ABSTRACT
Cholesterol is not only a major component of the cell membrane, but also plays an important role in a wide range of biological processes and pathologies. It is therefore crucial to develop appropriate tools for visualizing intracellular cholesterol transport. Here, we describe new cationic analogues of BODIPY-Cholesterol (TopFluor-Cholesterol, TF-Chol), which combine a positive charge on the sterol side chain and a BODIPY group connected via a C-4 linker. In contrast to TF-Chol, the new analogues TF-1 and TF-3 possessing acetyl groups on the A ring (C-3 position on steroid) internalized much faster and displayed slightly different levels of intracellular localization. Their applicability for cholesterol monitoring was indicated by the fact that they strongly label compartments with accumulated cholesterol in cells carrying a mutation of the Niemann-Pick disease-associated cholesterol transporter, NPC1.
Subject(s)
Boron Compounds/analysis , Cholesterol/analysis , Biological Transport , Boron Compounds/chemical synthesis , Boron Compounds/chemistry , Boron Compounds/metabolism , Cell Line , Cholesterol/analogs & derivatives , Cholesterol/chemical synthesis , Cholesterol/metabolism , Humans , Optical ImagingABSTRACT
Fluorescent sterol probes, comprising a fluorophore connected to a sterol backbone by means of a linker, are promising tools for enabling high-resolution imaging of intracellular cholesterol. In this study, we evaluated how the size of the linker, site of its attachment and nature of the fluorophore, affect the localization and trafficking properties of fluorescent sterol probes. Varying lengths of linker using the same fluorophore affected cell penetration and retention in specific cell compartments. A C-4 linker was confirmed as optimal. Derivatives of heterocyclic sterol precursors attached with identical C-4 linker to different fluorophores at diverse positions also showed significant differences in their binding properties to various intracellular compartments and kinetics of trafficking. Two novel red-emitting probes with good cell permeability, fast intracellular labelling and slightly different distribution displayed very promising characteristics for sterol probes. These probes also strongly labelled endo/lysosomal compartment in cells with pharmacologically disrupted cholesterol transport, or with a genetic mutation of cholesterol transporting protein NPC1, that overlapped with filipin staining of cholesterol. Overall, the present study demonstrates that the physicochemical properties of the fluorophore/linker pairing determine the kinetics of uptake and distribution and subsequently influence the applicability of final probes.
ABSTRACT
Purpurin 18 derivatives with a polyethylene glycol (PEG) linker were synthesized as novel photosensitizers (PSs) with the goal of using them in photodynamic therapy (PDT) for cancer. These compounds, derived from a second-generation PS, exhibit absorption at long wavelengths; considerable singlet oxygen generation and, in contrast to purpurin 18, have higher hydrophilicity due to decreased logP. Together, these properties make them potentially ideal PSs. To verify this, we screened the developed compounds for cell uptake, intracellular localization, antitumor activity and induced cell death type. All of the tested compounds were taken up into cancer cells of various origin and localized in organelles known to be important PDT targets, specifically, mitochondria and the endoplasmic reticulum. The incorporation of a zinc ion and PEGylation significantly enhanced the photosensitizing efficacy, decreasing IC50 (half maximal inhibitory compound concentration) in HeLa cells by up to 170 times compared with the parental purpurin 18. At effective PDT concentrations, the predominant type of induced cell death was apoptosis. Overall, our results show that the PEGylated derivatives presented have significant potential as novel PSs with substantially augmented phototoxicity for application in the PDT of cervical, prostate, pancreatic and breast cancer.
Subject(s)
Photochemotherapy/methods , Porphyrins/chemistry , Singlet Oxygen/chemistry , Cell Line, Tumor , Flow Cytometry , Humans , Microscopy, Fluorescence , SolubilityABSTRACT
Photodynamic therapy has become a feasible direction for the treatment of both malignant and non-malignant diseases. It has been in the spotlight since FDA regulatory approval was granted to several photosensitizers worldwide. Nevertheless, there are still strong limitations in the targeting specificity that is vital to prevent systemic toxicity. Here, we report the synthesis and biological evaluation of a novel bimodal oxime conjugate composed of a photosensitizing drug, red-emitting pheophorbide a, and nandrolone (NT), a steroid specifically binding the androgen receptor (AR) commonly overexpressed in various tumors. We characterized the physico-chemical properties of the NT-pheophorbide a conjugate (NT-Pba) and singlet oxygen generation. Because light-triggered therapies have the potential to provide important advances in the treatment of hormone-sensitive cancer, the biological potential of this novel specifically-targeted photosensitizer was assessed in prostatic cancer cell lines in vitro using an AR-positive (LNCaP) and an AR-negative/positive cell line (PC-3). U-2 OS cells, both with and without stable AR expression, were used as a second cell line model. Interestingly, we found that the NT-Pba conjugate was not only photodynamically active and AR-specific, but also that its phototoxic effect was more pronounced compared to pristine pheophorbide a. We also examined the intracellular localization of NT-Pba. Live-cell fluorescence microscopy provided clear evidence that the NT-Pba conjugate localized in the endoplasmic reticulum and mitochondria. Moreover, we performed a competitive localization study with the excess of nonfluorescent NT, which was able to displace fluorescent NT-Pba from the cell interior, thereby further confirming the binding specificity. The oxime ether bond degradation was assayed in living cells by both real-time microscopy and a steroid receptor reporter assay using AR U-2 OS cells. Thus, NT-Pba is a promising candidate for both the selective targeting and eradication of AR-positive malignant cells by photodynamic therapy.
Subject(s)
Antineoplastic Agents/pharmacology , Chlorophyll/analogs & derivatives , Oximes/pharmacology , Photochemotherapy , Photosensitizing Agents/pharmacology , Testosterone/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/chemistry , Cell Line, Tumor , Cell Proliferation/drug effects , Chlorophyll/chemistry , Chlorophyll/pharmacology , Dose-Response Relationship, Drug , Drug Screening Assays, Antitumor , Humans , Microscopy, Fluorescence , Molecular Structure , Optical Imaging , Oximes/chemistry , Particle Size , Photosensitizing Agents/chemical synthesis , Photosensitizing Agents/chemistry , Structure-Activity Relationship , Surface Properties , Testosterone/analogs & derivatives , Testosterone/chemistryABSTRACT
The phenolic compounds p-hydroxyacetophenone and catechin have been extracted from Norway spruce needles with pure methanol, 80 and 50% (v/v) aqueous methanol, pure acetonitrile, 80% (v/v) aqueous acetonitrile, and pure water. Extraction efficiency of the individual solvents was compared. Although 80% aqueous methanol is the solvent most frequently used for extraction of soluble phenolic compounds from needles, it was found that pure methanol is a more suitable extraction solvent. Surprisingly, a two-step procedure based on the extraction of crushed needles with water then re-extraction with methanol proved a good alternative to direct extraction with methanol. Extraction of uncrushed spruce needles might indicate that relatively more p-hydroxyacetophenone than catechin was located in the surface layer of the needle.
