T-lymphoblastic Cell Line Research Articles

Abstract 5865: Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines

Abstract Background: The enzyme purine nucleoside phosphorylase (PNP) is involved in the purine salvage pathway and is present at high levels in lymphoid tissues. Children born with PNP deficiency are known to have significantly reduced T-cell counts but relatively normal B-cell immunity, suggesting PNP as one of the most promising therapeutic targets for T-cell malignancies. Forodesine (For, BCX-1777), a novel PNP inhibitor, was approved in Japan for the treatment of relapsed/refractory PTCL. To understand the cytotoxic effect of For and its mechanism of action, we established a For-resistant (For-R) leukemia cell line and explored the mechanism of resistance. Methods: We established two For-R T-lymphoblastic leukemia cell lines (CCRF-CEM and MOLT4) through repeated escalating exposure to For and 2’-deoxyguanosined(dGuo). Gene expression analysis was performed to identify the mechanisms of resistance. Results: For-R cells showed very high resistance compared to parental cells (IC50 value, CEM/CEM-R 4.2nM/>5x105nM, MOLT4/MOLT4-R 8nM/>5x105nM). When cross-resistance was examined in these For-R cells showed mild resistance to cytarabine. The expression of hENT1, a nucleoside transporter, was examined and found to be upregulated in both CEM-R and MOLT4-R cells, while the expression level of deoxycytidine kinase (dCK) was unchanged in both cells. Next, high performance liquid chromatography was used to assess the intracellular dGTP in each parental and its For-R cells. In the parental cells, intracellular dGTP levels increased promptly 4-6 hours after fosteredin administration, whereas in resistant cells, intracellular dGTP levels were higher than before fosteredin administration, and there was little change in dGTP levels after administration. Conclusion: The established For-R cell line acquired a high resistance to For and showed high intracellular dGTP levels. Imbalance in intracellular GTP levels may be involved in the induction of cell death by For. IC50 value of each cell lines Citation Format: Naoko Hosono, Kana Oiwa, Toshiki Tasaki, Rie Nishi, Luigi Scott, Owen A. O’Connor, Takahiro Yamauchi. Characterization and resistance mechanisms of PNP inhibitor forodesin-resistant cell lines [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5865.

Design, Synthesis, Biological Evaluation, and Crystallographic Study of Novel Purine Nucleoside Phosphorylase Inhibitors.

Purine nucleoside phosphorylase (PNP) is a well-known molecular target with potential therapeutic applications in the treatment of T-cell malignancies and/or bacterial/parasitic infections. Here, we report the design, development of synthetic methodology, and biological evaluation of a series of 30 novel PNP inhibitors based on acyclic nucleoside phosphonates bearing a 9-deazahypoxanthine nucleobase. The strongest inhibitors exhibited IC50 values as low as 19 nM (human PNP) and 4 nM (Mycobacterium tuberculosis (Mt) PNP) and highly selective cytotoxicity toward various T-lymphoblastic cell lines with CC50 values as low as 9 nM. No cytotoxic effect was observed on other cancer cell lines (HeLa S3, HL60, HepG2) or primary PBMCs for up to 10 μM. We report the first example of the PNP inhibitor exhibiting over 60-fold selectivity for the pathogenic enzyme (MtPNP) over hPNP. The results are supported by a crystallographic study of eight enzyme-inhibitor complexes and by ADMET profiling in vitro and in vivo.

MiR-16-5p enhances sensitivity to RG7388 through targeting PPM1D expression (WIP1) in Childhood Acute Lymphoblastic Leukemia

