HUT-78 Cells Research Articles

Preclinical Efficacy and Biological Effects of Venetoclax and Ixazomib in Combination in Lymphoma Cells

Introduction. Bcl-2 family proteins comprise anti-apoptotic and pro-apoptotic proteins. Interaction between these proteins, as well as severe regulation of their expression, mediates cell survival and can quickly induce cell death. Venetoclax is Bcl-2-targeting that has shown preclinical and clinical activity in hematologic malignancies. Due to the development of resistance and the loss of dependence on the target protein, the monotherapy may be insufficient for maximal effectiveness. To circumvent the resistance mechanisms, many preclinical studies have shown that combination of venetoclax with other agents may represent a more effective therapeutic strategy. Ubiquitin-proteasome signaling pathway is a potential target that plays an important role in the proteolysis of key regulatory proteins. Proteasome inhibitors include ixazomib that inhibits cell growth and induces apoptosis in hematological malignancies cells resistant to conventional therapies and bortezomib. Objective: To analyze the preclinical efficacy and associated biological effects of venetoclax combined with ixazomib in a panel of lymphoma cell lines with diverse expression levels of Bcl-2 and other Bcl-2 family proteins. Methods: 12 lymphoma cell lines including FL (RL, WSU-NHL, Karpas422), MCL (Jeko1, Granta519), DLBCL (OCI-LY3, OCI-LY18), CTCL (Hut-78), ALCL (Karpas299), HL (L1236, L540), CLL (Mec1) and two MCL primary patient samples were exposed to venetoclax (0.01 - 8 µM) and ixazomib (10 - 2000 nM) alone for 24 - 72 hours to calculate IC50. Subsequently, lymphoma cells were exposed to venetoclax (0.015 - 25 nM) in combination with ixazomib (0.015 - 0.5 nM) for 24 hours. Cell viability was determined by MTT. Coefficient of synergy (combination index - CI) was calculated using CalcuSyn. Cell cycle and induction of apoptosis were evaluated by flow cytometry and changes in Bcl-2 family members, caspase activation and AKT phosphorylation were determined by western blotting. Results. In vitro, venetoclax and ixazomib alone induced cell death in a dose- and time-dependent manner against lymphoma cell lines. The IC50 is between 0.5 and 8 µM for venetoclax and between 12 and 1250 nM for ixazomib. The combination of venetoclax (0.03, 0.06, 12.5, 25 nM) with ixazomib (0.03, 0.06, 0.25, 0.5 nM) produced a synergistic effect (CI < 1) after 24 h of treatment in the most lymphoma cells lines leading to inhibition of cell growth and induction of apoptosis between 26 % and 59 % accompanied by increased with cleavage of caspases-3, -9 and PARP. We observed an additive effect (CI = 1) in Jeko1 (MCL) and MEC1 cells (CLL) and antagonist effect (CI > 1) Hut-78 cells (CTCL). Synergistic effect has been seen in two MCL primary patient samples (CI = 0.5 - 0.7). In sensitive lymphoma cells, the combination abrogated colony formation in the methylcellulose medium. When lymphoma cell lines were co-cultured with mesenchymal stromal cells with both drugs we observed a decrease of cell viability and a fraction of apoptotic cells indicating that drug combination may overcome the tumor promoting effects of stromal cells. The apoptosis induced in FL and Granta519 cells (MCL) by drug combination was accompanied by partial downregulation of Bcl-2 and strong upregulation of Bax, Bad, Bim and Noxa proteins. Jeko-1 cells were less sensitive to venetoclax-ixazomib combination-induced apoptosis. Western blot analysis showed a differential expression of Bcl-2, Mcl-1 and Bcl-XL proteins in FL, MCL and HL cell lines. Jeko-1 cells showed a normal expression of Bcl-2 and Mcl-1 proteins and high Bcl-xL protein level. Co-expression of related anti-apoptotic Bcl-2 family proteins could limit activity of treatment. Combined treatment induced G0/G1 cell cycle arrest and increased the sub-G1 population that was linked by the upregulation of p27 and p21. In addition, in RL, WSU-NHL and Granta519, enhanced cell death is associated with AKT inactivation and with a reduction of p-4EBP1, leading to decreased levels of c-MYC. Conclusion. Venetoclax exhibits strong synergistic activity with ixazomib in lymphoma cells. Studies are still ongoing and signaling pathways that promote the combination of venetoclax with ixazomib are to be analyzed. These data offer a rationale to continue exploring venetoclax-ixazomib combination and suggest that suppression of Bcl-2 family protein driven survival signaling may be one important mechanism for combination synergy. Disclosures No relevant conflicts of interest to declare.

Structural and biochemical modifications of model and native membranes of human immune cells in response to the action of zinc oxide nanoparticles.

