Human Lymphoblastic Cells Research Articles

Enzymatic reaction and competitive prereduction enable label-free and separation-free inductively coupled plasma mass spectrometry detection of leukemia cells

The enzyme terminal deoxynucleotidyl transferase (TdT) exhibits significant activity in various leukemia cells, including human acute lymphoblastic leukemia cells (CCRF). It demonstrates substantial potential as a diagnostic biomarker for acute lymphoblastic leukemia (ALL). Herein, a highly sensitive method for the homogeneous quantification of TdT and CCRF cells, without the need for labeling or separation, was established based on inductively coupled plasma mass spectrometry (ICP-MS). The simple sensor relied on the TdT-induced polymerization process, lengthening the DNA strand and producing large amounts of the by-product phosphates of pyrophosphate (PPi). The PPi formed complexes with Cu²⁺, altering its morphology in homogeneous systems. The remaining Cu²⁺ competed with Hg²⁺ in consuming ascorbic acid (AA) through the pre-reduction of Hg²⁺, affecting the ICP-MS signal of Hg²⁺. Under optimal conditions, this approach exhibited excellent specificity and ultrahigh sensitivity, with limits of detection (LODs) as low as 0.05 U/L for TdT and 5 cells/mL for CCRF cells, respectively. The proposed method offers significant advantages, including high sensitivity and precision, operation at room temperature without the involvement of proteases, and the ability to accurately quantify in complex biological matrices. Consequently, the proposed method shows great promise as a valuable tool for TdT-related biological research and leukemia diagnosis.

Inhibitory effects of progesterone on the human acute lymphoblastic leukemia cell line

IntroductionAcute lymphoblastic leukemia (ALL) is a rare form of blood cancer that can quickly advance if left untreated. Research has suggested that progesterone (P4) may be effective in treating certain types of tumors. Specifically, the membrane progesterone receptors may play a role in either enhancing or inhibiting cell growth in various tumors. This study aimed to investigate the impact of P4 on inhibition of NALM6 cells. MethodsNALM6 cells were exposed to different concentrations of P4 (ranging from 10 to 50 μM) at 24,48 and 72 h intervals. The cell survival rate was then evaluated using an MTT assay. Additionally, the study assessed the rate of mPR expression in the cells using flow cytometry at 48 and 72 h after P4 administration (at concentrations of 20 and 10 μM, respectively). Furthermore, the level of ROS was evaluated using the dichlorofluorescein diacetate (DCFDA) flow cytometry technique. ResultsThe study found that mPR-β was expressed in NALM6 cells and that P4 had a significant inhibitory effect on the growth of tumor cells in a time and concentration-dependent manner. Furthermore, P4 was found to reduce mPR-β expression at 48 and 72 h. The treatment also resulted in a decrease in ROS levels compared to untreated cells (P ≤ 0.05). ConclusionThe study suggests that p4 may be effective in growth-inhibiting NALM6 cells by decreasing cell viability and reducing ROS levels. However, further research is needed to understand the mechanism of action and interactions with various receptors and to confirm its effectiveness in treating NALM6 leukemia.

Acquired copy number amplification at the MYC enhancer in human B-precursor acute lymphoblastic leukemia cell lines.

Our study highlights the discovery of recurrent copy number alterations in noncoding regions, specifically blood enhancer cluster (BENC-CNA), in B-precursor acute lymphoblastic leukemia (BCP-ALL) cell lines. We demonstrate that BENC-CNA acts as a super-enhancer, driving MYC expression and possibly contributing to the immortalization and proliferative advantage of BCP-ALL cells invitro.

A Capillary-Force-Driven, Single-Cell Transfer Method for Studying Rare Cells.

