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The formation of acquired drug resistance is a major reason for the failure of anti-cancer therapies after initial response. Here, we introduce a novel model of acquired oxaliplatin resistance, a sub-line of the non-MYCN-amplified neuroblastoma cell line SK-N-AS that was adapted to growth in the presence of 4000 ng/mL oxaliplatin (SK-N-ASrOXALI4000). SK-N-ASrOXALI4000 cells displayed enhanced chromosomal aberrations compared to SK-N-AS, as indicated by 24-chromosome fluorescence in situ hybridisation. Moreover, SK-N-ASrOXALI4000 cells were resistant not only to oxaliplatin but also to the two other commonly used anti-cancer platinum agents cisplatin and carboplatin. SK-N-ASrOXALI4000 cells exhibited a stable resistance phenotype that was not affected by culturing the cells for 10 weeks in the absence of oxaliplatin. Interestingly, SK-N-ASrOXALI4000 cells showed no cross resistance to gemcitabine and increased sensitivity to doxorubicin and UVC radiation, alternative treatments that like platinum drugs target DNA integrity. Notably, UVC-induced DNA damage is thought to be predominantly repaired by nucleotide excision repair and nucleotide excision repair has been described as the main oxaliplatin-induced DNA damage repair system. SK-N-ASrOXALI4000 cells were also more sensitive to lysis by influenza A virus, a candidate for oncolytic therapy, than SK-N-AS cells. In conclusion, we introduce a novel oxaliplatin resistance model. The oxaliplatin resistance mechanisms in SK-N-ASrOXALI4000 cells appear to be complex and not to directly depend on enhanced DNA repair capacity. Models of oxaliplatin resistance are of particular relevance since research on platinum drugs has so far predominantly focused on cisplatin and carboplatin.
Background: Various kinase inhibitors are known to be ATP-binding cassette (ABC) transporter substrates and resistance acquisition to kinase inhibitors has been associated to increased ABC transporter expression. Here, we investigated the role of the ABC transporters ABCB1, ABCC1, and ABCG2 during melanoma cell resistance acquisition to the V600-mutant BRAF inhibitors PLX4032 (vemurafenib) and PLX4720. PLX4032 had previously been shown to interfere with ABCB1 and ABCG2. PLX4720 had been demonstrated to interact with ABCB1 but to a lower extent than PLX4032.
Findings: PLX4032 and PLX4720 affected ABCC1- and ABCG2-mediated drug transport in a similar fashion. In a panel of 16 V600E BRAF-mutated melanoma cell lines consisting of four parental cell lines and their sub-lines with acquired resistance to PLX4032, PLX4720, vincristine (cytotoxic ABCB1 and ABCC1 substrate), or mitoxantrone (cytotoxic ABCG2 substrate), we detected enhanced ABC transporter expression in 4/4 cytotoxic ABC transporter substrate-resistant, 3/4 PLX4720-resistant, and 1/4 PLX4032-resistant melanoma cell lines.
Conclusion: PLX4032 has the potential to induce ABC transporter expression but this potential is lower than that of PLX4720 or cytotoxic ABC transporter substrates. Since ABC transporters confer multi-drug resistance, this is of relevance for the design of next-line therapies.
Aurora kinase inhibitors displayed activity in pre-clinical neuroblastoma models. Here, we studied the effects of the pan-aurora kinase inhibitor tozasertib (VX680, MK-0457) and the aurora kinase inhibitor alisertib (MLN8237) that shows some specificity for aurora kinase A over aurora kinase B in a panel of neuroblastoma cell lines with acquired drug resistance. Both compounds displayed anti-neuroblastoma activity in the nanomolar range. The anti-neuroblastoma mechanism included inhibition of aurora kinase signalling as indicated by decreased phosphorylation of the aurora kinase substrate histone H3, cell cycle inhibition in G2/M phase, and induction of apoptosis. The activity of alisertib but not of tozasertib was affected by ABCB1 expression. Aurora kinase inhibitors induced a p53 response and their activity was enhanced in combination with the MDM2 inhibitor and p53 activator nutlin-3 in p53 wild-type cells. In conclusion, aurora kinases are potential drug targets in therapy-refractory neuroblastoma, in particular for the vast majority of p53 wild-type cases.
Purpose: To assess the levels of inflammatory and angiogenic cytokines in undiluted vitreous from treatment-naïve patients with macular edema secondary to nonischemic branch retinal vein occlusion (BRVO), with flow cytometric bead array (CBA) and to correlate the results with subjective and multiple spectral-domain optical coherence tomography (SD-OCT) parameters.
