Cisplatin-resistant Ovarian Carcinoma Cell Research Articles

Abstract 571: CRISPR/Cas9 screening identifies the proteasome subunit PSMC6 as key for the survival of cisplatin-resistant ovarian carcinoma cells

Abstract Ovarian carcinoma is a disease with poor prognosis, due to delays in diagnosis and the development of drug resistance. Since deregulation of the ubiquitin proteasome system may contribute to the development of drug resistance, the aim of this study was to investigate the interplay between expression of proteasome subunits and cisplatin resistance by utilizing a CRISPR/Cas9 dropout screen. Paired cisplatin-sensitive and -resistant cells (IGROV-1 and IGROV-1/Pt1) were transfected with Cas9 and transduced with gRNAs against proteasome components. Cells were collected at early (day 4) and late (day 21) time exposure and sequenced to identify genes necessary for survival. Various proteasome subunits were found to be essential for the survival of cisplatin-resistant ovarian carcinoma cells, including PSMA5, PSMC6, PSMD7, PSMD12 and PSMD14. Since PSMC6 knockout produced the greatest growth inhibitory effects, it was selected for functional studies utilizing conventional RNA interference. When IGROV-1 and IGROV-1/Pt1 cells were transfected with small interfering RNAs (siRNAs) targeting PSMC6, efficient down-regulation on the mRNA and protein level was observed. Phenotypic analyses of PSMC6 knockdown cells indicated reduced clonogenicity using both 2D and soft agar assays in parental and resistant cells associated with down-regulation of the canonical MAPK pathway. A slight but not significant increase in sensitivity to cisplatin was observed upon PSMC6 silencing in resistant cells using clonogenic assays. Finally, we carried out a pilot study using ovarian carcinoma specimens (n = 134) obtained through the institutional biobank. The samples were representative of the frequency occurrence of the various histological ovarian carcinoma subtypes. We found that PSMC6 expression, evaluated by qRT-PCR, was associated with tumor stage. These findings suggest that PSMC6 is linked to ovarian carcinoma aggressiveness and that PSMC6 targeting may be an interesting therapeutic strategy. Citation Format: Matteo Costantino, Padraig D'arcy, Luca Mirra, Cristina Corno, Giovanni L. Beretta, Johannes Gubat, Pietro Pratesi, Chiara M. Ciniselli, Paolo Verderio, Stig Linder, Paola Perego. CRISPR/Cas9 screening identifies the proteasome subunit PSMC6 as key for the survival of cisplatin-resistant ovarian carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 571.

Porphyrin metalla-assemblies coupled to cellulose nanocrystals for PDT and imaging applications

Photodynamic therapy (PDT) is an interesting and promising approach to tackle a broad spectrum of cancer. With the combination of a photosensitizer, light and oxygen, PDT achieves a unique selectivity by the production of localized reactive oxygen species (ROS) inside cells, which leads to their destruction. In addition, the luminescence properties of photosensitizers can be exploited to develop imaging tools. Unfortunately, the cancer selectivity and homogeneity of most photosensitizers are frequently limiting the performances of PDT and cancer detection/characterization by luminescence imaging. Consequently, our study aims to use cellulose nanocrystals to transport and deliver radiolabeled photo-responsive metalla-assemblies to create a new generation of theranostic agents for PDT and imaging applications. The synthesis, structural characterization, cytotoxicity evaluation, and in vivo biodistribution imaging of the compounds are presented. The best candidates show excellent biological activity and selectivity towards ovarian carcinoma cell line (A2780), cisplatin-resistant ovarian carcinoma cell line (A2780cis) versus normal human embryonic kidney cells (HEK293T), as well as efficient imaging properties, suggesting a potential use as multimodal theranostic agents.

Arene-ruthenium(II) complexes with pyrazole-based ligands bearing a pyridine moiety: Synthesis, structure, DFT calculations, and cytotoxicity