The characterization of individual cells within heterogeneous populations (e.g., rare tumor cells in healthy blood cells) has a great impact on biomedical research. To investigate the properties of these specific cells, such as genetic biomarkers and/or phenotypic characteristics, methods are often developed for isolating rare cells among a large number of background cells before studying their genetic makeup and others. Prior to using real-world samples, these methods are often evaluated and validated by spiking cells of interest (e.g., tumor cells) into a sample matrix (e.g., healthy blood) as model samples. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. People often circumvent the problem by diluting a solution of high-concentration cells, but the concentration becomes inaccurate after series dilution due to the fact that a cell suspension solution can be inhomogeneous, especially when the cell concentration is very low. We report on an alternative method for low-cost, accurate, and reproducible low-concentration cell spiking without the use of external pumping systems. By inducing a capillary force from sudden pressure drops, a small portion of the cellular membrane was aspirated into the reservoir tip, allowing for non-destructive single-cell transfer. We investigated the surface membrane tensions induced by cellular aspiration and studied a range of tip/tumor cell diameter combinations, ensuring that our method does not affect cell viability. In addition, we performed single-cell capture and transfer control experiments using human acute lymphoblastic leukemia cells (CCRF-CEM) to develop calibrated data for the general production of low-concentration samples. Finally, we performed affinity-based tumor cell isolation using this method to generate accurate concentrations ranging from 1 to 15 cells/mL.

A novel type IIb L-asparaginase from Latilactobacillus sakei LK-145: characterization and application.

We succeeded in homogeneously expressing and purifying L-asparaginase from Latilactobacillus sakei LK-145 (Ls-Asn1) and its mutated enzymes C196S, C264S, C290S, C196S/C264S, C196S/C290S, C264S/C290S, and C196S/C264S/C290S-Ls-Asn1. Enzymological studies using purified enzymes revealed that all cysteine residues of Ls-Asn1 were found to affect the catalytic activity of Ls-Asn1 to varying degrees. The mutation of Cys196 did not affect the specific activity, but the mutation of Cys264, even a single mutation, significantly decreased the specific activity. Furthermore, C264S/C290S- and C196S/C264S/C290S-Ls-Asn1 almost completely lost their activity, suggesting that C290 cooperates with C264 to influence the catalytic activity of Ls-Asn1. The detailed enzymatic properties of three single-mutated enzymes (C196S, C264S, and C290S-Ls-Asn1) were investigated for comparison with Ls-Asn1. We found that only C196S-Ls-Asn1 has almost the same enzymatic properties as that of Ls-Asn1 except for its increased stability for thermal, pH, and the metals NaCl, KCl, CaCl2, and FeCl2. We measured the growth inhibitory effect of Ls-Asn1 and C196S-Ls-Asn1 on Jurkat cells, a human T-cell acute lymphoblastic leukemia cell line, using L-asparaginase from Escherichia coli K-12 as a reference. Only C196S-Ls-Asn1 effectively and selectively inhibited the growth of Jurkat T-cell leukemia, which suggested that it exhibited antileukemic activity. Furthermore, based on alignment, phylogenetic tree analysis, and structural modeling, we also proposed that Ls-Asn1 is a so-called "Type IIb" novel type of asparaginase that is distinct from previously reported type I or type II asparaginases. Based on the above results, Ls-Asn1 is expected to be useful as a new leukemia therapeutic agent.

Biochips to measure ultraviolet light absorption by leukemia cells

During the treatment of leukemia, side effects create a great burden for patients. Therefore, new therapies to replace conventional chemotherapy and hematopoietic stem cell transplantation are highly desirable. In a previous study, I found that UV irradiation at 365 nm induced apoptosis in 98% of leukemia (Jurkat T) cells. To determine the most suitable wavelength for such treatments, it is necessary to measure the UV light absorption of cancer cells. In this study, a biochip was fabricated and evaluated for this purpose. Three types of leukemic cells (human acute myelogenous leukemia cells, human acute lymphoblastic leukemia cells, and human chronic myelogenous leukemia cells) were injected into the biochip, and light absorption was measured, revealing that individual leukemic cells have different light absorption characteristics. This variation is important in order to develop an implant care device to treat leukemia more effectively.

Regulating TKT activity inhibits proliferation of human acute lymphoblastic leukemia cells

Regulating TKT activity inhibits proliferation of human acute lymphoblastic leukemia cells

Thiophene derivative inflicts cytotoxicity via an intrinsic apoptotic pathway on human acute lymphoblastic leukemia cells.

