Cisplatin-resistant A2780cis Research Articles

The Induction of G2/M Phase Cell Cycle Arrest and Apoptosis by the Chalcone Derivative 1C in Sensitive and Resistant Ovarian Cancer Cells Is Associated with ROS Generation.

Ovarian cancer ranks among the most severe forms of cancer affecting the female reproductive organs, posing a significant clinical challenge primarily due to the development of resistance to conventional therapies. This study investigated the effects of the chalcone derivative 1C on sensitive (A2780) and cisplatin-resistant (A2780cis) ovarian cancer cell lines. Our findings revealed that 1C suppressed cell viability, induced cell cycle arrest at the G2/M phase, and triggered apoptosis in both cell lines. These effects are closely associated with generating reactive oxygen species (ROS). Mechanistically, 1C induced DNA damage, modulated the activity of p21, PCNA, and phosphorylation of Rb and Bad proteins, as well as cleaved PARP. Moreover, it modulated Akt, Erk1/2, and NF-κB signaling pathways. Interestingly, we observed differential effects of 1C on Nrf2 levels between sensitive and resistant cells. While 1C increased Nrf2 levels in sensitive cells after 12 h and decreased them after 48 h, the opposite effect was observed in resistant cells. Notably, most of these effects were suppressed by the potent antioxidant N-acetylcysteine (NAC), underscoring the crucial role of ROS in 1C-induced antiproliferative activity. Moreover, we suggest that modulation of Nrf2 levels can, at least partially, contribute to the antiproliferative effect of chalcone 1C.

Muscarinic Receptors as Targets for Metronomic Therapy in Ovarian Cancer

Aim: In this study, the effects of muscarinic acetylcholine receptor (mAChR) agonist carbachol on the proliferation of cisplatin-resistant (A2780cis) and cisplatin-free (SKOV-3) ovarian cancer cell line were for the first time investigated to further evaluate the potential therapeutic effect of metronomic chemotherapy.
 Material and Methods: The inhibitory effect of carbachol on cell proliferation was detected using the xCELLigence Real-Time Cell Analyzer (RTCA) dual plate (DP) system. A preliminary study was conducted to determine the dose of carbachol 100 µM, cisplatin 1 µM, and two combination studies were carried out with 100 µM carbachol + cisplatin 1 µM and 100 µM carbachol + 10 µM atropine, over cancer cells without drugs was used as the control group. The cell proliferation curve was monitored for 96 hours. The cell index value of inhibition in cell proliferation was automatically measured every hour for each well using RTCA 1.2.1 software.
 Results: Co-administration of carbachol with cisplatin caused a decrease in cell number in both A2780cis and SKOV-3 cell lines in a time-dependent manner (p

Cytotoxic effects of halophilic archaea metabolites on ovarian cancer cell lines

BackgroundOvarian cancer is one of the most frequent and deadly gynaecological cancers, often resistant to platinum-based chemotherapy, the current standard of care. Halophilic microorganisms have been shown to produce a large variety of metabolites, some of which show toxicity to various cancer cell lines. However, none have yet been shown to be active against ovarian cancer cells. Here, we examined the effects of metabolites secreted by the halophilic archaea Halorhabdus rudnickae and Natrinema salaciae on various cancer cell lines, including ovarian cancer cell lines.Results1H NMR analyses of Hrd. rudnickae and Nnm. salaciae culture supernatants contain a complex mixture of metabolites that differ between species, and even between two different strains of the same species, such as Hrd. rudnickae strains 64T and 66. By using the MTT and the xCELLigence RTCA assays, we found that the secreted metabolites of all three halophilic strains expressed cytotoxicity to the ovarian cancer cell lines, especially A2780, as well as its cisplatin-resistant derivative A2780cis, in a dose-dependent manner. The other tested cell lines A549, HepG2, SK-OV-3 and HeLa were only minimally, or not at all affected by the archaeal metabolites, and this was only seen with the MTT assay.ConclusionsThe halophilic archaea Hrd. rudnickae and Nnm. salaciae, isolated from a Polish salt mine and Lake Medee in the Mediterranean Sea, respectively, secrete metabolites that are active against ovarian cancer cells, including those that are resistant to cisplatin. This opens potential new possibilities for the treatment of these frequent and deadly gynaecological cancers.