Aim: Given the encouraging results of the p53-Mdm2 inhibitor RG7388 in clinical trials and the vital function of miR-16-5p in suppressing cell proliferation, the aim of the present study was to investigate the combined impact of RG7388 and miR-16-5p overexpression on the childhood acute lymphoblastic leukemia (chALL). Methods: miRTarBase and miRDB, along with KEGG and STRING databases, were used to predict miR-16-5p target genes and explore protein-protein interaction networks, respectively. B- and T-lymphoblastic cell lines, in addition to patient primary cells, were treated with RG7388. Ectopic overexpression of miR-16-5p in Nalm6 cell line was induced through cell electroporation and transfection of microRNA mimics was confirmed by qRT-PCR. Cell viability was evaluated using the MTT assay. Western blot analyses were performed to evaluate the effects of RG7388 and miR-16-5p upregulation on the protein levels of p53 and its downstream target genes in chALL cells. Paired sample t-test was employed for statistical analyses. Results: MTT assay showed RG7388-induced cytotoxicity in wild-type p53 Nalm6 cell line and p53 functional patient primary cells. However, CCRF-CEM and p53 non-functional leukemic cells indicated drug resistance. Western blot analyses validated the bioinformatics results, confirming the downregulation of WIP1, p53 stabilization, as well as overexpression of p21WAF1 and Mdm2 proteins in Nalm6 cells transfected with miR-16-5p. Moreover, enhanced sensitivity to RG7388 was observed in the transfected cells. Conclusion: This is the first study indicating the mechanistic importance of miR-16-5p overexpression in chALL and its inhibitory role in leukemia treatment when combined with the p53-Mdm2 antagonist, RG7388. These findings might be useful for researchers and clinicians to pave the way for better management of chALL.

Clinical Development of Senza5TM CART5: a Novel Dual Population CD5 CRISPR-Cas9 Knocked out Anti-CD5 Chimeric Antigen Receptor T Cell Product for Relapsed and Refractory CD5+ Nodal T-Cell Lymphomas

Introduction: Chimeric antigen receptor T-cell (CART) immunotherapy is FDA-approved for B-cell lymphoblastic leukemia, B-cell non-Hodgkin lymphomas, and multiple myeloma. However, CART immunotherapy for T-cell non-Hodgkin lymphomas (T-NHL) is lagging, with only limited clinical results. T-NHL have a poor prognosis and there is an unmet need for effective and long-lasting treatments. We previously presented preliminary preclinical results of anti-CD5 CAR T cells (CART5) that had CD5 knocked out (KO) (Blood (2020) 136 (Supplement 1): 51-52.). CD5 KO not only prevented CART fratricide but also dramatically enhanced CART function. Critical challenges to developing CART cells for T-NHL are CART fratricide and normal T-cell toxicity. In this study, we addressed these challenges by improving manufacturing and including a dual-population technology to mitigate toxicity. We present the development of Senza5TM CART5 an autologous good-manufacturing practice (GMP)-grade product (Fig.1) that will be tested in a phase I clinical trial for relapsed and refractory CD5+ nodal T-cell lymphomas. Methods and Results: We first identified a single guide RNA (sgRNA) that deletes CD5 in T cells with >90% KO efficiency by flow cytometry and ICE analysis. We then assessed the specificity of the KO by performing iGUIDE sequencing. We confirmed high on-target CD5 cleavage with minimal off-targets (CALCP, C20orf85, INPP4B, XPO7, and SLC10A7). These genes have little to no expression in naïve CD4+ and CD8+ T cells by RNA-seq (DICE). Targeted functional assays are ongoing. For the CAR construct, we selected a lead CAR5 among six 4-1BB-constructs based on its superior anti-tumor function in NOD-SCID IL2Rgnull (NSG) mice engrafted with a Jurkat T-lymphoblastic leukemia cell line. Furthermore, to confirm the specificity of the CAR5 for CD5, we screened CAR5 T cells for interactions with HEK293 cells expressing 5,828 human plasma membrane proteins or cell-surface tethered secreted proteins and 398 human heterodimers (Retrogenix assay). The lead CAR5 did not bind to any targets besides CD5. For an optimized GMP-grade product, we modified our manufacturing protocol, shortening it from 10 to 5 days. Moreover, KO efficiency and safety were optimized to reduce off-target activity without loss of efficiency. We also optimized the activation strategy to decrease cell loss. Functional studies were performed to demonstrate the comparability of these methods. With these improvements, we were able to achieve >90% CD5 KO with >30% CAR transduction, thereby allowing the generation of two main populations: one of CD5KO CART5 cells and the other of CD5KO normal untransduced T cells. CD5KO CART5 are expected to target CD5+ T-NHL cells but could also lead to normal T-cell toxicity. This toxicity will be mitigated by the CD5KO normal T cells that will have a survival advantage after infusion (Fig.1) and have been demonstrated to maintain reactivity against opportunistic pathogens such as CMV and EBV. To evaluate Senza5TMCART5 safety, we performed cytokine-independent CART proliferation and long- and short-term xenograft models. Senza5TM CART5 cells did not grow aberrantly without growth/survival-associated cytokines (IL2/7/15). We then used NSG mice to exclude uncontrolled Senza5TM CART5 growth and observed enhanced expansion and proliferation of Senza5TM CART5 with a xenogeneic GVHD rate similar to that of control mice with similar wild-type CART5 expansion. Since the CAR5 does not cross-react with murine CD5, we tested non-specific toxicity in tissues using an NSG xenograft model of Jurkat. On Day 10 post-CAR T injections, animals were euthanized, and a complete necropsy was performed on all animals. Senza5TM CART5 demonstrated tumor clearance with no detection of treatment-related lesions. Mice treated with untransduced T cells or CART19 demonstrated significant overall and organ-specific weight loss compared to Senza5TM CART5. Blood analyses determined no differences in their clinical chemistry profile or complete blood counts. Conclusion: In this study, we developed a novel dual-population, full-scale GMP-ready CD5 KO anti-CD5 CART product for T-cell lymphomas characterized by enhanced anti-tumor effect and a strategy to potentially mitigate toxicity. Autologous Senza5TM CART5 will be tested for CD5+ nodal T-cell lymphomas in a phase I clinical trial. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

