Cisplatin-resistant Variant Research Articles

The deubiquitinase USP8 regulates ovarian cancer cell response to cisplatin by suppressing apoptosis.

The identification of therapeutic approaches to improve response to platinum-based therapies is an urgent need for ovarian carcinoma. Deubiquitinases are a large family of ubiquitin proteases implicated in a variety of cellular functions and may contribute to tumor aggressive features through regulation of processes such as proliferation and cell death. Among the subfamily of ubiquitin-specific peptidases, USP8 appears to be involved in modulation of cancer cell survival by still poorly understood mechanisms. Thus, we used ovarian carcinoma cells of different histotypes, including cisplatin-resistant variants with increased survival features to evaluate the efficacy of molecular targeting of USP8 as a strategy to overcome drug resistance/modulate cisplatin response. We performed biochemical analysis of USP8 activity in pairs of cisplatin-sensitive and -resistant cells and found increased USP8 activity in resistant cells. Silencing of USP8 resulted in decreased activation of receptor tyrosine kinases and increased sensitivity to cisplatin in IGROV-1/Pt1 resistant cells as shown by colony forming assay. Increased cisplatin sensitivity was associated with enhanced cisplatin-induced caspase 3/7 activation and apoptosis, a phenotype also observed in cisplatin sensitive cells. Increased apoptosis was linked to FLIPL decrease and cisplatin induction of caspase 3 in IGROV-1/Pt1 cells, cisplatin-induced claspin and survivin down-regulation in IGROV-1 cells, thereby showing a decrease of anti-apoptotic proteins. Immunohistochemical staining on 65 clinical specimens from advanced stage ovarian carcinoma indicated that 40% of tumors were USP8 positive suggesting that USP8 is an independent prognostic factor for adverse outcome when considering progression free survival as a clinical end-point. Taken together, our results support that USP8 may be of diagnostic value and may provide a therapeutic target to improve the efficacy of platinum-based therapy in ovarian carcinoma.

Effect of the PARP inhibitor veliparib on germ cell tumor cell lines.

Germ cell tumors (GCTs) usually represent efficiently curable neoplasms due to their chemosensitivity to platinum-based therapeutic regimen. However, some patients develop therapeutic resistance and succumb to their disease. Novel therapeutic approaches are therefore needed for these patients. It has previously been demonstrated that poly (ADP-ribose) polymerase (PARP) expression is upregulated in GCTs compared with normal testis tissue. Therefore, PARP expression was analyzed in GCT cell lines and xenografts and it was examined whether its inhibition by veliparib can reverse cisplatin-resistance. Its expression was analyzed in sensitive and cisplatin-resistant variants (referred to as CisR throughout the manuscript) GCT cell lines and xenografts using quantitative PCR, western blotting and immunohistochemistry. The present study investigated whether the combination of cisplatin with the PARP inhibitor veliparib increased the cytotoxic effect of cisplatin in vitro using a luminescent viability assay and an immunodeficient mouse model in vivo. PARP expression was observed in all tested cell lines, with the highest expression in embryonal carcinoma (EC) cell lines and xenografts. Low or no expression was detected in the JEG-3 choriocarcinoma cell line pairs and xenografts. The combination of veliparib and cisplatin or carboplatin was examined in the cisplatin-resistant NTERA-2 CisR and NCCIT CisR EC cell lines and synergistic effects were observed in NTERA-2 CisR cells. However, in vivo analysis did not confirm this synergy. The present data indicated PARP expression in GCT cell lines and xenografts. However, veliparib failed to increase the cytotoxicity of platinum-based drugs. Therefore, further research is warranted to effectively inhibit PARP using different PARP inhibitors or other drug combinations.

Isothiocyanates (ITCs) 1-(Isothiocyanatomethyl)-4-phenylbenzene and 1-Isothiocyanato-3,5-bis(trifluoromethyl)benzene-Aldehyde Dehydrogenase (ALDH) Inhibitors, Decreases Cisplatin Tolerance and Migratory Ability of NSCLC.

One of the main treatment modalities for non-small-cell lung cancer (NSCLC) is cisplatin-based chemotherapy. However, the acquisition of cisplatin resistance remains a major problem. Existing chemotherapy regimens are often ineffective against cancer cells expressing aldehyde dehydrogenase (ALDH). As such, there is an urgent need for therapies targeting ALDH-positive cancer cells. The present study compares the anticancer properties of 36 structurally diverse isothiocyanates (ITCs) against NSCLC cells with the ALDH inhibitor disulfiram (DSF). Their potential affinity to ALDH isoforms and ABC proteins was assessed using AutoDockTools, allowing for selection of three compounds presenting the strongest affinity to all tested proteins. The selected ITCs had no impact on NSCLC cell viability (at tested concentrations), but significantly decreased the cisplatin tolerance of cisplatin-resistant variant of A549 (A549CisR) and advanced (stage 4) NSCLC cell line H1581. Furthermore, long-term supplementation with ITC 1-(isothiocyanatomethyl)-4-phenylbenzene reverses the EMT phenotype and migratory potential of A549CisR to the level presented by parental A549 cells, increasing E-Cadherin expression, followed by decreased expression of ABCC1 and ALDH3A1. Our data indicates that the ALDH inhibitors DSF and ITCs are potential adjuvants of cisplatin chemotherapy.