Synthesis, characterization and antiproliferative activity of platinum(II) complexes with 3-(2-pyridyl)-N1,2-methyl-1,2,4-triazoles

The present work describes the synthesis, investigation and in vitro cytotoxicity of Pt(II) complexes with 3-(2-pyridyl)-N1-methyl-1,2,4-triazole (L1) and 3-(2-pyridyl)–N2-methyl-1,2,4-triazole (L2). The crystal structures of PtL1Cl2 and PtL2Cl2 were established using X-Ray analysis, which revealed the square-planar PtN2Cl2 coordination geometry for the central metal ion. High-resolution mass spectrometry (HRMS), as well as one- and two-dimensional nuclear magnetic resonance (NMR) spectroscopy showed stability of the investigated complexes in DMF solution, in contrast to DMSO, where species were shown to partially dissociate. The cytotoxicity of the investigated compounds was determined by the colorimetric MTT assay against two human cancer cell lines, including cisplatin-sensitive human ovarian adenocarcinoma A2780 cells (IC50 = 26 ± 5 and 6.1 ± 0.6 for PtL1Cl2 and PtL2Cl2 correspondingly) and their cisplatin-resistant A2780cis analogue (IC50 = 24 ± 2 and 20 ± 3 for PtL1Cl2 and PtL2Cl2 correspondingly).

Exploring Structure-Activity Relationships in Photodynamic Therapy Anticancer Agents Based on Ir(III)-COUPY Conjugates.

Photodynamic therapy holds great promise as a non-invasive anticancer tool against drug-resistant cancers. However, highly effective, non-toxic, and reliable photosensitizers with operability under hypoxic conditions remain to be developed. Herein, we took the advantageous properties of COUPY fluorophores and cyclometalated Ir(III) complexes to develop novel PDT agents based on Ir(III)-COUPY conjugates with the aim of exploring structure-activity relationships. The structural modifications carried out within the coumarin scaffold had a strong impact on the photophysical properties and cellular uptake of the conjugates. All Ir(III)-COUPY conjugates exhibited high phototoxicity under green light irradiation, which was attributed to the photogeneration of ROS, while remaining non-toxic in the dark. Among them, two hit conjugates showed excellent phototherapeutic indexes in cisplatin-resistant A2780cis cancer cells, both in normoxia and in hypoxia, suggesting that photoactive therapy approaches based on the conjugation of far-red/NIR-emitting COUPY dyes and transition metal complexes could effectively tackle in vitro acquired resistance to cisplatin.

Ruthenium(II)–Arene Complexes with Glycosylated NHC-Carbene Co-Ligands: Synthesis, Hydrolytic Behavior, and Binding to Biological Molecules

Ruthenium(II) complexes with a three-legged piano-stool structure based on an arene ring, an N-heterocyclic carbene (NHC)-carbene ligand with a peracetylated glucose moiety and two chlorides or one bidentate ligand, were prepared and characterized by spectroscopy and crystallography. In one case, the sugar substituent is replaced by an ethyl. The chirality of the sugar results in the formation of two diastereomers that interconvert through rotations around the Ccarbene–Ru and Carene–Ru bonds. In water, the complexes undergo a series of equilibrium hydrolysis steps involving the Ru–Cl bonds. The Ru–arene bond and the sugar acetyls are also partially hydrolyzed, and the selectivity of the process is governed by the nature of the arene and the pH of the solution. The reactivity of the compounds was studied against model nucleophiles and biological macromolecules. In the former case, mass experiments demonstrated a variety of binding modes with a trend reflecting the stability in aqueous environment. In the latter case, protein crystallography was used to characterize the preferential binding sites of one of the complexes. The X-ray structure of the adduct formed upon reaction of one representative complex with hen egg white lysozyme contains a Ru center, which retains the carbene ligand, close to the side chain of Asp119. In the adduct with bovine pancreatic ribonuclease, there are two protein molecules in the asymmetric unit. In one molecule, two Ru centers are located close to the side chains of His105 and of His119, which is the protein active site. In the second molecule, only one Ru center was found in the proximity of the side chain of His105. The Ru complexes also interact with calf-thymus DNA, although without displacing the intercalating probe EB. Finally, the complexes are essentially inactive against the human ovarian carcinoma A2780 cells, the cisplatin-resistant A2780cis cells, and the human embryonic kidney HEK293T cells.