C9Mab-1: An Anti-Mouse CCR9 Monoclonal Antibody for Immunocytochemistry.

C-C motif chemokine receptor 9 (CCR9) is a G protein-coupled receptor, which is highly expressed in T-lymphocytes and different cancer cells. CCR9 aggravates immune diseases and cancer progression and is considered a biomarker and a therapeutic target of diseases. The development of specific monoclonal antibody (mAbs) for human CCR9 (hCCR9) is required to diagnose and treat immune diseases and cancers. Previously, we established the cell-based immunization and screening (CBIS) method, which does not need purified target proteins. Anti-hCCR9 mAb (clone C9Mab-1; mouse IgG1, kappa) was also developed using the CBIS method. C9Mab-1 is usable for flow cytometry against exogenously and endogenously expressing hCCR9. This study showed that C9Mab-1 and its recombinant antibody (recC9Mab-1) specifically detected exogenous hCCR9 stably overexpressed in Chinese hamster ovary (CHO)-K1 cells and endogenous hCCR9 expressed in a human T-lymphoblastic leukemia cell line MOLT-4 cells through immunocytochemistry. This study provides a new application of C9Mab-1 and recC9Mab-1 in immunocytochemistry.

Exposure to emamectin benzoate confers cytotoxic effects on human molt-4 T-cells and possible ameliorative role of vitamin E and dithiothreitol

Emamectin benzoate (EMB) is an avermectin insecticide that is extensively used for pest control, but there are few reports concerning its cytotoxic effects on human lymphocytes. In the current study, the hematotoxicity of EMB was evaluated in Molt-4 T-cells, a human T-lymphoblastic cell line with high motility, and the role of vitamin E (VitE) and dithiothreitol (DTT) in attenuating EMB cytotoxicity was characterized. Exposure of Molt-4 cells to EMB decreased cell viability and proliferation, induced a loss of cell clusters, and significantly increased membrane collapse and chromatin condensation. Moreover, EMB significantly increased cell death and suppressed transglutaminase activity. EMB treatment modulated the NF-κB signaling pathway, decreased the expression of p105, p50, and p65/RelA in cytosolic and nuclear fractions, and increased nuclear IκBα expression. EMB increased oxidative stress, as demonstrated by a significant increase in the levels of reactive oxygen species (ROS). Treatment with non-cytotoxic concentrations of VitE or DTT ameliorated the hematotoxicity induced by pretreatment with EMB, increased Molt-4 cell viability, raised the IC50 values of EMB, limited intracellular ROS generation, and mitigated EMB-mediated effects on NF-κB signaling. The results indicate the potential cytotoxicity of EMB on human lymphocytes, and demonstrate that VitE and DTT treatment can reduce the cytotoxic effects of EMB.

Characterization of newly established Pralatrexate-resistant cell lines and the mechanisms of resistance

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.