In an effort to identify novel anti-cancer agents, we employed a well-established High Throughput Screening (HTS) assay to assess the cytotoxic effect of compounds within the ChemBridge DIVERSet Library on a lymphoma cell line. This screen revealed a novel thiophene, F8 (methyl 5-[(dimethylamino)carbonyl]-4-methyl-2-[(3-phenyl-2-propynoyl) amino]-3-thiophenecarboxylate), that displays anti-cancer activity on lymphoma, leukemia, and other cancer cell lines. Thiophenes and thiophene derivatives have emerged as an important class of heterocyclic compounds that have displayed favorable drug characteristics. They have been previously reported to exhibit a broad spectrum of properties and varied uses in the field of medicine. In addition, they have proven to be effective drugs in various disease scenarios. They contain anti-inflammatory, anti-anxiety, anti-psychotic, anti-microbial, anti-fungal, estrogen receptor modulating, anti-mitotic, kinase inhibiting and anti-cancer activities, rendering compounds with a thiophene a subject of significant interest in the scientific community. Compound F8 consistently induced cell death at a low micromolar range on a small panel of cancer cell lines after a 48 h period. Further investigation revealed that F8 induced phosphatidylserine externalization, reactive oxygen species generation, mitochondrial depolarization, kinase inhibition, and induces apoptosis. These findings demonstrate that F8 has promising anti-cancer activity.

Zebrafish drug screening identifies Erlotinib as an inhibitor of Wnt/β-catenin signaling and self-renewal in T-cell acute lymphoblastic leukemia

The Wnt/β-catenin pathway’s significance in cancer initiation, progression, and stem cell biology underscores its therapeutic potential. However, the clinical application of Wnt inhibitors remains limited due to challenges posed by off-target effects and complex cross-talk of Wnt signaling with other pathways. In this study, we leveraged a zebrafish model to perform a robust and rapid drug screening of 773 FDA-approved compounds to identify Wnt/β-catenin inhibitors with minimal toxicity. Utilizing zebrafish expressing a Wnt reporter, we identified several drugs that suppressed Wnt signaling without compromising zebrafish development. The efficacy of the top hit, Erlotinib, extended to human cells, where it blocked Wnt/β-catenin signaling downstream of the destruction complex. Notably, Erlotinib treatment reduced self-renewal in human T-cell Acute Lymphoblastic Leukemia cells, which rely on active β-catenin signaling for maintenance of leukemia-initiating cells. Erlotinib also reduced leukemia-initiating cell frequency and delayed disease formation in zebrafish models. This study underscores zebrafish’s translational potential in drug discovery and repurposing and highlights a new use for Erlotinib as a Wnt inhibitor for cancers driven by aberrant Wnt/β-catenin signaling.

Novel wild edible mushroom Astraeus hygrometricus (Pers.) Morgan induces robust apoptosis on human acute lymphoblastic leukemia cells through a RONS-subsisted mitochondria-dependent pathway

ObjectiveTo explore the therapeutic effects of the novel wild edible mushroom Astraeus hygrometricus (Pers.) Morgan (A. hygrometricus) on human acute lymphoblastic leukemia cells. MethodsExtensive screening of the antiproliferative and chemopreventive potential of different extracts from 5 wild mushrooms, A. hygrometricus, Phallus sp., Lentinus sp., Tricholoma sp., and Serpula sp. was performed against a panel of 6 cancer cell lines and normal cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis determination, cell cycle profiling, intracellular reactive oxygen species (ROS) and reactive nitrogen species (RNS), and mitochondrial membrane potential were analyzed by flow cytometry. The activity of caspases was measured colorimetrically, and the expression pattern of mitochondrial proteins was analyzed. ResultsThe methanol extract of A. hygrometricus and MOLT-4 cells were identified as the most potent extract exhibiting antiproliferative activity and most sensitive cell line, respectively. The mushroom extract induced robust selective apoptosis in MOLT-4 cells and arrested cell cycle progression at the G0/G1 stage. The extract disrupted the mitochondrial membrane potential and enhanced ROS production in MOLT-4 cells. The methanol extract induced apoptosis by downregulating the expression of Bcl-2, increasing the expression of Bax, and activating the caspase cascade. ConclusionThe novel wild edible mushroom is a potential repository of biomolecules for the development of antileukemic drugs.

