CCRF-CEM Cells Research Articles

Novel Orally Available Degrader to Target Lck in T-Cell Acute Lymphoblastic Leukemia

Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive blood cancer that develops from abnormal growth of immature T-cell progenitors and constitutes about 15% of all pediatric ALL cases. Despite an overall survival rate of 80% in pediatric T-ALL patients, 20% of patients eventually experience relapse or succumb to refractory disease. Therefore, novel therapies for refractory/relapsed T-ALL patients are urgently needed, and they are currently being developed in clinical and preclinical studies. Based on recent literature, the over-activation of preTCR-LCK signaling has been revealed as a highly effective therapeutic target in T-ALL, with LCK being identified as a therapeutic vulnerability in ~44% of T-ALL cases. The LCK inhibitor, dasatinib, exhibited an anti-leukemia effect in a subset of T-ALL cases but was transient in most cases. Therefore, the development of new agents capable of sustaining inhibition of LCK signaling is needed. Method To validate the effects of the LCK degrader UBX-363, both in vitro and in vivo tests were conducted using dasatinib-sensitive HSB-2 cells (which have higher LCK activity compared to other T-ALL cell lines) and dasatinib-insensitive CCRF-CEM cells. UBX-363 is designed to target LCK through ubiquitin-proteasome system-mediated degradation. Cell proliferation was assessed using detection methods such as the CellTiter-Glo assay, and the degradation activity of the LCK protein in the tested cell lines was evaluated by immunoblotting. To examine the in vivo antitumor effects of UBX-363 or dasatinib, we performed HSB-2 and CCRF-CEM xenograft murine models. CB17/SCID mice were subcutaneously inoculated with 1x10 7 HSB-2 and 5x10 6 CCRF-CEM cells. UBX-363 was orally administered at doses of 0.2, 1, or 5 mg/kg (for HSB-2) and 2.5, 5, or 10 mg/kg (for CCRF-CEM), while dasatinib was orally administered at doses of 5 mg/kg (for HBS-2) and 10 mg/kg (for CCRF-CEM) once daily for 12 days. Results UBX-363 showed superior degradation activity against LCK proteins in the subnanomolar or single-digit nanomolar range of half-maximal degradation concentration in HSB-2 and CCRF-CEM cell lines (0.478 nM and 1.502 nM, respectively). In comparison to the LCK inhibitors dasatinib (~0.307 nM) and saracatinib (~17.41 nM), UBX-363 (~0.121 nM) demonstrated slightly superior anti-proliferative efficacy in HSB-2 cells. In CCRF-CEM cells, UBX-363 exhibited prominent anti-proliferative activity at lower treatment concentrations compared to the other LCK inhibitors (UBX-363: 11.12 nM, dasatinib: 3483 nM, saracatinib: 4383 nM). Furthermore, UBX-363 demonstrated faster tumor regression than dasatinib in the HSB-2 xenograft models. In the CCRF-CEM xenograft model, UBX-363 effectively suppressed tumor growth in a dose-dependent manner, whereas dasatinib showed no inhibitory effect on tumor growth. After 12 days of UBX-363 dosing on CCRF-CEM xenograft mice, the levels of LCK protein in tumor tissue significantly reduced. The degradation activity of UBX-363 on LCK showed about 100% reduction at dosages of 5 mg/kg and 10 mg/kg on day 12. Conclusion UBX-363 demonstrated strong anti-proliferative and anti-tumor effects, as well as degradation activity of LCK, in both in vitro and in vivo models of T-ALL. These results highlight UBX-363 as a promising therapeutic agent for LCK-activated T-ALL. In the future, we will conduct additional research to explore the advantages of this degrader, such as its ability to disrupt scaffolding of LCK, over LCK small molecule inhibitors.

Targeted Protein Degradation for c-MYC Overcomes Therapy Resistance in T-Cell Acute Lymphoblastic Leukemias