The Alcoholic Bark Extract of Terminalia Arjuna Exhibits Cytotoxic and Cytostatic Activity on Jurkat Leukemia Cells

Background: Natural products are characterized by a complex chemical composition and are capable of concurrently modulate several signalling pathways. Considering the biological complexity of carcinogenesis, natural products represent key components of the therapeutic armamentarium for oncological diseases. The bark of Terminalia arjuna is used in traditional Ayurvedic medicine for its astringent, expectorant, cardiotonic, styptic, and antidysenteric properties. Alongside its traditional uses, Terminalia arjuna exhibits different biological activities including antimutagenic and anticarcinogenic. Objective: This study was designed to evaluate the toxic effects of an alcoholic extract obtained from the bark of T. arjuna on a human T-lymphoblastic cell line (Jurkat). We explored the phytochemical composition and investigated the cytotoxic, cytostatic, genotoxic, and anti-genotoxic effects. Methods: The phytochemical composition was analyzed using spectrophotometric methods; all the biological endpoints were assessed through flow cytometry. Results: The phytochemical screening showed that polyphenols represent about 64% of the extract. Moreover, the extract was cytotoxic on Jurkat cells by inducing both apoptosis and necrosis, and blocked the cell cycle in the G2/M phase. Additionally, it was found that the extract lacks any genotoxic effect, but was not effective in protecting Jurkat cells from the DNA damage induced by H2O2 and etoposide. Conclusion: The results of our study show the toxic effects of Terminalia arjuna on Jurkat cells and confirm the pivotal role played by natural compounds in the oncological field. Further studies should be performed to better understand its clinical potential and deepen its toxicological profile.

Control of oxidative stress in Jurkat cells as a model of leukemia treatment

This paper describes proof-of-concept for antileukemia treatment based on the reactive oxygen species (ROS) induction supplemented with magnetic nanoparticles in the presence of moderate-intensity magnetic fields. The T-lymphoblastic leukemia cell line (Jurkat) was chosen as an in vitro model of acute lymphoblastic leukemia. The elevated ROS levels were generated by iron oxide magnetic nanoparticles. We found an enhanced cytotoxic effect of magnetic nanoparticles in the presence of a magnetic field on cancerous Jurkat cell line due to induced oxidative stress after cultivation in magnetic fields with an average strength of 0.26–0.36 T and the field spatial gradient up to 100 T/m. At the same conditions of nanoparticles intake, the viability of human peripheral blood mononuclear cells (model of healthy cells) was not inhibited. Thus, we demonstrated the feasibility of antileukemia treatment by magnetic nanoparticles controlled by a magnetic field.

Photoluminescent Detection of Human T-Lymphoblastic Cells by ZnO Nanorods.

The precise detection of cancer cells currently remains a global challenge. One-dimensional (1D) semiconductor nanostructures (e.g., ZnO nanorods) have attracted attention due to their potential use in cancer biosensors. In the current study, it was demonstrated that the possibility of a photoluminescent detection of human leukemic T-cells by using a zinc oxide nanorods (ZnO NRs) platform. Monoclonal antibodies (MABs) anti-CD5 against a cluster of differentiation (CD) proteins on the pathologic cell surface have been used as a bioselective layer on the ZnO surface. The optimal concentration of the protein anti-CD5 to form an effective bioselective layer on the ZnO NRs surface was selected. The novel biosensing platforms based on glass/ZnO NRs/anti-CD5 were tested towards the human T-lymphoblast cell line MOLT-4 derived from patients with acute lymphoblastic leukemia. The control tests towards MOLT-4 cells were performed by using the glass/ZnO NRs/anti-IgG2a system as a negative control. It was shown that the photoluminescence signal of the glass/ZnO NRs/anti-CD5 system increased after adsorption of T-lymphoblast MOLT-4 cells on the biosensor surface. The increase in the ZnO NRs photoluminescence intensity correlated with the number of CD5-positive MOLT-4 cells in the investigated population (controlled by using flow cytometry). Perspectives of the developed ZnO platforms as an efficient cancer cell biosensor were discussed.

