Sensing intracellular oxygen using near-infrared phosphorescent probes and live-cell fluorescence imaging

The development and application of a methodology for measurement of oxygen within single mammalian cells are presented, which employ novel macromolecular near infrared (NIR) oxygen probes based on new metalloporphyrin dyes. The probes, which display optimal spectral characteristics and sensitivity to oxygen, excellent photostability, and low cytotoxicity and phototoxicity, are loaded into cells by simple transfection procedures and subsequently analyzed by high-resolution fluorescence microscopy. The methodology is demonstrated by sensing intracellular oxygen in different mammalian cell lines, including A549, Jurkat, and HeLa, and monitoring rapid and transient changes in response to mitochondrial uncoupling by valinomycin and inhibition by antimycin A. Furthermore, the effect of ryanodine receptor-mediated Ca(2+) influx on cellular oxygen uptake is shown by substantial changes in the level of intracellular oxygen. The results demonstrate the ability of this technique to measure small, rapid, and transient changes in intracellular oxygen in response to different biological effectors. Moreover, this technique has wide ranging applicability in cell biology and is particularly useful in the study of low oxygen environments (cellular hypoxia), mitochondrial and cellular (dys)function, and for therapeutic areas, such as cardiovascular and neurological research, metabolic diseases, and cancer.     

Poly-adenine-mediated spherical nucleic acid probes for live cell fluorescence imaging of tumor-related microRNAs

Background

Accurately detecting and quantifying tumor-related microRNAs (miRNAs) in living cells is of great value for early cancer diagnosis. Herein, we present poly-adenine (polyA)-mediated spherical nucleic acid (SNA) nanoprobes for intracellular miRNA imaging in living cells.

Methods and results

polyA-mediated spherical nucleic acid (pASNA) nanoprobes consist of gold nanoparticles (AuNPs) anchored with fluorophore-labeled DNA molecules pre-hybridized with recognition sequences and polyA tails. The detection performance for miRNAs in vitro was studied to confirm the feasibility of pASNA nanoprobes for imaging live cell miRNAs. Before the pASNA nanoprobes were used for imaging intracellular miRNAs in MCF-7, HeLa, and LO2 cells, the stability and non-cytotoxicity were investigated using Dnase I and a standard colorimetric CCK8 assay. Flow cytometry, qRT-PCR analyses were conducted to confirm the different expression levels of miR-155 in live cells. Results showed that the pASNA nanoprobes had good detection sensitivity and specificity, excellent stability, and low toxicity. After incubating with pASNA nanoprobes, noticeable fluorescence signal enhancement could be clearly observed in MCF-7 and HeLa cells but not LO2 cells by confocal microscopy. Flow cytometry analysis and qRT-PCR indicated that MCF-7 and HeLa cells had higher miR-155 expression levels compared to LO2 cells.

Conclusions

The pASNA nanoprobes we developed had good sensitivity and specificity, excellent nuclease stability and low toxicity, thus representing a new approach to exquisitely reveal the distribution of endogenous miRNAs in live cells.

     

Comparative studies of intracellular Ca2+ in strongly and weakly metastatic rat prostate cancer cell lines

The metastatic ability of prostate cancer cells involves differential expression of ionic mechanisms. In the present study, using electrophysiological recordings and intracellular Ca2+ measurements, we investigated Ca2+ related signalling in two rat prostate cancer (MAT-LyLu and AT-2) cell lines of markedly different metastatic potential. Whole-cell voltage clamp experiments indicated the absence of an inward current carried through voltage-dependent Ca2+ channels in either cell line. A Ca2+-dependent component was also absent in the voltage-activated outward K+ currents. Indo-1 microfluorimetry confirmed these results and also revealed marked differences in the resting level of intracellular Ca2+ and the ability of the two cell lines to regulate intracellular Ca2+. The weakly metastatic AT-2 cells displayed a significantly higher resting intracellular Ca2+ than the related but strongly metastatic MAT-LyLu cell line. Increasing extracellular K+ decreased intracellular Ca2+ in the AT-2 but had no effect on intracellular Ca2+ levels in the MAT-LyLu cells. Furthermore, increasing extracellular Ca2+ increased intracellular Ca2+ in AT-2 but, again, had no effect on MAT-LyLu cells. These results suggested the presence of a tonic, voltage-independent Ca2+ permeation mechanism operating specifically in the AT-2 cells. The influx of Ca2+ into the AT-2 cells was suppressed by both CdCl2 (100-300 microM) and SKF-96365 (10-30 microM). It is concluded that the strongly metastatic MAT-LyLu cell line lacks a voltage-independent basal Ca2+ influx mechanism that is present in the weakly metastatic AT-2 cells.     