Two pyrazole-based ligands bearing a pyridine moiety, 3-methyl-1-(pyridin-2-yl)-5-pyrazolone (HLpy), and 3-methyl-1-(pyridin-2-yl)-4-trifluoroacetyl-5-pyrazolone (HQpy,CF3), were prepared and fully characterized in the solid-state and the tautomerism of both ligands in solution was also rationalized by using Density Functional Theory. Neutral ruthenium(II) arene complexes of composition [Ru(arene)(Lpy)Cl] and [Ru(arene)(Qpy,CF3)Cl] [arene = p-cymene (cym) or hexamethylbenzene (hmb)]) were synthesized and characterized by IR, 1H, 13C, 15N and 19F NMR spectroscopy, elemental analysis and ESI mass spectrometry. The structures of complexes [Ru(hmb)(Lpy)Cl] and [Ru(hmb)(Qpy,CF3)Cl] were determined by X-ray crystallography, showing κ2-N,N’-coordination not only for Lpy but also for Qpy,CF3 ligand. DFT studies confirm that κ2O,O’-coordination, generally observed in metal complexes with 4-acyl-5-pyrazolone ligands, is not favored in this case. The cytotoxicity of ligands and complexes was evaluated against human ovarian carcinoma cells (A2780 and A2780cisR, cisplatin sensitive and cisplatin-resistant, respectively), and non-tumorous human embryonic kidney SV40 transformed (HEK293T) cells to reveal essentially equivalent activity in the cisplatin sensitive and cisplatin-resistant ovarian carcinoma cells.

Targeted biologic inhibition of both tumor cell-intrinsic and intercellular CLPTM1L/CRR9-mediated chemotherapeutic drug resistance

Recurrence of therapy-resistant tumors is a principal problem in solid tumor oncology, particularly in ovarian cancer. Despite common complete responses to first line, platinum-based therapies, most women with ovarian cancer recur, and eventually, nearly all with recurrent disease develop platinum resistance. Likewise, both intrinsic and acquired resistance contribute to the dismal prognosis of pancreatic cancer. Our previous work and that of others has established CLPTM1L (cleft lip and palate transmembrane protein 1-like)/CRR9 (cisplatin resistance related protein 9) as a cytoprotective oncofetal protein that is present on the tumor cell surface. We show that CLPTM1L is broadly overexpressed and accumulated on the plasma membrane of ovarian tumor cells, while weakly or not expressed in normal tissues. High expression of CLPTM1L is associated with poor outcome in ovarian serous adenocarcinoma. Robust re-sensitization of resistant ovarian cancer cells to platinum-based therapy was achieved using human monoclonal biologics inhibiting CLPTM1L in both orthotopic isografts and patient-derived cisplatin resistant xenograft models. Furthermore, we demonstrate that in addition to cell-autonomous cytoprotection by CLPTM1L, extracellular CLPTM1L confers resistance to chemotherapeutic killing in an ectodomain-dependent fashion, and that this intercellular resistance mechanism is inhibited by anti-CLPTM1L biologics. Specifically, exosomal CLPTM1L from cisplatin-resistant ovarian carcinoma cell lines conferred resistance to cisplatin in drug-sensitive parental cell lines. CLPTM1L is present in extracellular vesicle fractions of tumor culture supernatants and in patients’ serum with increasing abundance upon chemotherapy treatment. These findings have encouraging implications for the use of anti-CLPTM1L targeted biologics in the treatment of therapy-resistant tumors.

Hsa-miR-105-1 Regulates Cisplatin-Resistance in Ovarian Carcinoma Cells by Targeting ANXA9.

Purpose Cisplatin is one of the most effective drugs for treating ovarian carcinoma (OC), which is among the most lethal types of carcinoma. However, the chemoresistance to cisplatin that develops over time leads to a poor clinical outcome for many OC patients. Therefore, it is necessary to clearly understand the molecular mechanisms of chemoresistance. In this study, we examined how Hsa-miR-105-1 functions in cisplatin-resistant OC cells. Methods The levels of Hsa-miR-105-1 expression in cisplatin-sensitive and resistant OC cell lines were detected by qRT-PCR. The target gene of Hsa-miR-105-1 was predicted by using the TargetScan and Starbase databases and verified by the double luciferase reporter gene assay. The target gene of Hsa-miR-105-1 was identified as ANXA9, and ANXA9 expression was evaluated by qRT-PCR, western blotting, and immunofluorescence. To validate the function of Hsa-miR-105-1 in OC cells, we silenced or overexpressed Hsa-miR-105-1 in cisplatin-sensitive or resistant OC cell lines, respectively. Furthermore, the expression levels of several apoptosis-related proteins, including P53, P21, E2F1, Bcl-2, Bax, and caspase-3, were examined by western blot analysis. Results The levels of Hsa-miR-105-1 expression were abnormally downregulated in cisplatin-resistant OC cells, while ANXA9 expression was significantly upregulated in those cells. Treatment with an Hsa-miR-105-1 inhibitor promoted the expression of ANXA9 mRNA and protein, enhanced the resistance to cisplatin, and attenuated the cell apoptosis induced by cisplatin in cisplatin-sensitive OC cells. Moreover, treatment with Hsa-miR-105-1 mimics inhibited ANXA9 expression, which further increased the levels of P53, P21, and Bax expression and decreased the levels of E2F1 and Bcl-2 expression, finally resulting in an increased sensitivity to cisplatin in cisplatin-resistant OC cells. Conclusion We found that a downregulation of Hsa-miR-105-1 expression enhanced cisplatin-resistance, while an upregulation of Hsa-miR-105-1 restored the sensitivity of OC cells to cisplatin. The Hsa-miR-105-1/ANXA9 axis plays an important role in the cisplatin-resistance of OC cells.