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy characterized by diffuse infiltration of the bone marrow by immature lymphoblasts of thymic origin expressing T-cell surface markers. Early T cell precursor (ETP)-ALL is a distinct subtype of T-ALL characterized by a lack of CD1a and CD8; low expression of CD5 and the presence of myeloid or stem cell markers, and recent studies have shown resistance to conventional chemotherapies and poor outcome in ETP-ALL compared to thymic or mature T-ALL. A recent study revealed distinct gene expression profiles in adult compared to pediatric-adolescent ETP-ALL, representing higher expression levels of BCL2 and CD34 (Dai et al. Proc Natl Acad Sci 2022), however, detailed proteomic profiling in ETA-ALL has yet to be determined. Amplification of overexpression of MYC oncogene or stabilization of c-MYC protein occurs up to 70% in human cancers. Multifaceted activities of MYC include rapid proliferation of malignant cells supported by increased mitochondrial and ribosome biogenesis, dysregulated protein translation, and altered metabolism. NOTCH1-MYC signaling axis activation is one of the hallmarks that drives T-ALL leukemogenesis (Weng et al, Science 2004; Sanchez-Martin and Ferrando, Blood 2017). We have reported that targeting of BRD4 proteins induces suppression of leukemia initiating cells (LICs) in T-ALL by inhibiting the NOTCH1-MYC-CD44 axis, providing rationale to target MYC in therapy-resistant T-ALL (Piya et al. Leukemia 2022). We recently reported targeted protein degradation of c-MYC utilizing GT19715, the first-in-class cereblon modulator (CELMoD) for c-MYC exhibited promising anti-leukemia efficacy in acute myeloid leukemias (Nishida et al. ASH 2022). Here we employ GT19715 to investigate efficacy of targeting protein degradation of c-MYC in T-ALL. GT19715 induced dose-dependent apoptosis and cytoreduction in T-ALL cell lines with IC50 values below 10 nM except HPB-ALL cells. We found substantial decrease of c-MYC protein levels in sensitive but not in resistant cells (HPB-ALL). GT19715 induced apoptosis and ³ 90% cytoreduction at nanomolar concentrations in primary, therapy-resistant T-ALL. GT19715 also enhanced cell death induced by dexamethasone. In a xenograft model of CCRF-CEM cells carrying NOTCH1, PTEN, FBXW7, KRAS and TP53 mutations, GT19715 (3 mg/kg, three IP injections per week, two weeks on and one week off) reduced > 99% circulating human CD45+ leukemia cells compared to vehicle on day 16 after engraftment ( Fig. A), suggesting promising anti-leukemia efficacy in T-ALL in vivo. ETP-ALL cells from an adult patient exhibited distinct clustering compared to those from an adolescent patient as determined by single-cell mass cytometry (CyTOF) (N = 2). Adult ETP-ALL cells were characterized by increased protein levels of BCL2, CD34, CD44 and p-S6 compared to adolescent ETP-ALL cells, suggesting more stem-like properties. On the other hand, adolescent ETP-ALL cells showed higher CD7, CD33 and CD38 levels compared to the adult sample ( Fig. B). Interestingly, GT19715 treatment predominantly reduced p-S6 high clusters in adult ETP-ALL cells, potentially suggesting c-MYC degradation-induced suppression of the AKT-mTOR pathway. Conclusion: Targeted protein degradation of c-MYC induces promising anti-leukemia efficacy in T-ALL cells in vitro and in vivo. Further mechanistic and in vivo studies are ongoing.

RNA-binding protein NOVA1 promotes acute T-lymphocyte leukemia progression by stabilizing USP44 mRNA.

Acute T-lymphocyte leukemia (T-ALL) is a malignant tumor disease. RNA-binding protein neotumor ventral antigen-1 (NOVA1) is highly expressed in bone marrow mononuclear cells of T-ALL patients, while the role of NOVA1 in T-ALL progression remains unknown. The gain- and loss-of-function studies for NOVA1 were performed in Jurkat and CCRF-CEM cells. NOVA1 overexpression promoted cell proliferation and cell cycle progression. NOVA1 knockdown increased the apoptosis rate of T-ALL cells. Ubiquitin-specific protease 44 (USP44), a nuclear protein with deubiquitinase catalytic activity, has been reported to play an oncogene role in human T-cell leukemia. USP44 expression was positively associated with NOVA1, and RNA immunoprecipitation assay verified the binding of NOVA1 to the mRNA of USP44. USP44 knockdown partially abolished NOVA1-induced cell proliferation and inhibition of apoptosis. The in vivo xenograft experiment was performed by injection of T-ALL tumor cells into the tail vein of NOD/SCID mice. The knockdown of NOVA1 had lower tumorigenicity. NOVA1 knockdown alleviated pathological changes in lung and spleen tissues, and increased the overall survival period and the weight of T-ALL mice. Thus, NOVA1 acts as an accelerator in T-ALL, and its function might be achieved by binding to and stabilizing USP44 mRNA.

Targeted delivery of AZD5363 to T-cell acute lymphocytic leukemia by mSiO2-Au nanovehicles.