Targeting IGF2BP3 Enhances Anti-Leukemic Effects of Menin-MLL Inhibition in MLL-AF4 Leukemia

Background: MLL-rearranged leukemias are a clinically challenging and biologically unique subtype of leukemias associated with poor prognosis. While novel therapeutic strategies have been primarily directed at epigenetic dysregulation, post-transcriptional gene regulatory mechanisms have emerged as important mediators in leukemogenesis and have the unexplored potential to be potent combinatorial therapeutic targets. Previously, we found that the RNA-binding protein IGF2BP3 is a critical regulator of MLL-AF4 leukemogenesis and represents a promising therapeutic target. Methods: We studied the combined effects of targeting IGF2BP3 and the Menin-MLL interaction in MLL-AF4 driven leukemia in vitro and in vivo, using genetic inhibition through CRISPR-Cas9 mediated deletion of Igf2bp3 and pharmacologic inhibition of the Menin-MLL interaction with commercially available inhibitors MI-503, MI-463, and MI-538. In vitro, we tested the human B-cell acute lymphoblastic leukemia cell lines, SEM, RS4;11 and NALM6, and MLL-Af4-transformed murine hematopoietic stem and progenitor cells (herein referred to as MLL-Af4 Lin-), derived from bone marrow of Cas9 mice. Results: Depletion of Igf2bp3 sensitized MLL-AF4 leukemia to the negative effects of Menin-MLL inhibition on leukemic cell growth, colony formation and leukemic initiating cells in vitro. Mechanistically, we found that both Igf2bp3 depletion and Menin-MLL inhibition led to increased differentiation in vitro and in vivo in functional readouts and by gene expression analyses. Both MI-503 treatment and IGF2BP3 knockdown in MLL-Af4 Lin- cells showed a shift towards more differentiated colony morphologies in colony formation assays, decreased expression of c-Kit and increased expression of maturation markers by flow cytometry, and morphologic changes consistent with increased differentiation. To gain insight into these phenotypic findings, we examined gene expression from MLL-Af4 Lin- cells with IGF2BP3 knockdown and treated with MI-503. Metascape analysis revealed significant enrichment in pathways involved in cell differentiation and activation, particularly in leukocytes. This was observed in both I3KO and MI-503 treated cells, with significant overlap in shared differentially expressed genes in both conditions. Next, we looked at the overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 knockdown with targets identified from MLL-AF4 ChIP and IGF2BP3 CLIP in CD11b+ and MLL-Af4 Lin- cells. We found significant overlap between differentially expressed genes with MI-503 treatment and IGF2BP3 CLIP targets, suggesting that IGF2BP3 directly regulates genes that are affected by Menin-MLL inhibition. Furthermore, we saw overlap between differentially expressed genes with IGF2BP3 knockdown and MLL-AF4 ChIP targets identified in SEM and RS4;11 cells. These patterns highlight the interaction between the MLL-AF4 and IGF2BP3 transcriptomes and suggest a mechanism for the synergistic inhibition of leukemia by targeting both MLL-AF4 and IGF2BP3. To examine the combined effects of IGF2BP3 knockdown and Menin-MLL inhibition on leukemic engraftment and survival in vivo, we transplanted MLL-Af4 Lin- cells, depleted (or non-depleted) for IGF2BP3 and treated with MI-503 at 0.5 μM for 5 days, to initiate leukemia. We observed a significant decrease in peripheral blood leukemic engraftment with MI-503 treatment and IGF2BP3 knockdown. IGF2BP3 knockdown had a greater effect on survival and attenuating disease than MI-503 alone and showed enhanced anti-leukemic effects in combination. Conclusions: Our work shows that IGF2BP3 is an oncogenic amplifier of MLL-AF4 mediated leukemogenesis and is a potent therapeutic target and suggests a novel combinatorial approach to targeting leukemia at both the transcriptional and post-transcriptional level.