Ionic mononuclear [Fe] and heterodinuclear [Fe,Ru] bis(diphenylphosphino)alkane complexes: Synthesis, spectroscopy, DFT structures, cytotoxicity, and biomolecular interactions

Iron(II) and Ru(II) half-sandwich compounds encompass some promising pre-clinical anticancer agents whose efficacy may be tuned by structural modification of the coordinated ligands. Here, we combine two such bioactive metal centres in cationic bis(diphenylphosphino)alkane-bridged heterodinuclear [Fe2+, Ru2+] complexes to delineate how ligand structural variations modulate compound cytotoxicity. Specifically, Fe(II) complexes of the type [(η5–C5H5)Fe(CO)2(κ1–PPh2(CH2)nPPh2)]{PF6} (n = 1–5), compounds 1–5, and heterodinuclear [Fe2+, Ru2+] complexes, [(η5–C5H5)Fe(CO)2(μ–PPh2(CH2)nPPh2))(η6–p–cymene)RuCl2]{PF6} (n = 2–5) (compounds 7–10), were synthesized and characterised. The mononuclear complexes were moderately cytotoxic against two ovarian cancer cell lines (A2780 and cisplatin resistant A2780cis) with IC50 values ranging from 2.3 ± 0.5 μM to 9.0 ± 1.4 μM. For 7–10, the cytotoxicity increased with increasing Fe⋅⋅⋅Ru distance, consistent with their DNA affinity. UV–visible spectroscopy suggested the chloride ligands in heterodinuclear 8–10 undergo stepwise substitution by water on the timescale of the DNA interaction experiments, probably affording the species [RuCl(OH2)(η6-p-cymene)(PRPh2)]2+ and [Ru(OH)(OH2)(η6-p-cymene)(PRPh2)]2+ (where PRPh2 has R = [−(CH2)5PPh2–Fe(C5H5)(CO)2]+). One interpretation of the combined DNA-interaction and kinetic data is that the mono(aqua) complex may interact with dsDNA through nucleobase coordination. Heterodinuclear 10 reacts with glutathione (GSH) to form stable mono- and bis(thiolate) adducts, 10-SG and 10-SG2, with no evidence of metal ion reduction (k1 = 1.07 ± 0.17 × 10−1 min−1 and k2 = 6.04 ± 0.59 × 10−3 min−1 at 37 °C). This work highlights the synergistic effect of the Fe2+/Ru2+ centres on both the cytotoxicity and biomolecular interactions of the present heterodinuclear complexes.

The copper transporter CTR1 and cisplatin accumulation at the single-cell level by LA-ICP-TOFMS.

More than a decade ago, studies on cellular cisplatin accumulation via active membrane transport established the role of the high affinity copper uptake protein 1 (CTR1) as a main uptake route besides passive diffusion. In this work, CTR1 expression, cisplatin accumulation and intracellular copper concentration was assessed for single cells revisiting the case of CTR1 in the context of acquired cisplatin resistance. The single-cell workflow designed for in vitro experiments enabled quantitative imaging at resolutions down to 1µm by laser ablation-inductively coupled plasma-time-of-flight mass spectrometry (LA-ICP-TOFMS). Cisplatin-sensitive ovarian carcinoma cells A2780 as compared to the cisplatin-resistant subline A2780cis were investigated. Intracellular cisplatin and copper levels were absolutely quantified for thousands of individual cells, while for CTR1, relative differences of total CTR1 versus plasma membrane-bound CTR1 were determined. A markedly decreased intracellular cisplatin concentration accompanied by reduced copper concentrations was observed for single A2780cis cells, along with a distinctly reduced (total) CTR1 level as compared to the parental cell model. Interestingly, a significantly different proportion of plasma membrane-bound versus total CTR1 in untreated A2780 as compared to A2780cis cells was observed. This proportion changed in both models upon cisplatin exposure. Statistical analysis revealed a significant correlation between total and plasma membrane-bound CTR1 expression and cisplatin accumulation at the single-cell level in both A2780 and A2780cis cells. Thus, our study recapitulates the crosstalk of copper homeostasis and cisplatin uptake, and also indicates a complex interplay between subcellular CTR1 localization and cellular cisplatin accumulation as a driver for acquired resistance development.