Immunostimulatory and anti-tumor metronomic cyclophosphamide regimens assessed in primary orthotopic and metastatic murine breast cancer

The impressive successes of immune checkpoint blockade antibodies to treat various types of cancer are limited to minor subsets of patients. Combination therapy strategies, including with chemotherapy, are being explored to possibly improve the efficacy of immunotherapies. Here we report results regarding the use of an immunostimulatory regimen of metronomic cyclophosphamide (CTX). We show that in orthotopic models of syngeneic murine triple-negative breast cancer (EMT6), CTX administered at 140 mg/kg every 6 days (CTX140 1q6d) is superior at inhibiting primary tumor growth when compared to maximum tolerated dose or daily oral (continuous) low-dose CTX. In SCID or SCID beige mice, anti-tumor effects of CTX140 1q6d are reduced, reinforcing the therapeutic contribution of the adaptive and innate immune systems. In a second breast cancer model (SP1-AC2M2), CTX140 1q6d again showed clear superiority in anti-tumor effects, causing complete tumor regressions; however, these mice were not protected from subsequent tumor re-challenge, suggesting absence of immune memory. We also show that in an aggressive and metastatic cisplatin-resistant variant (EMT6-CDDP), CTX140 1q6d is superior and invokes an influx of intra-tumoral CD4+ and CD8+ T cells. CTX increases expression of tumor cell PD-L1; however, when combined with concomitant PD-L1 antibody therapy none of the CTX regimens showed increased benefit. This work sheds light on the potential use of metronomic CTX for the treatment of breast cancer, in particular using the quasi-weekly regimen, but also underscores the complexity of the anti-tumor mechanisms and potential to improve immune checkpoint therapy efficacy.

Exosome-mediated delivery of miR-30a sensitize cisplatin-resistant variant of oral squamous carcinoma cells via modulating Beclin1 and Bcl2

Exosomes facilitate cross-talk amongst tumor cells, and thus also possess the potential to influence tumor-microenvironment and chemo-resistance. miRNAs, the important constituent of exosomes, are often dysregulated in cancer. They have been shown to play an essential role in tumor progression, metastasis, invasion, and resistance developed against different therapies. Acquisition of cisplatin-chemoresistance remains a major hurdle in the effective treatment of oral squamous cell carcinoma (OSCC). In this study, we demonstrate the importance of exosome-mediated miR-30a transfer in conferring cisplatin sensitivity in the otherwise resistant OSCC cells. Notably, miR-30a was found to be significantly reduced in exosomes isolated from the serum of OSCC patients, especially those having disease-recurrence, post cisplatin treatment. In conjunction with the findings in clinical samples, decreased miR-30a expression was observed in vitro in the cisplatin-resistant cultured OSCC cells compared to the cisplatin-sensitive cells. Besides, we identified Beclin1, an autophagy-related marker, as a target of miR-30a and found it to be overexpressed in cisplatin-resistant OSCC cells, thus indicating at its possible negative-regulation by miR30a. Exosomes from the cisplatin-resistant cells that have been transfected with miR-30a mimics, when delivered to the naïve cisplatin-resistant cells, caused not only the significant enhancements in miR-30a expression but also a concomitant decrease in Beclin1 and Bcl2 expression (autophagic and anti-apoptotic marker). More importantly, this together resulted in the sensitization of cisplatin-resistant cells. Thus, our study highlighted the role of exosomal-mediated miR-30a transfer in regaining sensitivity of the cisplatin-resistant OSCC cells via Beclin1 and Bcl2 regulation and hence suggests at its potential therapeutic role.

Evaluating the immunoproteasome as a potential therapeutic target in cisplatin-resistant small cell and non-small cell lung cancer.

