Proliferation Of Raji Cells Research Articles

Urtica dioica agglutinin (a plant lectin) has a caspase-dependent apoptosis induction effect on the acute lymphoblastic leukemia cell line

Urtica dioica agglutinin (UDA) is a very small plant lectin with anti-prostatic activity. In this study, we investigated the effect of UDA on proliferation and apoptosis induction in human acute lymphoid leukemia (ALL) cell lines. The effect of UDA on Jurkat and Raji cell proliferation was examined by MTS assay. Distribution of cell cycle phases was determined by PI staining and apoptosis was examined with annexin V/PI and western blot. Results showed UDA treatment reduced cell proliferation in cells by inducing apoptosis. PI staining was associated with a higher percentage of the cell population in sub G1. Caspase-8 and caspase-9 dependent apoptosis occurred in Jurkat cells. Generally, UDA treatment resulted in cell death in ALL cell lines and induced apoptosis in the T-ALL cell line, Jurkat, through extrinsic and intrinsic pathways. These results may be considered as a guide to working on UDA as an anti-leukemic drug in the future.

Silymarin Inhibits Proliferation and Induces Apoptosis in Epstein-Barr Virus-Positive Lymphoma Cells by Suppressing Nuclear Factor-Kappa B�Pathway

Epstein Barr virus-positive lymphoma results from the loss of homeostatic balance between Epstein Barr virus and immune cells. In this study, we investigated the mechanism underlying reduction in Epstein Barr virus-positive lymphoma cell proliferation and apoptosis by silymarin, an anti-inflammatory/antioxidant molecule extracted from Silybum�marianum (milk thistle). We examined the effect of silymarin on Raji cell proliferation, apoptosis, the expression of latent membrane protein�1, the proteins related to apoptosis, and the activation of nuclear factor kappa-B. Results showed that silymarin inhibited the proliferation and promoted apoptosis of Raji cells in a concentration-dependent manner. Also, silymarin reduced phosphorylated inhibitor of nuclear factor kappa-B and p65 levels. Silymarin treatment elevated latent membrane protein 1 expression and knockdown of this protein led to increased proliferation inhibition and apoptosis by silymarin. In conclusion, nuclear factor kappa-B and latent membrane protein 1 knockdown could work in concert with silymarin in suppressing Epstein Barr virus-positive lymphoma cell proliferation and inducing apoptosis.

Role and mechanism of PTEN in Burkitt's lymphoma.

The aim of the present study was to explore the possible mechanisms of phosphatase and tensin homolog (PTEN) in the pathogenesis of Burkitt's lymphoma, and provide novel information that can be used in the targeted treatment of this disease. PTEN lentiviral overexpression vector and short-hairpin PTEN silencing vectors were constructed. The effect of PTEN on the growth and proliferation of CA46 and RAJI cells was analyzed using a Cell Counting Kit-8 assay. Apoptosis was detected by Hoechst 33342 and propidium iodide double staining. Flow cytometry was used to analyze the cell cycle. A Transwell chamber was used to detect cell migration and invasion abilities. Western blot analysis was used to detect related protein changes. The mechanism of the effect of PTEN on the biological characteristics of Burkitt's lymphoma cells was subsequently analyzed. The results revealed that PTEN inhibited the proliferation of CA46 and RAJI cells by downregulating the expression of p-AKT, It was indicated that the upregulation of proapoptotic proteins (including Bad and Bax) induced apoptosis, regulated cyclin (including P53, P21, CDK4, CDK6, cyclin D3 and cyclin H) to inhibit cell cycle progression, and mediated epithelial-mesenchymal transition-like cell markers (including E-cadherin, N-cadherin, β-catenin, TCF-8, vimentin, Slug and Snail) to inhibit cell migration and invasion. In conclusion, the tumor-suppressor gene PTEN inhibited the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) signaling pathway and inhibited the proliferation and migration of Burkitt's lymphoma cells, induced apoptosis and cell cycle arrest, thus playing a crucial role in the pathogenesis of Burkitt's lymphoma.

Preclinical safety evaluation of chimeric antigen receptor-modified T cells against CD19 in NSG mice.

With the increase of chimeric antigen receptor-modified T (CAR-T) cell therapy, serious complications initiated by CAR-T cells have garnered wide attention. We have previously developed a 4-1BB/CD3-ζ-costimulated CAR-T cells against CD19 (CART19) for adult acute lymphoblastic leukemia (ALL). In this study, a preclinical safety assessment of CART19 was performed on NSG mice, to evaluate the preclinical toxicity along with its efficacy and tissue distribution. A total of 120 NSG mice were used for a combined pharmacodynamics and toxicity study for 56 days. Ninety-six mice of which were single dosed with Raji-Luc (5×105 per animal, i.p.) and different concentrations of CART19 (0.2×107, 0.6×107 and 1.8×107 per animal, i.v.), while the rest were assigned to the Untreated group. Optical intensity of Raji-Luc in mice, clinical symptoms, body mass, hematological analysis, humanized cytokine, lymphocyte subset counting, necropsy and histopathological examinations were performed. In addition, a single dose of 0.6×107 CART19 was intravenously administered to 48 NSG mice, and the distribution of CART19 in different tissues was analyzed using quantitative PCR. CART19 is widely distributed in organs well-perfused with blood, including the lungs, blood, bone marrow, liver and spleen. Significant proliferation of CART19 was also found in the blood by through recognition using humanized CD3+ for T lymphocytes. The survival rate and leukemia related clinical symptoms in mice administered CART19 were markedly ameliorated, and the proliferation of Raji cells in mice was effectively inhibited. However, CART19 had no obvious effects on either the mean body mass or the blood cell counts, and no cytokine release syndrome and graft versus host disease were observed. NSG mice given CART19 treatment demonstrated a longer survival period without significant immunotoxicity, suggesting encouraging clinical prospects for CART19 in patients with R/R ALL. Our study shed light on evaluation and supervision strategies for CAR-T products for the treatment of hematological diseases or leukemia.