Glycosylation potential of human prostate cancer cell lines

Altered glycosylation is a universal feature of cancer cells and altered glycans can help cancer cells escape immune surveillance, facilitate tumor invasion, and increase malignancy. The goal of this study was to identify specific glycoenzymes, which could distinguish prostate cancer cells from normal prostatic cells. We investigated enzymatic activities and gene expression levels of key glycosyl- and sulfotransferases responsible for the assembly of O- and N-glycans in several prostatic cells. These cells included immortalized RWPE-1 cells derived from normal prostatic tissues, and prostate cancer cells derived from metastasis in bone (PC-3), brain (DU145), lymph node (LNCaP), and vertebra (VCaP). We found that all cells were capable of synthesizing complex N-glycans and O-glycans with the core 1 structure, and each cell line had characteristic biosynthetic pathways to modify these structures. The in vitro measured activities corresponded well to the mRNA levels of glycosyltransferases and sulfotransferases. Lectin and antibody binding to whole cells supported these results, which form the basis for the development of tumor cell-specific targeting strategies.     

Molecular probes for fluorescence image-guided cancer surgery

In cancer surgery, complete surgical resection of tumor lesions is critical to optimize the outcome. However, it is sometimes difficult to distinguish the boundary between tumor and normal tissues, and residual tumor tissue can result in cancer recurrence. Intraoperative imaging with fluorescent molecular probes can assist surgeons to visualize tumor lesions and their boundaries during surgery. Here, we review molecular probes for fluorescence image-guided cancer surgery, focusing especially on recent developments in high-performance tumor-imaging probes and the strategies used for their design.     

Bone morphogenetic protein signaling in prostate cancer cell lines

Prostate cancer is the most commonly diagnosed malignancy in men and is often associated with bone metastases. Prostate cancer bone lesions can be lytic or schlerotic, with the latter predominating. Bone morphogenetic proteins (BMPs) are a family of growth factors, which may play a role in the formation of prostate cancer osteoblastic bone metastases. This study evaluated the effects of BMPs on prostate cancer cell lines. We observed growth inhibitory effects of BMP-2 and -4 on LNCaP, while PC-3 was unaffected. Flow cytometric analysis determined that LNCaP cell growth was arrested in G(1) after bone morphogenetic protein-2 treatment. Treatment of LNCaP and PC-3 with BMP-2 and -4 activated downstream signaling pathways involving SMAD-1, up-regulation of p21(CIP1/WAF1) and changes in retinoblastoma (Rb) phosphorylation. Interestingly, bone morphogenetic protein-2 treatment stimulated a 2.7-fold increase in osteoprotegerin (OPG), a molecule, which inhibits osteoclastogenesis, production in PC-3.     

Phycobiliprotein-Fab conjugates as probes for single particle fluorescence imaging

BACKGROUND AND METHODS: Single particle fluorescence imaging (SPFI) is a recently developed method that has provided a powerful approach to observing receptor movement and associations at high spatial resolution. It provides a noninvasive alternative to the existing biochemical techniques. It can also quantify and resolve molecular interactions at the cell surface at a nanometer scale. Probes that have been used in the past to study mobility and associations of cell surface receptors have many limitations. These include concerns about the specificity of the probes, the possibility that their size interferes with the receptor once bound to it, the nonuniform fluorescence, and the questionable stoichiometry. RESULTS: In this study, we have generated phycobiliprotein-Fab conjugates, and have shown that they are a significant advance on existing probes for SPFI studies. They are small in size, highly specific, highly fluorescent, of known stoichiometry, photostable, emit uniform fluorescence, and are generally well defined. CONCLUSIONS: It is highly important that when studying receptor mobility or associations, fully characterized probes are used. Phycoerythrin(PE)-Fab probes provide us with the perfect tool for SPFI, and a system with a wide range of applicability to study any cell surface receptor against which a monoclonal antibody exists.     

In vivo fluorescence lifetime imaging monitors binding of specific probes to cancer biomarkers

One of the most important factors in choosing a treatment strategy for cancer is characterization of biomarkers in cancer cells. Particularly, recent advances in Monoclonal Antibodies (MAB) as primary-specific drugs targeting tumor receptors show that their efficacy depends strongly on characterization of tumor biomarkers. Assessment of their status in individual patients would facilitate selection of an optimal treatment strategy, and the continuous monitoring of those biomarkers and their binding process to the therapy would provide a means for early evaluation of the efficacy of therapeutic intervention. In this study we have demonstrated for the first time in live animals that the fluorescence lifetime can be used to detect the binding of targeted optical probes to the extracellular receptors on tumor cells in vivo. The rationale was that fluorescence lifetime of a specific probe is sensitive to local environment and/or affinity to other molecules. We attached Near-InfraRed (NIR) fluorescent probes to Human Epidermal Growth Factor 2 (HER2/neu)-specific Affibody molecules and used our time-resolved optical system to compare the fluorescence lifetime of the optical probes that were bound and unbound to tumor cells in live mice. Our results show that the fluorescence lifetime changes in our model system delineate HER2 receptor bound from the unbound probe in vivo. Thus, this method is useful as a specific marker of the receptor binding process, which can open a new paradigm in the "image and treat" concept, especially for early evaluation of the efficacy of the therapy.     