We evaluated the expression of proteasome subunits to assess whether the proteasome could be a therapeutic target in cisplatin-resistant lung cancer cells. Cisplatin-resistant (CR) variants were established from three non-small cell lung cancer (NSCLC) cell lines (A549, H1299, and H1975) and two small cell lung cancer (SCLC) cell lines (SBC3 and SBC5). The expression of proteasome subunits, the sensitivity to immunoproteasome inhibitors, and 20S proteasomal proteolytic activity were examined in the CR variants of the lung cancer cell lines. All five CR cell lines highly expressed one or both of the immunoproteasome subunit genes, PSMB8 and PSMB9, while no clear trend was observed in the expression of constitutive proteasome subunits. The CR cells expressed significantly higher levels of PSMB8 and PSMB9 proteins, as well. The CR variants of the H1299 and SBC3 cell lines were more sensitive to immunoproteasome inhibitors, and had significantly more proteasomal proteolytic activity than their parental counterparts. The immunoproteasome may be an effective therapeutic target in a subset of CR lung cancers. Proteasomal proteolytic activity may be a predictive marker for the efficacy of immunoproteasome inhibitors in cisplatin-resistant SCLC and NSCLC.

Cisplatin Resistance in Osteosarcoma: In vitro Validation of Candidate DNA Repair-Related Therapeutic Targets and Drugs for Tailored Treatments.

Treatment of high-grade osteosarcoma, the most common malignant tumor of bone, is largely based on administration of cisplatin and other DNA damaging drugs. Altered DNA repair mechanisms may thus significantly impact on either response or resistance to chemotherapy. In this study, by using a panel of human osteosarcoma cell lines, either sensitive or resistant to cisplatin, we assessed the value as candidate therapeutic targets of DNA repair-related factors belonging to the nucleotide excision repair (NER) or base excision repair (BER) pathways, as well as of a group of 18 kinases, which expression was higher in cisplatin-resistant variants compared to their parental cell lines and may be indirectly involved in DNA repair. The causal involvement of these factors in cisplatin resistance of human osteosarcoma cells was validated through gene silencing approaches and in vitro reversal of CDDP resistance. This approach highlighted a subgroup of genes, which value as promising candidate therapeutic targets was further confirmed by protein expression analyses. The in vitro activity of 15 inhibitor drugs against either these genes or their pathways was then analyzed, in order to identify the most active ones in terms of inherent activity and ability to overcome cisplatin resistance. NSC130813 (NERI02; F06) and triptolide, both targeting NER factors, proved to be the two most active agents, without evidence of cross-resistance with cisplatin. Combined in vitro treatments showed that NSC130813 and triptolide, when administered together with cisplatin, were able to improve its efficacy in both drug-sensitive and resistant osteosarcoma cells. This evidence may indicate an interesting therapeutic future option for treatment of osteosarcoma patients who present reduced responsiveness to cisplatin, even if possible effects of additive collateral toxicities must be carefully considered. Moreover, our study also showed that targeting protein kinases belonging to the mitogen-activated protein kinase (MAPK) or fibroblast growth factor receptor (FGFR) pathways might indicate new promising therapeutic perspectives in osteosarcoma, demanding for additional investigation.

Dynamic analysis of N-glycomic and transcriptomic changes in the development of ovarian cancer cell line A2780 to its three cisplatin-resistant variants.

BackgroundPlatinum resistance development is a dynamic process that occurs during continuous chemotherapy and contributes to high mortality in ovarian cancer. Abnormal glycosylation has been reported in platinum resistance. Many studies on platinum resistance have been performed, but few of them have investigated platinum resistance-associated glycans based on N-glycomics. Moreover, glycomic alterations during platinum resistance development in ovarian cancer are rarely reported. Therefore, the objective of this study was to determine platinum resistance-related N-glycans in ovarian cancer cells during continuous exposure to cisplatin. These glycans might be involved in the mechanism of platinum resistance and serve as biomarkers to monitor its development.MethodsThis study mimicked the development of platinum resistance in ovarian cancer by continuously exposing A2780 cells to cisplatin. Cisplatin-resistant variants were confirmed by higher half maximal inhibitory concentration (IC50) values and increased P-glycoprotein (ABCB1, P-gp) expression compared to A2780 cells. Analysis of dynamic N-glycomic changes during the development of platinum resistance in cisplatin-resistant variants was performed with MALDI-time-of-flight (TOF)-MS combined with ethyl esterification derivatization, which were used to discriminate between α2,3- and α2,6-linkage N-acetylneuraminic acid. N-glycan alterations were further validated on a glycotransferase level via transcriptome sequencing and real-time PCR (RT-PCR).ResultsCompared to the A2780 cells, MS analysis indicated that α2,3-linked sialic structures and N-glycan gal-ratios were significantly higher, while fucosylated glycans were lower in three cisplatin-resistant variants. Transcriptome sequencing and RT-PCR showed that gene expression of ST3GAL6 and MGAT4A increased, while gene expression of FUT11, FUT1, GMDS, and B4GALT5 decreased in three cisplatin-resistant variants.ConclusionsAnalysis of N-glycans and glycogene expression showed that α2,3-linked sialic structures might serve as biomarkers to monitor the development of platinum resistance and to guide individualized treatment of ovarian cancer patients.