Three induced pluripotent stem cell lines (TRNDi033-A, TRNDi034-A, TRNDi035-A) generated from lymphoblasts of three apparently healthy individuals

Expanded human lymphoblast cells from three different aged healthy individuals, 8-year-old male, 0-year-old newborn (NB) male, and 26-year-old female, were used to generate induced pluripotent stem cell (iPSC) lines TRNDi033-A, TRNDi034-A and TRNDi035-A, respectively, by exogenous expression of five reprogramming factors, human OCT4, SOX2, KLF4, L-MYC and LIN28. The authenticity of established iPSC lines was confirmed by the expressions of stem cell markers, karyotype analysis, embryoid body formation, and scorecard analysis. These iPSC lines could serve as healthy donor controls that are age and sex matched for the studies involving patient-specific iPSCs.

ATF4 renders human T-cell acute lymphoblastic leukemia cell resistance to FGFR1 inhibitors through amino acid metabolic reprogramming.

Abnormalities of FGFR1 have been reported in multiple malignancies, suggesting FGFR1 as a potential target for precision treatment, but drug resistance remains a formidable obstacle. In this study, we explored whether FGFR1 acted a therapeutic target in human T-cell acute lymphoblastic leukemia (T-ALL) and the molecular mechanisms underlying T-ALL cell resistance to FGFR1 inhibitors. We showed that FGFR1 was significantly upregulated in human T-ALL and inversely correlated with the prognosis of patients. Knockdown of FGFR1 suppressed T-ALL growth and progression both in vitro and in vivo. However, the T-ALL cells were resistant to FGFR1 inhibitors AZD4547 and PD-166866 even though FGFR1 signaling was specifically inhibited in the early stage. Mechanistically, we found that FGFR1 inhibitors markedly increased the expression of ATF4, which was a major initiator for T-ALL resistance to FGFR1 inhibitors. We further revealed that FGFR1 inhibitors induced expression of ATF4 through enhancing chromatin accessibility combined with translational activation via the GCN2-eIF2α pathway. Subsequently, ATF4 remodeled the amino acid metabolism by stimulating the expression of multiple metabolic genes ASNS, ASS1, PHGDH and SLC1A5, maintaining the activation of mTORC1, which contributed to the drug resistance in T-ALL cells. Targeting FGFR1 and mTOR exhibited synergistically anti-leukemic efficacy. These results reveal that FGFR1 is a potential therapeutic target in human T-ALL, and ATF4-mediated amino acid metabolic reprogramming contributes to the FGFR1 inhibitor resistance. Synergistically inhibiting FGFR1 and mTOR can overcome this obstacle in T-ALL therapy.

50 Human Hazard Assessment of Nanomaterials: Supporting Risk Decision Making Through Interlaboratory Trial Data

Abstract The prevalent application of engineered nanomaterials (ENMs) has led to an extensive effort to develop tools facilitating their risk assessment and management. This interlaboratory trial (encompassing two laboratories) sought to investigate the genotoxic impact of ENMs using the hypoxanthine phosphoribosyltransferase (HPRT) forward mutation assay and in vitro cytokinesis-blocked micronucleus (CBMN) assay, utilising harmonised protocols. In the CBMN assay, human lymphoblast (TK6) cells were exposed to zinc oxide (ZnO), titanium dioxide (TiO2) and tungsten carbide-cobalt (WC/Co) for 24-hours after which 1000 binucleated cells were scored for micronuclei (n=2). For the HPRT assay, TK6 and mouse fibroblast (V79) cells were exposed to ZnO, TiO2, WC/Co, CuO and Nanocyl-CNTs for 24-hours after which 600 wells were scored for point mutations (n=2). Significant cytotoxicity and micronuclei frequency was reported following ZnO exposure (20µg/ml). Significant micronuclei induction was reported following WC/Co exposures (2-fold over control, 100µg/ml). No significant mutagenicity was detected with ZnO or TiO2; both laboratories observed significant cytotoxicity following exposure to ZnO (20µg/ml; 37% reduction in cell viability). In the second interlaboratory trial CuO induced significant cytotoxicity (36% reduction in viability: 0.5µg/ml), and a 32-fold increase in mutagenicity. The Nanocyl induced a 6-fold increase in mutagenicity. The data generated here has shown good statistical concordance between laboratories and has highlighted a promising route forward in supporting risk decision making for ENMs. The authors would like to acknowledge this research has received funding from the European Union’s Horizon 2020 research and innovation program for the RiskGONE project, grant agreement #814425.

