Abstract Background Switch-associated protein 70 (SWAP-70) is a guanine nucleotide exchange factor that is involved in cytoskeletal rearrangement and regulation of migration and invasion of malignant tumors. However, the mechanism by which SWAP-70 regulates the migration and invasion of glioblastoma (GB) cells has not been fully elucidated. Methods This study used an online database to analyze the relationship between SWAP-70 expression and prognosis in GB patients. The in vitro wound healing assay and transwell invasion assay were used to determine the role of SWAP-70 in GB cell migration and invasion as well as the underlying mechanism. Results We found that patients with high SWAP-70 expression in the GB had a poor prognosis. Downregulation of SWAP-70 inhibited GB cell migration and invasion, whereas SWAP-70 overexpression had an opposite effect. Interestingly, SWAP-70 expression was positively correlated with the expression of the standard form of CD44 (CD44s) in GB tissues. Downregulation of SWAP-70 also reduced CD44s protein expression, whereas SWAP-70 overexpression enhanced CD44s protein expression. However, downregulation of SWAP-70 expression did not affect the mRNA expression of CD44s. Reversal experiments showed that overexpressing CD44s in cell lines with downregulated SWAP-70 partially abolished the inhibitory effects of downregulated SWAP-70 on GB cell migration and invasion. Conclusions These results suggest that SWAP-70 may promote GB cell migration and invasion by regulating the expression of CD44s. SWAP-70 may serve as a new biomarker and a potential therapeutic target for GB.
This study was aimed to investigate the effect of MEK inhibitor AZD8330 on proliferation and apoptosis of multiple myeloma IM9 and NCI-H929 cell lines and its possible mechanism. These two cell line cells were exposed to different concentrations of AZD8330 for 48 h. The CCK-8 assay was used to detect cell viability and the IC50 value at 48 h. These above-mentioned IM9 and NCI-H929 cells were treated with 5,10 and 100 nmol/L of AZD8330, then the change of cell cycle was analysed by flow cytometry with PI staining. The Wester blot was used to detect the expression levels of cyclin D and cyclin E, and multiple myeloma cells were treated with 10, 100, 1000 and 2000 nmol/L of AZD8330, the AnnexinV/7-AAD double staining was used to analyse cell apoptosis and the Western blot was used to detect the expression level of caspase-3. The results showed that AZD8330 could significantly inhibit the cell viability of IM9 and NCI-H929 cell lines in a time-and dose-dependent manner, the IC50 value (48 h) of IM9 and NCI-H929 were 19.88 ± 2.7 nmol/L and 29.3 ± 2.03 nmol/L respectively, these two cell lines were arrested on G1 phase of cell cycle, the apoptosis cells increased along with enhancement of AZD8330 concentration, and the expression level of cleaved caspase-3 protein was up-regulated. It is concluded that AZD8330 can efficiently inhibit the proliferation of NCI-H929 and IM9 cell lines, and induce apoptosis, suggesting that the AZD8330 may be a potential chemotherapeutic candidate for multiple myeloma therapy.