T-cell acute lymphocytic leukemia (T-ALL) is the most common cancer in children, with a low survival rate because of drug resistance and a high recurrence rate. Targeted delivery of chemotherapy drugs can reduce their side effects and improve their efficacy. The abnormality of phosphatidylinositol-3-kinase/protein kinase B/ mammalian target of rapamycin (PI3K/Akt/mTOR) pathway plays a key role in T-ALL occurrence. AZD5363 is a selective Akt inhibitor with promising therapeutic potential for tumors encoded by the PI3K/Akt/mTOR pathway. However, the toxicity and side effects have limited its application in treating T-ALL. This study aimed to design a delivery system for targeting AZD5363 to T-ALL by sgc8c aptamer designed as mesoporous silica (mSiO2) decorated with Au nanoparticles. The cell-specific targeting and cytotoxicity of mSiO2-Au-AZD5363-Apt were investigated. The mSiO2-Au nanovehicles were found feasible for AZD5363 delivery, with high loading efficiency and pH-responsive release in the acidic lysosome. More importantly, mSiO2-Au-AZD5363-Apt nanovehicles could specifically recognize and enter T-ALL cells in vitro and in vivo, effectively inhibiting the proliferation of CCRF-CEM cells. In conclusion, mSiO2-Au-AZD5363-Apt provided an effective therapeutic method for the targeted treatment of T-ALL.

Cytotoxicity, acute and sub-chronic toxicities of the leaves of Bauhinia thonningii (Schumach.) Milne-Redh. (Caesalpiniaceae)

BackgroundBauhinia thonningii is a plant traditionally used against many human diseases such as gastric ulcers, fever, inflammations, coughs, dysentery, diarrhea, and malaria. In the present investigation, the cytotoxicity of methanol extract of Bauhinia thonningii leaves (BTL), fractions and the isolated phytoconstituents was determined in a panel of 9 human cancer cell lines including drug sensitive and multidrug-resistant (MDR) phenotypes. The acute and sub-chronic oral toxicity of BTL was investigated as well.MethodsCompounds were isolated using chromatographic techniques while their chemical structures were determined using spectroscopic methods. The resazurin reduction assay (RRA) was used to evaluate the cytotoxicity of samples, propidium iodide (PI) for apoptosis, 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazolylcarbocyanine iodide (JC-1) staining for mitochondrial membrane potential (MMP) analysis, 2´,7´-dichlorodihydrofluoresceine diacetate (H2DCFH-DA) staining for the quantification of reactive oxygen species (ROS), whereas Caspase Glo assays were combined by means of flow cytometry. Furthermore, the toxicological investigations were performed as recommended by the Organization for Economic Cooperation and Development (OECD).ResultsThe botanicals as well as 6-C-methylquercetin-3,7-dimethyl ether (2), quercetin-3-O-L-rhamnopyranoside (5), quercetin-3-O-β-glucopyranoside (6), 6,8-C-dimethylkaempferol 3,7-dimethyl ether (7), and 6,8-C-dimethylkaempferol-3-methyl ether (8) had promising cytotoxic effects in the 9 tested cancer cell lines. The IC50 values below 20 µg/mL (botanicals) or 10 µM (compounds) on at least 1/9 tested cancer cell lines were considered. The best cytotoxic effects with IC50 values below 5 µM were achieved with compounds 7 against CEM/ADR5000 leukemia cells (2.86 µM) and MDA-MB-231-pcDNA breast adenocarcinoma cells (1.93 µM) as well as 8 against CCRF-CEM leukemia cells (3.03 µM), CEM/ADR5000 cells (2.42 µM), MDA-MB-231-pcDNA (2.34 µM), and HCT116 p53−/− cells (3.41 µM). BTL and compound 8 induced apoptotic cell death in CCRF-CEM cells through caspase activation, alteration of MMP, and increased ROS production. BTL did not cause any adverse effects in rats after a single administration at 5000 mg/kg or a repeated dose of 250 mg/kg body weight (b. w.).ConclusionBauhinia thonningii and its constituents are sources of cytotoxic drugs that deserve more in-depth studies to develop novel antiproliferative phytomedicine to fight cancer including resistant phenotypes.

GingerenoneA overcomes dexamethasone resistance by activating apoptosis and inhibiting cell proliferation in pediatric T-ALL cells.

Plant-based combination strategies have been widely considered in cancer therapy to attenuate chemotherapeutics side effects. The anti-leukemic effect of the whole ginger extract was previously portrayed by our team, and the current study is centered around the cytotoxicity and mechanism of action of a phenolic subsidiary of ginger, GingerenoneA, on pediatric acute lymphoblastic leukemia. GingernoneA imposed, dose-dependently, inhibitory effects on the viability of T and B leukemia cell lines confirmed by MTT assays. Resistance to Dexamethasone, a mostly used chemotherapeutic in acute lymphoblastic leukemia treatments, was overcome by GingernoneA. A synergistic effect of Dexamethasone and GingrenoneA on T leukemia cell lines and patient primary cells was confirmed. Annexin-V/PI and acridine orange/ethidium bromide staining illustrated dose-dependent apoptosis in CCRF-CEM cells developed by GingerenoneA. The intrinsic and extrinsic apoptosis induction and antiproliferative attribution of GingerenoneA were validated by western blot and qPCR. Despite the supposed loss of function in CCRF-CEM cells, TP53 showed increased expression levels and functional activity upon treatment with GingernoneA. Bioinformatic studies revealed the conceivable impact of GingerenoneA on the reactivity of mutant P53 through its binding to Cys124. Our findings may provide novel strategies for therapeutic intervention to ameliorate pALL outcomes.