Enhanced efficacy in drug-resistant cancer cells through synergistic nanoparticle mediated delivery of cisplatin and decitabine.

There are several limitations with monodrug cancer therapy, including poor bioavailability, rapid clearance and drug resistance. Combination therapy addresses these by exploiting synergism between different drugs against cancer cells. In particular, the combination of epigenetic therapies with conventional chemotherapeutic agents can improve the initial tumour response and overcome acquired drug resistance. Co-encapsulation of multiple therapeutic agents into a single polymeric nanoparticle is one of the many approaches taken to enhance therapeutic effect and improve the pharmacokinetic profile. In this study, different types of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs), matrix and core–shell (CS), were investigated for simultaneous encapsulation of a demethylating drug, decitabine, and a potent anticancer agent, cisplatin. It was shown that by altering the configuration of the CS structure, the release profile could be tuned. In order to investigate whether this could enhance the anticancer effect compared to cisplatin, human ovarian carcinoma cell line (A2780) and its cisplatin resistant variant (A2780cis) were exposed to free cisplatin and the CS–NPs. A better response was obtained in both cell lines (11% and 51% viability of A2780 and A2780cis, respectively) using CS–NPs than cisplatin alone (27%, 82% viability of A2780 and A2780cis, respectively) or in combination with decitabine (22%, 96% viability of A2780 and A2780cis, respectively) at equivalent doses (10 μM).

Glucosamine reverses drug resistance in MRP2 overexpressing ovarian cancer cells

Glucosamine (GlcN), a natural amino sugar in human body, was reported to exhibit anticancer activity against some tumors. In the present study, we evaluated the cytotoxicity and multi-drug resistance (MDR) reversal activity of GlcN on resistant MRP2-overexpressing ovarian cancer A2780RCIS cells. The cytotoxicity and MDR reversal activity of GlcN on cancer cells were measured by MTT assay. The effects of GlcN on MRP1 and MRP2 mRNA expression and function were evaluated by qRT-PCR and flow cytometry, respectively. The cell migration capacity of ovarian cancer cells were assessed in the presence or absence of GlcN using wound healing migration assay. Furthermore, the effects of GlcN on the mRNA expression of E-cadherin, vimentin and α-smooth muscle actin as Epithelial-Mesenchymal Transition (EMT)-related markers were evaluated by qRT-PCR. Our results indicated that glucosamine reduced the proliferation of human ovarian cancer cell lines (A2780) and its cisplatin resistant variant (A2780RCIS) in a dose-dependent manner. The IC50 values for A2780RCIS cells treated with cisplatin in the presence of different concentrations of GlcN (0, 1, 2 and 3 mM) for 72 h were 44.463 ± 1.603, 35.17 ± 0.025, 22.25 ± 0.018, 17.78 ± 0.012 μM respectively. Also GlcN decreased the expression of MRP1 and MRP2 mRNA in ovarian cancer cells. Our results further demonstrated that although GlcN had no significant effects on the expression of studied EMT-related markers in invasive A2780RCIS cells, it was able to inhibit their migration in vitro. According to these findings, GlcN could effectively enhance cisplatin cytotoxicity in resistant A2780RCIS cells.

Abstract 4361: Kynurenine - Aryl hydrocarbon receptor axis: A crucial modulator of immunometabolism in cisplatin resistant lung cancer