A novel tumor-bearing humanized mouse model for evaluating the efficacy and predictive safety of bispecific T cell engager

Abstract Bispecific T cell engager (BiTE) is one of the most promising therapeutic approaches for treatment of various malignancies. Humanized mice engrafted with human peripheral blood mononuclear cells (PBMCs) have currently become the preclinical platform for evaluating the effectiveness of various cancer immunotherapies including BiTE. However, the wide application of this humanized animal model is limited by a strong xenoreaction of human T cells against the mouse tissues. In this study, we used humanized murine major histocompatibility class I and class II double knock-out NSG mice (referred to DKO) to evaluate the antitumor effect of BiTE. The DKO mice were implanted with human pre-B acute lymphoblastic leukemia cell line (Nalm6). Five days later, the mice were reconstituted with PBMCs. Following CD19xCD3 BiTE treatment whole-body tumor burden was quantified by a in vivo imaging system. Our data demonstrated that the mice developed consistent clinical signs of leukemia-related body weight loss and hind-limb paralysis within 4 weeks. With serial dose of CD19xCD3 BiTE treatment, the tumor cell growing was significantly suppressed. A lower incidence of hind-limb paralysis with prolonged survival were observed in BiTE-treated mice compared with controls. In addition, after treatment with CD19xCD3 BiTE, the humanized mice exhibited elevated serum levels of human IFN-γ, TNF-a, IL-6, IL-10, IL-2, and IL-4. The cytokine production was dose-dependent and associated with the increase of CD19xCD3 BiTE effectiveness. This tumor-bearing humanized model provides a promising preclinical platform for evaluating the efficacy, toxic cytokine release, and dosing requirements of any T cell-based immunotherapies for cancer treatment.

Brevetoxin induces a shift in the redox state of the proteome and unfolded protein response in human lymphoblast cells that can be alleviated with the acrolein scavenger MESNA

Human lymphoblast cells were treated with the marine algal toxin, brevetoxin-2 (PbTx-2), and its effects on the proteome were assessed by redox proteomics using cysteine reactive tandem mass tags (TMT). Additionally, cells were simultaneously treated with PbTx-2 and the antioxidant and acrolein scavenger sodium 2-mercaptoethylsulfonate (MESNA) to determine if MESNA could prevent the proteomic effects of brevetoxin-2. A massive shift in the redox state of the proteome of brevetoxin-2 treated cells was observed. The main pathway affected was genetic information processing. Significantly oxidized proteins included Trx-1, peroxyredoxins (Prxs), ribosomal proteins, and the eukaryotic initiation factor 2 β subunit (eIF2β). Proteins that were overexpressed in brevetoxin-treated cells included four folding chaperones. These effects were diminished in the presence of MESNA indicating that MESNA may act through its antioxidant properties or as a brevetoxin scavenger. These studies provide novel insights into new prophylactics for brevetoxicosis in humans and wildlife.

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.

Exploration of anti-leukemic effect of soft coral-derived 13-acetoxysarcocrassolide: Induction of apoptosis via oxidative stress as a potent inhibitor of heat shock protein 90 and topoisomerase II.