Methods: A total of 43 eyes from 43 patients (mean age 69.7 years, 23 male) were divided into groups of new, "fresh" (n = 28; mean duration after onset 4.1 months) and older BRVO (n = 15; 11.6 months). Because of macular edema, these patients underwent an intravitreal therapy combining a single-site 23 g core vitrectomy with bevacizumab and dexamethasone. Undiluted vitreous was then analyzed for interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor isoform A (VEGF-A) levels with CBA and correlated with visual acuity (VA), clinical parameters of BRVO (type and perfusion status), and morphologic parameters, such as central macular thickness, central retinal thickness, thickness of the neurosensory retina, thickness of the serous retinal detachment, and the disruption of the ellipsoid line (photoreceptor inner and outer segments) and the external limiting membrane, as measured with SD-OCT. Twenty-eight undiluted vitreous samples from patients with idiopathic, nonuveitis vitreous floaters served as the controls.
Results: The mean IL-6 was 23.2 pg/mL (standard deviation, ±48.8), MCP-1 was 602.6 (±490.3), and VEGF-A was 161.8 (±314.3), and this was higher than in the control group, which had a mean IL-6 of 6.2 ± 3.4 pg/mL (P = 0.17), MCP-1 of 253.2 ± 73.5 (P < 0.0000001), and VEGF-A of 7.0 ± 4.9 (P < 0.003). In all BRVO samples, IL-6 correlated positively with MCP-1 and VEGF-A (correlation coefficient r = 0.79 and r = 0.46, respectively). VEGF-A was the only cytokine to correlate significantly with SD-OCT parameters (thickness of the neurosensory retina r = 0.31; disruption of the ellipsoid line r = 0.33). In the older BRVO group, there was a positive correlation between cytokines (IL-6 with MCP-1, r = 0.77; Il-6 with VEGF-A, r = 0.68; MCP-1 and VEGF-A, r = 0.68), whereas only IL-6 correlated with MCP-1 in the fresh group (r = 0.8).
Conclusion: The inflammatory markers and VEGF-A were elevated in the vitreous fluid of patients with BRVO, and these correlated with one another. VEGF-A was more often correlated with the morphologic changes assessed by SD-OCT, whereas the inflammatory markers had no significant influence on SD-OCT changes.
Purpose: To correlate inflammatory and proangiogenic key cytokines from undiluted vitreous of treatment-naïve central retinal vein occlusion (CRVO) patients with SD-OCT parameters.
Methods: Thirty-five patients (age 71.1 years, 24 phakic, 30 nonischemic) underwent intravitreal combination therapy, including a single-site 23-gauge core vitrectomy. Twenty-eight samples from patients with idiopathic, non-uveitis floaterectomy served as controls. Interleukin 6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), and vascular endothelial growth factor (VEGF-A) levels were correlated with the visual acuity (logMar), category of CRVO (ischemic or nonischemic) and morphologic parameters, such as central macular thickness-CMT, thickness of neurosensory retina-TNeuro, extent of serous retinal detachment-SRT and disintegrity of the IS/OS and others.
Results: The mean IL-6 was 64.7pg/ml (SD ± 115.8), MCP-1 1015.7 ( ± 970.1), and VEGF-A 278.4 ( ± 512.8), which was significantly higher than the control IL-6 6.2 ± 3.4pg/ml (P=0.06), MCP-1 253.2 ± 73.5 (P<0.0000001) and VEGF-A 7.0 ± 4.9 (P<0.0006). All cytokines correlated highly with one another (correlation coefficient r=0.82 for IL-6 and MCP-1; r=0.68 for Il-6 and VEGF-A; r=0.64 for MCP-1 and VEGF-A). IL-6 correlated significantly with CMT, TRT, SRT, dIS/OS, and dELM. MCP-1 correlated significantly with SRT, dIS/OS, and dELM. VEGF-A correlated not with changes in SD-OCT, while it had a trend to be higher in the ischemic versus the nonischemic CRVO group (P=0.09).
Conclusions: The inflammatory cytokines were more often correlated with morphologic changes assessed by SD-OCT, whereas VEGF-A did not correlate with CRVO-associated changes in SD-OCT. VEGF inhibition alone may not be sufficient in decreasing the inflammatory response in CRVO therapy.