Background: miR-302 cluster has been reported as a tumor suppressor in many human cancers; yet, its function in chronic myeloid leukemia (CML) tumorigenesis remains largely unclear. The study was aimed to explore the functional roles of miR-302 cluster in CML progression. Materials and methods: Quantitative reverse transcriptase PCR and Western blot were performed to evaluate miR-302 cluster and vascular endothelial growth factor A (VEGFA) expression levels. Cell Counting Kit-8 assay, colony formation assay and human umbilical vein endothelial cell line capillary tube formation were used to determine the influence of miR-302 cluster on the growth and angiogenesis of K562 cells, respectively. Luciferase reporter assay was employed to confirm the direct target interaction between miR-302 cluster and VEGFA. Results: This study demonstrated that miR-302 cluster was frequently downregulated in CML samples and cell lines and high level of miR-302 cluster was significantly associated with good prognosis of CML patients. Compared with miRNA negative control, miR-302 cluster mimics obviously suppressed cell growth, colony formation and angiogenesis. Further studies revealed that VEGFA was a direct target gene of miR-302 cluster. Moreover, overexpression of VEGFA dramatically abated the inhibition of miR-302 cluster on cell growth and angiogenesis. Conclusion: The present study, for the first time, identified miR-302 cluster as a tumor suppressor, and overexpression of miR-302 cluster inhibited growth and angiogenesis in K562 cells. miR-302 cluster may be a potential therapeutic target in CML to develop the adjuvant antiangiogenic therapy based on VEGFA. Keywords: chronic myeloid leukemia, angiogenesis, miR-302, VEGFA
To investigate the effect of alantolactone on perliferation and apoptosis of multiple myeloma (MM) RPMI-8226 cells, and to explore its possible mechism in vitro and in vivo.The RPMI-8226 cells were treated with alantolactone (1, 2.5, 5, 7.5 and 10 µmol/L) for 48 h, cell viability was detected by CCK-8 assay and the value of IC50 was calculated; The RPMI-8226 cells were treated with alantolactone (2.5, 5 and 7.5 µmol/L) for 48 h, the apoptotic rate was detected by flow cytmetry with Annexin V/PI staining; the expression level of cleaved caspase-3 and phosphorylation of ERK were measured by Western blot; the nude mice was used to further confirm the proapoptotic effect of alantolactone on MM cells in vivo.The alantolactone inhibited RPMI-8226 cell viability remarkably with a dose-dependent manner; the IC50 value of RPMI-8226 cells at 48 h was 4.32 ± 0.15 µmol/L; the apoptotic rate increased observably with a dose-dependent manner; the levels of cleaved-caspase-3 increased and the phosphorylation of ERK decreased significantly; as compared to control, the volum of tumor was much smaller, the expression levels of Ki67 and p-ERK decreased.The alantolactone can efficiently inhibit the proliferation and induce the apoptosis of multiple myeloma RPMI-8226 cells in vitro and in vivo through inhibiting the activation of ERK signal pathway.
Clonal heterogeneity and co‐occurring mutations contribute to drug resistance in AML. Leukemia samples from patients progressing on gilteritinib, a FLT3 inhibitor, were selected for RAS mutant clones, which are known to contribute preclinically to resistance (McMahon CM, Cancer Discov 2019). Previously, we demonstrated that TP‐0930, a multi‐kinase inhibitor, targets FLT3‐ITD and resistance‐conferring tyrosine kinase domain mutations in preclinical models. Here, we present the activity of TP‐0903 in RAS pathway mutant models of AML. KiNativ assay was performed in OCI‐AML3 cells ( NRAS Q61L) treated with TP‐0903 (100 nM, 2h). Inhibition of cell viability was evaluated in AML cell lines (MTT) and primary samples (CellTiter Glo). Inhibition of AURKA/B, AKT, ERK, MCL‐1, and BCL‐2 were determined by western blot. Apoptosis, cell cycle, and differentiation assays were performed in OCI‐AML3 using annexin V, DAPI and CD11b‐APC staining. In vivo activity of TP‐0903 was evaluated in a systemic OCI‐AML3‐Luc+ xenograft model; 5e5 luciferase expressing cells were tail vein injected to NSG mice and TP‐0903 50 mg/kg or vehicle was given orally once dailyx5/week. Progression of leukemia was monitored via bioluminescence imaging and Kaplan Meier was used for survival analysis. In Kinativ assay, inhibition of native kinases were similar to those observed in a previous binding assay. We observed that TP‐0903 inhibited ACK1 (activated CDC42 kinase 1 or