Two palladium (II) complexes derived from halogen-substituted Schiff bases and 2-picolylamine induce parthanatos-type cell death in sensitive and multi-drug resistant CCRF-CEM leukemia cells

The use of cisplatin and its derivatives in cancer treatment triggered the interest in metal-containing complexes as potential novel anticancer agents. Palladium (II)-based complexes have been synthesized in recent years with promising antitumor activity. Previously, we described the synthesis and cytotoxicity of palladium (II) complexes containing halogen-substituted Schiff bases and 2-picolylamine. Here, we selected two palladium (II) complexes with double chlorine-substitution or double iodine-substitution that displayed the best cytotoxicity in drug-sensitive CCRF-CEM and multidrug-resistant CEM/ADR5000 leukemia cells for further biological investigation. Surprisingly, these compounds did not significantly induce apoptotic cell death. This study aims to reveal the major mode of cell death of these two palladium (II) complexes. We performed annexin V-FITC/PI staining and flow cytometric mitochondrial membrane potential measurement followed by western blotting, immunofluorescence microscopy, and alkaline single cell electrophoresis (comet assay). J4 and J6 still induced neither apoptosis nor necrosis in both leukemia cell lines. They also insufficiently induced autophagy as evidenced by Beclin and p62 detection in western blotting. Interestingly, J4 and J6 induced a novel mode of cell death (parthanatos) as mainly demonstrated in CCRF-CEM cells by hyper-activation of poly(ADP-ribose) polymerase 1 (PARP) and poly(ADP-ribose) (PAR) using western blotting, flow cytometric measurement of mitochondrial membrane potential collapse, nuclear translocation of apoptosis-inducing factor (AIF) by immunofluorescence microscopy, and DNA damage by alkaline single cell electrophoresis (comet assay). AIF translocation was also observed in CEM/ADR5000 cells. Thus, parthanatos was the predominant mode of cell death induced by J4 and J6, which explains the high cytotoxicity in CCRF-CEM and CEM/ADR5000 cells. J4 and J6 may be interesting drug candidates and deserve further investigations to overcome resistance of tumors against apoptosis. This study will promote the design of further novel palladium (II)-based complexes as chemotherapeutic agents.

Identification of compounds that preferentially suppress the growth of T-cell acute lymphoblastic leukemia-derived cells.

T-cell acute lymphoblastic leukemia (T-ALL) is one of the most frequently occurring cancers in children and is associated with a poor prognosis. Here, we performed large-scale screening of natural compound libraries to identify potential drugs against T-ALL. We identified three low-molecular-weight compounds (auxarconjugatin-B, rumbrin, and lavendamycin) that inhibited the proliferation of the T-ALL cell line CCRF-CEM, but not that of the B lymphoma cell line Raji in a low concentration range. Among them, auxarconjugatin-B and rumbrin commonly contained a polyenyl 3-chloropyrrol in their chemical structure, therefore we chose auxarconjugatin-B for further analyses. Auxarconjugatin-B suppressed the in vitro growth of five human T-ALL cell lines and two T-ALL patient-derived cells, but not that of adult T-cell leukemia patient-derived cells. Cultured normal T cells were several-fold resistant to auxarconjugatin-B. Auxarconjugatin-B and its synthetic analogue Ra#37 depolarized the mitochondrial membrane potential of CCRF-CEM cells within 3 h of treatment. These compounds are promising seeds for developing novel anti-T-ALL drugs.

Modes of Action of a Novel c-MYC Inhibiting 1,2,4-Oxadiazole Derivative in Leukemia and Breast Cancer Cells.

