In Vitro and In Vivo Evaluation of the Effects of Drug 2c and Derivatives on Ovarian Cancer Cells.

BACKGROUND: The identification of novel therapeutic strategies for ovarian cancer (OC), the most lethal gynecological neoplasm, is of utmost urgency. Here, we have tested the effectiveness of the compound 2c (4-hydroxy-2,6-bis(4-nitrobenzylidene)cyclohexanone 2). 2c interferes with the cysteine-dependent deubiquitinating enzyme (DUB) UCHL5, thus affecting the ubiquitin-proteasome-dependent degradation of proteins. METHODS: 2c phenotypic/molecular effects were studied in two OC 2D/3D culture models and in a mouse xenograft model. Furthermore, we propose an in silico model of 2c interaction with DUB-UCHL5. Finally, we have tested the effect of 2c conjugated to several linkers to generate 2c/derivatives usable for improved drug delivery. RESULTS: 2c effectively impairs the OC cell line and primary tumor cell viability in both 2D and 3D conditions. The effectiveness is confirmed in a xenograft mouse model of OC. We show that 2c impairs proteasome activity and triggers apoptosis, most likely by interacting with DUB-UCHL5. We also propose a mechanism for the interaction with DUB-UCHL5 via an in silico evaluation of the enzyme-inhibitor complex. 2c also reduces cell growth by down-regulating the level of the transcription factor E2F1. Eventually, 2c activity is often retained after the conjugation with linkers. CONCLUSION: Our data strongly support the potential therapeutic value of 2c/derivatives in OC.

Unveiling the promising anticancer activity of palladium(II)-aryl complexes bearing diphosphine ligands: a structure-activity relationship analysis.

In continuation of our previous works on the cytotoxic properties of organopalladium compounds, in this contribution we describe the first systematic study of the anticancer activity of Pd(II)-aryl complexes. To this end, we have prepared and thoroughly characterized a wide range of palladium derivatives bearing different diphosphine, aryl and halide ligands, developing, when necessary, specific synthetic protocols. Most of the synthesized compounds showed remarkable cytotoxicity towards ovarian and breast cancer cell lines, with IC50 values often comparable to or lower than that of cisplatin. The most promising complexes ([PdI(Ph)(dppe)] and [PdI(p-CH3-Ph)(dppe)]), characterized by a diphosphine ligand with a low bite angle, exhibited, in addition to excellent cytotoxicity towards cancer cells, low activity on normal cells (MRC5 human lung fibroblasts). Specific immunofluorescence tests (cytochrome c and H2AX assays), performed to clarify the possible mechanism of action of this class of organopalladium derivatives, seemed to indicate DNA as the primary cellular target, whereas caspase 3/7 assays proved that the complex [PdI(Ph)(dppe)] was able to promote intrinsic apoptotic cell death. A detailed molecular docking analysis confirmed the importance of a diphosphine ligand with a reduced bite angle to ensure a strong DNA-complex interaction. Finally, one of the most promising complexes was tested towards patient-derived organoids, showing promising ex vivo cytotoxicity.

Microfluidic production of amiodarone loaded nanoparticles and application in drug repositioning in ovarian cancer.

Amiodarone repositioning in cancer treatment is promising, however toxicity limits seem to arise, constraining its exploitability. Notably, amiodarone has been investigated for the treatment of ovarian cancer, a tumour known for metastasizing within the peritoneal cavity. This is associated with an increase of fatty acid oxidation, which strongly depends on CPT1A, a transport protein which has been found overexpressed in ovarian cancer. Amiodarone is an inhibitor of CPT1A but its role still has to be explored. Therefore, in the present study, amiodarone was tested on ovarian cancer cell lines with a focus on lipid alteration, confirming its activity. Moreover, considering that drug delivery systems could lower drug side effects, microfluidics was employed for the development of drug delivery systems of amiodarone obtaining simultaneously liposomes with a high payload and amiodarone particles. Prior to amiodarone loading, microfluidics production was optimized in term of temperature and flow rate ratio. Moreover, stability over time of particles was evaluated. In vitro tests confirmed the efficacy of the drug delivery systems.

Rational Design of Palladium(II) Indenyl and Allyl Complexes Bearing Phosphine and Isocyanide Ancillary Ligands with Promising Antitumor Activity.