TNK2) and GCK (germinal center kinase or MAP4K2) by 87 and 46%, respectively. Inhibition of ACK1 (Kd = 7.3 nM, IC50 = 30.7 nM) and GCK (Kd = 1.8 nM, IC50 = 2.5 nM) was confirmed in binding and kinase assays. In RAS mutant AML cell lines (OCI‐AML3, THP1, HL60, and U937), TP‐0903 inhibited viability with IC50 values of 26 – 99 nM. In OCI‐AML3, TP‐0903 at 100 nM inhibited pAURKA/B, pAKT and MCL‐1; caused a G2‐M arrest and polyploidy (20 nM, 24h); and induced apoptosis (20–50 nM, 72h). TP‐0903, compared to gilteritinib, was more potent in inhibiting 4 ex vivo human primary AML samples with N/KRAS mutations co‐occurring with wt FLT3 or FLT3 ‐ITD, along with other mutations (IC50 range, 21 – 60 vs 260 – 1370 nM). In a 14‐day CFU assay, TP‐0903 inhibited colony formation of a FLT3 ‐ITD and NRAS mutant primary sample compared to DMSO (mean, 65 vs 188 CFUs). In an OCI‐AML3‐Luc+ xenograft study, TP‐0903 suppressed the outgrowth of leukemia at day 21 (P<0.0001) and prolonged median survival by 9 days compared to veh‐treated mice (P<0.0001). At study endpoint, spleen weight was higher in veh‐treated mice than in TP‐0903‐treated mice (mean ± SD, 333 ± 23 vs 181 ± 21 mg, P=0.0029). TP‐0903, a novel multi‐kinase inhibitor, shows promising in vitro and in vivo activity in RAS mutant AML. In a previous report (Nonami A, Blood 2015), an integrated approach involving cell‐based pharmacologic screening combined with KiNativ, gene expression profiling and mechanism studies, identified 2 kinase signaling pathways (ACK1/AKT and GCK) that synergistically contributed to the growth of NRAS mutant leukemia cells, including OCI‐AML3. Based on our data, TP‐0903 may exert its activity in RAS mutant AML via inhibiting ACK1 and GCK.
Chimeric antigen receptor T (CAR-T) cell therapy has shown promising results for relapsed/refractory (R/R) acute lymphoblastic leukemia (ALL). The immune response induced by murine single-chain variable fragment (scFv) of the CAR may limit CAR-T cell persistence and thus increases the risk of leukemia relapse. In this study, we developed a novel humanized scFv from the murine FMC63 antibody. A total of 18 R/R ALL patients with or without prior murine CD19 CAR-T therapy were treated with humanized CD19-targeted CAR-T cells (hCART19s). After lymphodepletion chemotherapy with cyclophosphamide and fludarabine, the patients received a single dose (1 × 106 /kg) of autologous hCART19s infusion. Among the 14 patients without previous CAR-T therapy, 13 (92.9%) achieved complete remission (CR) or CR with incomplete count recovery (CRi) on day 30, whereas 1 of the 3 patients who failed a second murine CAR-T infusion achieved CR after hCART19s infusion. At day 180, the overall and leukemia-free survival rates were 65.8% and 71.4%, respectively. The cumulative incidence of relapse was 22.6%, and the nonrelapse mortality rate was 7.1%. During treatment, 13 patients developed grade 1-2 cytokine release syndrome (CRS), 4 patients developed grade 3-5 CRS, and 1 patient experienced reversible neurotoxicity. These results indicated that hCART19s could induce remission in patients with R/R B-ALL, especially in patients who received a reinfusion of murine CAR-T.
Malignant glioma is the most common primary brain tumor in adults and has a poor prognosis. However, there are no effective targeted therapies for glioma patients. Thus, the development of novel targeted therapeutics for glioma is urgently needed. In this study, we examined the prognostic significance BTK expression in patients with glioma. Furthermore, we investigated the mechanism and therapeutic potential of ibrutinib in the treatment of human glioma in vitro and in vivo. Our data demonstrate that high expression of BTK is a novel prognostic marker for poor survival in patients with glioma. BTK-specific inhibitor ibrutinib effectively inhibits the proliferation, migration and invasion ability of glioma cells. Furthermore, ibrutinib can induce G1 cell-cycle arrest by regulating multiple cell cycle-associated proteins. More importantly, we found that BTK inhibition significantly blocks the degradation of IκBα and prevents the nuclear accumulation of NF-κB p65 subunit induced by EGF in glioma cells. Taken together, our study suggests that BTK is a novel prognostic marker and molecular therapeutic target for glioma. BTK is required for EGFR-induced NF-κB activation in glioma cells. These findings provide the basis for future clinical studies of ibrutinib for the treatment of glioma.
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