The development of nanotechnology has led to the increased production of zinc oxide nanoparticles (ZnO-NPs) and their application in a wide variety of everyday products. It creates the need for a full assessment of their safety for humans. The aim of the study was to assess the toxic effects of ZnO-NPs on model human cells of the immune system: U-937, HL-60, HUT-78, and COLO-720L. Particular attention was paid to the direct interaction of the nanoparticles with membrane lipids and the role of zinc ions in the mechanism of their toxicity. Cell viability, lipid peroxidation, cell membrane integrity, and the amount of zinc ions released from nanoparticles were tested. Disruption in cell metabolism was noted for ZnO-NPs concentrations from 6.25 mg/L. Contact with ZnO-NPs caused lipid peroxidation of all cells and correlated with membrane disruption of HL-60, HUT-78, and COLO-720L cells. Model monolayers (Langmuir technique) were used to assess the interaction of the nanoparticles with the studied lipids. Physicochemical parameters, such as the area per molecule at maximal layer compression, the pressure at which the monolayer collapses, and the static compression modulus, were calculated. The models of the HL-60 and U-937 cell membranes under ZnO-NPs treatment reacted in a different way. It has also been shown that Zn2+ are not the main causative factor of ZnO-NPs toxicity. Investigating the early stages of mechanism of nanoparticles toxicity will allow for a more complete risk assessment and development of methods for a safer synthesis of engineering nanomaterials.

Design of PEG-modified magnetic nanoporous silica based miltefosine delivery system: Experimental and theoretical approaches

Silica nanoparticles incorporating magnetic iron oxides (MS) with sizes around 20 nm were developed. The MS material was modified with NH 2 -groups and then grafted by PEG chains. The anticancer drug miltefosine was loaded into the NH 2 -modified and PEGylated MS nanoparticles by impregnation procedure resulting in 11–28% of drug content. PEG chains were also conjugated to the miltefosine loaded MS-NH 2 nanocarrier. The amount of loaded drug and the release properties depend on whether PEGylation of the nanoparticles was performed before or after the drug loading step. The parent and drug loaded samples were characterized by XRD, N 2 physisorption, thermal gravimetric analysis, magnetic measurements, XPS and ATR FT-IR spectroscopy. ATR FT-IR spectroscopic data and DFT calculations supported the interaction between the mesoporous silica surface and miltefosine molecules. The in vitro release profile at pH = 7.0 showed sustained drug release and dependence on the experimental design. Free and formulated miltefosine samples were further investigated for antiproliferative activity against malignant HUT-78 cell line. Formulated miltefosine showed superior cytotoxic effect on the tested cells as compared to the free drug. In addition, the MTT assay of the cytotoxic potential of non-loaded silica nanocomposites showed no intrinsic cytotoxicity associated with the carriers. • Novel spherical PEGylated magnetic mesoporous silica nanoparticles were developed • The obtained nanoparticles were successfully loaded with miltefosine • Miltefosine loading and the release rate were affected by carrier modification • The cytotoxicity of the drugs loaded formulations on human tumor cells was preserved

Prolonging the Half-Life of Histone Deacetylase Inhibitor Belinostat via 50 nm Scale Liposomal Subcutaneous Delivery System for Peripheral T-Cell Lymphoma.

Simple SummaryBelinostat is the novel histone deacetylase inhibitors (HDACis) for treatment for peripheral T-cell lymphoma (PTCL). However, the half-life of belinostat is only 1.1 h. The aim of the study was to improve the half-life and it’s in vivo circulation behavior by using liposome encapsulation technology. The 50 nm scale liposomes were prepared, which showed the sustained release behavior, decrease the burst effect and improving the drug’s toxicity and had similar power for HuT-78 cells. Moreover, we proposed that phospholipid types are crucial factors for size forming and in vivo circulation behavior. We found that DOPC phospholipid material increased the half-life of belinostat, decreased clearance and presented a higher area under curve exposure. Due to the lymphatic delivery complexation, the localized at the lymphatic organs study is necessary to evaluate in the near future.Lymph node metastasis is an aggressive condition characterized by poor treatment outcomes and low overall survival. Belinostat is a novel histone deacetylase (HDAC) inhibitor approved by the Food and Drug Administration (FDA) for the treatment of relapsed peripheral T-cell lymphoma (PTCL). However, the major problem is that belinostat has a short half-life of 1.1 h. In this study, we successfully prepared 50 nm liposomal colloids, which showed a controlled release pattern and excellent pharmacokinetics. The results showed that the particle size of liposomes consisting of dioleoylphosphatidylcholine (DOPC) was larger than that of those consisting of dioleoylglycerophosphoserine (DOPS). In terms of release kinetics of belinostat, the free drug was rapidly released and showed lower area under curve (AUC) exposure for in vivo pharmacokinetics. When liposomal formulations were employed, the release pattern was fitted with Hixson–Crowell models and showed sustained release of belinostat. Moreover, HuT-78 cells were able to take up all the liposomes in a concentration-dependent manner. The safety assessment confirmed hemocompatibility, and the platelet count was increased. Furthermore, the liposomes consisting of DOPC or DOPS had different behavior patterns, and their delivery to lymphatic regions should be thoroughly investigated in the future.