Pharmacokinetic evaluation and antitumor potency of liposomal nanoparticle encapsulated cisplatin targeted to CD24-positive cells in ovarian cancer.

CD24, which is upregulated in several human malignancies, is related to Epithelial-mesenchymal-transition (EMT) and has characteristics of cancer stem-like cells, especially in cisplatin-resistant ovarian carcinoma cells. Drug delivery systems represent a promising therapeutic approach for diseases with treatment resistance, and the present study investigated a novel CD24-targeted drug delivery system for advanced ovarian carcinoma. We produced liposomal cisplatin with a red fluorescent substance - cyanine 5.5 (GL-CDDP-Cy5.5). In order to target CD24-positive cells, an anti-CD24 monoclonal antibody was modified to the above drug (CD24-GL-CDDP-Cy5.5). Specific uptake of CD24-GL-CDDP-Cy5.5 was confirmed using a therapeutically resistant ovarian cancer cell line, Caov-3 cells. Antitumor effects of CD24-GL-CDDP-Cy5.5 were then evaluated in Caov-3 ×enograft mice. CD24-GL-CDDP-Cy5.5 showed more specific uptake by flow cytometry than GL-CDDP-Cy5.5. In xenograft mice, GL-CDDP-Cy5.5 and CD24-GL-CDDP-Cy5.5 treatment had significantly higher platinum concentration in disseminated tumor cells than cisplatin (P<0.05). Moreover, CD24-GL-CDDP-Cy5.5 suppressed tumor growth and prolonged survival time compared with other treatments. Median survival times of the control, cisplatin, GL-CDDP-Cy5.5 and CD24-GL-CDDP-Cy5.5 groups were 37, 36, 46 and 54 days after inoculation, respectively. Immunohistochemical analysis showed that CD24-GL-CDDP-Cy5.5 treatment, compared with GL-CDDP-Cy5.5, decreased the number of CD24-positive cells and suppressed the EMT phenomenon significantly (P<0.05). The present study demonstrated that CD24-GL-CDDP-Cy5.5, compared with other treatments, improved therapeutic efficacy. The present results suggested the potential for targeting anticancer therapeutics for CD24-positive cells to prevent disease progression.

Anticancer Potential of Diiron Vinyliminium Complexes.

Although ferrocene derivatives have attracted considerable attention as possible anticancer agents, the medicinal potential of diiron complexes has remained largely unexplored. Herein, we describe the straightforward multigram-scale synthesis and the antiproliferative activity of a series of diiron cyclopentadienyl complexes containing bridging vinyliminium ligands. IC50 values in the low-to-mid micromolar range were determined against cisplatin sensitive and resistant human ovarian carcinoma (A2780 and A2780cisR) cell lines. Notable selectivity towards the cancerous cells lines compared to the non-tumoral human embryonic kidney (HEK-293) cell line was observed for selected compounds. The activity seems to be multimodal, involving reactive oxygen species (ROS) generation and, in some cases, a fragmentation process to afford monoiron derivatives. The large structural variability, amphiphilic character and good stability in aqueous media of the diiron vinyliminium complexes provide favorable properties compared to other widely studied classes of iron-based anticancer candidates.

In vitro anticancer active cis-Pt(II)-diiodido complexes containing 4-azaindoles.

