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Determination of a minimal postmortem interval via age estimation of necrophagous diptera has been restricted to the juvenile stages and the time until emergence of the adult fly, i.e. up until 2–6 weeks depending on species and temperature. Age estimation of adult flies could extend this period by adding the age of the fly to the time needed for complete development. In this context pteridines are promising metabolites, as they accumulate in the eyes of flies with increasing age. We studied adults of the blow fly Lucilia sericata at constant temperatures of 16 °C and 25 °C up to an age of 25 days and estimated their pteridine levels by fluorescence spectroscopy. Age was given in accumulated degree days (ADD) across temperatures. Additionally, a mock case was set up to test the applicability of the method. Pteridine increases logarithmically with increasing ADD, but after 70–80 ADD the increase slows down and the curve approaches a maximum. Sex had a significant impact (p < 4.09 × 10−6) on pteridine fluorescence level, while body-size and head-width did not. The mock case demonstrated that a slight overestimation of the real age (in ADD) only occurred in two out of 30 samples. Age determination of L. sericata on the basis of pteridine levels seems to be limited to an age of about 70 ADD, but depending on the ambient temperature this could cover an extra amount of time of about 5–7 days after completion of the metamorphosis.
The survivin suppressant YM155 is a drug candidate for neuroblastoma. Here, we tested YM155 in 101 neuroblastoma cell lines (19 parental cell lines, 82 drug-adapted sublines). 77 cell lines displayed YM155 IC50s in the range of clinical YM155 concentrations. ABCB1 was an important determinant of YM155 resistance. The activity of the ABCB1 inhibitor zosuquidar ranged from being similar to that of the structurally different ABCB1 inhibitor verapamil to being 65-fold higher. ABCB1 sequence variations may be responsible for this, suggesting that the design of variant-specific ABCB1 inhibitors may be possible. Further, we showed that ABCC1 confers YM155 resistance. Previously, p53 depletion had resulted in decreased YM155 sensitivity. However, TP53-mutant cells were not generally less sensitive to YM155 than TP53 wild-type cells in this study. Finally, YM155 cross-resistance profiles differed between cells adapted to drugs as similar as cisplatin and carboplatin. In conclusion, the large cell line panel was necessary to reveal an unanticipated complexity of the YM155 response in neuroblastoma cell lines with acquired drug resistance. Novel findings include that ABCC1 mediates YM155 resistance and that YM155 cross-resistance profiles differ between cell lines adapted to drugs as similar as cisplatin and carboplatin.
Background: MDM2 inhibitors are under investigation for the treatment of acute myeloid leukaemia (AML) patients in phase III clinical trials. To study resistance formation to MDM2 inhibitors in AML cells, we here established 45 sub-lines of the AML TP53 wild-type cell lines MV4-11 (15 sub-lines), OCI-AML-2 (10 sub-lines), OCI-AML-3 (12 sub-lines), and SIG-M5 (8 sub-lines) with resistance to the MDM2 inhibitor nutlin-3.
Methods: Nutlin-3-resistant sub-lines were established by continuous exposure to stepwise increasing drug concentrations. The TP53 status was determined by next generation sequencing, cell viability was measured by MTT assay, and p53 was depleted using lentiviral vectors encoding shRNA.
Results: All MV4-11 sub-lines harboured the same R248W mutation and all OCI-AML-2 sub-lines the same Y220C mutation, indicating the selection of pre-existing TP53-mutant subpopulations. In concordance, rare alleles harbouring the respective mutations could be detected in the parental MV4-11 and OCI-AML-2 cell lines. The OCI-AML-3 and SIG-M5 sub-lines were characterised by varying TP53 mutations or wild type TP53, indicating the induction of de novo TP53 mutations. Doxorubicin, etoposide, gemcitabine, cytarabine, and fludarabine resistance profiles revealed a noticeable heterogeneity among the sub-lines even of the same parental cell lines. Loss-of-p53 function was not generally associated with decreased sensitivity to cytotoxic drugs.
Conclusion: We introduce a substantial set of models of acquired MDM2 inhibitor resistance in AML. MDM2 inhibitors select, in dependence on the nature of a given AML cell population, pre-existing TP53-mutant subpopulations or induce de novo TP53 mutations. Although loss-of-p53 function has been associated with chemoresistance in AML, nutlin-3-adapted sub-lines displayed in the majority of experiments similar or increased drug sensitivity compared to the respective parental cells. Hence, chemotherapy may remain an option for AML patients after MDM2 inhibitor therapy failure. Even sub-lines of the same parental cancer cell line displayed considerable heterogeneity in their response to other anti-cancer drugs, indicating the need for the detailed understanding and monitoring of the evolutionary processes in cancer cell populations in response to therapy as part of future individualised treatment protocols.