Abstract The effect of tumor metabolism on the tumor microenvironment is not well established. Our previous data have shown that cisplatin resistant (CR) lung cancer cells increased secretion of thioredoxin-1 (TRX1) and kynurenine (KYN). Interestingly, high TRX1 and KYN levels in tumor microenvironment can enhance immunosuppressive environment. We have demonstrated that CR cells possessed lower levels of hypoxia-inducible factor-1α (HIF1α) due to metabolic re-programming. ARNT or HIF1β is a known binding partner of both HIF1α and aryl hydrocarbon (AHR). Importantly, recent study indicates that KYN can serve as an endogenous ligand for AHR in cancer cells. Thus, we hypothesize that in the absence of HIF1α, ARNT is now available to bind and form a new partner with KYN/AHR and initiate the transcription of genes which favor survival/proliferation of CR cells. Four pairs of NSCLC cell lines and their CR variants (ALC, FC, H460R, A549R) were used. Using immunofluorescence technique, our result showed that AHR localized primary in the nucleus of CR cells when compared with parental cell counterparts. We further determined that KYN was specific to AHR activation in CR cells by inhibiting AHR translocation using 10µM of dimethoxyflavone (DMF; an AHR antagonist). AHR was less accumulated inside nuclease after treatment. Importantly, treatment of DMF also resulted in suppression of Indoleamine 2,3-Dioxygenase-1 (IDO1) activities. To further verify these findings, we assayed the expression of AHR-target gene (LAT1 and CYP1B1) with and without adding KYN. Both genes expressions were higher in CR cells and were further augmented upon an addition of KYN. In contrast, we did not find an increase in LAT1 after exposure to KYN in parental cells. Using flow cytometer, we found that CR cells possessed higher surface-LAT1 levels when compared to parental cell counterparts, and treatment of KYN resulted in significant tryptophan uptake in CR cells. Importantly, treatment with IDO1 inhibitor (5.5uM) significantly suppressed LAT1 and CYP1B1 expression in CR cells and reverse cisplatin resistance in CR from 2.5 to 0.75 μg/ml (3.3 fold). Thus, our data strongly indicate that KYN/AHR/ARNT axis plays a unique modulator role in CR cells metabolism. Overall these results will have potential impact on (i) how one can effectively exploit the KP pathway to treat CR tumors, and (ii) improving understanding on how CR cells evade immune surveillance. Supported by Department of Veterans Affairs (BLR&D Merit review and CDA2) Citation Format: Medhi Wangpaichitr, Dan JM Nguyen, Ying-Ying Li, Chunjing Wu, Lynn G. Feun, Niramol Savaraj. Kynurenine - Aryl hydrocarbon receptor axis: A crucial modulator of immunometabolism in cisplatin resistant lung cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4361.

Brd4 inhibition suppresses HPV16 E6 expression and enhances chemoresponse: A potential new target in cervical cancer therapy.

Although a vast amount of research underlines the roles of the HR HPV E6 and E7 oncogenes in HPV-induced carcinogenesis of cervical cancer, it remains unclear whether these oncogenes are also involved in the resistance of the cancer against chemotherapy. We examined the role of the HPV16 E6 oncogene in cisplatin resistance by analyzing its expression in newly established cisplatin-sensitive versus -resistant cervical cancer cell lines (CC7, CC10). Resistant variants were obtained by interval exposure treatment with 1-2 μM cisplatin for 8-9 months. Our results demonstrate that the expression level of HPV16 E6 directly correlates with the extent of cisplatin resistance in novel as well as established (SiHa) drug resistant cervical cancer cell lines. Overexpression of HPV16 E6 in cisplatin-naïve cells rendered these cells more resistant to cisplatin. Reducing E6 expression by JQ1 treatment reversed the drug resistant phenotype and strongly enhanced chemoresponse only in HPV-positive cisplatin-resistant variants and not in HPV-negative C33A cervical cancer cells. The level of E6 directly correlated with the extent of cisplatin sensitivity and was shown to be increased in newly established drug-resistant cell line variants, while reducing E6 expression using Brd4-inhibitors enhanced chemoresponse when co-delivered with cisplatin. Inhibition of Brd4 could represent a new therapeutic option by increasing treatment response in cervical cancer cells and might allow lower cisplatin dosages, thus reducing negative side effects.

Half-Sandwich Ir(III) Complex of N1-Pyridyl-7-azaindole Exceeds Cytotoxicity of Cisplatin at Various Human Cancer Cells and 3D Multicellular Tumor Spheroids

The half-sandwich iridium(III) complexes [Ir(η5-Cpx)(phaza–)Cl] (1, 2), [Ir(η5-Cpx)(thaza–)Cl] (3, 4), and [Ir(η5-Cpx)(pyaza)Cl]PF6 (5, 6) containing deprotonated 1-phenyl-7-azaindole (phaza–) and 1-(thiophen-2-yl)-7-azaindole (thaza–) and electroneutral 1-(pyridin-2-yl)-7-azaindole (pyaza), were prepared; Cpx = pentamethylcyclopentadienyl (Cp*; for 1, 3, and 5) or 1,2,3,4-tetramethyl-5-phenylcyclopentadienyl (Cpph; for 2, 4, and 6). The complexes were thoroughly characterized, including a single-crystal X-ray analysis of complexes 1, 5, and 6. All of the complexes were screened for their in vitro cytotoxicity at the A2780 human ovarian carcinoma cell line and its A2780R cisplatin-resistant variant (2D culture cells). The best-performing complex 6 was further studied against the human DU-145 prostatic carcinoma, A549 lung carcinoma, HCT116 colon carcinoma, HeLa cervix adenocarcinoma, and MCF7 breast adenocarcinoma cell lines (2D culture cells). Complex 6 showed a cytotoxic profile different from that of c...

Cationic and Neutral N-Heterocyclic Carbene Gold(I) Complexes: Cytotoxicity, NCI-60 Screening, Cellular Uptake, Inhibition of Mammalian Thioredoxin Reductase, and Reactive Oxygen Species Formation.