Exosomes released from cisplatin-resistant ovarian cancer cells modulate the reprogramming of cells in tumor microenvironments toward the cancerous cells

Exosomes released from cancer cells are involved in the reorganization of the tumor microenvironment which is the essential aspect of cancer pathogenesis. The intercommunications between cancer cells and diverse cell types in the microenvironment are accomplished by exosomes in ovarian cancer. Internalization pathway, intracellular fate, and biological functions in recipient cells mediated by exosomes released from cisplatin-resistant A2780cis have been studied. Also, histopathological evaluation of tumor, ovary, liver tissues and lymph nodes in vivo studies have been performed. The recipient cells internalized the exosomes via active uptake mechanisms, as shown by confocal microscopy. However, inhibitor studies and flow cytometry analysis showed that each recipient cell line used different uptake pathways. Also, confocal microscopy imaging indicated that the internalized exosomes trapped in the endosomes or phagosomes were distributed to the different cellular compartments including ER, Golgi, and lysosome. The transfer of exosomal oncogenic cargo into the cells modified the intracellular signaling of recipient cells including invasion and metastasis by Boyden-Chamber assay, proliferation by ATP analysis, epithelial-mesenchymal transition (EMT) markers at protein and mRNA levels by western blotting and real-time PCR, and protein kinases in the phospho-kinase array. This remodeling contributed to the initiation of carcinogenesis in ovarian epithelial and peritoneal mesothelial cells, and the progression of carcinogenesis in ovarian cancer cells. In addition, intraperitoneal tumor model studies show that exosomes released from cisplatin-resistant A2780cis cells may play role in the enlargement of lymph nodes, and tumor formations integrated with the liver, attached to the stomach and in the ovarian tissues.

Effect of Tumor Targeted-Anthracycline Nanomedicine, HPMA Copolymer-Conjugated Pirarubicin (P-THP) against Gynecological Malignancies

Anthracyclines are important for the treatment of gynecological malignancies, but their effects are modest, and one of the major reasons is the lack of a tumor-targeting property. To overcome this drawback, a poly (hydroxypropyl meta-acrylamide) conjugated with tetrahydropyraryl doxorubicin (P-THP) has been developed, which exhibits a highly tumor-specific accumulation owing to the enhanced permeability and retention effect. The effect of P-THP has been confirmed by using various cell lines and solid tumor models, while its effect on gynecological malignancies have not been investigated. In this regard, human uterine sarcoma cell line with metastatic potential MEA-SA C9 high, epithelial ovarian cancer cell line A2780 and its cisplatin-resistant line A2780cis, and DOX-resistant line A2780ADR were used in this study, and the therapeutic effect as well as the safety profiles of P-THP were investigated compared to native THP, cisplatin, and paclitaxel, which are commonly used for gynecological malignancies, both in vitro and in vivo. Similar to native THP, a dose-dependent toxicity of P-THP was identified in all cell lines. Moreover, the IC50 values in the 3 h following P-THP were 1.5–10 times higher than those at 72 h, though the intracellular uptake of P-THP in all cells were 2–10-fold less than THP. In vivo studies using xenograft tumor models revealed that P-THP significantly suppressed the MES-SA C9 high, A2780, and A2780cis tumor growth at the dose of 15 mg/kg (THP equivalent), which is three times above the maximal tolerance dose of native THP, while no body weight loss or acute death occurred. However, in A2780ADR cells and the xenograft model, no significant difference in the therapeutic effect was observed between THP and P-THP, suggesting that P-THP exhibits its effect depending on the release of the active free THP in tumor tissues, and thus the internalization into tumor cells. These findings indicates that P-THP has the potential as a therapeutic for gynecological malignancies to improve the therapeutic outcomes and survival rates of patients, even in refractory patients.

Synthesis and In Vitro Studies of Photoactivatable Semisquaraine-type Pt(II) Complexes.