LW-213 induces cell apoptosis in human cutaneous T-cell lymphomas by activating PERK-eIF2α-ATF4-CHOP axis.

Cutaneous T-cell lymphoma (CTCL) is characterized by a heterogeneous group of extranodal non-Hodgkin lymphomas, in which monoclonal T lymphocytes infiltrate the skin. LW-213, a derivative of wogonin, was found to induce cell apoptosis in chronic myeloid leukemia (CML). In this study, we investigated the effects of LW-213 on CTCL cells and the underlying mechanisms. We showed that LW-213 (1-25 μM) dose-dependently inhibited human CTCL cell lines (Hut-102, Hut-78, MyLa, and HH) with IC50 values of around 10 μM, meanwhile it potently inhibited primary leukemia cells derived from peripheral blood of T-cell lymphoma patients. We revealed that LW-213-induced apoptosis was accompanied by ROS formation and the release of calcium from endoplasmic reticulum (ER) through IP3R-1channel. LW-213 selectively activated CHOP and induced apoptosis in Hut-102 cells via activating PERK-eIF2α-ATF4 pathway. Interestingly, the degree of apoptosis and expression of ER stress-related proteins were alleviated in the presence of either N-acetyl cysteine (NAC), an ROS scavenger, or 2-aminoethyl diphenylborinate (2-APB), an IP3R-1 inhibitor, implicating ROS/calcium-dependent ER stress in LW-213-induced apoptosis. In NOD/SCID mice bearing Hut-102 cell line xenografts, administration of LW-213 (10 mg/kg, ip, every other day for 4 weeks) markedly inhibited the growth of Hut-102 derived xenografts and prolonged survival. In conclusion, our study provides a new insight into the mechanism of LW-213-induced apoptosis, suggesting the potential of LW-213 as a promising agent against CTCL.

Estrogen signaling differentially alters iron metabolism in monocytes in an Interleukin 6-dependent manner

The ability of monocytes to release or sequester iron affects their role in cancer and inflammation. Previous work has shown that while IL-6 upregulates hepcidin synthesis and enhances iron sequestration, E2 reduces hepcidin synthesis and increases iron release. Given that E2 upregulates IL-6 production in monocytes, it is likely that the exact effect of E2 on iron metabolism in monocytes is shaped by its effect on IL-6 expression. To address this issue, the expression of key iron regulatory proteins was assessed in E2-treated U937, HuT-78, THP-1 and Hep-G2 cells. Iron status was also evaluated in U937 cells treated with the ERα agonist PPT, the ER antagonist ICI-182780, dexamethasone + E2, IL-6 + E2 and in IL-6-silenced U937 cells. E2 treatment reduced hepcidin synthesis in HuT-78, THP-1 and Hep-G2 cells but increased hepcidin synthesis and reduced FPN expression in U937 cells. E2-treated U937 cells also showed reduced HIF-1α and FTH expression and increased TFR1 expression, which associated with increased labile iron content as compared with similarly treated Hep-G2 cells. While treatment of U937 cells with interleukin 6 (IL-6) resulted in increased expression of hepcidin, dexamethasone treatment resulted in reduced hepcidin synthesis relative to E2- or dexamethasone + E2-treated cells; IL-6 silencing also resulted in reduced hepcidin synthesis in U937 cells. Lastly, while iron depletion resulted in increased cell death in U937 cells, E2 treatment resulted in enhanced cell survival and reduced apoptosis. These findings suggest that E2 differentially alters iron metabolism in monocytes in an IL-6 dependent manner.

Antiretroviral and cytotoxic activities of Tityus obscurus synthetic peptide.

New drugs are constantly in demand, and nature's biodiversity is a rich source of new compounds for therapeutic applications. Synthetic peptides based on the transcriptome analysis of scorpion venoms of Tityus obscurus, Opisthacanthus cayaporum, and Hadrurus gertschi were assayed for their cytotoxic and antiretroviral activity. The Tityus obscurus scorpion-derived synthetic peptide (FFGTLFKLGSKLIPGVMKLFSKKKER), in concentrations ranging from 6.24 to 0.39 μM, proved to be the most active one against simian immunodeficiency virus (SIV) replication in the HUT-78 cell line and in primary human leukocytes, with the lowest toxicity for these cells. The immune cellular response evaluated in primary human leukocytes treated with the most promising peptide and challenged with SIV infection exhibited production of cytokines such as interleukin (IL)-4, IL-6, IL-8, IL-10, and interferon-γ, which could be involved in cell defense mechanisms to overcome viral infection through proinflammatory and anti-inflammatory pathways, similar to those evoked for triggering the mechanisms exerted by antiviral restriction factors.

WNT974 Inhibits Proliferation, Induces Apoptosis, and Enhances Chemosensitivity to Doxorubicin in Lymphoma Cells by Inhibiting Wnt/β-Catenin Signaling.