077 RNA interference screening for novel cytokine and chemokine factors regulating HIV-1 trans-infection from dendritic cells to CD4+ T-cells

The propagation of HIV-1 is driven in part by cell-to-cell transmission most frequently observed in dendritic cells (DC) and their subsets - one of the earliest immune cells likely to encounter HIV-1 during acute infection at mucosal surfaces. DCs are capable of highly effective viral transfer to target CD4+ T-cells across the virological synapse, a specialised virus-induced cell junction, which enables wide-spread viral dissemination and accelerated disease progression. Our previous findings have implicated a major role for cytokines and chemokines in the infection and transmission of HIV-1 from DC subsets, though a global study of their molecular functions has yet to be completed. In this study, we screened 319 individual genes using the Human ON-TARGET plus SMARTpool cytokine & chemokine siRNA library to investigate the differential effects on HIV-1 transfer from monocyte-derived DCs (MDDC) to a CD4+ SupT1 T-lymphoblastic cell line. Using integrative, data-driven approaches we successfully identified several cytokine superfamilies with potent restrictive properties against HIV-1 trans-infection from MDDC to SupT1. The activities of these candidates were validated using three key loss-of-function assays including genetic downregulation, neutralisation by biologics and pharmacological inhibition in trans- to both SupT1 and autologous CD4+ T-cells. Disruption of specific cytokine-driven mechanisms in MDDC results in dramatic changes in the capacity for cell-to-cell transfer to CD4+ T-cells. These findings add to a growing body of evidence linking the cytokine network which will inform novel therapeutic strategies against early-stage HIV-1 infection and transmission

Changes in the Radiation Toxicity of Human Lymphoblastic T-cell Line (Jurkat) by a Common Pesticide: Diazinon.

Background: Diazinon is one of the most common pesticides in the world playing a similar role to radiation and it could cause DNA breaks and genetic effects.Objective: In this study, radiosensitivity of a lymphoblastic cell line pretreated by Diazinon was investigated.Material and Methods: In this case-control study, the human lymphoblastic T-cell line was divided into 6 groups based on receiving radiation or/and Diazinon. After that, the DNA damage, in all of the groups, were counted by cytokinesis-block micronuclei assay using different indices. Results: The mean frequency of micronuclei, nuclear bridges and nuclear buds in cell groups exposed by both Diazinon and radiation were remarkably higher than the other groups which just received radiation or Diazinon alone. The interaction between radiation and Diazinon treatment was statistically significant for NBUDS index.Conclusion: The results indicated that the Diazinon contamination could affect the radiosensitivity index of cancerous cells while further molecular and in-vivo studies are needed to investigate genetic and toxic effects of Diazinon on DNA and its repair system.

2-Deoxyglycoside Conjugates of Lupane Triterpenoids with High Cytotoxic Activity-Synthesis, Activity, and Pharmacokinetic Profile.

A set of 41 glycosidic conjugates of pentacyclic triterpenes was synthesized in order to improve the solubility of highly cytotoxic parent compounds. Their in vitro cytotoxic activity was evaluated in 25 cancer cell lines and 2 noncancer fibroblasts. Fifteen compounds had high cytotoxicity on the T-lymphoblastic leukemia cell line CCRF-CEM and 6 of them were active in multiple cell lines of various histogenic origin and not toxic in fibroblasts. Compound 11a had IC50 of 0.64 μM in CCRF-CEM cells, 0.60 μM in K-562 cells, and 0.37 μM in PC-3 cells; compound 12a had IC50 of 0.64 μM in CCRF-CEM cells and 0.71 μM in SW620 cells; compound 17b had IC50 of 0.86 μM in HCT116 cells and 0.92 μM in PC-3 cells. Compounds 11b and 12b were slightly less active than the previously mentioned derivatives; however, their solubility was significantly better, and therefore they were selected for the in vivo evaluation of the pharmacokinetic profile in mice. In both compounds, the maximum concentration in plasma was achieved very rapidly-the highest level in plasma was found 1 h after administration (22.2, respectively, 6.4 μM). For compound 12b, the resorption was followed with fast elimination, and 12 h after administration, the compound was not detected in plasma. In contrast, compound 11b was eliminated more slowly; it was still present in plasma after 12 h, but its concentration dropped below the detection limit after 24 h. The elimination half-time determined for compound 11b was 2.4 h and for compound 12b just about 1.4 h. These values are reasonable for further drug development.