The c-MYC oncogene regulates multiple cellular activities and is a potent driver of many highly aggressive human cancers, such as leukemia and triple-negative breast cancer. The oxadiazole class of compounds has gained increasing interest for its anticancer activities. The aim of this study was to investigate the molecular modes of action of a 1,2,4-oxadiazole derivative (ZINC15675948) as a c-MYC inhibitor. ZINC15675948 displayed profound cytotoxicity at the nanomolar range in CCRF-CEM leukemia and MDA-MB-231-pcDNA3 breast cancer cells. Multidrug-resistant sublines thereof (i.e., CEM/ADR5000 and MDA-MB-231-BCRP) were moderately cross-resistant to this compound (<10-fold). Molecular docking and microscale thermophoresis revealed a strong binding of ZINC15675948 to c-MYC by interacting close to the c-MYC/MAX interface. A c-MYC reporter assay demonstrated that ZINC15675948 inhibited c-MYC activity. Western blotting and qRT-PCR showed that c-MYC expression was downregulated by ZINC15675948. Applying microarray hybridization and signaling pathway analyses, ZINC15675948 affected signaling routes downstream of c-MYC in both leukemia and breast cancer cells as demonstrated by the induction of DNA damage using single cell gel electrophoresis (alkaline comet assay) and induction of apoptosis using flow cytometry. ZINC15675948 also caused G2/M phase and S phase arrest in CCRF-CEM cells and MDA-MB-231-pcDNA3 cells, respectively, accompanied by the downregulation of CDK1 and p-CDK2 expression using western blotting. Autophagy induction was observed in CCRF-CEM cells but not MDA-MB-231-pcDNA3 cells. Furthermore, microarray-based mRNA expression profiling indicated that ZINC15675948 may target c-MYC-regulated ubiquitination, since the novel ubiquitin ligase (ELL2) was upregulated in the absence of c-MYC expression. We propose that ZINC15675948 is a promising natural product-derived compound targeting c-MYC in c-MYC-driven cancers through DNA damage, cell cycle arrest, and apoptosis.

The cardiac glycoside ZINC253504760 induces parthanatos-type cell death and G2/M arrest via downregulation of MEK1/2 phosphorylation in leukemia cells

Overcoming multidrug resistance (MDR) represents a major obstacle in cancer chemotherapy. Cardiac glycosides (CGs) are efficient in the treatment of heart failure and recently emerged in a new role in the treatment of cancer. ZINC253504760, a synthetic cardenolide that is structurally similar to well-known GCs, digitoxin and digoxin, has not been investigated yet. This study aims to investigate the cytotoxicity of ZINC253504760 on MDR cell lines and its molecular mode of action for cancer treatment. Four drug-resistant cell lines (P-glycoprotein-, ABCB5-, and EGFR-overexpressing cells, and TP53-knockout cells) did not show cross-resistance to ZINC253504760 except BCRP-overexpressing cells. Transcriptomic profiling indicated that cell death and survival as well as cell cycle (G2/M damage) were the top cellular functions affected by ZINC253504760 in CCRF-CEM cells, while CDK1 was linked with the downregulation of MEK and ERK. With flow cytometry, ZINC253504760 induced G2/M phase arrest. Interestingly, ZINC253504760 induced a novel state-of-the-art mode of cell death (parthanatos) through PARP and PAR overexpression as shown by western blotting, apoptosis-inducing factor (AIF) translocation by immunofluorescence, DNA damage by comet assay, and mitochondrial membrane potential collapse by flow cytometry. These results were ROS-independent. Furthermore, ZINC253504760 is an ATP-competitive MEK inhibitor evidenced by its interaction with the MEK phosphorylation site as shown by molecular docking in silico and binding to recombinant MEK by microscale thermophoresis in vitro. To the best of our knowledge, this is the first time to describe a cardenolide that induces parthanatos in leukemia cells, which may help to improve efforts to overcome drug resistance in cancer.Graphical A cardiac glycoside compound ZINC253504760 displayed cytotoxicity against different multidrug-resistant cell lines. ZINC253504760 exhibited cytotoxicity in CCRF-CEM leukemia cells by predominantly inducing a new mode of cell death (parthanatos). ZINC253504760 downregulated MEK1/2 phosphorylation and further affected ERK activation, which induced G2/M phase arrest.

Exploring the chemical space of functionalized [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the bromodomain of BRD9

Here we report a detailed structure–activity relationship (SAR) study related to [1,2,4]triazolo[4,3-a]quinoxaline-based compounds targeting the reader module of bromodomain containing-protein 9 (BRD9). 3D structure-based pharmacophore models, previously introduced by us, were here employed to evaluate a second generation of compounds, exploring different substitution patterns on the heterocyclic core. Starting from the promising data obtained from our previously identified [1,2,4]triazolo[4,3-a]quinoxaline-based compounds 1–4, the combination of in silico studies, chemical synthesis, biophysical and in vitro assays led to the identification of a new set of derivatives, selected for thoroughly exploring the chemical space of the bromodomain binding site. In more details, the investigation of different linkers at C-4 position highlighted the amine spacer as mandatory for the binding with the protein counterpart and the crucial role of the alkyl substituents at C-1 for increasing the selectivity toward BRD9. Additionally, the importance of a hydrogen bond donor group, critical to anchor the ZA region and required for the interaction with Ile53 residue, was inferred from the analysis of our collected results. Herein we also propose an optimization and an update of our previously reported “pharm-druglike2” 3D structure-based pharmacophore model, introducing it as “pharm-druglike2.1”. Compounds 24-26, 32, 34 and 36 were identified as new valuable BRD9 binders featuring IC50 values in the low micromolar range. Among them, 24 and 36 displayed an excellent selectivity towards BRD9 and a good antiproliferative effect on a panel of leukemia models, especially toward CCRF-CEM cell line, with no cytotoxicity on healthy cells. Notably, the interaction of 24 and 36 with the bromodomain and PHD finger-containing protein 1 (BRPF1) also emerged, disclosing them as new and unexplored dual inhibitors for these two proteins highly involved in leukemia. These findings highlight the potential for the identification of new attractive dual epidrugs as well as a promising starting point for the development of chemical degraders endowed with anticancer activities.