The synthesis, full characterization, photochemical properties, and cytotoxic activity toward cisplatin-resistant cancer cell lines of new semisquaraine-type Pt(II) complexes are presented. The synthesis of eight semisquaraine-type ligands has been carried out by means of an innovative, straightforward methodology. A thorough structural NMR and X-ray diffraction analysis of the new ligands and complexes has been done. Density functional theory calculations have allowed to assign the trans configuration of the platinum center. Through the structural modification of the ligands, it has been possible to synthesize some complexes, which have turned out to be photoactive at wavelengths that allow their activation in cell cultures and, importantly, two of them show remarkable solubility in biological media. Photodegradation processes have been studied in depth, including the structural identification of photoproducts, thus justifying the changes observed after irradiation. From biological assessment, complexes C7 and C8 have been demonstrated to behave as promising photoactivatable compounds in the assayed cancer cell lines. Upon photoactivation, both complexes are capable of inducing a higher cytotoxic effect on the tested cells compared with nonphotoactivated compounds. Among the observed results, it is remarkable to note that C7 showed a PI > 50 in HeLa cells, and C8 showed a PI > 40 in A2780 cells, being also effective over cisplatin-resistant A2780cis cells (PI = 7 and PI = 4, respectively). The mechanism of action of these complexes has been studied, revealing that these photoactivated platinum complexes would actually present a combined mode of action, a therapeutically potential advantage.

Synthesis, crystal structure, and in vitro evaluation of the anticancer activity of new Pt (Pd) complexes with 1-[(dimethylamino)methyl]-2-naphthol ligand.

The synthesis of new Pt(II) and Pd(II) complexes with 1-aminomethyl-2-naphtol ligands has been first performed. The adducts of [PtCl4]2- and [PdCl4]2- anions with the 1-aminomethyl-2-naphtol NH cation were synthesized. The structure for four Pt (Pd)-containing compounds was investigated using X-ray diffraction. The obtained compounds were examined for in vitro cytotoxic activity against Jurkat and K562 human leukemia cells, lymphoma U937cells, A2780 and the cisplatin-resistant A2780cis lines of human ovarian cancer, and normal fibroblasts. Study of induction of apoptosis and the effect of new palladium and platinum complexes on the cell cycle was carried out. The cells showed a higher sensitivity to Pt(II) compounds than to Pd(II) ones. All the synthesized metal complexes show much more antitumor activity compared with a platinum-containing cisplatin drug.

Ex vivo expanded allogeneic natural killer cells have potent cytolytic activity against cancer cells through different receptor-ligand interactions

BackgroundRecently, allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics. Although many NK expansion strategies have been reported previously, a deeper understanding of cryopreserved allogeneic NK cells is needed for specific therapeutic approaches.MethodsWe isolated CD3−CD56+ primary natural killer (pNK) cells from healthy donors and expanded them ex vivo using a GMP-compliant method without any feeder to generate large volumes of therapeutic pNK cells and cryopreserved stocks. After validation for high purity and activating phenotypes, we performed RNA sequencing of the expanded and cryopreserved pNK cells. The pNK cells were used against various cancer cell lines in 7-AAD/CFSE cytotoxicity assay. For in vivo efficacy study, NSG mice bearing subcutaneous cisplatin-resistant A2780cis xenografts were treated with our pNK cells or cisplatin. Antitumor efficacy was assessed by measuring tumor volume and weight.ResultsCompared to the pNK cells before expansion, pNK cells after expansion showed 2855 upregulated genes, including genes related to NK cell activation, cytotoxicity, chemokines, anti-apoptosis, and proliferation. Additionally, the pNK cells showed potent cytolytic activity against various cancer cell lines. Interestingly, our activated pNK cells showed a marked increase in NKp44 (1064-fold), CD40L (12,018-fold), and CCR5 (49-fold), and did not express the programmed cell death protein 1(PD-1). We also demonstrated the in vitro and in vivo efficacies of pNK cells against cisplatin-resistant A2780cis ovarian cancer cells having a high programmed death-ligand 1(PD-L1) and low HLA-C expression.ConclusionsTaken together, our study provides the first comprehensive genome wide analysis of ex vivo-expanded cryopreserved pNK cells. It also indicates the potential use of expanded and cryopreserved pNK cells as a highly promising immunotherapy for anti-cancer drug resistant patients.

Impact of Selected Signaling Proteins on SNAIL 1 and SNAIL 2 Expression in Ovarian Cancer Cell Lines in Relation to Cells' Cisplatin Resistance and EMT Markers Level.