BackgroundUpregulation of the Wnt/β-catenin pathway has been demonstrated to promote tumor proliferation and chemoresistance in lymphoma. Our objective was to evaluate the effect of the Wnt/β-catenin pathway inhibitor WNT974 in lymphoma cells.Material/MethodsHuman lymphoma cell lines HUT-78 and BJAB were treated with or without 1 μM WNT974±0.15 μg/L doxorubicin (Dox). Cell viability and proliferation were evaluated by CCK-8 and colony formation assay. Expression of proliferating cell nuclear antigen (PCNA), KI67, and apoptotic-related proteins including Bcl-2, Bax, cleaved-caspase3, and cleaved-caspase9, together with Wnt pathway proteins Wnt, β-catenin, Axin2, and c-Myc, were detected by Western blot analysis. Flow cytometry was used to calculate the ratio of apoptotic cells.ResultsIn HUT-78 and BJAB cells, 1 μM WNT974 significantly reduced viability and colony formation. The expression of 2 markers of tumor cell proliferation, protein PCNA and KI67, was also reduced by WNT974. Treatment with 1 μM WNT974 for 48 h increased the rate of cell apoptosis, inhibited the expression of anti-apoptotic protein Bcl-2, and enhanced pro-apoptotic proteins Bax, cleaved-caspase3, and cleaved-caspase9 expression in both cell lines. After treatment with WNT974 plus Dox, cell viability was markedly decreased compared with Dox treatment alone. Mechanistically, WNT974 prevented the expression of Wnt, Axin2, β-catenin, and its target gene c-Myc.ConclusionsWNT974 effectively treats lymphoma by inhibiting cell proliferation, inducing cell apoptosis, and enhancing chemosensitivity to Dox, and these effects are dependent on blocking Wnt/β-catenin signaling.

589 Effects of CCR4 antagonists in cutaneous T-cell lymphoma cells

CC chemokine receptor 4 (CCR4) is responsible for T-cell skin homing. It is overexpressed in type 2 helper T-cells (Th2) as well as cutaneous T-cell lymphoma (CTCL) cells. Mycosis fungoides (MF) and Sézary syndrome (SS) are two most common types of CTCL. Currently, Mogamulizumab, a humanized anti-CCR4 antibody, has been FDA-approved to treat MF and SS. However, the clinical response rates for SS patients was 30% and for MF patients was 22% and severe drug eruptions following treatment were reported. Thus, a more effective and less toxic strategy is needed for CCR4-targeted therapy. CCR4 antagonists have been studied in diseases where Th2 cells participate, such as asthma and atopic dermatitis, but little has been done for CTCL. Here, we tested two CCR4 antagonists, C021 (class I antagonist) and AZD2098 (class II antagonist), in MF derived cell line (MJ) and SS derived cell line (Hut78). The effects of two compounds on cell chemotaxis, proliferation, apoptosis, and Th2 cytokine secretion were assessed. As expected, CCR4 was highly expressed on MJ and Hut78 cells. MJ and Hut78 cells showed chemotactic responses to TARC/CCL17 and MDC/CCL22. Both C021 and AZD2098 inhibited chemotactic responses to TARC/CCL17 and MDC/CCL22 in MJ and Hut78 cells. Of note, C021 inhibited chemotaxis to CCL17 at a much lower concentration (IC50: 0.186 μM) than to CCL22 (1.3 μM) in MJ cells. AZD2098 inhibited chemotaxis to CCL17 at a much lower concentration (IC50: 0. 12 μM) than to CCL22 (0.866 μM) in Hut78 cells. Interestingly, only C021 downregulated CCR4 expression on cell surfaces. Only C021 inhibited cell proliferation, at a higher concentration (IC50: 3.21 μM in MJ cells; 5.98 μM in Hut78 cells), and induced cell apoptosis. AZD2098 had no such effect at any concentrations tested. In addition, the expressions of IL-5 and IL-13 proteins were decreased after treatment with C021 but not with AZD2098. Our results suggest that both CO21 and AZD2098 have inhibitory effects on chemotaxis of CTCL cells, but only C021 exerts other effects on CTCL cells. The in vivo study is on the way.

Apoptotic and anti-proliferative effect of guanosine and guanosine derivatives in HuT-78 T lymphoma cells