4-Azaindole (1H-pyrrolo[3,2-b]pyridine; 4aza) and its N1-alkylated derivative N1-isopropyl-4-azaindole (1-(propan-2-yl)-1H-pyrrolo[3,2-b]pyridine; ip4aza) have been used for the preparation of the cis-diiodido-platinum(II) complexes cis-[Pt(4aza)2I2] (1), cis-[PtI2(ip4aza)2] (2), cis-[Pt(4aza)I2(NH3)] (3) and cis-[PtI2(ip4aza)(NH3)] (4). The prepared complexes were thoroughly characterized (e.g., multinuclear NMR spectroscopy and ESI mass spectrometry) and their in vitro cytotoxicity was assessed at human ovarian carcinoma (A2780), cisplatin-resistant ovarian carcinoma (A2780R) and colon carcinoma (HT-29) cell lines, where they showed, in some cases, significantly higher activity than the used reference-drug cisplatin. The results of in vitro cytotoxicity testing at the A2780 and A2780R cells indicated that alkylation of the 4-azaindole moiety at the position of the N1 atom had a positive biological effect, because the ip4aza-containing complexes 2 and 4 showed significantly (p < 0.005) higher cytotoxicity than 4aza-containing analogues 1 and 3. The resistance factors (A2780R/A2780 model) equalled 0.8-1.4, indicating the ability of complexes 1-4 to overcome the acquired resistance of the A2780 cells against cisplatin. Complexes 1 and 2 revealed low toxicity against primary culture of human hepatocytes. The flow cytometry studies of the A2780 cell cycle modification showed that complexes 1-4 induce different cell cycle perturbations as compared with cisplatin, thus suggesting a different mechanism of their antitumor action.

Histidine Targeting Heterobimetallic Ruthenium(II)-Gold(I) Complexes.

Inspired by the preferential, allosteric binding of RAPTA-T and auranofin to the nucleosome core particle , we describe the design and synthesis of a series of heterobimetallic ruthenium(II)-gold(I) complexes with varying spacer lengths ranging from four to eight polyethylene glycol units. Evaluation of their cytotoxicity reveals IC50 values in the low micromolar range against cisplatin sensitive and resistant human ovarian carcinoma (A2780, A2780cisR) and nontumoral human embryonic kidney (HEK293) cell lines. Binding studies monitored via mass spectrometry revealed an affinity for histidine residues on a fragment of the amyloid β-protein (residues 1-16, employed as a model system), which is in accordance with the binding sites of parent drugs, RAPTA-C and auranofin, to the nucleosome core particle.

Transporter-Mediated Interaction Between Platinum Drugs and Sorafenib at the Cellular Level.

Combining the multikinase inhibitor sorafenib with the platinum-based chemotherapy of solid tumors was expected to improve treatment outcome. However, in many clinical trials, no benefit from sorafenib addition to the platinum-containing regimen could be demonstrated. Moreover, in some studies, decreased survival of ovarian cancer patients as well as non-small cell lung cancer patients with squamous cell histology was observed. The aim of this study was to investigate the cellular mechanisms of the pharmacological interaction between platinum drugs and sorafenib in different cancer cell lines. The interaction was characterized by combination index analysis, platinum accumulation and DNA platination were determined using flameless atomic absorption spectrometry, and protein expression was assessed with Western blot. In the sensitive A2780 ovarian carcinoma and H520 squamous cell lung carcinoma cell lines, sorafenib induced downregulation of Na+,K+-ATPase. In A2780 cells, the kinase inhibitor also decreased the expression of copper transporter 1 (CTR1). As a result, sorafenib treatment led to a diminished cellular accumulation of cisplatin and carboplatin and to a decrease in DNA platination in these cell lines. This was not the case in the cisplatin-resistant A2780cis ovarian carcinoma and H522 lung adenocarcinoma cell lines featuring lower basal expression of the above-mentioned transporters. In all cell lines studied, an antagonistic interaction between platinum drugs and sorafenib was found. Our results suggest that sorafenib impairs cisplatin and carboplatin uptake through downregulation of CTR1 and/or Na+,K+-ATPase resulting in reduction of DNA platination. This effect is not observed in cancer cells with defects in platinum accumulation.