It has been increasingly recognized that SNAIL1 and SNAIL2, as major EMT-inducers, might also be involved in drug resistance of cancer cells. We sought to determine a relation between SNAIL1/2, E-cadherin and N-cadherin expression, as well as ovarian cancer cells’ resistance to cisplatin and EMT markers’ level. Thus, four ovarian cancer cell lines, were used: A2780, A2780cis, SK-OV-3 and OVCAR-3. We assessed the impact of ERK1/2, AKT and STAT3 proteins (chosen by the profiling activity of over 40 signaling proteins) on SNAIL1/2 expression, along with E-cadherin and N-cadherin levels. We showed that expression of SNAIL1 and N-cadherin are the highest in cisplatin-resistant A2780cis and SK-OV-3 cells, while high SNAIL2 and E-cadherin levels were observed in cisplatin-sensitive A2780 cells. The highest E-cadherin level was noticed in OVCAR-3 cells. SNAIL1/2 expression was dependent on ERK1/2 activity in cisplatin-resistant and potentially invasive SK-OV-3 and OVCAR-3 cells. STAT-3 regulates expression of SNAIL1/2 and leads to the so-called “cadherin switch” in cancer cells, independently of their chemoresistance. In conclusion, SNAIL1, but not SNAIL2, seems to be involved in ovarian cancer cells’ cisplatin resistance. STAT3 is a universal factor determining the expression of SNAIL1/2 in ovarian cancer cells regardless of their chemoresitance or invasive capabilities.

Further Approaches in the Design of Antitumor Agents with Response to Cell Resistance: Looking toward Aza Crown Ether-dtc Complexes.

The dianionic aza crown ether-dtc N,N'-bis(dithiocarbamate)-1,10-diaza-18-crown-6 (L2-) is a versatile ligand capable of yielding binuclear complexes with group 10 elements, also known as Ni-triade, [μ-(κ2-S,-S'-L)M2(PPh3)4]Cl2 (M = Pd (1), Pt (2)), [μ-(κ2-S,-S'-L)M2(PPh3)4](BPh4)2 (M = Pd (3), Pt (4)), and μ-(κ-S,-S'-L)Ni2(PPh3)2Cl2 (5), and has proven to be an excellent option to the design of metal-based drugs able to provide multiple response to cell resistance. Palladium and platinum complexes, 1 and 2, were tested for cytotoxicity in the human cervix carcinoma cell line HeLa-229, the human ovarian carcinoma cell line A2780, and the cisplatin-resistant mutant A2780cis, finding significant activity toward all three cancer cell lines, with low micromolar IC50 values, comparable to cisplatin. Markedly, against the cisplatin resistant cell line A2780cis, compound 2 exhibits better cytotoxic activity than the clinical drug (IC50 = 2.3 ± 0.2 μM for 2 versus 3.6 ± 0.5 μM for cisplatin). Moreover, an enhancement of the antitumor response is achieved when adding an equimolar amount of alkali metal chloride (NaCl or KCl) to the medium, for instance, testing compound 1 against the cisplatin-resistant A2780cis cells, the IC50 decreases from 9.3 ± 0.4 to 7.4 ± 0.3 and 5.4 ± 0.1 μM, respectively, after addition of the salt solution. For the platinum derivative 2, the IC50 improves by ca. 40% reaching 1.3 ± 0.1 μM when potassium chloride is added. Likewise, the resistant factor found for 2 (RF = 1) confirms that this complex circumvents cisplatin-resistance in A2780cis and is improved with the addition of potassium chloride (RF = 0.65). The presence of the aza crown ether moiety as linker in the systems studied herein is a key point since, in addition to allowing and facilitating interaction with alkali metal ions, this unit is flexible enough to adapt to a variety of environments, as confirmed by the X-ray crystal structures described, where different conformations and ways to fold in are found. In order to gain insight into the electronic and structural facts involved in the interaction of complex 2 with the alkali metal ions, a DFT study was performed, and the description of the molecular electrostatic potentials (MEPs) is also presented.