THE EFFECT OF HOMALANTHUS POPULNEUS (GIESEL.) PAX. EXTRACT IN EXPRESSION OF T-CELL RECEPTOR: INHIBITION STUDY OF HIV INFECTION

Objective: The aim of this study is to analyze the effect of Homalanthus populneus’s extract toward the expression of CD4 and CD8 which both areimportant in body’s defense mechanism against HIV.Methods: Leaves and barks of H. populneus were extracted with 70% ethanol. Freeze dry method had been used in order to get the final extract.PBMCs were extracted and it has been used for CD4 and CD8 expression test using flow cytometry. In oreder to analyze the effect of the extract to theHIV type 1, gp41 and gp120 expression was tested using talicytometry and using enzyme-linked immunosorbent assay, respectively.Results: This study reported that H. populneus’s extract reduced the expression of CD4 receptor in both peripheral blood mononuclear cell (PBMC)and T-lymphoblast cell line (CEM). In contract, this extract increased CD8 expression in PBMC. It was also able to reduce the percentage of proteingp41 and gp120 in CEM cultures.Conclusion: Those results show that H. populneus’s extract is potentially developed as an HIV drug from Indonesia. However, further study needs tobe done including analyzing the effect of variety of concentrations and also exposure periods.

Pterostilbene increases Fas expression in T-lymphoblastic leukemia cell lines.

Treatment of acute lymphoblastic leukemia (ALL) has been promising in last decades, but side effects still persist and searching for the least toxic agents continue. Pterostilbene (PTE) is a natural compound with several anti-cancer and anti-oxidant properties. Fas, as a member of death inducing family of tumor necrosis factor (TNF) receptors with an intracellular death domain, can initiate the extrinsic apoptosis signaling pathway. Here after the half maximal inhibitory concentration (IC50) determination in cell lines, we searched for PTE effects on Fas, both in mRNA and surface levels in two ALL cell lines, Jurkat and Molt-4. After harvesting cells in optimum situations, MTS assay was used to determine IC50 concentrations. Real-time polymerase chain reaction (RT-PCR) and flow cytometry were performed for Fas mRNA and surface expression variations after exposure to PTE. The findings showed that PTE decreases cell viability with different extent in two ALL cell lines. In addition to inducing apoptosis, it can increase Fas in both gene and cell surface expression in the same concentrations. Pterostilbene as a natural anti-cancer agent can increase Fas expression both in mRNA and surface levels that results in apoptosis signal transduction improvement which sensitizes cells to apoptosis by immune effector cells. As a result, abnormal cells removal would be more efficiently with the minimum side effects on normal cells.

Pralatrexate-Resistant Cell Lines Show Intact Internalization Via RFC1 but Collateral Sensitivity to Nucleoside Analogs