Effect of low dose fractionated radiation on reversing cisplatin resistance in ovarian carcinoma via VEGF and mTOR ⁎

Abstract Objective To investigate the mechanism of low-dose fractionated radiation on reversing cisplatin resistance in ovarian carcinoma via vascular endothelial growth factor (VEGF) and mammalian target of rapamycin (mTOR) in vivo. Methods Human cisplatin-resistant ovarian carcinoma cells (SKOV3/DDP) were injected into nude mice to establish ovarian cancer xenografts. The mice were randomly divided into three groups: a control group, a low-dose fractionated radiation (LDRFT) group, and a conventional-dose radiation group. Each group was exposed to 0 cGy, 50 cGy, and 200 cGy radiation, respectively, for 4 weeks, up to a total of 8.0 Gy. Mice in the LDFRT group were irradiated twice daily with 6 hour intermissions on day 1 and 2 of every week for a total of 4 weeks. Conventional-dose group mice were given a single 200 cGy radiation dose on the first day each week for a total of 4 weeks. Maximum horizontal and vertical diameters of the tumors were measured every other day and used to create a tumor growth curve. After 4 weeks of irradiation, we dissected the tumor tissue and calculated the tumor inhibition rate. RT-PCR detected the expression of VEGF and mTOR, and Western blots detected the expression of corresponding proteins. Results Both LDRFT and conventional-dose radiation inhibited the growth of tumor cells, and growth of tumors in the two radiation groups compared with growth in the control group were significantly different (P &lt; 0.05). The rate of tumor inhibition in the LDFRT group (37.5603%) was lower than in the conventionaldose group (47.4446%), but there was no significant difference (P &gt; 0.05). Compared with the other two groups, the mRNA expression of VEGF was significantly lower in the LDFRT group (P &lt; 0.05), but there was no obvious difference between the conventional-dose and control groups. There was no obvious difference in the mRNA expression of mTOR among the three groups, but the expression of the protein p-mTOR was lower in the LDFRT group (P &lt; 0.05), as confirmed by Western blotting. Conclusion LDFRT is as effective at inhibiting the growth of tumor cells as conventional-dose radiation. In addition, LDFRT could deregulate the expression of VEGF and p-mTOR, and may therefore play a vital role in reversing cisplatin resistance in ovarian cancer.

Abstract 2315: Overcoming treatment resistance in cisplatin-resistant ovarian carcinoma cells by tissue-specific miRNAs

Abstract Ovarian cancer is a common human cancer with a poor outcome and a high risk of death. Due to resistances to current standard therapies (carboplatin and paclitaxel for 3 to 6 cycles) additional therapeutic approaches are required and novel specific biomarkers for ovarian cancer patients are necessary. MicroRNAs (miRNA) are conserved, small noncoding RNAs regulating gene expression by binding to their target mRNA which have been shown to regulate biological processes including apoptosis. Apoptosis is a conserved, irreversible process that allows cells to undergo a highly regulated form of cell death. However, the final role of miRNAs in apoptotic signaling has not yet been fully determined. Modulating the expression of key molecular components of the cell death machinery is an attractive strategy for cancer therapy overcoming chemo drug resistance. To extend the pool of potential miRNA as anticancer agents and biomarkers, we transferred a high content miRNA screening for pro-apoptotic miRNAs to human cancer cell lines. Based on the high recovery rate of apoptosis inducing miRNAs in SKOV3 cells analyses were extended to multiple ovarian carcinoma cell lines with different genetic background. After transfection of miR-1912-5p the apoptosis rate increased strongly in cisplatin-resistant cells. In combination treatment with carboplatin the apoptotic effect increased after transfection of miR-1912-5p in the cisplatin-resistant A2780 cells. Furthermore, miR-1912-5p, miR-147b and miR-3073a showed significant apoptosis induction in the various ovarian cancer cell lines alone and in combined therapy with carboplatin. Western Blot analysis revealed an enhanced expression of the pro-apoptotic proteins Bak1 and Bax and a decrease inBcl2 and Bcl-xL. We characterized the miRNAs in regard to their endogenous expression and detected an enhanced expression after apoptosis induction by chemotherapeutic drugs. Finally, analysis of The Cancer Genome Atlas data showed an influence on the median survival of ovarian cancer patients (&amp;gt; 69 years). Our data indicate a potential benefit of tissue specific pro-apoptotic miRNAs in regard to cisplatin-resistant cells. Furthermore the study identified novel pro-apoptotic miRNAs with their potential use as novel therapeutic entities or prognostic biomarkers for ovarian cancer. Citation Format: Michael Kleemann, Jeremias Bereuther, Simon Fischer, Kim Marquart, Simon Hänle, Kristian Unger, Verena Jendrossek, Christian U. Riedel, René Handrick, Kerstin Otte. Overcoming treatment resistance in cisplatin-resistant ovarian carcinoma cells by tissue-specific miRNAs [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2315. doi:10.1158/1538-7445.AM2017-2315

Raman micro-spectroscopy applied to treatment resistant and sensitive human ovarian cancer cells.