A structurally diverse library of 14 gold(I) cationic bis(NHC) and neutral mono(NHC) complexes (NHC: N-heterocyclic carbene) was synthesized and characterized in this work. Four of them were new cationic gold(I) complexes containing functionalized NHCs, and their X-ray crystal structures are presented herein. All of the complexes were investigated for their anticancer activities in four cancer cell lines, including a cisplatin-resistant variant, and a noncancerous cell line. Seven of the cationic gold(I) complexes were found to display high and specific cytotoxic activities toward cancer cells. Two of them were even able to overcome cisplatin resistance. Two highly potent cationic complexes (11 and 15) were also submitted to the NCI-60 cancer panel for further cytotoxicity evaluation. Complex 15 showed a surprisingly high potency toward leukemia among the nine examined cancer subtypes, particularly toward the CCRF-CEM leukemia cell line with a concentration for 50 % inhibition of growth down to 79.4 nm. In addition, cationic complex 13, which demonstrated a remarkable cytotoxicity against hepatocellular carcinoma, was selected to obtain insight into the mechanistic aspects in HepG2 cells. Cellular uptake measurements were indicative of good bioavailability. By various biochemical assays, this complex was found to effectively inhibit thioredoxin reductase and its cytotoxicity toward HepG2 cells was found to be reactive oxygen species dependent.

Copper(II) complexes based on tripodal pyrazolyl amines: Synthesis, structure, magnetic properties and anticancer activity

The Cu(II) complexes [Cu(bpdmpz)Cl]ClO4 (1), [Cu(bdmpzp)Cl]ClO4 (2-ClO4), [Cu(bdmpzp)Cl]PF6 (2-PF6) and [Cu(tdmpza)Cl]ClO4 (3), bpdmpzp=[bis[((2-pyridylmethyl)-di(3,5-dimethyl-1H-pyrazolyl)methyl)]amine, bdmpzp=[bis((di(3,5-dimethyl-1H-pyrazolyl)methyl)-(2-pyridylmethyl)]amine and tdmpza=tris[di(3,5-dimethyl-1H-pyrazolyl)-methyl)]amine were synthesized and characterized by elemental analysis, magnetic and conductivity measurements, electrospray-ionization mass spectrometry, infrared and electronic spectroscopy, and X-ray crystallography. The magnetic properties of the complexes, measured at variable temperature, revealed weak antiferromagnetic intermolecular interactions. The cytotoxicity of the complexes 1, 2-ClO4, 3, and 4 ([Cu(bedmpzp)Cl]PF6, where bedmpzp=[bis(3,5-dimethyl-1H-pyrazol-1-yl-1-ethyl)-(2-pyridylmethyl)]amine), was investigated against four human cancer cell lines: A2780 (ovarian), A2780R (cisplatin-resistant variant), HOS (aggressive bone tumors), CaCo2 (epithelial colorectal adenocarcinoma) and on healthy human hepatocytes. The complex 4 was the most cytotoxic one, with IC50=1.4μM (A2780), 8.3μM (A2780R), 4.7μM (HOS) and 10.8μM (CaCo2). The mass spectrometry-based interaction studies, involving selected sulfur-containing biomolecules and small model proteins, revealed pro-oxidant effects of complexes 1 and 4 and differences in stability of both complexes in the mixtures containing the model protein cytochrome c after 24h incubation, complex 1 formed 1:1 adduct, the formation of which was accompanied by the loss of one dimethylpyrazole pendant arm from the bpdmpz ligand, while the complex 4 composition remained intact and the complex formed both 1:1 and 1:2 adducts (cytochrome c vs. Cu(II)-complex).

Copper(II) Complexes of Phenanthroline and Histidine Containing Ligands: Synthesis, Characterization and Evaluation of their DNA Cleavage and Cytotoxic Activity.

Copper(II) complexes have been intensely investigated in a variety of diseases and pathological conditions due to their therapeutic potential. The development of these complexes requires a good knowledge of metal coordination chemistry and ligand design to control species distribution in solution and tailor the copper(II) centers in the right environment for the desired biological activity. Herein we present the synthesis and characterization of two ligands HL1 and H2L2 containing a phenanthroline unit (phen) attached to the amino group of histidine (His). Their copper(II) coordination properties were studied using potentiometry, spectroscopy techniques (UV-vis and EPR), mass spectrometry (ESI-MS) and DFT calculations. The data showed the formation of single copper complexes, [CuL1]+ and [CuL2], with high stability within a large pH range (from 3.0 to 9.0 for [CuL1]+ and from 4.5 to 10.0 for [CuL2]). In both complexes the Cu2+ ion is bound to the phen unit, the imidazole ring and the deprotonated amide group, and displays a distorted square pyramidal geometry as confirmed by single crystal X-ray crystallography. Interestingly, despite having similar structures, these copper complexes show different redox potentials, DNA cleavage properties and cytotoxic activity against different cancer cell lines (human ovarian (A2780), its cisplatin-resistant variant (A2780cisR) and human breast (MCF7) cancer cell lines). The [CuL2] complex has lower reduction potential (Epc= -0.722 V vs -0.452 V for [CuL1]+) but higher biological activity. These results highlight the effect of different pendant functional groups (carboxylate vs amide), placed out of the coordination sphere, in the properties of these copper complexes.