The effects of 100 μM of 3′,5′-cGMP, cAMP, cCMP, and cUMP as well as of the corresponding membrane-permeant acetoxymethyl esters on anti-CD3-antibody (OKT3)-induced IL-2 production of HuT-78 cutaneous T cell lymphoma (Sézary lymphoma) cells were analyzed. Only 3′,5′-cGMP significantly reduced IL-2 production. Flow cytometric analysis of apoptotic (propidium iodide/annexin V staining) and anti-proliferative (CFSE staining) effects revealed that 3′,5′-cGMP concentrations > 50 μM strongly inhibited proliferation and promoted apoptosis of HuT-78 cells (cultured in the presence of αCD3 antibody). Similar effects were observed for the positional isomer 2′,3′-cGMP and for 2′,-GMP, 3′-GMP, 5′-GMP, and guanosine. By contrast, guanosine and guanosine-derived nucleotides had no cytotoxic effect on peripheral blood mononuclear cells (PBMCs) or acute lymphocytic leukemia (ALL) xenograft cells. The anti-proliferative and apoptotic effects of guanosine and guanosine-derived compounds on HuT-78 cells were completely eliminated by the nucleoside transport inhibitor NBMPR (S-(4-Nitrobenzyl)-6-thioinosine). By contrast, the ecto-phosphodiesterase inhibitor DPSPX (1,3-dipropyl-8-sulfophenylxanthine) and the CD73 ecto-5′-nucleotidase inhibitor AMP-CP (adenosine 5′-(α,β-methylene)diphosphate) were not protective. We hypothesize that HuT-78 cells metabolize guanosine-derived nucleotides to guanosine by yet unknown mechanisms. Guanosine then enters the cells by an NBMPR-sensitive nucleoside transporter and exerts cytotoxic effects. This transporter may be ENT1 because NBMPR counteracted guanosine cytotoxicity in HuT-78 cells with nanomolar efficacy (IC50 of 25–30 nM). Future studies should further clarify the mechanism of the observed effects and address the question, whether guanosine or guanosine-derived nucleotides may serve as adjuvants in the therapy of cancers that express appropriate nucleoside transporters and are sensitive to established nucleoside-derived cytostatic drugs.

Curcumin circumvent lactate-induced chemoresistance in hepatic cancer cells through modulation of hydroxycarboxylic acid receptor-1.

Curcumin has been demonstrated to affect the chemoresistance in cancer cells of various origins. However, its ability to modulate lactate-induced chemoresistance remains unclear. The Present investigation demonstrates that curcumin inhibits the survival of HepG2 and HuT78 cells and can modulate chemo-susceptibility of HepG2 cells. Experimental simulation of simultaneous and pre-treatment suggest cooperatively between curcumin and anticancer drugs as well as the modulation of molecular regulators. Inhibition of glucose consumption, lactate production, extracellular acidity and augmented level of Nitric oxide were observed. DAPI staining revealed hyper condensation of chromatin in curcumin-treated HepG2 cells. Curcumin also diminished the lactate-induced chemoresistance against doxorubicin in hepatic cancer cells along with down regulation of lactate receptor (hydroxycarboxylic acid receptor-1; HCAR-1/GPR81). Alteration of the extracellular milieu along with inhibited expression of genes (hif-1α, ldh-a, mct-1, mdr-1 and stat-3) and proteins (HIF-1α and HCAR-1) are indicated to be involved in curcumin-induced reversal of chemoresistance in HepG2 cells. Findings of present investigation contribute to knowledge of curcumin mediated chemosensitization and its mechanism.

Synergistic cytotoxic activity of cannabinoids from cannabis sativa against cutaneous T-cell lymphoma (CTCL) in-vitro and ex-vivo.

Cannabis sativa produces hundreds of phytocannabinoids and terpenes. Mycosis fungoides (MF) is the most common type of cutaneous T-cell lymphoma (CTCL), characterized by patches, plaques and tumors. Sézary is a leukemic stage of CTCL presenting with erythroderma and the presence of neoplastic Sézary T-cells in peripheral blood. This study aimed to identify active compounds from whole cannabis extracts and their synergistic mixtures, and to assess respective cytotoxic activity against CTCL cells. Ethanol extracts of C. sativa were analyzed by high-performance liquid chromatography (HPLC) and gas chromatography/mass spectrometry (GC/MS). Cytotoxic activity was determined using the XTT assay on My-La and HuT-78 cell lines as well as peripheral blood lymphocytes from Sézary patients (SPBL). Annexin V assay and fluorescence-activated cell sorting (FACS) were used to determine apoptosis and cell cycle. RNA sequencing and quantitative PCR were used to determine gene expression. Active cannabis compounds presenting high cytotoxic activity on My-La and HuT-78 cell lines were identified in crude extract fractions designated S4 and S5, and their synergistic mixture was specified. This mixture induced cell cycle arrest and cell apoptosis; a relatively selective apoptosis was also recorded on the malignant CD4+CD26- SPBL cells. Significant cytotoxic activity of the corresponding mixture of pure phytocannabinoids further verified genuine interaction between S4 and S5. The gene expression profile was distinct in My-La and HuT-78 cells treated with the S4 and S5 synergistic mixture. We suggest that specifying formulations of synergistic active cannabis compounds and unraveling their modes of action may lead to new cannabis-based therapies.