BackgroundPralatrexate (PDX) is a novel dihydrofolate reductase (DHFR) inhibitor which is designed to have a high affinity for reduced folate carrier (RFC1), DHFR, and folylpolyglutamate synthetase (FPGS), resulting in greater internalization and accumulation in tumor cells than methotrexate. PDX has been approved by the U.S. FDA and many international health care ministries for the treatment of relapsed and refractory peripheral T-cell lymphoma (R/R PTCL). A recent case match control analysis has confirmed a survival advantage for patients enrolled on PROPEL compared to a well-matched population of patients receiving standard of care. Recently, PDX has demonstrated compelling activity in combination with romidepsin in R/R PTCL patients, where is role is likely to expand. In this study we explored the mechanisms of resistance to PDX and approaches to overcome them, including combination strategies using PDX-resistant (PDX-R) leukemic cell lines.MethodsT-lymphoblastic leukemia cell lines, CCRF-CEM (CEM), MOLT-4 (MOLT4), and MOLT-3 (MOLT3), were used. To establish PDX-R cell lines, the cells were initially incubated with low-concentrations of PDX, and then concentrations of the drug were gradually increased. After acquisition of PDX-R, the single-cell separation was performed by a limiting dilution. Microarray analysis was performed in CEM, MOLT4 and each PDX-R counterpart.ResultsThe 50% inhibitory concentration (IC50) values of PDX against CEM, MOLT4 and MOLT3 were 0.6 nM, 2.4 nM and 1.4 nM, respectively, while the PDX-R cell lines exhibited IC50 values of 20 nM, 80 nM and 40 nM, respectively. After 48-hour treatment with PDX, annexin-V positivity was significantly reduced in PDX-R cells (in CEM, MOLT4 and MOLT3 was 78.7%, 77.7%, 97.4% vs 4.4%, 5.9%, 6.6%, respectively). The expression of RFC1 was not altered in PDX-R cells, nor was any acquired mutation of RFC1 detected in cell lines. Intracellular uptake of [14C]-PDX was modestly decreased in CEM/PDX-R, while there was no difference of it in MOLT4/PDX-R, suggesting that the resistance mechanism was not due to the inability to internalize drug. Gene expression array revealed that the expression of DNA-methyltransferase 3 beta (DNMT3b) was significantly elevated in both CEM/PDX-R (12.8 times) and MOLT4/PDX-R (2.8 times) cells, whereas there was no significant difference in that of DHFR, FPGS and thymidylate synthase. When we explored the profile of cross-resistances in PDX-R cells, it was surprising that PDX-R cells were more sensitive to nucleoside analogs, including cytarabine (AraC) and forodesine (FDS). After 72 hours of FDS-treatment, induction of apoptosis was seen in both parental and PDX-R cells, suggesting that the mechanisms of PDX-R is not cross resistant. In addition, there was no cross-resistance to bortezomib or panobinostat in PDX-R cell lines.DiscussionWe established PDX-R cell lines which were around 30-fold resistant to PDX compared to their parental counterparts. The characteristics of PDX-R cells are as follows: (i) intact internalization of PDX, (ii) intact apoptotic mechanisms, (iii) no cross-resistance to BOR or LBH, (iv) collateral sensitive to nucleoside analogs, such as AraC and FDS. These data suggest that the mechanism of acquired resistance to PDX may be associate with not only cellular uptake of PDX, but also DNA methylation and/or nucleic-acid metabolism. These findings may be underscored by the fact that nucleoside analogs exhibited greater potency in the PDX-R cells in vitro. Next steps are focused on exploring the methylation status of these genes involved in PDX activity, and their merits of combining with hypomethylating agents.ConclusionsWe established PDX-R T-lymphoblastic leukemia cell lines which show collateral sensitivity to nucleoside analogs. The mechanisms of PDX resistance is not always cellular uptake of PDX, epigenetic alteration might contribute to the development PDX-resistance. Nevertheless, the new T-lymphoma-targeting agent, FDS can be effective in this setting. Our results may encourage further investigation to overcome resistance of PDX for patients with R/R PTCL. DisclosuresO'Connor:ADC Therapeutics: Research Funding; Seattle Genetics: Research Funding; Celgene: Research Funding.

GLI1 and CTNNB1 Knockdown Activates NOTCH and mTOR Signalling in NB4 Myeloid Leukaemia Cells.

Hedgehog (HH), WNT, NOTCH, and mechanistic target of rapamycin (mTOR) signalling pathways are known to regulate the progression of cancer; however, their interaction in leukaemia cells is not fully clarified. Myeloid and T-lymphoblastic leukaemia cell lines (NB4, THP-1, Jurkat, and DND-41) were transfected with small interfering RNAs targeting the glioma-associated oncogene homolog 1 (GLI1) and catenin beta-1 (CTNNB1) genes involved in the regulation of HH and WNT pathways, respectively, and we examined cell proliferation and gene expression. The knockdown of GLI1 and CTNNB1 did not significantly affect proliferation of any cell line; however, it up-regulated the expression of NOTCH1, cleaved NOTCH1 fragment, and phosphorylated mTOR in NB4 cells, but not in the other cell lines. Our data suggest that HH and WNT act upstream of NOTCH and mTOR pathways and negatively regulate them in myeloid NB4 cells. Further studies are required to determine the biological significance of this signalling crosstalk in leukaemia.

Modulation of multidrug resistant in cancer cells by EGCG, tannic acid and curcumin