A new class of palladium-indenyl complexes characterized by the presence of one bulky alkyl isocyanide and one aryl phosphine serving as ancillary ligands has been prepared, presenting high yields and selectivity. All the new products were completely characterized using spectroscopic and spectrometric techniques (NMR, FT-IR, and HRMS), and, for most of them, it was also possible to define their solid-state structures via X-ray diffractometry, revealing that the indenyl fragment always binds to the metal centre with a hapticity intermediate between ƞ3 and ƞ5. A reactivity study carried out using piperidine as a nucleophilic agent proved that the indenyl moiety is the eligible site of attack rather than the isocyanide ligand or the metal centre. All complexes were tested as potential anticancer agents against three ovarian cancer cell lines (A2780, A2780cis, and OVCAR-5) and one breast cancer cell line (MDA-MB-231), displaying comparable activity with respect to cisplatin, which was used as a positive control. Moreover, the similar cytotoxicity observed towards A2780 and A2780cis cells (cisplatin-sensitive and cisplatin-resistant, respectively) suggests that our palladium derivatives presumably act with a mechanism of action different than that of the clinically approved platinum drugs. For comparison, we also synthesized Pd-ƞ3-allyl derivatives, which generally showed a slightly higher activity towards ovarian cancer cells and lower activity towards breast cancer cells with respect to their Pd-indenyl congeners.

3D dynamic cultures of HGSOC organoids to model innovative and standard therapies.

High-grade serous ovarian cancer (HGSOC) needs new technologies for improving cancer diagnosis and therapy. It is a fatal disease with few options for the patients. In this context, dynamic culture systems coupling with patient-derived cancer 3D microstructures could offer a new opportunity for exploring novel therapeutic approaches. In this study, we optimized a passive microfluidic platform with 3D cancer organoids, which allows a standardized approach among different patients, a minimum requirement of samples, multiple interrogations of biological events, and a rapid response. The passive flow was optimized to improve the growth of cancer organoids, avoiding the disruption of the extracellular matrix (ECM). Under optimized conditions of the OrganoFlow (tilting angle of 15° and an interval of rocking every 8 min), the cancer organoids grow faster than when they are in static conditions and the number of dead cells is reduced over time. To calculate the IC 50 values of standard chemotherapeutic drugs (carboplatin, paclitaxel, and doxorubicin) and targeted drugs (ATRA), different approaches were utilized. Resazurin staining, ATP-based assay, and DAPI/PI colocalization assays were compared, and the IC 50 values were calculated. The results showed that in the passive flow, the IC 50 values are lower than in static conditions. FITC-labeled paclitaxel shows a better penetration of ECM under passive flow than in static conditions, and cancer organoids start to die after 48 h instead of 96 h, respectively. Cancer organoids are the last frontiers for ex vivo testing of drugs that replicate the response of patients in the clinic. For this study, organoids derived from ascites or tissues of patients with Ovarian Cancer have been used. In conclusion, it was possible to develop a protocol for organoid cultures in a passive microfluidic platform with a higher growth rate, faster drug response, and better penetration of drugs into ECM, maintaining the samples' vitals and collecting the data on the same plate for up to 16 drugs.

A Green Synthesis of Carbene-Metal-Amides (CMAs) and Carboline-Derived CMAs with Potent in†vitro and ex vivo Anticancer Activity.

The modularity and ease of synthesis of carbene-metal-amide (CMA) complexes based on the coinage metals (Au, Ag, Cu) and N-heterocyclic carbenes (NHCs) as ancillary ligands pave the way for the expansion of their applications beyond photochemistry and catalysis. Herein, we further improve the synthesis of such compounds by circumventing the use of toxic organic solvents which were previously required for their purification, and we expand their scope to include complexes incorporating carbolines as the amido fragments. The novel complexes are screened both in vitro and ex vivo, against several cancer cell lines and high-grade serous ovarian cancer (HGSOC) tumoroids, respectively. Excellent cytotoxicity values are obtained for most complexes, while the structural variety of the CMA library screened thus far, provides promising leads for future developments. Variations of all three components (NHC, metal, amido ligand), enable the establishment of trends regarding cytotoxicity and selectivity towards cancerous over normal cells.

A simple synthetic entryway into new families of NHC-gold-amido complexes and their in vitro antitumor activity.

A simple synthetic pathway to Au-NHC amido complexes is described. Syntheses and isolation of [Au(NHC)(NR1R2)] complexes, bearing various NHC ligands and NH-containing heterocycles under mild conditions are reported. The in vitro anticancer activity of these gold-complexes was investigated on three human cancer cell lines. A number of these show comparable or even better antiproliferative activity than cisplatin. Noteworthy is the non-toxicity of most of the complexes on normal cells.