西达本胺联合苦参碱对皮肤T细胞淋巴瘤细胞系增殖、凋亡的影响及可能的凋亡机制

Objective To evaluate the effect of chidamide combined with matrine on proliferation and apoptosis of cutaneous T-cell lymphoma (CTCL) cell lines HH and Hut78, and to explore their apoptotic mechanisms. Methods Both HH and Hut78 cells were treated with 0.4 μmol/L chidamide and 0.6 g/L matrine alone or in combination for 24, 48 and 72 hours, with those treated with dimethyl sulfoxide (DMSO) serving as control groups. MTS assay was performed to detect cellular proliferation rates of HH and Hut78 cells at each time point. After 48-hour treatment, flow cytometry was conducted to detect cell apoptosis, and Western blot analysis to determine expression of apoptosis-related proteins in these cells. Statistical analysis was carried out by using repeated measures analysis of variance, one-way analysis of variance, and least significant difference (LSD) -t test for multiple comparisons. Results Compared with DMSO, chidamide and matrine alone or in combination could inhibit the proliferation of HH and Hut78 cells to different extents (F = 15.88, 558.26, P 0.05) , while the combination group (20.94% ± 0.64%) showed a significantly higher apoptosis rate compared with the control group, matrine group and chidamide group (LSD-t = 7.37, 5.40, 5.69 respectively, all P 0.05) . Conclusion Chidamide in combination with matrine can inhibit proliferation and induce apoptosis of HH and Hut78 cells, likely by regulating the expression of apoptosis-related proteins E-cadherin, NF-κB, p-Bad, Bcl-2 and cleaved caspase-3. Key words: Lymphoma, T-cell, cutaneous; Matrine; Apoptosis; Chidamide; Hut78 cells; HH cells

The retinoid derivant ECPIRM selectively exhibited anti-proliferation effects in cutaneous T-Cell lymphoma via ITK-mediated signaling pathway

BackgroundThe treatment of Cutaneous T-cell lymphoma (CTCL) met huge challenges because of the heterogeneity and the scarcity of targeted drugs. ECPIRM derived from isotretinoin exhibited strong anti-proliferation effects in Hut78 and MJ cells rather than Myla cells. However, there was no data regarding the potential target of ECPIRM for its selective activity. ObjectivesTo investigate the potential target of ECPIRM for its selective anti-proliferation activity. MethodsWe evaluated the cell viability of CTCL cells after ECPIRM treatment, and detected the effects of ECPIRM on the biomarker genes of CTCL. Subsequently, the mRNA and protein level of Interleukin-2-inducible T-cell kinase (ITK) was determined. Then the induction of apoptosis triggered by ITK inhibitor BMS-509744 and ITK siRNAs were detected, and the docking of ECPIRM interacted with ITK and the effects of ECPIRM on ITK-mediated signaling pathway were analyzed. Finally, we evaluated the anti-growth activity of ECPIRM in Hut78-xenografted nude mice, and the relative expression of cleaved caspase-3, ITK, p-ERK and p-Akt were determined. ResultsITK was highly expressed in Hut78 and MJ cells rather than Myla cells, and targeted inhibition of ITK triggered cell apoptosis. ECPIRM efficiently bound the hydrophobic active pocket of ITK, and significantly inhibited ITK-mediated signaling pathway. In addition, ECPIRM suppressed tumor growth in Hut78-xenografted model, and upregulated the expression of cleaved caspase 3 and inhibited the expression of ITK, p-ERK and p-Akt in tumor tissues, which was consistent with in vitro study. ConclusionECPIRM might provide a novel strategy for CTCL by inhibiting ITK-mediated signaling pathway.

Antiproliferative Activity of Extracts and Fractions from Irradiated Curcuma zanthorrhiza Rhizomes Against Mouse Leukemia and Human Cancer Cell Lines

Curcuma zanthorrhiza Roxb. is a medicinal plant that is used as a raw material in the herbal medicine and pharmaceutical industries. The main content of C. zanthorrhiza is curcuminoid, which is used as an antioxidant and an anticancer agent. The aim of this research was to study the effect of gamma radiation used for preserving simplicia or herbal drugs through the examination of their cytotoxicity against mouse leukemia L1210 cells and antiproliferative activity against human cancer cell lines HUT78, A549, HeLa, and THP1. The samples of curcuma rhizome were irradiated by gamma ray emitted by Cobalt-60 as a source at doses of 0 (control), 5, 7.5, 10, and 15 kGy. After irradiation, the samples were macerated using n -hexane, ethyl acetate, and ethanol, respectively. Preliminary cytotoxicity test toward extract from control sample against mouse leukemia L1210 cells revealed that the ethyl acetate extract was the most active extract inhibiting the growth of cells with an IC 50 value of 16.6 µg/mL, followed by ethanol extract (18.8 µg/mL) and n -hexane extract (42.7 μg/mL). Fractionation using a chromatography column of the ethyl acetate extract resulted in seven fractions denoted as F1-F7. The cytotoxicity test of the seven fractions against mouse leukemia L1210 cells showed that fraction 3 (F3) was the most active fraction with an IC 50 value of 10.0 μg/mL, followed by F7 (11.2 μg/mL), F6 (11.8 μg/mL), F5 (12.0 μg/mL), F1 (13.2 μg/mL), F4 (14.5 μg/mL), and F2 (27.8 μg/mL), respectively. Based on these results, all irradiated samples were then extracted, fractionated, and tested for cytotoxicity in a similar manner. The result showed that irradiation of samples under doses up to 10 kGy can be used to preserve Curcuma zanthorrhiza simplicia without damaging its efficacy. To ensure that the irradiation dose of 10 kGy did not reduce anticancer activity, the F3 from the irradiated sample at a dose of 10 kGy was also examined of its in-vitro antiproliferative activity using HUT78, A549, HeLa, and THP1 human cancer cell lines. The results showed that irradiation of the sample at a dose of 10 kGy reduced the antiproliferative activity of F3 against HUT78 (32 %), A549 (48 %), HeLa (42 %), and THP1 (31 %). However, its reduction did not eliminate its antiproliferative activities. These results indicated that the preservation of simplicia using radiation can be done at a maximum radiation dose of 10 kGy by modifying the concentration of simplicia in the fabrication process of herbal medicine formulation.

