection of 120 surgical specimens from prostatectomy cases was established, among which 54 were adenocarcinomas. Hormone-free cell culture conditions were developed allowing routine propagation of cells expressing prostate basal cell markers and stem/progenitor cell markers, and which proliferated as spheres/spheroids in suspension cultures. Colonies of androgen-independent epithelial cells grew out from 30/43 (70%) of the adenocarcinoma cases studied in detail. Fluorescence microscopy and flow cytometry showed that CR-PrCa cells were positive for CD44, CD133, CK5/14, c-kit, integrin ?2?1, SSEA4, E-Cadherin and Aldehyde Dehydrogenase (ALDH). All 30 CR-PrCa cell cultures were also TERT-positive, but negative for TMPRSS2-ERG. Additionally, a subset of 22 of these CR-PrCa cell cultures was examined by orthotopic xenografting in intact and castrated SCID mice, generating histologically typical locally-invasive human PrCa or undifferentiated cancers, respectively, in 6-8 weeks. Cultured PrCa cells and orthotopically-induced in vivo cancers lacked PSA expression. We report here the propagation of Cancer Initiating Cells (CIC) directly from Stage I human PrCa tissue without selection or genetic manipulation. The propagation of stem/progenitor-like CR-PrCa cells derived from early human prostate carcinomas suggests the existence of a subpopulation of cells resistant to androgen-deprivation therapy and which may drive the subsequent emergence of disseminated CR-PrCa.
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Project description:The surface-enhanced Raman spectroscopy (SERS) of blood serum was investigated to differentiate between prostate cancer (PCa) and benign prostatic hyperplasia (BPH) in males with a prostate-specific antigen level of 4-10 ng/mL, so as to reduce unnecessary biopsies. A total of 240 SERS spectra from blood serum were acquired from 40 PCa subjects and 40 BPH subjects who had all received prostate biopsies and were given a pathological diagnosis. Multivariate statistical techniques, including principal component analysis (PCA) and linear discriminant analysis (LDA) diagnostic algorithms, were used to analyze the spectra data of serum from patients in control (CTR), PCa and BPH groups; results offered a sensitivity of 97.5%, a specificity of 100.0%, a precision of 100.0% and an accuracy of 99.2% for CTR; a sensitivity of 90.0%, a specificity of 97.5%, a precision of 94.7% and an accuracy of 98.3% for BPH; a sensitivity of 95.0%, a specificity of 93.8%, a precision of 88.4% and an accuracy of 94.2% for PCa. Similarly, this technique can significantly differentiate low- and high-risk PCa with an accuracy of 92.3%, a specificity of 95% and a sensitivity of 89.5%. The results suggest that analyzing blood serum using SERS combined with PCA-LDA diagnostic algorithms is a promising clinical tool for PCa diagnosis and assessment.
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Project description:BackgroundMucosal healing (MH) is a key treatment target in the management of inflammatory bowel disease (IBD) and is defined in endoscopic terms by the newly published PICaSSO score. Raman Spectroscopy (RS) is based on the scattering of inelastic light giving spectra that are highly specific for individual molecules. We aimed to establish spectral changes before and after treatment and whether Raman Spectroscopy is able to accurately differentiate between inflammation and MH.MethodsBiopsies were taken for ex vivo RS analysis alongside biopsies for histological analysis from IBD patients undergoing optical diagnosis endoscopic assessment. We compared pre- vs. post-biological treatment in IBD patients and healthy controls and active vs. MH in UC and CD. For spectral analysis, we used supervised self-organising maps for separation and classification.ResultsA total of 23 patients (14 IBD, 9 HC) were recruited for comparison of pre- vs. post-biologic treatment and 74 IBD patients were included for the assessment of MH in IBD, giving 9700 Raman Spectra. Spectral differences were seen between pre- and post-treatment which were observed comparing MH vs. active inflammation. Reductions in intensity at 1003cm-1 and 1252cm-1 when a reduction in inflammation was seen post-treatment and when MH was present. MH was associated with an increase in intensity at 1304cm-1. The trained neural network differentiated MH from active inflammation with a sensitivity, specificity, PPV, NPV and accuracy in UC of 96.29% (sd 0.94), 95.03% (sd 1.52), 94.89% (sd 1.59), 96.33 (sd 0.97) and 95.65 (sd 0.99) and 96.19% (sd 1.46), 88% (sd 4.20), 86.60% (sd 5.39), 96.55% (sd 1.32) and 91.6% (sd 2.75) in CD respectively.ConclusionWe demonstrated RS can demonstrate biochemical changes following treatment of IBD and accurately differentiates MH from active inflammation in IBD and might be a future tool to personalise therapeutic management in IBD.
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