The human immunodeficiency virus (HIV) protease inhibitor saquinavir shows anticancer activity. Although its nitric oxide-modified derivative saquinavir-NO (saq-NO) was less toxic to normal cells, it exerted stronger inhibition of B16 melanoma growth in syngeneic C57BL/6 mice than saquinavir did. Saq-NO has been shown to block proliferation, upregulate p53 expression, and promote differentiation of C6 glioma and B16 cells. The anticancer activity of substances is frequently hampered by cancer cell chemoresistance mechanisms. Therefore, we here investigated the roles of p53 and the ATP-binding cassette (ABC) transporters P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and breast cancer resistance protein 1 (BCRP1) in cancer cell sensitivity to saq-NO to get more information about the potential of saq-NO as anticancer drug. Saq-NO exerted anticancer effects in lower concentrations than saquinavir in a panel of human cancer cell lines. Neither p53 mutation or depletion nor expression of P-gp, MRP1, or BCRP1 affected anticancer activity of saq-NO or saquinavir. Moreover, saq-NO sensitized P-gp-, MRP1-, or BCRP1-expressing cancer cells to chemotherapy. Saq-NO induced enhanced sensitization of P-gp- or MRP1-expressing cancer cells to chemotherapy compared with saquinavir, whereas both substances similarly sensitized BCRP1-expressing cells. Washout kinetics and ABC transporter ATPase activities demonstrated that saq-NO is a substrate of P-gp as well as of MRP1. These data support the further investigation of saq-NO as an anticancer drug, especially in multidrug-resistant tumors.
Combination chemotherapy with gemcitabine and cisplatin in patients with metastatic urothelial cancer of the bladder frequently results in the development of acquired drug resistance. Availability of cell culture models with acquired resistance could help to identify candidate treatments for an efficient second-line therapy. Six cisplatin- and six gemcitabine-resistant cell lines were established. Cell viability assays were performed to evaluate the sensitivity to 16 different chemotherapeutic substances. The activity of the drug transporter ATP-binding cassette transporter, subfamily B, member 1 (ABCB1, a critical mediator of multidrug resistance in cancer) was evaluated using fluorescent ABCB1 substrates. For functional assessment, cells overexpressing ABCB1 were generated by transduction with a lentiviral vector encoding for ABCB1, while zosuquidar was used for selective inhibition. In this study, 8 of 12 gemcitabine- or cisplatin-resistant cell lines were cross-resistant to carboplatin, 5 to pemetrexed, 4 to methotrexate, 3 to oxaliplatin, 5-fluorouracil, and paclitaxel, and 2 to cabazitaxel, larotaxel, docetaxel, topotecan, doxorubicin, and mitomycin c, and 1 of 12 cell lines was cross-resistant to vinflunine and vinblastine. In one cell line with acquired resistance to gemcitabine (TCC-SUPrGEMCI20), cross-resistance seemed to be mediated by ABCB1 expression. Our model identified the vinca alkaloids vinblastine and vinflunine, in Europe an already approved second-line therapeutic for metastatic bladder cancer, as the most effective compounds in urothelial cancer cells with acquired resistance to gemcitabine or cisplatin. These results demonstrate that this in vitro model can reproduce clinically relevant results and may be suitable to identify novel substances for the treatment of metastatic bladder cancer.
The nucleoside analogue nelarabine, the prodrug of arabinosylguanine (AraG), has been known for decades to be effective against acute lymphoblastic leukaemias of T-cell (T-ALL), but not of B-cell (B-ALL) origin. The mechanisms underlying this lineage-specific drug sensitivity have remained elusive. Data from pharmacogenomics studies and from a panel of ALL cell lines revealed an inverse correlation of SAMHD1 expression and nelarabine sensitivity. SAMHD1 can hydrolyse and thus inactivate triphosphorylated nucleoside analogues. Transcriptomic and protein expression profiling of cell lines and patient-derived leukaemic blasts revealed lower SAMHD1 abundance in T-ALL than in B-ALL. Mechanistically, SAMHD1 promoter methylation strongly correlated with suppressed SAMHD1 expression, while T-ALL cells did not display increased global DNA methylation. Targeted SAMHD1 degradation using virus-like particles containing Vpx sensitised B-ALL cells to AraG, while ectopic SAMHD1 expression in SAMHD1-null T-ALL cells induced AraG resistance. SAMHD1 had a larger impact on cytarabine activity than on nelarabine/ AraG activity in acute myeloid leukaemia (AML) cells, but more strongly affected nelarabine/ AraG activity in ALL cells. This indicates a critical role of the cancer entity. In conclusion, lineage-specific differences in SAMHD1 promoter methylation and, in turn, SAMHD1 expression levels determine ALL cell response to nelarabine. SAMHD1 is a potential biomarker for the identification of ALL patients likely to benefit from nelarabine therapy and a therapeutic target to overcome nelarabine resistance.