The effects of Astragalus polysaccharides, tragacanthin, and bassorin on methotrexate-resistant acute lymphoblastic leukemia.

One strategy to overcome methotrexate (MTX) resistance in acute lymphoblastic leukemia is suppressing MDR1 expression. It has been proved Astragalus polysaccharides (APS) exert their anticancer effect by reversing drug resistance. Due to the structural similarity of tragacanthin and bassorin with APS, we aimed to investigate the effects of the aforementioned polysaccharides on the expression of the MDR1 gene in the MTX-treated CCRF-CEM cells. Cytotoxicity of APS, bassorin, and tragacanthin on CCRF-CEM, CCRF-CEM/MTX (cells treated with MTX at IC50), and CCRF-CEM/R cells (CCRF-CEM cells resistant to MTX) was evaluated by MTT assay. The effect of all three compounds on MDR1 expression was evaluated using RT-PCR. All the concentrations of tragacanthin, bassorin, and APS (except at 0.8-100 μg/mL in CCRF-CEM) decreased the viability of all the cells compared to the negative control group; and against the positive control (MTX-treated cells), only bassorin at 20-100 μg/mL in CCRF-CEM/R and tragacanthin at 50 and 100 μg/mL in CCRF-CEM/MTX and at 2-100 μg/mL in CCRF-CEM/R decreased cell viability. Tragacanthin diminished MDR1 expression in CCRF-CEM/MTX and CCRF-CEM/R cells, which MTX had already induced. According to the results of this study, tragacanthin was a potent cytotoxic agent against CCRF-CEM cells and enhanced the chemosensitivity of CCRF-CEM/MTX and CCRF-CEM/R cells to MTX by down-regulation of MDR1 gene expression. Therefore, it could be a promising compound against cancer. Other possible mechanisms of action of tragacanthin should be evaluated and further in vitro and in vivo investigations are required.

CD7 protein plays a crucial role in T cell infiltration in tumors

CD7 protein as a target is being used to treat CD7+ lymphoma; however, the role of CD7 in the hematopoietic system remains largely unknown. Therefore, we evaluated the effects of CD7 KO in mice. The differentiation of the hematopoietic system in the bone marrow and the number of various cell types in the thymus and spleen did not differ between CD7 KO and WT mice. After subcutaneous inoculation of B16–F10 melanoma cells, tumors from CD7 KO mice grew more rapidly, and the proportion of CD8+ T cells in the spleen and tumors decreased. In vitro, the infiltration and adhesion of CD8+ T cells from the spleen of CD7 KO mice were weakened. Blocking CD7 in normal T cells did not alter the migration and infiltration, but in Jurkat, CCRF-CEM, and KG-1a tumor cell lines, migration and invasion were significantly reduced after blocking CD7. Therefore, CD7 does not affect hematopoietic system development but plays a crucial role in T cell infiltration into tumors.

Study on the Mechanism of Multi-Drug Resistance of Agaricus Blazei Extract FA-2-b-β Mediated Wnt Signaling Pathway to Reverse Acute T Lymphoblastic Leukemia

To investigate the mechanism of drug reversing resistance of Agaricus blazei extract FA-2-b-β on T cell acute lymphoblastic leukemia (T-ALL) cell lines. Cell proliferation was detected by CCK-8 assay; the apoptosis, cell cycle mitochondrial membrane potential, and intracellular rhodamine accumulation were detected by flow cytometry, and apoptosis-related gene and protein expression were detected by qPCR and Western blot; the membrane surface protein MDR1 was observed by immunofluorescence microscopy. Different concentrations of FA-2-b-β significantly inhibited proliferation and induced apoptosis of CCRF-CEM and CEM/C1 (P<0.05), and CCRF-CEM cell cycle were arrested at S phase, and CEM/C1 cells were arrested at G0/G1 phase. Western blot and qPCR results show that FA-2-b-β inhibited ABCB1、ABCG2、CTNNB、MYC and BCL-2 expression, but upregulated Bax expression. In addition, FA-2-b-β reversed the resistance characteristics of CEM/C1 drug-resistance cells, which decreased mitochondrial membrane potential, and significantly increased the intracellular rhodamine accumulation, and weakening of the expression of the membrane surface protein MDR1. With the Wnt/β-catenin inhibitor (ICG001), the process was further intensified. Agaricus Blazei Extract FA-2-b-β inhibits cell proliferation, promotes apoptosis, regulates the cell cycle, reduces mitochondrial energy supply, and down-regulate MDR1 expression to reverse the resistance of CEM/C1, which all suggest it is through regulating the Wnt signaling pathway in T-ALL.

