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Characterization of the Newly Established Homoharringtonine- (HHT-) Resistant Cell Lines and Mechanisms of Resistance

ABSTRACT: Homoharringtonine- (HHT-) based HHT, aclarubicin, and cytarabine (HAA) induction regimen is the first-line therapy for nonelder acute myeloid leukemia (AML) patients in China. However, drug resistance is a new challenge, and little attention has been devoted to excavating resistant mechanisms. This study used the classic method to construct six HHT-resistant cell lines with a gradually increasing resistance index (RI) to discover HHT drug resistance mechanisms dynamically. After HHT resistance, the cell growth rate decreased, cell cycle delayed, and P-glycoprotein (p-gp, CD243) expression levels increased. Furthermore, we explored the changes in transcriptomics between HHT-sensitive and HHT-resistant cells using RNA-sequence. Through Kyoto Encyclopedia of Genes and Genomes (KEGG), Gene Ontology (GO), and hub gene analyses, we found that immune activity, especially G-protein coupled receptor (GPR) and related molecules, may mediate HHT resistance. Moreover, Calcitonin Receptor-Like (CALCRL) and G Protein Subunit Alpha I1 (GNAI1), which belong to GPRs, were stimulated in HHT-resistant cell strains in vitro and vivo, indicating that they may play a critical role in HHT resistance. In addition, these two genes have prognostic significance for AML patients. Taken together, we successfully constructed HHT-resistant cell lines with dynamic RIs and explored the resistance mechanisms, which will help identify new drugs for HHT-resistant AML patients.

SUBMITTER: Li F

PROVIDER: S-EPMC9448541 | biostudies-literature | 2022 Jan

REPOSITORIES: biostudies-literature

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Project description:BackgroundPralatrexate (PDX) is a novel antifolate approved for the treatment of patients with relapsed/refractory peripheral T-cell lymphoma, but some patients exhibit intrinsic resistance or develop acquired resistance. Here, we evaluated the mechanisms underlying acquired resistance to PDX and explored potential therapeutic strategies to overcome PDX resistance.MethodsTo investigate PDX resistance, we established two PDX-resistant T-lymphoblastic leukemia cell lines (CEM and MOLT4) through continuous exposure to increasing doses of PDX. The resistance mechanisms were evaluated by measuring PDX uptake, apoptosis induction and folate metabolism-related protein expression. We also applied gene expression analysis and methylation profiling to identify the mechanisms of resistance. We then explored rational drug combinations using a spheroid (3D)-culture assay.ResultsCompared with their parental cells, PDX-resistant cells exhibited a 30-fold increase in half-maximal inhibitory concentration values. Induction of apoptosis by PDX was significantly decreased in both PDX-resistant cell lines. Intracellular uptake of [14C]-PDX decreased in PDX-resistant CEM cells but not in PDX-resistant MOLT4 cells. There was no significant change in expression of dihydrofolate reductase (DHFR) or folylpolyglutamate synthetase (FPGS). Gene expression array analysis revealed that DNA-methyltransferase 3β (DNMT3B) expression was significantly elevated in both cell lines. Gene set enrichment analysis revealed that adipogenesis and mTORC1 signaling pathways were commonly upregulated in both resistant cell lines. Moreover, CpG island hypermethylation was observed in both PDX resistant cells lines. In the 3D-culture assay, decitabine (DAC) plus PDX showed synergistic effects in PDX-resistant cell lines compared with parental lines.ConclusionsThe resistance mechanisms of PDX were associated with reduced cellular uptake of PDX and/or overexpression of DNMT3B. Epigenetic alterations were also considered to play a role in the resistance mechanism. The combination of DAC and PDX exhibited synergistic activity, and thus, this approach might improve the clinical efficacy of PDX.

Project description:Drug resistance is an urgent problem to be solved in the treatment of non‑small‑cell lung cancer (NSCLC). Osimertinib is a third‑generation EGFR‑tyrosine kinase inhibitor, which can improve the efficacy and quality of life of patients; however, the inevitable resistance after long‑term use of osimertinib often leads to treatment failure. Cell lines are key tools for basic and preclinical studies. At present, few osimertinib‑resistant cell lines (HCC827‑OR and H1975‑OR) have been established. In the present study, osimertinib‑resistant cell lines were established by gradually increasing the drug concentration. Half‑maximal inhibitory concentration (IC50), cell morphology, whole exon sequencing, Cell Counting Kit‑8 assay, EdU staining and flow cytometry were used to evaluate the osimertinib‑resistant cell lines. Western blot analysis was used to detect the expression levels of key proteins involved in osimertinib resistance. The circular RNA (circRNA) expression profile was identified by RNA sequencing (RNA‑seq) analysis of HCC827, HCC827‑OR, H1975 and H1975‑OR cells. Subsequently, the biological roles of differentially expressed circRNAs were explored in in vitro studies. Osimertinib‑resistant cell lines were successfully established via treatment with an increasing concentration of osimertinib. Osimertinib IC50 and proliferation of resistant cells were much higher than those of sensitive cells. Notably, phosphorylated (p)‑AKT and p‑ERK were markedly activated in resistant cells, and the inhibitory effect of osimertinib on p‑AKT and p‑ERK was weaker in resistant cells than that in parental cells. RNA‑seq analysis identified differentially expressed circRNAs in HCC827, HCC827‑OR, H1975 and H1975‑OR cells. The most dysregulated circRNAs (circPDLIM5 and circPPP4R1) were selected for further functional study. Kyoto Encyclopedia of Genes and Genomes pathway analysis showed that the host genes of differentially expressed circRNAs were associated with 'endocytosis' and 'regulation of autophagy'. In conclusion, the present study established osimertinib‑resistant cell lines and revealed that circRNAs may serve as a promising biomarker in NSCLC osimertinib resistance.

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Characterization of the Newly Established Homoharringtonine- (HHT-) Resistant Cell Lines and Mechanisms of Resistance

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