Bifunctionality and Antitumor Efficacy of ZG-126, a Vitamin D Receptor Agonist/Histone Deacetylase Inhibitor Hybrid Molecule.

Analogues of hormonal vitamin D, 1,25-dihydroxyvitamin D (1,25D), signal through the nuclear vitamin D receptor (VDR). They have potential in combination therapies with other anticancer agents such as histone deacetylase inhibitors (HDACi's). Here, we characterize the ZG series of hybrid compounds that combine HDACi within the backbone of a VDR agonist. All display improved solubility, with ZG-126 being the most robustly bifunctional molecule in multiple cell lines. ZG-126 is well tolerated and strongly induces VDR target gene expression in vivo at therapeutic doses. Its antitumor efficacy is superior to 1,25D and the HDACi SAHA, separately or together, in mouse models of melanoma and triple-negative breast cancer (TNBC). Notably, ZG-126 treatment reduces metastases almost 4-fold in an aggressive TNBC model. ZG-126 also reduces total macrophage infiltration and the proportion of immunosuppressive M2-polarized macrophages in TNBC tumors by 2-fold. ZG-126 thus represents a bifunctional and efficacious anticancer agent with improved physicochemical properties.

Molecular mechanisms of bifunctional vitamin D receptor agonist-histone deacetylase inhibitor hybrid molecules in triple-negative breast cancer.

The active form of vitamin D, 1,25-dihydroxyvitamin D (1,25D), and its analogues signal through the nuclear vitamin D receptor (VDR), a ligand-regulated transcription factor, and have been extensively investigated as anticancer agents. 1,25D and its analogs have potential in combination therapies because they exhibit synergistic activities with other anticancer agents such as histone deacetylase inhibitors (HDACi). We have developed a series of hybrid molecules that combine HDACi within the backbone of a VDR agonist and thus represent fully integrated bifunctional molecules. They exhibit anti-tumor efficacy in reducing tumor growth and metastases in an aggressive model of triple-negative breast cancer. However, their solubility is limited by their hydrophobic diarylpentane cores. Our goals here were two-fold: (1) to improve the solubility of hybrids by introducing nitrogen into diarylpentane cores, and (2) to investigate the molecular mechanisms underlying their anti-tumor efficacy by performing comparative gene expression profiling studies with 1,25D and the potent HDACi suberoylanilide hydroxamic acid (SAHA). We found that substituting aryl with pyrydyl rings did not sacrifice bifunctionality and modestly improved solubility. Notably, one compound, AM-193, displayed enhanced potency as a VDR agonist and in cellular assays of cytotoxicity. RNAseq studies in triple negative breast cancer cells revealed that gene expression profiles of hybrids were very similar to that of 1,25D, as was that observed with 1,25D and SAHA combined. The effects of SAHA alone on gene expression were limited and distinct from those 1,25D or hybrids. The combined results suggest that efficacy of hybrids arises from targeting HDACs that do not have a direct role in gene regulation. Moreover, pathways analysis revealed that hybrids regulate numerous genes controlling immune cell infiltration into tumors and suppress the expression of several secreted molecules that promote breast cancer growth and metastasis.

Thermally conductive graphene-based nanofluids, a novel class of cryosolutions for mouse blastocysts vitrification.

Limited heating and cooling rates have long been recognized as bottlenecks in improving embryo cryopreservation. As a result, efforts to achieve higher heat transfer rates gave rise to milestones like open cryodevices and minimal media loading. A crucial but commonly ignored variable is heat conduction by cryosolutions. The low heat conductivity of the aqueous media surrounding embryos slows down cooling and heating rates of the embryo, imposing the risk of preventable damages. In this study, we introduce a novel thermally conductive cryosolution based on graphene oxide nanoparticles and test its performance against conventional sucrose-based solutions for vitrification of mouse blastocysts. Replacing sucrose with graphene oxide brought about similar re-expansion, hatching, and implantation rates of post-vitrification embryos while also preventing an array of cellular and molecular stresses. Our results showed significantly reduced oxidative stress, characterized by control-level expression of Sod1 and significant downregulation of Sod2 transcription when graphene oxide was used instead of sucrose. This molecular response was in agreement with the reduced level of reactive oxygen species produced in vitrified/warmed embryos using graphene-based solutions. The downstream impacts of this stress reduction manifested in significant downregulation of two major pro-apoptotic genes, Bax and Trp53, down to the same level as fresh embryos. Interestingly, embryos maintained their spherical shape during dehydration in graphene-based solutions and did not "collapse" when shrinking, like in sucrose-based solutions. These results provide new insights into the benefits of thermally conductive cryosolutions and showcase the potential of graphene oxide as a cryoprotectant in embryo vitrification.

Toward embryo cryopreservation-on-a-chip: A standalone microfluidic platform for gradual loading of cryoprotectants to minimize cryoinjuries.

Embryo vitrification is a fundamental practice in assisted reproduction and fertility preservation. A key step of this process is replacing the internal water with cryoprotectants (CPAs) by transferring embryos from an isotonic to a hypertonic solution of CPAs. However, this applies an abrupt osmotic shock to embryos, resulting in molecular damages that have long been a source of concern. In this study, we introduce a standalone microfluidic system to automate the manual process and minimize the osmotic shock applied to embryos. This device provides the same final CPA concentrations as the manual method but with a gradual increase over time instead of sudden increases. Our system allows the introduction of the dehydrating non-permeating CPA, sucrose, from the onset of CPA-water exchange, which in turn reduced the required time of CPA loading for successful vitrification without compromising its outcomes. We compared the efficacy of our device and the conventional manual procedure by studying vitrified-warmed mouse blastocysts based on their re-expansion and hatching rates and transcription pattern of selected genes involved in endoplasmic reticulum stress, oxidative stress, heat shock, and apoptosis. While both groups of embryos showed comparable re-expansion and hatching rates, on-chip loading reduced the detrimental gene expression of cryopreservation. The device developed here allowed us to automate the CPA loading process and push the boundaries of cryopreservation by minimizing its osmotic stress, shortening the overall process, and reducing its molecular footprint.