Mitotic catastrophe and p53-dependent senescence induction in T-cell malignancies exposed to nonlethal dosage of GL-V9.

Mitotic catastrophe of cancer cells induced by drugs is characterized by low dosage and low toxicity, representing a significant advantage in the cancer treatment. Effective therapeutic options are limited for T-cell malignancies patients who are still treated by high-dose multiagent chemotherapy, potentially followed by hematopoietic stem cell transplantation, highlighting the urgency for identification of more effective anti-T-cell malignancies drugs. The use of antineoplastic drugs which induced tumor cell mitotic catastrophe would be a new strategy for cancer therapy. Nevertheless, there is still no effective mitotic catastrophe agent in T-cell malignancies. Our study showed that nonlethal dosage (ND) of GL-V9 (5-hydroxy-8-methoxy-2-phenyl-7-(4-(pyrrolidin-1-yl) butoxy) 4 H-chromen-4-one) (2µM), a potential anticancer drug, not only attenuated cell growth and survival, but also arrested the cell cycle in G2/M phase and induced multipolar spindles, nuclear alterations (micronucleation and multinucleation), which are the most prominent morphological characteristics of mitotic catastrophe, in T-cell malignancies cell lines including Jurkat, HuT-102, and HuT-78. Moreover, ND GL-V9 could trigger DNA damage, and significantly influence several mitosis-associated proteins. Besides, results showed that ND GL-V9 increased the activity of senescence-associated β-galactosidase (SA-β-Gal) following the induction of mitotic catastrophe in Jurkat and HuT-102 cells with intact p53, while causing apoptosis in p53-deficient HuT-78 cells. We concluded that the anti-T-cell malignancies effects of ND GL-V9 and clarified the precise regulation in the process of mitosis under the action of GL-V9 in T-cell malignancies. Our data provided new evidence for the study of T-cell malignancies treatment associated with mitotic catastrophe and cellular senescence induction.

TPD7 inhibits the growth of cutaneous T cell lymphoma H9 cell through regulating IL‐2R signalling pathway

IL‐2R pathway is a key regulator in the development of immune cells and has emerged as a promising drug target in cancer treatment, but there is a scarcity of related inhibitors. TPD7 is a novel biphenyl urea taspine derivate, which has been shown anti‐cancer effect. Here, we demonstrated the anti‐cancer activity of TPD7 in cutaneous T cell lymphoma and investigated the underlying mechanism of TPD7 through IL‐2R signalling. The inhibitory effect of TPD7 on cell viability exhibited a strong correlation with the expression level of IL‐2R, and cutaneous T cell lymphoma H9 and HUT78 cells were most sensitive to TPD7. TPD7 was nicely bound to IL‐2R and down‐regulated the mRNA and protein levels of IL‐2R. Furthermore, TPD7 suppressed the downstream cascades of IL‐2R including JAK/STAT, PI3K/AKT/mTOR and PLCγ/Raf/MAPK signalling, resulting in Bcl‐2 mitochondrial apoptosis pathway and cell cycle proteins CDK/Cyclins regulation. And, these were verified by flow cytometry analysis that TPD7 facilitated cell apoptosis in H9 cells via mitochondrial pathway and impeded cell cycle progression at G2/M phase. TPD7 is a novel anti‐cancer agent and may be a potential candidate for cutaneous T cell lymphoma treatment by regulating IL‐2R signalling pathway.

Thrombospondin-1 promotes tumor progression in cutaneous T-cell lymphoma via CD47.