Anti-tumor effects of mevalonate pathway inhibition in ovarian cancer

BackgroundOvarian cancer remains the most fatal gynecological malignancy. Current therapeutic options are limited due to late diagnosis in the majority of the cases, metastatic spread to the peritoneal cavity and the onset of chemo-resistance. Thus, novel therapeutic approaches are required. Statins and amino-bisphosphonates are inhibitors of the mevalonate pathway, which is a fundamental pathway of cellular metabolism, essential for cholesterol production and posttranslational protein farnesylation and geranylgeranylation. While this pathway has emerged as a promising treatment target in several human malignancies, its potential as a therapeutic approach in ovarian cancer is still not fully understood.MethodsHuman ovarian cancer cell lines (IGROV-1, A2780, A2780cis) were treated with increasing concentrations (0.5-100 μM) of statins (simvastatin, atorvastatin, rosuvastatin) and zoledronic acid. Effects on cell vitality and apoptosis were assessed using Cell Titer Blue®, Caspase 3/7 Glo®, clonogenic assays as well as cleaved poly (ADP-ribose) polymerase (cPARP) detection. The inhibition of the mevalonate pathway was confirmed using Western Blot of unprenylated Ras and Rap1a proteins. Quantitative real-time PCR and ELISA were used to analyze modulations on several key regulators of ovarian cancer tumorigenesis.ResultsThe treatment of IGROV-1 and A2780 cells with statins and zoledronic acid reduced vitality (by up to 80%; p < 0.001) and induced apoptosis by up to 8-folds (p < 0.001) in a dose-dependent fashion. Rescue experiments using farnesyl pyrophosphate or geranylgeranyl pyrophosphate evidenced that blocked geranylgeranylation is the major underlying mechanism of the pro-apoptotic effects. Gene expression of the tumor-promoting cytokines and mediators, such as transforming growth factor (TGF)-β1, vascular endothelial growth factor (VEGF), interleukin (IL)-8, and IL-6 were significantly suppressed by statins and zoledronic acid by up to 90% (p < 0.001). For all readouts, simvastatin was most potent of all agents used. Cisplatin-resistant A2780cis cells showed a relative resistance to statins and zoledronic acid. However, similar to the effects in A2780 cells, simvastatin and zoledronic acid significantly induced caspase 3/7 activation (6-folds; p < 0.001).ConclusionOur in vitro findings point to promising anti-tumor effects of statins and zoledronic acid in ovarian cancer and warrant additional validation in preclinical and clinical settings.

Dual targeting of GSK3B and HDACs reduces tumor growth and improves survival in an ovarian cancer mouse model

ObjectiveTo investigate the anti-tumor effect of a newly-developed dual inhibitor (APCS-540) of glycogen synthase kinase 3 beta (GSK3B) and histone deacetylases (HDACs) in ovarian cancer cells. MethodsThe effects of APCS-540 on cancer cell proliferation, migration, invasion and cancer stemness were investigated in vitro in human (KURAMOCHI, OVCA420, OVSAHO) and mouse (BR-Luc, ID8, MOSE-HRas-Myc) ovarian cancer cells. Cisplatin-sensitive (A2780) and cisplatin-resistant (A2780cis) cell lines were used to evaluate APCS-540's effect on chemoresistance. The immunocompetent syngeneic mouse model BR-Luc was used to test the effect of APCS-540 on ovarian cancer progression and survival. ResultsAPCS-540 showed significant anti-tumor effects in vitro in both human and mouse ovarian cancer cells. Importantly, APCS-540 demonstrated marked cytotoxicity against cisplatin-resistant cancer cells and reversed cisplatin-resistance when used in combination with platinum. APCS-540 significantly decreased cancer cell invasion. A significant 66% increase in survival was observed in mice treated with APCS-540 compared to control mice. ConclusionDual inhibition of GSK3B and HDACs via APCS-540 showed potent anti-tumor activity in vitro and in vivo, suggesting that APCS-540 may provide a novel treatment option for ovarian cancer, including the platinum-resistant disease.

Cisplatin Protein Binding Partners and Their Relevance for Platinum Drug Sensitivity.