Identification of differentially expressed proteins in curcumin-treated prostate cancer cell lines

Due to high prevalence and slow progression of prostate cancer, primary prevention appears to be attractive strategy for its eradication. During the last decade, curcumin (diferuloylmethane), a natural compound from the root of turmeric (Curcuma longa), was described as a potent chemopreventive agent. Curcumin exhibits anti-inflammatory, anticarcinogenic, antiproliferative, antiangiogenic, and antioxidant properties in various cancer cell models. This study was designed to identify proteins involved in the anticancer activity of curcumin in androgen-dependent (22Rv1) and -independent (PC-3) human prostate cancer cell lines using two-dimensional difference in gel electrophoresis (2D-DIGE). Out of 425 differentially expressed spots, we describe here the MALDI-TOF-MS analysis of 192 spots of interest, selected by their expression profile. This approach allowed the identification of 60 differentially expressed proteins (32 in 22Rv1 cells and 47 in PC-3 cells). Nineteen proteins are regulated in both cell lines. Further bioinformatic analysis shows that proteins modulated by curcumin are implicated in protein folding (such as heat-shock protein PPP2R1A; RNA splicing proteins RBM17, DDX39; cell death proteins HMGB1 and NPM1; proteins involved in androgen receptor signaling, NPM1 and FKBP4/FKBP52), and that this compound could have an impact on miR-141, miR-152, and miR-183 expression. Taken together, these data support the hypothesis that curcumin is an interesting chemopreventive agent as it modulates the expression of proteins that potentially contribute to prostate carcinogenesis.     

Epigenetic inactivation of PCDH10 in human prostate cancer cell lines

PCDH10 (protocadherin-10), a novel tumour suppressor gene, is down-regulated in several human cancers due to hypermethylation of promoter CGIs (CpG islands). Here, we investigated the expression of PCDH10 in different normal adult tissues and in a panel of prostate cancer cell lines. PCDH10 was widely expressed in normal tissues with higher levels in the prostate. The expression of PCDH10 was markedly reduced or silenced in prostate cancer cell lines compared with normal adult prostate tissue. Decreased PCDH10 expression was correlated with the methylation status of the PCDH10 promoter. Furthermore, the DNA demethylating agent 5'-azacytidin restored PCDH10 expression by suppressing PCDH10 promoter methylation in prostate cancer cell lines. Treatment with Trichostatin A alone had no significant effect on the expression of PCDH10 but enhanced the effect of 5'-azacytidin. In conclusion, we found that the decreased PCDH10 expression in prostate cancer cells was associated with the aberrant methylation of PCDH10 promoter CGI. Our results may contribute to the understanding of the role of PCDH10 inactivation in the progression of prostate cancers.     

Regulation of intracellular pH in cancer cell lines under normoxia and hypoxia

Acid‐extrusion by active transport is important in metabolically active cancer cells, where it removes excess intracellular acid and sets the intracellular resting pH. Hypoxia is a major trigger of adaptive responses in cancer, but its effect on acid‐extrusion remains unclear. We studied pH‐regulation under normoxia and hypoxia in eight cancer cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A, HT29, HT1080, MiaPaca2, HeLa) using the pH‐sensitive fluorophore, cSNARF‐1. Hypoxia responses were triggered by pre‐incubation in low O₂ or with the 2‐oxoglutarate‐dependent dioxygenase inhibitor dimethyloxalylglycine (DMOG). By selective pharmacological inhibition or transport‐substrate removal, acid‐extrusion flux was dissected into components due to Na⁺/H⁺ exchange (NHE) and Na⁺‐dependent HCO transport. In half of the cell‐lines (HCT116, RT112, MDA‐MB‐468, MCF10A), acid‐extrusion on NHE was the dominant flux during an acid load, and in all of these, bar one (MDA‐MB‐468), NHE‐flux was reduced following hypoxic incubation. Further studies in HCT116 cells showed that <4‐h hypoxic incubation reduced NHE‐flux reversibly with a time‐constant of 1–2 h. This was not associated with a change in expression of NHE1, the principal NHE isoform. Following 48‐h hypoxia, inhibition of NHE‐flux persisted but became only slowly reversible and associated with reduced expression of the glycosylated form of NHE1. Acid‐extrusion by Na⁺‐dependent HCO transport was hypoxia‐insensitive and comparable in all cell lines. This constitutive and stable element of pH‐regulation was found to be important for setting and stabilizing resting pH at a mildly alkaline level (conducive for growth), irrespective of oxygenation status. In contrast, the more variable flux on NHE underlies cell‐specific differences in their dynamic response to larger acid loads. J. Cell. Physiol. 228: 743–752, 2013. © 2012 Wiley Periodicals, Inc.     