CD47 is highly expressed on various hematopoietic malignancies, and enables cancer cells to avoid immunosurveillance. Its ligand, thromobospondin-1 (TSP-1) is a multifunctional protein, and CD47/TSP-1 interactions promote tumor progression in various malignancies. In this study, we investigated roles of TSP-1 and CD47 in cutaneous T-cell lymphoma (CTCL). Flow cytometric analysis and immunohistochemistry showed that CTCL tumor cells and CTCL cell lines (Hut78, HH, and MyLa cells) overexpressed CD47 compared with normal CD4+ T cells. Overexpression of CD47 was partially induced by high c-Myc expression in CTCL tumor cells. TSP-1 mRNA expression levels in CTCL lesional skin were higher than those in normal skin and correlated with increased risk of disease-related death. Moreover, TSP-1 was expressed on CTCL tumor cells by immunohistochemistry. Serum soluble TSP-1 levels in patients with Sézary syndrome were significantly elevated. TSP-1 promotes proliferation and survival of CTCL tumor cells, which is inhibited by anti-CD47 neutralizing antibody or CD47 knockdown. Stimulation with TSP-1 also induces cell migration and in vivo growth. These effects were mediated by phosphorylation of ERK1/2 and AKT and expression of survivin. Collectively, our findings prompt a novel therapeutic approach to CTCL based on discovery that CD47/TSP-1 interactions play important roles in progression of CTCL.

YKL-40 Promotes Proliferation of Cutaneous T-Cell Lymphoma Tumor Cells through Extracellular Signal–Regulated Kinase Pathways

YKL-40, one of the chitinase-like proteins, is associated with the pathogenesis of a wide variety of human diseases through modulation of inflammation and tissue remodeling by its diverse roles in cell proliferation, differentiation, and survival. Emerging evidence shows that aberrantly expressed YKL-40 promotes the development of malignancies by inducing proliferation of tumor cells, cytokine production, and angiogenesis by acting on various stromal cells, immune cells, and tumor cells. In this study, we investigated the expression and function of YKL-40 in cutaneous T-cell lymphoma (CTCL). We first revealed that serum YKL-40 levels were increased in patients with CTCL and correlated with disease severity markers. We also found that YKL-40 was expressed by epidermal keratinocytes and tumor cells in lesional skin of CTCL by immunohistochemistry. Although YKL-40 did not affect cytokine production from CTCL cell lines, YKL-40 promoted the proliferation of Hut78 cells and HH cells invitro, which was dependent on extracellular signal-regulated kinase 1/2 pathways. Moreover, exogenous YKL-40 administration enhanced tumor growth of HH cells invivo. Our study has suggested that YKL-40 produced from epidermal keratinocytes and CTCL cells promoted the proliferation of CTCL cells through extracellular signal-regulated kinase 1/2 pathways in autocrine and paracrine manners, leading to development of CTCL.

Growth inhibition and transcriptional effects of ribavirin in lymphoma.

Ribavirin exhibits inhibitory effects on the epigenetic enzyme enhancer of zeste homolog2 (EZH2), which participates in lymphomagenesis. Additionally, preclinical and clinical studies have demonstrated the anti‑lymphoma activity of this drug. To further investigate the potential of ribavirin as an anticancer treatment for lymphoma, the tumor‑suppressive effects of ribavirin were analyzed in lymphoma cell lines. The effects of ribavirin on the viability and clonogenicity of the B‑cell lymphoma cell line Pfeiffer (EZH2‑mutant), Toledo (EZH2 wild‑type) and cutaneous T‑cell lymphoma Hut78 cell line were assessed. Expression of EZH2 and trimethylation status of histone3, lysine27 trimethylated (H3K27m3) was also determined in response to ribavirin. The transcriptional effects of ribavirin on Hut78 cells were analyzed by microarray expression and the results were validated by reverse transcription‑quantitative polymerase chain reaction, western blotting and knockout of signal transducer and activator of transcription1 (STAT1). The results of the present study demonstrated that ribavirin suppressed the growth and clonogenicity of cells in a dose‑dependent manner. Ribavirin did not affect the expression of EZH2 nor altered its activity as evaluated by H3K27 trimethylation status. Furthermore, the results of transcriptome analysis indicated that the majority of the canonical pathways affected by ribavirin were associated with the immune system, including 'antigen presentation', 'communication between innate and adaptive immune cells' and 'cross‑talk between dendritic and natural killer cells'. The results of gene expression analysis were confirmed, by demonstrating at the RNA and protein levels, downregulation of stearoyl‑CoA desaturase and upregulation of STAT1. Depletion of STAT1, which was proposed as a key regulator of the aforementioned pathways, exerted growth inhibitory effects almost to the same extent as ribavirin. In conclusion, ribavirin was proposed to exert growth inhibitory effects on lymphoma cell lines, particularly Hut78 cells, a cutaneous T‑cell lymphoma cell line. Of note, these effects may depend on, at least in part, the activation of canonical immune pathways regulated by the key factors STAT1 and interferon‑γ. Our results provide insight into the anti‑lymphoma potential of ribavirin; however, further investigations in preclinical and clinical studies are required to determine the effectiveness of ribavirin as a therapeutic agent for treating lymphoma.