Cisplatin is a widely used drug in the treatment of various solid tumors, such as ovarian cancer. However, while the acquired resistance significantly limits the success of therapy, some tumors, such as colorectal cancer, are intrinsically insensitive to cisplatin. Only a small amount of intracellular platinum binds to the target—genomic DNA. The fate of the remaining drug is largely obscure. This work aimed to identify the cytosolic protein binding partners of cisplatin in ovarian and colorectal cancer cells and to evaluate their relevance for cell sensitivity to cisplatin and oxaliplatin. Using the fluorescent cisplatin analog BODIPY-cisplatin, two-dimensional gel electrophoresis, and mass spectrometry, we identified the protein binding partners in A2780 and cisplatin-resistant A2780cis ovarian carcinoma, as well as in HCT-8 and oxaliplatin-resistant HCT-8ox colorectal cell lines. Vimentin, only identified in ovarian cancer cells; growth factor receptor-bound protein 2, only identified in colorectal cancer cells; and glutathione-S-transferase π, identified in all four cell lines, were further investigated. The effect of pharmacological inhibition and siRNA-mediated knockdown on cytotoxicity was studied to assess the relevance of these binding partners. The silencing of glutathione-S-transferase π significantly sensitized intrinsically resistant HCT-8 and HCT-8ox cells to cisplatin, suggesting a possible involvement of the protein in the resistance of colorectal cancer cells to the drug. The inhibition of vimentin with FiVe1 resulted in a significant sensitization of A2780 and A2780cis cells to cisplatin, revealing new possibilities for improving the chemosensitivity of ovarian cancer cells.

Novel Phenothiazine-Bridged Porphyrin-(Hetero)aryl dyads: Synthesis, Optical Properties, In Vitro Cytotoxicity and Staining of Human Ovarian Tumor Cell Lines.

We report here the synthetic procedure applied for the preparation of new AB3-type and trans-A2B2 type meso-halogenophenothiazinyl-phenyl-porphyrin derivatives, their metal core complexation and their peripheral modification using Suzuki–Miyaura cross coupling reactions with various (hetero)aryl (phenothiazinyl, 7-formyl-phenothiazinyl, (9-carbazolyl)-phenyl and 4-formyl-phenyl, phenyl) boronic acid derivatives. The meso-phenothiazinyl-phenyl-porphyrin (MPP) dyes family was thus extended by a series of novel phenothiazine-bridged porphyrin-(hetero)aryl dyads characterized by UV–Vis absorption/emission properties typical to the porphyrin chromophore, slightly modulated by increasing the size of peripheral substituents. Three phenothiazine-bridged porphyrin-heteroaryl dyads with fluorescence emission above 655 nm were selected as fluorophores in red spectral region for applications in cellular staining of human ovarian tumors. In vitro experiments of cell metabolic activity displayed a moderate toxicity on human ovarian tumor cell lines (OVCAR-3, cisplatin-sensitive A2780 and cisplatin-resistant A2780cis respectively). Visualization of the stained living cells was performed both by fluorescence microscopy imaging and by fluorescence lifetime imaging under two photon excitation (TPE-FLIM), confirming their cellular uptake and the capability of staining the cell nucleus.

Promotion of Cell Death in Cisplatin-Resistant Ovarian Cancer Cells through KDM1B-DCLRE1B Modulation.

Ovarian cancer is the gynecological malignancy with the poorest prognosis, in part due to its high incidence of recurrence. Platinum agents are widely used as a first-line treatment against ovarian cancer. Recurrent tumors, however, frequently demonstrate acquired chemo-resistance to platinum agent toxicity. To improve chemo-sensitivity, combination chemotherapy regimens have been investigated. This study examined anti-tumor effects and molecular mechanisms of cytotoxicity of Oldenlandia diffusa (OD) extracts on ovarian cancer cells, in particular, cells resistant to cisplatin. Six ovarian cancer cells including A2780 and cisplatin-resistant A2780 (A2780cis) as representative cell models were used. OD was extracted with water (WOD) or 50% methanol (MOD). MOD significantly induced cell death in both cisplatin-sensitive cells and cisplatin-resistant cells. The combination treatment of MOD with cisplatin reduced viability in A2780cis cells more effectively than treatment with cisplatin alone. MOD in A2780cis cells resulted in downregulation of the epigenetic modulator KDM1B and the DNA repair gene DCLRE1B. Transcriptional suppression of KDM1B and DCLRE1B induced cisplatin sensitivity. Knockdown of KDM1B led to downregulation of DCLRE1B expression, suggesting that DCLRE1B was a KDM1B downstream target. Taken together, OD extract effectively promoted cell death in cisplatin-resistant ovarian cancer cells under cisplatin treatment through modulating KDM1B and DCLRE1B.