Six p53 wild-type cancer cell lines from infrequently p53-mutated entities (neuroblastoma, rhabdomyosarcoma, and melanoma) were continuously exposed to increasing concentrations of the murine double minute 2 inhibitor nutlin-3, resulting in the emergence of nutlin-3-resistant, p53-mutated sublines displaying a multi-drug resistance phenotype. Only 2 out of 28 sublines adapted to various cytotoxic drugs harboured p53 mutations. Nutlin-3-adapted UKF-NB-3 cells (UKF-NB-3rNutlin10 μM, harbouring a G245C mutation) were also radiation resistant. Analysis of UKF-NB-3 and UKF-NB-3rNutlin10 μM cells by RNA interference experiments and lentiviral transduction of wild-type p53 into p53-mutated UKF-NB-3rNutlin10 μM cells revealed that the loss of p53 function contributes to the multi-drug resistance of UKF-NB-3rNutlin10 μM cells. Bioinformatics PANTHER pathway analysis based on microarray measurements of mRNA abundance indicated a substantial overlap in the signalling pathways differentially regulated between UKF-NB-3rNutlin10 μM and UKF-NB-3 and between UKF-NB-3 and its cisplatin-, doxorubicin-, or vincristine-resistant sublines. Repeated nutlin-3 adaptation of neuroblastoma cells resulted in sublines harbouring various p53 mutations with high frequency. A p53 wild-type single cell-derived UKF-NB-3 clone was adapted to nutlin-3 in independent experiments. Eight out of ten resulting sublines were p53-mutated harbouring six different p53 mutations. This indicates that nutlin-3 induces de novo p53 mutations not initially present in the original cell population. Therefore, nutlin-3-treated cancer patients should be carefully monitored for the emergence of p53-mutated, multi-drug-resistant cells.
Adaptation of wild-type p53 expressing UKF-NB-3 cancer cells to the murine double minute 2 inhibitor nutlin-3 causes de novo p53 mutations at high frequency (13/20) and multi-drug resistance. Here, we show that the same cells respond very differently when adapted to RITA, a drug that, like nutlin-3, also disrupts the p53/Mdm2 interaction. All of the 11 UKF-NB-3 sub-lines adapted to RITA that we established retained functional wild-type p53 although RITA induced a substantial p53 response. Moreover, all RITA-adapted cell lines remained sensitive to nutlin-3, whereas only five out of 10 nutlin-3-adapted cell lines retained their sensitivity to RITA. In addition, repeated adaptation of the RITA-adapted sub-line UKF-NB-3rRITA10 μM to nutlin-3 resulted in p53 mutations. The RITA-adapted UKF-NB-3 sub-lines displayed no or less pronounced resistance to vincristine, cisplatin, and irradiation than nutlin-3-adapted UKF-NB-3 sub-lines. Furthermore, adaptation to RITA was associated with fewer changes at the expression level of antiapoptotic factors than observed with adaptation to nutlin-3. Transcriptomic analyses indicated the RITA-adapted sub-lines to be more similar at the gene expression level to the parental UKF-NB-3 cells than nutlin-3-adapted UKF-NB-3 sub-lines, which correlates with the observed chemotherapy and irradiation sensitivity phenotypes. In conclusion, RITA-adapted cells retain functional p53, remain sensitive to nutlin-3, and display a less pronounced resistance phenotype than nutlin-3-adapted cells.
The human cytomegalovirus (HCMV) is suspected to increase tumour malignancy by infection of cancer and/or stroma cells (oncomodulation). So far, oncomodulatory mechanisms have been attributed to the presence of HCMV and direct action of its gene products on cancer cells. Here, we investigated whether the prolonged presence of HCMV can result in the irreversible selection of a cancer cell population with increased malignancy. The neuroblastoma cell line UKF-NB-4 was long-term (200 passages) infected with the HCMV strain Hi91 (UKF-NB-4Hi) before virus eradication using ganciclovir (UKF-NB-4HiGCV). Global gene expression profiling of UKF-NB-4, UKF-NB-4Hi and UKF-NB-4HiGCV cells and subsequent bioinformatic signal transduction pathway analysis revealed clear differences between UKF-NB-4 and UKF-NB-4Hi, as well as between UKF-NB-4 and UKF-NB-4HiGCV cells, but only minor differences between UKF-NB-4Hi and UKF-NB-4HiGCV cells. Investigation of the expression of a subset of five genes in different chronically HCMV-infected cell lines before and after virus eradication suggested that long-term HCMV infection reproducibly causes specific changes. Array comparative genomic hybridisation showed virtually the same genomic differences for the comparisons UKF-NB-4Hi/UKF-NB-4 and UKF-NB-4HiGCV/UKF-NB-4. UKF-NB-4Hi cells are characterised by an increased invasive potential compared with UKF-NB-4 cells. This phenotype was completely retained in UKF-NB-4HiGCV cells. Moreover, there was a substantial overlap in the signal transduction pathways that differed significantly between UKF-NB-4Hi/UKF-NB-4HiGCV and UKF-NB-4 cells and those differentially regulated between tumour tissues from neuroblastoma patients with favourable or poor outcome. In conclusion, we present the first experimental evidence that long-term HCMV infection can result in the selection of tumour cell populations with enhanced malignancy.