Ginsenoside 24-OH-PD from red ginseng inhibits acute T-lymphocytic leukaemia by activating the mitochondrial pathway.

Ginsenoside 24-hydroxy-ginsengdiol (24-OH-PD), extracted from red ginseng, is a novel diol-type ginsenoside, strongly inhibits the growth of human T-cell acute lymphoblastic leukaemia (T-ALL) CCRF-CEM cells. Our research aimed at investigating the mechanism underlying this inhibition. Cell viability was determined using the cell counting kit-8 (CCK-8) assay, and NOD/SCID mice bearing CCRF-CEM cells were used to verify the therapeutic effect of 24-OH-PD on T-ALL in vivo. We equally analysed pathways related to 24-OH-PD in CCRF-CEM cells using RNA-Seq analysis. Cell apoptosis, reactive oxygen species (ROS), mitochondrial membrane potential (ΔΨm), and mitochondrial permeability transition pore (mPTP) levels were detected by flow cytometry. The activity of caspase3 and caspase9 was detected by enzyme activity detection kits. The expression levels of apoptosis-related proteins and mRNA were determined through western blotting and quantitative reverse-transcription PCR assays (qRT-PCR). CCK-8 assay and animal xenograft experiments confirmed that 24-OH-PD significantly inhibited T-ALL in a dose-dependent manner, both in vivo and in vitro. RNA-Seq results suggest that mitochondria-mediated apoptosis pathway plays an important role in this process. Furthermore, intracellular ROS levels increased, mPTP opened, and ΔΨm decreased following 24-OH-PD treatment. Pretreatment with the antioxidant, NAC, reversed the effects of 24-OH-PD on apoptosis and ROS generation. Moreover, 24-OH-PD treatment increased the expression of Bax and caspase family members, thereby releasing cytochrome c (Cytc) and inducing apoptosis. Our findings showed that, 24-OH-PD induces apoptosis in CCRF-CEM cells by activating the mitochondrial-dependent apoptosis pathway through ROS accumulation. This inhibitory effect implies that 24-OH-PD could be further developed as treatment of T-ALL.

Identification of Piperlongumine as Potent Inhibitor of Necroptosis.

Excessive necroptosis contributes to the pathogenesis of several inflammatory and neurodegenerative diseases. Here, using a high-throughput screening approach, we investigated the anti-necroptosis effects of piperlongumine, an alkaloid isolated from the long pepper plant, in vitro and in a mouse model of systemic inflammatory response syndrome (SIRS). A natural compound library was screened for anti-necroptosis effects in cellular. The underlying mechanism of action of the top candidate piperlongumine was explored by quantifying the necroptosis marker phosphorylated receptor-interacting protein kinase 1 (p-RIPK1) by Western blotting. The anti-inflammatory effect of piperlongumine was assessed in a tumor necrosis factor α (TNFα)-induced SIRS model in mice. Among the compounds investigated, piperlongumine significantly rescued cell viability. The half maximal effective concentration (EC50) of piperlongumine for inhibiting necroptosis was 0.47 μM in HT-29 cells, 6.41 μM in FADD-deficient Jurkat cells, and 2.33 µM in CCRF-CEM cells, while the half maximal inhibitory concentration (IC50) was 95.4 µM in HT-29 cells, 93.02 µM in FADD-deficient Jurkat cells, and 161.1 µM in CCRF-CEM cells. Piperlongumine also significantly inhibited TNFα-induced intracellular RIPK1 Ser166 phosphorylation in cell lines and significantly prevented decreases in body temperature and improved survival in SIRS mice. As a potent necroptosis inhibitor, piperlongumine prevents phosphorylation of RIPK1 at its activation residue Ser166. Piperlongumine thus potently inhibits necroptosis at concentrations safe enough for human cells in vitro and inhibits TNFα-induced SIRS in mice. Piperlongumine has potential clinical translational value for the treatment of the spectrum of diseases associated with necroptosis, including SIRS.

Development and evaluation of novel artemisinin-isatin hybrids with potential anti-leukemic cytotoxicity.

