Investigation of Specific Targeting of Triptorelin-Conjugated Dextran-Coated Magnetite Nanoparticles as a Targeted Probe in GnRH+ Cancer Cells in MRI

In recent years, the conjugation of superparamagnetic iron oxide nanoparticles (SPIONs), as tumor-imaging probes for magnetic resonance imaging (MRI), with tumor targeting peptides possesses promising advantages for specific delivery of MRI agents. The objective of the current study was to design a targeted contrast agent for MRI based on Fe3O4 nanoparticles conjugated triptorelin (SPION@triptorelin), which has a great affinity to the GnRH receptors. The SPIONs-coated carboxymethyl dextran (SPION@CMD) conjugated triptorelin (SPION@CMD@triptorelin) were synthesized using coprecipitation method and characterized by DLS, TEM, XRD, FTIR, Zeta, and VSM techniques. The relaxivities of synthetized formulations were then calculated using a 1.5 Tesla clinical magnetic field. MRI, quantitative cellular uptake, and cytotoxicity level of them were estimated. The characterization results confirmed that the formation of SPION@CMD@triptorelin has been conjugated with a suitable size. Our results demonstrated the lack of cellular cytotoxicity of SPION@CMD@triptorelin, and it could increase the cellular uptake of SPIONs to MDA-MB-231 cancer cells 6.50-fold greater than to SPION@CMD at the concentration of 75 μM. The relaxivity calculations for SPION@CMD@triptorelin showed a suitable r2 and r2/r1 with values of 31.75 mM−1·s−1 and 10.26, respectively. Our findings confirm that triptorelin-targeted SPIONs could provide a T2-weighted probe contrast agent that has the great potential for the diagnosis of GnRH-positive cancer in MRI.

Role of Gonadotropin-Releasing Hormone (GnRH) in Ovarian Cancer

The hypothalamus–pituitary–gonadal (HPG) axis is the endocrine regulation system that controls the woman’s cycle. The gonadotropin-releasing hormone (GnRH) plays the central role. In addition to the gonadotrophic cells of the pituitary, GnRH receptors are expressed in other reproductive organs, such as the ovary and in tumors originating from the ovary. In ovarian cancer, GnRH is involved in the regulation of proliferation and metastasis. The effects on ovarian tumors can be indirect or direct. GnRH acts indirectly via the HPG axis and directly via GnRH receptors on the surface of ovarian cancer cells. In this systematic review, we will give an overview of the role of GnRH in ovarian cancer development, progression and therapy.

Plasticity in medaka gonadotropes via cell proliferation and phenotypic conversion

Follicle-stimulating hormone (Fsh) and luteinizing hormone (Lh) produced by the gonadotropes play a major role in control of reproduction. Contrary to mammals and birds, Lh and Fsh are mostly produced by two separate cell types in teleost. Here, we investigated gonadotrope plasticity, using transgenic lines of medaka (Oryzias latipes) where DsRed2 and hrGfpII are under the control of the fshb and lhb promotors respectively. We found that Fsh cells appear in the pituitary at 8 dpf, while Lh cells were previously shown to appear at 14 dpf. Similar to Lh cells, Fsh cells show hyperplasia from juvenile to adult stages. Hyperplasia is stimulated by estradiol. Both Fsh and Lh cells show hypertrophy during puberty with similar morphology. They also share similar behavior, using their cellular extensions to make networks. We observed bi-hormonal gonadotropes in juveniles and adults but not in larvae where only mono-hormonal cells are observed, suggesting the existence of phenotypic conversion between Fsh and Lh in later stages. This is demonstrated in cell culture, where some Fsh cells start to produce Lhβ, a phenomenon enhanced by gonadotropin-releasing hormone (Gnrh) stimulation. We have previously shown that medaka Fsh cells lack Gnrh receptors, but here we show that with time in culture, some Fsh cells start responding to Gnrh, while fshb mRNA levels are significantly reduced, both suggestive of phenotypic change. All together, these results reveal high plasticity of gonadotropes due to both estradiol-sensitive proliferation and Gnrh promoted phenotypic conversion, and moreover, show that gonadotropes lose part of their identity when kept in cell culture.

Gonadotropin Releasing Hormone Agonists Have an Anti-apoptotic Effect on Cumulus Cells

Background: Ovaries are sensitive to chemotherapy, which may lead to early depletion of primordial follicle reserve. One strategy for gonadal function preservation is temporary ovarian suppression with Gonadotropin Releasing Hormone agonists (GnRHa) during chemotherapy. To date, GnRHa protective mechanism of action remains not fully elucidated. Methods: We collected 260 immature cumulus cell-oocyte complexes (COC) from 111 women < 38 years old, with a normal ovarian reserve. The COC were randomly assigned to the following groups: a) control; culture with the addition of b) GnRHa; c) cyclophosphamide; d) cyclophosphamide plus GnRHa. After in vitro treatments, RNA and proteins were extracted from oocytes and cumulus cells (CC), separately. Potential effects of drugs were evaluated on GnRH receptors, apoptosis pathways, ceramide pathway, and glutathione synthesis by quantitative PCR and, whenever possible, by Western blot. Results: Cyclophosphamide triggered activation of the extrinsic pathway of apoptosis mediated by BAX in CC. The co-administration of GnRHa inhibited the apoptosis pathway in CC. According to our model, the GnRHa does not directly act on oocytes, which do not express GnRH receptors. Moreover, glutathione synthesis was decreased after GnRHa treatment both in CC and oocytes. Conclusion: Our data suggest that the protective mechanisms induced by GnRHa is mediated by an anti-apoptotic effect on CC.