Background: Cancer is one of the most common life-threatening diseases worldwide; many patients develop multidrug resistance after treatment with anticancer drugs. The main mechanism leading to multidrug resistance is the overexpression of ABC transporters in cancer cells. Chemosensitizers are needed to inhibit the activity of ABC transporters, resulting in higer intracellular concentration of anticancer drugs. Some secondary metabolites have been reported to be chemosensitizers by inhibiting ABC transporters. Epigallocatechin gallate (EGCG), tannic acid, and curcumin were employed in this study. Different assays were used to detect whether they have the ability to inhibit P-gp activity and overcome multidrug resistance in cancer cells overexpressing P-gp. Hypothesis/Purpose: CEM/ADR 5000 and Caco-2 cell lines, which overexpress P-gp, are multidrug resistant cell lines. We first detected whether the combination of polyphenols (EGCG, tannic acid, curcumin) and doxorubicin, an anticancer drug, is synergistic or not. To further understand the potential mechanism, EGCG, tannic acid, and curcumin were tested to check whether they have the ability to inhibit P-gp activity. When P-gp activity is inhibited, the intracellular concentration of doxorubicin is higher, resulting in enhanced cytotoxicity of doxorubicin. Study design: The P-gp overexpressing human colon cancer cell line Caco-2 and human T-lymphoblastic leukemia cell line CEM/ADR 5000 were used in this study. Two-drug combinations (doxorubicin + polyphenol) and three-drug combinations (doxorubicin + polyphenol + digitonin) were tested to examine potential synergism. The potential mechanism leading to synergism would be the inhibition of P-gp activity. A Rhodamine 123 assay and Calcein-AM assay in Caco-2 and CEM/ADR 5000, respectively, were used to detect P-gp inhibition by EGCG, curcumin, and tannic acid. Methods: MTT assay was used to determine the cytotoxicity of doxorubicin, polyphenols and digitonin alone, and then their combinations. Furthermore, Rhodamine 123 and Calcein-AM were used to detect the effects of polyphenols on the activity of P-gp. Results: The results demonstrated that a combination of non-toxic concentrations of each polyphenol with doxorubicin synergistically sensitized Caco-2 and CEM/ADR 5000 cells. Furthermore, three-drug combinations (doxorubicin + polyphenol + digitonin) were much more effective. In addition, the activity of P-gp in Caco-2 and CEM/ADR 5000 cells was measured. Consistent with the combination results, tannic acid and curcumin decreased the activity of P-gp both in Caco-2 and CEM/ADR 5000. EGCG, which weakly affected the activity of P-gp in CEM/ADR 5000, only had an effect on P-gp under higher concentration in Caco-2 cells. Conclusion: Our results show that EGCG, curcumin, and tannic acid, when combined with doxorubicin, can exert synergism, mediated by a reduced activity of P-gp. This study suggests that polyphenols, by modulating the activity of P-gp, may be used as chemosensitisers.

Enniatin B induces expression changes in the electron transport chain pathway related genes in lymphoblastic T-cell line

Enniatin B is a ionophoric and lipophilic mycotoxin which reaches the bloodstream and has the ability to penetrate into cellular membranes. The purpose of this study was to reveal changes in the gene expression profile caused by enniatin B in human Jurkat lymphoblastic T-cells after 24 h of exposure at 1.5, 3 and 5 μM by next generation sequencing. It was found that up to 27% of human genome expression levels were significantly altered (5750 genes for both down-regulation and up-regulation). In the three enniatin B concentrations studied 245 differentially expressed genes were found to be overlapped, 83 were down and 162 up-regulated. ConsensusPathDB analysis of over-representation of differentially expressed genes provided a list of gene ontology terms in which several biological processes related to nucleoside monophosphate metabolic process, respiratory chain complex, electron transport chain, oxidative phosphorylation and cellular respiration were the most altered. Also, an interesting correlation was found between enniatin B toxicity and the up-regulation of the UCP protein complex. In summary, the transcriptomic analysis revealed that mitochondria are the organelles showing more related differentially expressed genes. Consequently, differentially expressed genes involved in biological processes, molecular functions and pathways related to mitochondrial metabolism and respiration were significantly changed.

Triterpenic azines, a new class of compounds with selective cytotoxicity to leukemia cells CCRF-CEM.

From betulinic acid (1a), we synthesized 30-oxobetulinic acid (2a) that is highly cytotoxic against many cancer cell lines; however, its generic toxicity is the main obstacle in further development as cytostatic. Methodology & results: From 2a, we prepared a new class of compounds - nonsymmetrical azines and tested their in vitro cytotoxicity. All new azines with a free 28-COOH group (4a-4e) were highly and selectively cytotoxic against the T-lymphoblastic leukemia cell line CCRF-CEM and exhibited dose-dependent inhibition of RNA and DNA synthesis and other cell-cycle alterations, including the M-phase block. The potential use of azines (4a-4e) in drug development focused on hematological cancers is significantly higher than that of previously studied acids 1a and 2a.