Despite the many advances intended to enhance the response to treatment, the survival rate of patients with ovarian cancer has only marginally improved in the past few decades. One major cause for this, is the lack of diagnostics for platinum-resistant disease. The goal of this study was to determine whether Raman micro-spectroscopy in conjunction with multivariate statistical analysis could discriminate between chemically fixed cisplatin-resistant (A2780cp) and cisplatin-sensitive (A2780s) human ovarian carcinoma cells. Raman spectra collected from individual cells were pre-processed and subsequently analyzed with Principal Component Analysis - Linear Discriminant Analysis (PCA-LDA). Statistically significant differences (P< 0.0001) were observed between the Raman spectra of A2780s and A2780cp cells. A diagnostic accuracy of 82% was obtained using the PCA-LDA classifier model for the discrimination between the A2780s and A2780cp cells. The loading plot analysis suggests that relative increases in proteins and glutathione in the cisplatin-resistant cells compared to the cisplatin-sensitive cells are most likely the major source of discrimination between the two types of cells. These results support the potential application of Raman spectroscopy in the identification of chemo-resistant tumors prior to treatment.

Enhanced anti-cancer activities of a gold(III) pyrrolidinedithiocarbamato complex incorporated in a biodegradable metal-organic framework

An anti-cancer active gold(III) pyrrolidinedithiocarbamato complex [(PDTC)AuIIICl2] (1) has been synthesized and characterized by means of X-ray crystallography. Compared to the pyrrolidinedithiocarbamate ligand itself, this gold(III) complex displays an up to 33-fold higher anti-cancer potency towards a panel of cancer cell lines including the cisplatin-resistant ovarian carcinoma cell line (A2780cis). As demonstrated by a set of Transwell® assay-based cytotoxicity experiments, incorporating this gold(III) complex in a zinc-based biodegradable metal-organic framework (MOF) displays a significant enhancement in anti-cancer activity towards A2780cis than the gold(III) complex alone.

Rapamycin enhanced the antitumor efficacy of oxaliplatin in cisplatin-resistant ovarian cancer cells A2780cis both in vitro and in vivo

Objective: This study aimed to investigate the efficacy of combination of rapamycin, an mammalian target of rapamycin (mTOR) inhibitor for treating rejection after organ transplantation, and oxaliplatin, a third-generation of platinum drug usually used to treat chemoresistant or progressive ovarian cancer, in cisplatin-resistant ovarian carcinoma cells A2780cis.Methods/Materials: Expressions of mTOR and its target molecules p70S6K and 4E-BP1 were determined in cisplatin-sensitive and -resistant cells A2780 and A2780cis, respectively, using Western blotting. Proliferation of A2780cis exposure to oxaliplatin or oxaliplatin plus rapamycin was examined using MTT assay in vitro as well as a nude mice model in vivo. Cell apoptosis and proapoptosis proteins including caspase-8 and -3 and PARP were determined using flow cytometry and Western blotting.Results: We found that A2780cis cells had partial cross-resistance between cisplatin and oxaliplatin. The levels of phosphorylated mTOR (p-mTOR), p70S6K, and 4E-BP1 were significantly increased in A2780cis cells compared to A2780 cells, which might be implicated in cisplatin-induced chemoresistance. Rapamycin obviously enhanced the inhibitory effect of oxaliplatin on the growth of A2780cis both in vitro and in vivo. Rapamycin slightly induced cell apoptosis but significantly enhanced the effect of oxaliplatin in soliciting apoptosis of A2780cis cells, which might be ascribed to its ability in further increasing the levels of cleaved caspase-8 and -3 and PARP induced by oxaliplatin.Conclusion: These results suggested that combination of oxaliplatin and rapamycin enhanced the antitumour efficacy of oxaliplatin in A2780cis cells and therefore might have a role in treating cisplatin-resistant ovarian carcinoma.

HOTAIR is a potential target for the treatment of cisplatin‑resistant ovarian cancer.