Twenty-one novel ester tethered artemisinin-isatin hybrids were designed, synthesized and screened against human myeloid leukemia cell lines (K562 and K562/ADR), human acute lymphoblastic leukemia cell line (CCRF-CEM) as well as normal human peripheral blood mononuclear cells (PBMCs) for their cytotoxicity by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The structure-activity relationships (SARs) were also discussed to facilitate further rational design of more effective candidates. The preliminary results showed that most of the ester tethered artemisinin-isatin hybrids (IC50: 0.32-29.35 µM) exhibited promising activity against CCRF-CEM cells, and some of them (IC50: 1.23-49.84 µM) were also active against K562 and K562/ADR human myeloid leukemia cell lines. Among them, hybrid 7d (IC50: 0.32, 2.67 and 1.23 µM) not only possessed profound activity against the three tested leukemia cell lines and excellent safety and selectivity profiles, but also showed promising pharmacokinetic properties. Accordingly, hybrid 7d could be considered as a potential lead molecule for the development of novel anti-leukemic agents with minimal untoward events to normal human cells.

Design, synthesis, anticancer, and antibacterial evaluation of some quinazolinone-based derivatives as DHFR inhibitors.

Two series of quinazolinone derivatives were designed and synthesized as dihydrofolate reductase (DHFR) inhibitors. All compounds were evaluated for their antibacterial and antitumor activities. Antibacterial activity was evaluated against three strains of Gram-positive and Gram-negative bacteria. Compound 3d exhibited the highest inhibitory activity against Staphylococcus aureus DHFR (SaDHFR) with IC50 of 0.769 ± 0.04 μM compared to 0.255 ± 0.014 μM for trimethoprim. Compound 3e was also more potent than trimethoprim against Escherichia coli DHFR (EcDHFR) with IC50 of 0.158 ± 0.01 μM and 0.226 ± 0.014 μM, respectively. Compound 3e exhibited a promising antiproliferative effect against most of the tested cancer cells. It also showed potent activity against leukemia (CCRF-CEM, and RPMI-8226); lung NCI-H522, and CNS U251 with GI% of 65.2, 63.22, 73.28, and 97.22, respectively. The cytotoxic activity of compound 3e was almost half the activity of doxorubicin against CCRF-CEM cell line with IC50 of 1.569 ± 0.06 μM and 0.822 ± 0.03 µM, respectively. In addition, compound 3e inhibited human DHFR with IC50 value of 0.527 ± 0.028 µM in comparison to methotrexate (IC50 = 0.118 ± 0.006 µM). Compound 3e caused an arrest of the cell cycle mainly at the S phase and caused a rise in the overall apoptotic percentage from 2.03% to 48.51%. (23.89-fold). Treatment of CCRF-CEM cells with compound 3e produced a significant increase in the active caspase-3 level by 6.25-fold compared to untreated cells. Molecular modeling studies were performed to evaluate the binding pattern of the most active compounds in the bacterial and human DHFR.

Ferroptosis Inhibitory Compounds from the Deep-Sea-Derived Fungus Penicillium sp. MCCC 3A00126

Two new xanthones (1 and 2) were isolated from the deep-sea-derived fungus Penicillium sp. MCCC 3A00126 along with 34 known compounds (3-36). The structures of the new compounds were established by spectroscopic data. The absolute configuration of 1 was validated by comparison of experimental and calculated ECD spectra. All isolated compounds were evaluated for cytotoxicity and ferroptosis inhibitory activities. Compounds 14 and 15 exerted potent cytotoxicity against CCRF-CEM cells, with IC50 values of 5.5 and 3.5 μM, respectively, whereas 26, 28, 33, and 34 significantly inhibited RSL3-induced ferroptosis, with EC50 values of 11.6, 7.2, 11.8, and 2.2 μM, respectively.

Shotgun Proteomics of Co-Cultured Leukemic and Bone Marrow Stromal Cells from Different Species as a Preliminary Approach to Detect Intercellular Protein Transfer

Cellular interactions within the bone marrow microenvironment modulate the properties of subsets of leukemic cells leading to the development of drug-resistant phenotypes. The intercellular transfer of proteins and organelles contributes to this process but the set of transferred proteins and their effects in the receiving cells remain unclear. This study aimed to detect the intercellular protein transfer from mouse bone marrow stromal cells (OP9 cell line) to human T-lymphoblasts (CCRF-CEM cell line) using nanoLC-MS/MS-based shotgun proteomics in a 3D co-culture system. After 24 h of co-culture, 1513 and 67 proteins from human and mouse origin, respectively, were identified in CCRF-CEM cells. The presence of mouse proteins in the human cell line, detected by analyzing the differences in amino acid sequences of orthologous peptides, was interpreted as the result of intercellular transfer. The transferred proteins might have contributed to the observed resistance to vincristine, methotrexate, and hydrogen peroxide in the co-cultured leukemic cells. Our results suggest that shotgun proteomic analyses of co-cultured cells from different species could be a simple option to get a preliminary survey of the proteins exchanged among interacting cells.