Survivin is a drug target and the survivin suppressant YM155 a drug candidate for high-risk neuroblastoma. Findings from one YM155-adapted subline of the neuroblastoma cell line UKF-NB-3 had suggested that increased ABCB1 (mediates YM155 efflux) levels, decreased SLC35F2 (mediates YM155 uptake) levels, decreased survivin levels, and TP53 mutations indicate YM155 resistance. Here, the investigation of ten additional YM155-adapted UKF-NB-3 sublines only confirmed the roles of ABCB1 and SLC35F2. However, cellular ABCB1 and SLC35F2 levels did not indicate YM155 sensitivity in YM155-naïve cells, as indicated by drug response data derived from the Cancer Therapeutics Response Portal (CTRP) and the Genomics of Drug Sensitivity in Cancer (GDSC) databases. Moreover, the resistant sublines were characterised by a remarkable heterogeneity. Only seven sublines developed on-target resistance as indicated by resistance to RNAi-mediated survivin depletion. The sublines also varied in their response to other anti-cancer drugs. In conclusion, cancer cell populations of limited intrinsic heterogeneity can develop various resistance phenotypes in response to treatment. Therefore, individualised therapies will require monitoring of cancer cell evolution in response to treatment. Moreover, biomarkers can indicate resistance formation in the acquired resistance setting, even when they are not predictive in the intrinsic resistance setting.
Novel treatment options are needed for the successful therapy of patients with high-risk neuroblastoma. Here, we investigated the cyclin-dependent kinase (CDK) inhibitor SNS-032 in a panel of 109 neuroblastoma cell lines consisting of 19 parental cell lines and 90 sublines with acquired resistance to 14 different anticancer drugs. Seventy-three percent of the investigated neuroblastoma cell lines and all four investigated primary tumor samples displayed concentrations that reduce cell viability by 50% in the range of the therapeutic plasma levels reported for SNS-032 (<754 nM). Sixty-two percent of the cell lines and two of the primary samples displayed concentrations that reduce cell viability by 90% in this concentration range. SNS-032 also impaired the growth of the multidrug-resistant cisplatin-adapted UKF-NB-3 subline UKF-NB-3rCDDP1000 in mice. ABCB1 expression (but not ABCG2 expression) conferred resistance to SNS-032. The antineuroblastoma effects of SNS-032 did not depend on functional p53. The antineuroblastoma mechanism of SNS-032 included CDK7 and CDK9 inhibition-mediated suppression of RNA synthesis and subsequent depletion of antiapoptotic proteins with a fast turnover rate including X-linked inhibitor of apoptosis (XIAP), myeloid cell leukemia sequence 1 (Mcl-1), baculoviral IAP repeat containing 2 (BIRC2; cIAP-1), and survivin. In conclusion, CDK7 and CDK9 represent promising drug targets and SNS-032 represents a potential treatment option for neuroblastoma including therapy-refractory cases.
Whereas the lack of biomarkers in penile cancer (PeCa) impedes the development of efficacious treatment protocols, preliminary evidence suggests that c-MET and associated signaling elements may be dysregulated in this disorder. In the following study, we investigated whether c-MET and associated key molecular elements may have prognostic and therapeutic utility in PeCa. Formalin-fixed, paraffin-embedded tumor tissue from therapy-naïve patients with invasive PeCa was used for tissue microarray (TMA) analysis. Immunohistochemical staining was performed to determine the expression of the proteins c-MET, PPARg, β-catenin, snail, survivin, and n-MYC. In total, 94 PeCa patients with available tumor tissue were included. The median age was 64.9 years. High-grade tumors were present in 23.4%, and high-risk HPV was detected in 25.5%. The median follow-up was 32.5 months. High expression of snail was associated with HPV-positive tumors. Expression of β-catenin was inversely associated with grading. In both univariate COX regression analysis and the log-rank test, an increased expression of PPARg and c-MET was predictive of inferior disease-specific survival (DSS). Moreover, in multivariate analysis, a higher expression of c-MET was independently associated with worse DSS. Blocking c-MET with cabozantinib and tivantinib induced a significant decrease in viability in the primary PeCa cell line UKF-PeC3 isolated from the tumor tissue as well as in cisplatin- and osimertinib-resistant sublines. Strikingly, a higher sensitivity to tivantinib could be detected in the latter, pointing to the promising option of utilizing this agent in the second-line treatment setting.