Previous studies have demonstrated that the presence of Hox transcript antisense intergenic RNA (HOTAIR) is correlated with poor survival in several types of cancer, including breast cancer, and promotes tumor metastasis. Currently, little is known regarding the correlation between HOTAIR and chemoresistance in cancer. The current study aimed to investigate the role of HOTAIR in epithelial ovarian cancer, and the correlation between HOTAIR and cisplatin resistance. Reverse transcription-quantitative polymerase chain reaction was conducted to detect HOTAIR expression in the ovarian specimens and ovarian carcinoma cell lines. The results indicated that the expression level of HOTAIR was higher in epithelial ovarian cancer tissues than the level in the benign ovarian tissues. The expression level was also higher in late-stage malignant ovarian tumors compared with the level in early-stage tumors. Levels of HOTAIR were also higher in the SKOV-3CDDP/R cisplatin-resistant ovarian carcinoma cell line than in the SKOV-3 cisplatin-sensitive cell line. The knockdown of HOTAIR using siRNAs with transfection reagent suppressed cell proliferation, reduced the invasion ability of the cells and notably, it restored cisplatin-sensitivity of the cisplatin-resistant cells specifically by enhancing cisplatin-induced cytotoxicity and apoptosis in SKOV-3CDDP/R cells. In conclusion, HOTAIR may be used in the development of novel treatments for ovarian cancer, particularly those that are resistant to conventional therapies.

Cross-reacting material 197, a heparin-binding EGF-like growth factor inhibitor, reverses the chemoresistance in human cisplatin-resistant ovarian cancer.

Cross-reacting material 197 (CRM197), a specific HB-EGF inhibitor, has been proven to be a promising antitumor agent for ovarian cancer therapy. Our previous studies have shown that CRM197 has potent antitumor activity in human cisplatin-resistant ovarian cancer. However, the relationship between CRM197 and the resistance to cisplatin remains unclear. Here, we report that CRM197 significantly reverses the resistance to cisplatin in cisplatin-resistant ovarian carcinoma cell line (A2780/CDDP). We established xenograft nude mice models with A2780 and A2780/CDDP cells. Notably, we observed that CRM197 suppresses the expression of HB-EGF and epidermal growth factor receptor in A2780/CDDP cells and xenografts harboring the overexpression of HB-EGF and epidermal growth factor receptor. Experiments conducted in vitro and in vivo suggest that CRM197 markedly downregulates the expression of excision repair cross-complementing group 1 (P = 0.002) and DNA repair capacity in A2780/CDDP tumor (P < 0.001) by inactivation of extracellular signal-regulated kinase signaling, providing novel possible mechanisms for the ability of CRM197 to restore drug sensitivity. These results suggest that CRM197 as an HB-EGF inhibitor might be a cisplatin-chemosensitizing agent for the treatment of ovarian carcinoma with resistance to cisplatin.

Induction of autophagy contributes to cisplatin resistance in human ovarian cancer cells.

Cisplatin resistance is a major challenge in the clinical treatment of ovarian cancer, of which the underlying mechanisms remain unknown. The aim of the present study was to explore the role of autophagy in cisplatin resistance in ovarian cancer cells. A2780cp cisplatin-resistant ovarian carcinoma cells and the A2780 parental cell line, were used as a model throughout the present study. The cell viability was determined using a water soluble tetrazolium salt-8 assay, and western blot analysis was performed to determine the protein expression levels of microtubule-associated protein 1 light chain 3 (LC3 I and LC3 II), and Beclin 1. Beclin 1 small interfering (si)RNA and 3-methyladenine (3-MA) were used to determine whether inhibition of autophagy may re-sensitize cisplatin-resistant cells to cisplatin. The ultrastructural analysis of autophagosomes was performed using transmission electron microscopy, and apoptosis was measured by flow cytometry. In both A2780cp and A2780 cells, cisplatin induced the formation of autophagosomes and upregulated the expression levels of autophagy protein markers, LC3 II and Beclin 1. However, the levels of autophagy were significantly higher in A2780cp cells, as compared with the A2780 cells. The combined treatment of cisplatin with 3-MA, the autophagy pharmacological inhibitor, increased the cell death rate, but had no effects on apoptosis, as compared with cisplatin treatment alone in A2780cp cells. However, inhibition of autophagy by siRNA knockdown of Beclin 1 expression enhanced cisplatin-induced cell death and apoptosis. The findings of the present study suggest that autophagy has a protective role in human ovarian cancer cells, and that targeting autophagy may promote chemotherapeutic sensitivity.