Abstract 3261: Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth

Abstract Although we previously reported that metronomic cyclophosphamide can augment the anti-tumor impact of anti-CTLA-4 immunotherapy, the effectiveness of this strategy to treat drug resistant tumors remains to be evaluated. To that end, the parental, drug sensitive, EMT-6/P murine breast cancer, or its cisplatin (DDP) or cyclophosphamide (CTX) resistant variants, were implanted into Balb/c mice (n = 5-8 mice per group), either subcutaneously or orthotopically. Established tumors where monitored for relative growth following treatment with anti-CTLA-4 antibody (mouse anti-CD152, clone 9H10, given at 100ug on day 1 and 35ug on day 6, i.p.), alone or in combination with; a) metronomic CTX (ldCTX; 20mg/kg/day), b) Bolus (150mg/kg) plus ldCTX, or c) sequential treatment with gemcitabine (160mg/kg every 3 days). EMT-6/P tumors responded to anti-CTLA-4 therapy, but this response was less effective when combined with Bolus plus ldCTX. Anti-CTLA-4 could be effectively combined with either ldCTX (without a bolus), or with a regimen of sequential gemcitabine. The EMT-6/P tumor showed consistent results in subcutaneous tumors and in those that were orthotopically implanted into the mammary fat pad. Tumor responses were less pronounced in drug resistant EMT-6/CTX or EMT-6/DDP tumor models than in the EMT-6/P tumor, but nonetheless even in the drug resistant models CTLA-4 targeting could be effectively combined with metronomic CTX or sequential gemcitabine (p<0.05). A number mice implanted with the parent or with the drug resistant tumors developed spontaneous metastases under continuous therapy, including mice bearing subcutaneous tumors, and about 20% of the mice on the combination therapies showed complete tumor responses. Of these mice, over 90% rejected subsequent EMT/6 tumor grafts, indicating that the anti-CTLA-4 plus metronomic chemotherapy regimens did not impair immunological memory. Furthermore, the efficacy of a therapy of anti-CTLA-4 with sequential continuous gemcitabine was confirmed in an independent syngeneic CT-26 colon cancer model. We conclude that metronomic chemotherapy can combined with anti-CTLA-4 therapy, both in syngeneic breast and colon cancer models, and in breast tumors selected in vivo for resistance to alkylating agents. Sequential therapy of anti-CTLA-4 followed by gemcitabine is effective in both chemotherapy naïve tumors and in tumors selected in vivo for resistance to CTX or to DDP, although tumor relapses can occur, in some cases accompanied by the development spontaneous metastases. Our results suggest a strategy for improving the anti-tumor efficacy of anti-CTLA-4 based therapies. Citation Format: Karla Parra, Paloma Valenzuela, Alejandra E. Gallegos, Natzidielly Lerma, Luis Reza, Georgialina Rodriguez, Urban Emmenegger, Marian Manciu, Teresa Di Desidero, Guido Bocci, Mitchell S. Felder, Robert A. Kirken, Giulio Francia. Impact of CTLA-4 blockade in conjunction with metronomic chemotherapy on preclinical breast cancer growth. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3261.

Berberine modulates cisplatin sensitivity of human gastric cancer cells by upregulation of miR-203.

Chemotherapeutic resistance is the main reason of the failure in clinical treatment of gastric cancer. Berberine (BER) is the active compound of traditional Chinese medicine Huang Lian. The aim of this present study is to evaluate the effect of BER on cisplatin resistance in gastric cancer cells and to investigate its possible mechanism. Gastric cancer cell lines SGC-7901 and BGC-823 and their respective cisplatin-resistant variants SGC-7901/DDP and BGC-823/DDP were used in this study. We found that BER treatment significantly reversed cisplatin sensitivity and induced caspase-dependent apoptosis in SGC-7901/DDP and BGC-823/DDP cells; BER treatment induced miR-203 expression, and overexpression of miR-203 mimicked the cisplatin-sensitizing effect of BER. Importantly, we showed that miR-203 was able to target the 3'UTR of Bcl-w. Therefore, we conclude that BER treatment reduces cisplatin resistance of gastric cancer cells by modulating the miR-203/Bcl-w apoptotic axis. BER may be a novel agent to enhance chemotherapeutic responses in cisplatin-resistant gastric cancer patients.