Plasticity in medaka gonadotropes via cell proliferation and phenotypic conversion

ABSTRACTFollicle stimulating hormone (Fsh) and luteinizing hormone (Lh) produced by the gonadotropes, play a major role in control of reproduction. Contrary to mammals and birds, Lh and Fsh are mostly produced by two separate cell types in teleost. Here, we investigated gonadotrope plasticity, using transgenic lines of medaka (Oryzias latipes) where DsRed2 and hrGfpII are under the control of fshb and lhb promotors respectively. We found that Fsh cells first appear in the pituitary at 8 dpf. Similar to in Lh cells, Fsh cells show hyperplasia from juvenile to adult stages. Hyperplasia is stimulated by estradiol exposure. Both Fsh and Lh cells show hypertrophy during puberty with similar morphology. They also share similar behavior, using their cellular extensions to make networks. We observed bi-hormonal gonadotropes in juvenile and adult fish but not during larval stage where only mono-hormonal cells are observed, suggesting the existence of phenotypic conversion between Fsh and Lh in later stages. This is demonstrated in cell culture, where some Fsh start to produce lhb, a phenomenon enhanced by gonadotropin releasing hormone (Gnrh) stimulation. We have previously shown that medaka Fsh cells lack Gnrh receptors, but here we show that with time in culture, some Fsh cells start responding to Gnrh, while fshb mRNA levels are significantly reduced, both suggestive of phenotypic change. All together, these results reveal high plasticity of gonadotropes due to both estradiol sensitive proliferation and Gnrh promoted phenotypic conversion, and also shows that gonadotropes lose part of their identity when kept in cell culture.

Generation of Reactive Oxygen Species by Photosensitizers and their Modes of Action on Proteins

In this review, we first survey the mechanisms underlying the chemical modification of amino acid residues in proteins by singlet oxygen elicited by photosensitizers. Singlet oxygen has the capacity to cause widespread chemical damage to cellular proteins. Its use in photodynamic therapy of tumors thus requires the development of methodologies for specific addressing of the photosensitizer to malignant cells while sparing normal tissue. We describe three targeting paradigms for achieving this objective. The first involves the use of a photosensitizer with a high affinity for its target protein; in this case, the photosensitizer is methylene blue for acetylcholinesterase. The second paradigm involves the use of the hydrophobic photosensitizer hypericin, which has the capacity to interact selectively with partially unfolded forms of proteins, including nascent species in rapidly dividing or virus-infected and cancer cells, acting preferentially at membrane interfaces. In this case, partially unfolded molten globule species of acetylcholinesterase serve as the model system. In the third paradigm, the photodynamic approach takes advantage of a general approach in ‘state-of-the-art’ chemotherapy, by coupling the photosensitizer emodin to a specific peptide hormone, GnRH, which recognizes malignant cells via specific GnRH receptors on their surface.

Synthesis and in vitro biochemical evaluation of oxime bond-linked daunorubicin–GnRH-III conjugates developed for targeted drug delivery

Gonadotropin releasing hormone-III (GnRH-III), a native isoform of the human GnRH isolated from sea lamprey, specifically binds to GnRH receptors on cancer cells enabling its application as targeting moieties for anticancer drugs. Recently, we reported on the identification of a novel daunorubicin–GnRH-III conjugate (GnRH-III–[4Lys(Bu), 8Lys(Dau=Aoa)] with efficient in vitro and in vivo antitumor activity. To get a deeper insight into the mechanism of action of our lead compound, the cellular uptake was followed by confocal laser scanning microscopy. Hereby, the drug daunorubicin could be visualized in different subcellular compartments by following the localization of the drug in a time-dependent manner. Colocalization studies were carried out to prove the presence of the drug in lysosomes (early stage) and on its site of action (nuclei after 10 min). Additional flow cytometry studies demonstrated that the cellular uptake of the bioconjugate was inhibited in the presence of the competitive ligand triptorelin indicating a receptor-mediated pathway. For comparative purpose, six novel daunorubicin–GnRH-III bioconjugates have been synthesized and biochemically characterized in which 6Asp was replaced by D-Asp, D-Glu and D-Trp. In addition to the analysis of the in vitro cytostatic effect and cellular uptake, receptor binding studies with 125I-triptorelin as radiotracer and degradation of the GnRH-III conjugates in the presence of rat liver lysosomal homogenate have been performed. All derivatives showed high binding affinities to GnRH receptors and displayed in vitro cytostatic effects on HT-29 and MCF-7 cancer cells with IC50 values in a low micromolar range. Moreover, we found that the release of the active drug metabolite and the cellular uptake of the bioconjugates were strongly affected by the amino acid exchange which in turn had an impact on the antitumor activity of the bioconjugates.