Effect of sodium butyrate on human breast cancer cell lines

We have investigated the effects exerted by sodium butyrate (NaBu) on the growth and cell cycle perturbations of four human breast cancer cell lines (MCF7, T47D, MDA-MB231 and BT20) with different steroid receptor profiles. Moreover, since one of the supposed mechanisms of action for NaBu activity involves the induction of apoptosis, we have studied the effects of NaBu on DNA fragmentation by agarose gel electrophoresis and flow cytometry. In all investigated cell lines, NaBu exerted a time- and dose-dependent inhibition of growth and caused a maximum inhibitory effect (85% to 90%) at the concentration of 2.5 m m . The inhibition was already evident after 3 days of treatment. The antiproliferative effect of NaBu was associated with a persistent block of cells in the G₂M phase. The block was associated with apoptosis only in oestrogen-receptor positive cell lines. The inhibiting effect of NaBu in hormone-dependent and independent cell lines and its ability to induce apoptosis through a cell cycle perturbation in hormone-dependent cell lines may have important implications in the treatment of human tumours including breast cancer.     

Fluorescent sterol probes for intracellular transport,imaging, and therapeutics

Sterols play a significant role in many physiological processes affecting membrane organization, transport, permeability, and signal transduction. The development of fluorescent sterol analogs that have immediate functional relevance to the natural biomolecules is one approach to understanding the sterol-driven physiological processes. Visualizing cellular compartments with tailor-made fluorescent molecules through specific labeling methods enables organelle targeting and reveals dynamic information. In this review, we focus on the recent literature published between 2020 and 2022, with particular emphasis on extrinsic fluorophores and their investigations of sterol-driven biological processes involving sterol transport, biomolecular interactions, and biological imaging.     

Raman tags: Novel optical probes for intracellular sensing and imaging

Optical labels are needed for probing specific target molecules in complex biological systems. As a newly emerging category of tags for molecular imaging in live cells, the Raman label attracts much attention because of the rich information obtained from targeted and untargeted molecules by detecting molecular vibrations. Here, we list three types of Raman probes based on different mechanisms: Surface Enhanced Raman Scattering (SERS) probes, bioorthogonal Raman probes, and Resonance Raman (RR) probes. We review how these Raman probes work for detecting and imaging proteins, nucleic acids, lipids, and other biomolecules in vitro, within cells, or in vivo. We also summarize recent noteworthy studies, expound on the construction of every type of Raman probe and operating principle, sum up in tables typically targeting molecules for specific binding, and provide merits, drawbacks, and future prospects for the three Raman probes.     

Chromosome compartmentalization alterations in prostate cancer cell lines model disease progression

Prostate cancer aggressiveness and metastatic potential are influenced by gene expression and genomic aberrations, features that can be influenced by the 3D structure of chromosomes inside the nucleus. Using chromosome conformation capture (Hi-C), we conducted a systematic genome architecture comparison on a cohort of cell lines that model prostate cancer progression, from normal epithelium to bone metastasis. We describe spatial compartment identity (A-open versus B-closed) changes with progression in these cell lines and their relation to gene expression changes in both cell lines and patient samples. In particular, 48 gene clusters switch from the B to the A compartment, including androgen receptor, WNT5A, and CDK14. These switches are accompanied by changes in the structure, size, and boundaries of topologically associating domains (TADs). Further, compartment changes in chromosome 21 are exacerbated with progression and may explain, in part, the genesis of the TMPRSS2-ERG translocation. These results suggest that discrete 3D genome structure changes play a deleterious role in prostate cancer progression.      

On the use of 2,1,3-benzothiadiazole derivatives as selective live cell fluorescence imaging probes

Newly designed 2,1,3-benzothiadiazole-containing fluorescent probes with four excited state intramolecular proton transfer (ESIPT) sites were successfully tested in live cell-imaging assays using a confluent monolayer of human stem-cells (tissue). All tested dyes were compared with the commercially available DAPI and gave far better results.     

Recent advances in HDAC-targeted imaging probes for cancer detection

Histone Deacetylases (HDACs) are abnormally high expressed in various cancers and play a crucial role in regulating gene expression. While HDAC-targeted inhibitors have been rapidly developed and approved in the last twenty years, noninvasive monitoring and visualizing the expression levels of HDACs in tumor tissues might help to early diagnosis in cancer and predict the response to HDAC-targeted cancer therapy. In this review, we summarize the recent advancements in the development of HDAC-targeted probes and their applications in cancer imaging and image-guided surgery. We also discuss the design strategies, advantages and disadvantages of these probes. We hope that this review will provide guidance for the design of HDAC-targeted imaging probes and clinical applications in future.