Using natural honey as an anti-oxidant and thermodynamically efficient cryoprotectant in embryo vitrification.

Embryo cryopreservation is a common practice in reproductive biology and infertility treatments. Despite major improvements over years, the cryoprotectant solutions are still a major source of concern, mostly due to their chemical toxicity and suboptimal protection against cryoinjuries. In this work, we introduced natural honey as a non-permeating cryoprotectant to replace traditionally used sucrose in embryo vitrification. The proposed media were compared with conventional ones by evaluating vitrified/warmed mouse embryos based on their re-expansion, hatching rate and transcription pattern of selected genes involved in heat-shock response, apoptosis and oxidative stress. Despite the similar high re-expansion rate, molecular fingerprint of the cryopreservation is remarkably reduced when honey is used instead of sucrose. The biological response of the proposed media was explained from a fundamental point of view using antioxidant analysis, DSC and GC techniques. It was found that the proposed honey-based medium is less thermodynamically prone to ice formation, which along with its antioxidant capacity can control the production of oxygen radicals and minimize the stress-induced transcriptional response. Furthermore, this work tries to correlate the physico-chemical properties of the vitrification solutions with the cellular and molecular aspects of the cryopreservation and proposes the application of natural cryoprotectants in cryobiology.

In vitro versus In vivo: Development-, Apoptosis-, and Implantation- Related Gene Expression in Mouse Blastocyst.

BACKGROUND: While mammalian embryos can adapt to their environments, their sensitivity overshadows their adaptability in suboptimal in vitro conditions. Therefore, the environment in which the gametes are fertilized or to which the embryo is exposed can greatly affect the quality of the embryo and consequently its implantation potential. OBJECTIVES: Since providing an optimal culture condition needs a deep understanding of the environmental effects, and regarding the fact that normal morphology fails to be a reliable indicator of natural embryo development, the current study aimed at comparing in vivo- and in vitro-derived blastocysts at the molecular level. MATERIALS AND METHODS: In vivo and in vitro mouse blastocysts were obtained by flushing the uterine horns and in vitro fertilization/culture, respectively. Normal blastocysts of both groups were evaluated in terms of hatching rate and expression of three lineage-differentiation-, apoptosis-, and implantation-related genes. RESULTS: The hatching rate was lower in In vitro fertilization (IVF)-produced blastocysts in comparison with that of the in vivo counterparts. More importantly, the study results indicated significant changes in the expression levels of eight out of ten selected genes, especially Mmp-9 (about -10.7-fold). The expression of Mmp-9 in trophoblast cells is required for successful implantation and trophoblast invasion. CONCLUSIONS: The current study, in addition to confirming that the altered gene expression pattern of in vitro-produced embryos resulted in normal morphology, provided a possible reason for lower implantation rate of in vitro-produced blastocysts regarding the Mmp-9 expression.

Different Origin, Different Response: Gene Expression Pattern in Collapsed Vitrified Blastocyst.

There is a large body of animal experimental data about assisted reproductive techniques that could be applied to improve clinical outcomes. The great part of this information was obtained from research on in vivo-derived embryos. But whether these results are always similar with those we expect from embryos having in vitro origin in the clinical cases is a critical question. The present study was designed to compare the effects of vitrification (VIT) and artificial collapse (AC) as two commonly used techniques on in vivo- and in vitro-derived mouse embryos. In this regard, both origins of blastocysts were produced and randomly divided into three experimental groups, including control (non-vitrified), VIT, and AC-VIT. The survival and hatching rates and the expression of development-related genes were assessed in all groups and compared with their control counterpart. According to our results, although in vivo and in vitro origins followed the same pattern in the hatching rate, the real-time PCR data showed two distinct patterns of gene expression. Compared to the control, vitrification increased the expression of pluripotency genes in in vivo group. While in vitro vitrified blastocysts showed a significant reduction in the transcripts of these genes. More interestingly, although AC resulted in a sharp decrease of Gata6 and Grb2 in post warmed in vivo blastocysts, it could not affect the vitrified IVP ones. In conclusion, it seems that vitrification and artificial collapse techniques have different effects on embryo fate depending on in vivo or in vitro origins of the embryos.

Low oxygen tension promotes invasive ability and embryo implantation rate.

Low oxygen concentrations during in vitro embryo development not only improving the embryo quality but also can lead to successful implantation. Yet, there is no investigation at the molecular level to indicate the association between increased implantation rate and invasive ability of blastocyst and its inner cell mass quality with in vitro culture under a hypoxic condition. Therefore, the present study was designed to investigate blastocyst formation, total cell number, hatching and implantation rates. In addition we assessed the transcription levels of invasion-(Mmp-9 and uPA) and pluripotency-related genes (Pou5f1, Nanog) in mouse blastocyst under hypoxic condition. In vivo two-cell embryos were randomly divided into two groups; 5% O2 and 20% O2. Embryos were then cultured to the blastocyst stage and evaluated in terms of cellular parameters. The expression levels of selected genes were also analyzed both in experimental group and in vivo blastocysts recovered from uteri as control group. Results indicated the blastocyst formation, hatching and implantation rates were improved when the embryos were cultured in hypoxic condition. Furthermore, the expression levels of Mmp-9, Nanog and Pou5f1 showed an increase in 5% O2 in comparison with 20% O2 group. In conclusion, it seems that hypoxic condition by increasing the quality and invasion ability of the blastocyst can improve implantation rate.