Abstract 1801: APR-246, a clinical-stage mutant p53-reactivating compound, resensitizes ovarian cancer cells to platinum compounds and doxorubicin

Abstract Background: Platinum-based drugs are since decades used as first-line treatment for many solid tumors. Patients with ovarian cancer often respond well to platinum compounds but a majority of patients rapidly develop resistance and die of chemotherapy refractory disease. The mechanisms underlying resistance are multifactorial, but two of the main causes are mutations in the tumor suppressor p53 and elevated intracellular glutathione (GSH) levels. Mutations in p53 occur in about 60% of ovarian tumors. APR-246 (PRIMA-1MET) is the first clinical-stage compound that reactivates mutant p53. APR-246 is a prodrug that accumulates in cancer cells and is converted to the active form MQ, a Michael acceptor that binds to mutant p53, refolds it to wild type conformation and triggers apoptosis (Lambert et al. Cancer Cell 15, 2009). APR-246 has been tested in a Phase I/IIa clinical trial with promising results (Lehmann et al. J Clin Oncol 30, 2012), and a Phase Ib/II study with platinum-based combination therapy in recurrent p53 mutant ovarian cancer is underway. Methods: Cell viability was assessed with WST-1, MTT or FMCA assay. p53 gene status was determined by Sanger sequencing and single strand conformation analysis, and p53 protein expression by Western blotting. Intracellular GSH levels were assessed with GSH kit (Cayman). Results: We have previously shown outstanding synergistic anticancer effects with APR-246 in combination with platinum compounds in p53 mutant solid cancer cell lines, including cisplatin resistant ovarian cancer cells. Synergistic effects were also observed ex vivo as well as in vivo in mice carrying human tumor xenografts. Here we show that APR-246 not only reactivates mutant p53 but also decreases intracellular GSH levels in a dose-dependent manner, presumably via adduct formation between MQ and GSH. APR-246 resensitized cisplatin resistant p53 mutant ovarian A2780-CP20 carcinoma cells to cisplatin, as shown by a decrease in the IC50 value from 52 to 2.9 µM, similar to the IC50 in parental A2780 cells. APR-246 also restored the sensitivity of resistant A2780ADR cells to doxorubicin. A2780-CP20 and A2780ADR were developed from the parental A2780 line with wild type p53 by chronic exposure to the respective drug. Moreover, APR-246 resensitized the p53 mutant OVCAR-3 cell line, established from a drug resistant patient, to cisplatin. Conclusions: Our results show that APR-246 not only reactivates mutant p53 but also decreases intracellular glutathione levels. We propose that this unique dual mechanism of action accounts for the resensitization and strong synergistic effects with APR-246 and platinum drugs. Our results provide strong rationale for the planned clinical study in ovarian cancer and suggest that combination treatment with APR-246 and DNA damaging drugs could have broad applicability in the treatment of drug resistant p53 mutant human tumors. Citation Format: Nina Mohell, Jessica Alfredsson, Åsa Fransson, Vladimir Bykov, Mikael von Euler, Klas Wiman, Ulf Björklund. APR-246, a clinical-stage mutant p53-reactivating compound, resensitizes ovarian cancer cells to platinum compounds and doxorubicin. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1801. doi:10.1158/1538-7445.AM2014-1801

Synthesis and In Vitro Pharmacological Behavior of Platinum(II) Complexes Containing 1,2-Diamino-1-(4-fluorophenyl)-2-alkanol Ligands

In continuation of our effort to optimize the pharmacological profile of [1,2-diamino-1,2-bis(4-fluorophenyl)ethane]dichloridoplatinum(II) complexes, we synthesized [1,2-diamino-1-(4-fluorophenyl)alkanol]dichloridoplatinum(II) analogs. The aim of this study was to evaluate the influence of hydroxyl groups at the C2 moiety on aqueous solubility, lipophilicity, cellular platinum accumulation, and cytotoxicity against MDA-MB-231, U-937, RAJI, and SC-1 cells as well as against cisplatin-sensitive and cisplatin-resistant A2780 and A2780cisR ovarian carcinoma cells. As expected, the OH groups improved the water solubility and decreased the lipophilicity of the neutral ligands, resulting in complexes with favorable pharmacokinetic properties. The cellular uptake of the compounds in MDA-MB-231 and U-937 cells proved to depend on the configuration and showed only a slight correlation with lipophilicity. The most active complexes were R,R/S,S configured, which points to a carrier-mediated mode of action. [threo-1,2-Diamino-1-(4-fluorophenyl)propan]dichloridoplatinum(II) and [threo-2,3-diamino-3-(4-fluorophenyl)propan-1-ol]dichloridoplatinum(II) possessed only low cross-resistance to cisplatin and were up to 10-fold more active in lymphoma cell lines.