13-Acetoxysarcocrassolide (13-AC) is a marine cembranoid derived from the aquaculture soft coral of Lobophytum crassum. The cytotoxic effect of 13-AC against leukemia cells was previously reported but its mechanism of action is still unexplored. In the current study, we showed that 13-AC induced apoptosis of human acute lymphoblastic leukemia Molt4 cells, as evidenced by the cleavage of PARP and caspases, phosphatidylserine externalization, as well as the disruption of mitochondrial membrane potential. The use of N-acetylcysteine (NAC), a reactive oxygen species (ROS) scavenger, attenuated the cytotoxic effect induced by 13-AC. Molecular docking and thermal shift assay indicated that the cytotoxic mechanism of action of 13-AC involved the inhibition of heat shock protein 90 (Hsp 90) activity by eliciting the level of Hsp 70 and topoisomerase IIα in Molt4 cells. 13-AC also exhibited potent antitumor activity by reducing the tumor volume (48.3%) and weight (72.5%) in the in vivo Molt4 xenograft mice model. Our findings suggested that the marine cembranoid, 13-AC, acted as a dual inhibitor of Hsp 90 and topoisomerase IIα, exerting more potent apoptotic activity via the enhancement of ROS generation.

Abnormal expression of H-Ras induces S-phase arrest and mitotic catastrophe in human T-lymphocyte leukemia.

Leukemia is a neoplasm with high incidence and mortality rates. Mitotic death has been observed in tumor cells treated with chemotherapeutic agents. Ras family proteins participate in the transduction of signals involved in different processes, such as proliferation, differentiation, survival, and paradoxically, initiation of cell death. This study investigated the effect of H-Ras expression on human T-cell acute lymphoblastic leukemia MOLT-4 cells. Cells were electroporated with either wild-type (Raswt) or oncogenic mutant in codon 12 exon 1 (Rasmut) versions of H-Ras gene and stained for morphological analysis. Cell viability was assessed using trypan blue staining and cell cycle analysis using flow cytometry. H-Ras gene expression was determined using quantitative real-time reverse transcription polymerase chain reaction. The t, ANOVA, and Scheffe tests were used for statistical analysis. Human T-cell acute lymphoblastic leukemia MOLT-4 cells showed nuclear fragmentation and presence of multiple nuclei and micronuclei after transfection with either wt or mutant H-Ras genes. Cell cycle analysis revealed a statistically significant increase in cells in the S phase when transfected with either wt (83.67%, P<0.0005) or mutated (81.79%, P<0.0001) H-Ras genes. Although similar effects for both versions of H-Ras were found, cells transfected with the mutated version died at 120 h of mitotic catastrophe. Transfection of human T-cell acute lymphoblastic leukemia MOLT-4 cells with either normal or mutated H-Ras genes induced alterations in morphology, arrest in the S phase, and death by mitotic catastrophe.

In Vitro Investigation of the Anticancer Properties of Ammodaucus Leucotrichus Coss. & Dur.

Little is known about the pharmacological activity of Ammodaucus leucotrichus&amp;nbsp;Coss. & Dur., a small annual species that grows in the Saharan and sub-Saharan countries. In the present study, we investigated whether the standardized ethanolic extract of A. leucotrichus fruits and R-perillaldehyde, a monoterpenoid isolated from A. leucotrichus fruits, are able to affect different processes involved in different phases of cancer development. In particular, we explored their genoprotective, proapoptotic, antiproliferative, and cytodifferentiating potential on different human cell models. We analyzed the genoprotective and proapoptotic activity on human lymphoblast cells (TK6) using the micronucleus test, and the cytodifferentiation effects on human promyelocytic cells (HL60) through the evaluation of different markers of differentiation forward granulocytes or monocytes. The results showed that the extract and perillaldehyde were able to induce apoptosis and protect from clastogen-induced DNA damage. To our best knowledge, this is the first report on the ability of A. leucotrichus and perillaldehyde to induce apoptosis and protect DNA from the toxicity of different compounds. Data reported in this work are the starting point for their pharmacological use. Going forward, efforts to determine their effects on other events associated with cancer development, such as angiogenesis and metastasization, will provide important information and improve our understanding of their potential in cancer therapy.