YAP induces cisplatin resistance through activation of autophagy in human ovarian carcinoma cells.

ObjectiveTo identify the role of YAP in cisplatin resistance in human ovarian cancer cells and in the regulation of autophagy in these cancer cells.Materials and methodsThe cisplatin-sensitive OV2008 parental cell line and its cisplatin-resistant variant C13K were cultured. RNA interference was used to knock down the YAP gene. Accumulation of GFP-LC3 puncta was performed by fluorescence microscopy. The formation of autophagosomes was observed by transmission electron microscopy. Drug sensitivity was examined using CCK-8 assay, while apoptosis, the level of intracellular rhodamine 123 and lysosomal acidification were analyzed by fluorescence-activated cell sorting. Acid phosphatase activity was measured using an acid phosphatase-assay kit. Real-time polymerase chain reaction, Western blotting, and immunofluorescence detection were used to detect the protein and messenger RNA expression of YAP, YAP target genes, CCND1, cleaved PARP, and caspase 3, Atg-3 and -5, and the LC3B protein.ResultsYAP signaling may regulate cisplatin resistance in ovarian cancer cells by augmenting cellular autophagic flux. After knockdown of YAP-sensitized C13K cells to cisplatin by inducing a decrease in autophagy, YAP led to an increase in autophagy via enhancement of autolysosome degradation.ConclusionYAP-mediated autophagy may play a protective role in cisplatin-resistant human ovarian cancer cells. Therefore, YAP-mediated autophagy should be explored as a new target for enhancing the efficacy of cisplatin against ovarian cancer and other types of malignancies.

Abstract B18: Association between apoptotic response and KiSS1 modulation in NSCLC preclinical models treated with SAHA and cisplatin

Abstract Non-Small-Cell Lung Cancer (NSCLC) is an aggressive disease with low responsiveness to chemotherapy. Platinum-based therapy is the standard of care for NSCLC with limited benefits due to the occurrence of resistance. Since histone deacetylases (HDAC) have been implicated in silencing of apoptotic pathways and failure to undergo apoptosis in response to antitumor agents may result in drug resistance, in the present study we examined the capability of SAHA (vorinostat), a pan-HDAC inhibitor to improve cisplatin-induced apoptosis in NSCLC models, focusing on the possible role of KiSS1, a well-known metastasis suppressor, which could be involved in apoptosis regulation. Preclinical pharmacology approaches were employed including growth inhibition assays, western blot analyses, ELISA and antitumor activity tests in murine xenografts. The drug interaction was examined using the Bliss method. We examined the effect of the combination of SAHA and cisplatin in the NSCLC H460 and A549 cell lines, including the cisplatin-resistant variants (H460/Pt, A549/Pt), which were found to exhibit enhanced invasive potential as compared to parental cells, besides reduced susceptibility to cisplatin-induced apoptosis. We observed that a synergistic interaction between SAHA and cisplatin occurred in cisplatin-sensitive H460 cells and in cisplatin-resistant H460/Pt cells, where it was less marked. The synergism was schedule-dependent because it was not observed following a simultaneous drug exposure, but when cells were pre-treated with SAHA for 24 h, a circumstance that we previously showed to up-regulate KiSS1 mRNA level. Under such a condition, increased levels of the pro-apoptotic protein Bax were detected by western blot analysis in cells exposed to the drug combination as compared to untreated and singly treated cells in both H460 and H460/Pt cells. Such a behaviour was associated with increased levels of apoptotic cells. Moreover, intracellular and extracellular levels of the KiSS1 protein tended to increase upon SAHA and combined treatment. In the A549 and A549/Pt cells an additive interaction was mainly observed. The efficacy of the combination was tested in vivo after subcutaneous inoculum of H460/Pt cells in immunodeficient mice. A significant tumor volume inhibition was found when mice bearing advanced tumors were treated with the combination of SAHA and cisplatin according to the best schedule identified in cellular studies. Our findings indicate that the efficacy of the drug combination is schedule-dependent and may be influenced by the molecular background of the experimental models. The evidence of increased apoptosis in relation to KiSS1 up-modulation, together with previous findings of the involvement of KiSS1 in counteracting the invasive ability of NSCLC cells suggest a multi-tasking role for this factor. Citation Format: Laura Gatti, Michelandrea De Cesare, Cristina Corno, Nives Carenini, Elisabetta Corna, Nadia Zaffaroni, Emilio Ciusani, Paola Perego. Association between apoptotic response and KiSS1 modulation in NSCLC preclinical models treated with SAHA and cisplatin. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B18.