Study of the mechanism by which dinaciclib induces apoptosis and cell cycle arrest of lymphoma Raji cells through a CDK1-involved pathway.

ObjectiveThis study aimed to identify and evaluate the mechanism by which apoptosis and cell cycle arrest were induced by dinaciclib in lymphoma Raji cells.MethodsThe colony formation assay was used to detect cell proliferation of Raji cells. Cell cycle arrest and cell apoptosis were determined by flow cytometry and TUNEL assays, respectively. Protein expression related to the Raji cell state was evaluated by Western blot. The Raji/Dinaciclib drug‐resistant cell line was established, where the regulating functions of CDK1‐involved pathway were verified. In addition, the effect of dinaciclib in vivo was examined in orthotopically implanted tumors in nude mice.ResultsCell apoptosis was induced, and DNA synthesis ability was decreased in a time‐dependent manner in dinaciclib‐treated lymphoma Raji cells. Furthermore, the cell cycle was found to be blocked in the G2/M Phase. Further study indicated that CDK1‐involved pathway played a key regulatory role in this process. It was revealed by cell transfection that the expression of cell cycle proteins was downregulated after treatment with dinaciclib through a CDK1‐involved pathway, which eventually led to apoptosis. Knockdown of CDK1 restored the sensitivity of the Raji/Dinaciclib cells to dinaciclib. Xenograft model of nude mice showed that dinaciclib treatment in vivo could effectively inhibit tumor growth, consistent with the experiment results mentioned before.ConclusionIn this study, we clarified the mechanisms through which dinaciclib induces Raji cell apoptosis and blocks the cell cycle through a CDK1‐involved pathway, which supported that dinaciclib had potential values in the treatment of lymphoma.

Knockdown of long non-coding RNA PVT1 inhibits the proliferation of Raji cells through cell cycle regulation.

Long non-coding RNA plasmacytoma variant translocation 1 (PVT1) has been reported to be associated with oncogenesis. However, the functional role of PVT1 in Burkitt lymphoma has not yet been addressed. The purpose of the present study was to investigate the effect of PVT1 knockdown by small interfering RNA (siRNA) on the proliferation of Burkitt lymphoma Raji cells and to explore its possible mechanism of action. An effective siRNA targeting PVT1 was screened and the corresponding short hairpin RNA (shRNA) was reconstructed into a lentiviral vector. Cell proliferation and cell cycle distribution were assessed by Cell Counting kit-8 assay and flow cytometry, respectively. Protein expression levels of c-Myc, cyclin-dependent kinase inhibitor1A (CDKN1A, P21) and cyclin E1 (CCNE1) were detected by western blotting. A polymerase chain reaction (PCR) array was used to analyse the expression of genes associated with the cell cycle. PVT1 knockdown markedly suppressed proliferation, and induced cell cycle arrest at the G0/G1 phase in Raji cells. Protein expression levels of c-Myc and CCNE1 were reduced, whereas P21 protein expression was markedly increased following downregulation of PVT1 in Raji cells. The cell cycle PCR array revealed that 54 genes were upregulated and 26 genes were downregulated in Raji cells following PVT1 knockdown. Reverse transcription-quantitative PCR demonstrated that cyclin G2 (CCNG2), CDKN1A, Retinoblastoma-like 2 (RBL2, p130), HUS1 checkpoint homolog, cyclin dependent kinase inhibitor 3 (CDKN3) and cyclin dependent kinase inhibitor 1B (CDKN1B) expression were upregulated, whereas the expression levels of CCNE1, cyclin D1 (CCND1) and cell division cycle 20 (CDC20) were downregulated in Raji cells with PVT1 knockdown. In conclusion, PVT1 knockdown may inhibit the proliferation of Raji cells by arresting cells in G0/G1 phase. Furthermore, inhibition of cell proliferation may be associated with a reduction inc-Myc expression and alterations in the expression levels of cell cycle-associated genes.

\u03b2-Asarone increases doxorubicin sensitivity by suppressing NF-\u03baB signaling and abolishes doxorubicin-induced enrichment of stem-like population by destabilizing Bmi1

BackgroundLymphoma is one of the most common hematologic malignancy. Drug resistance is the main obstacle faced in lymphoma treatment. Cancer stem cells are considered as the source of tumor recurrence, metastasis and drug resistance. The β-Asarone, a low-toxicity compound from the traditional medical herb Acorus calamus, has been shown to act as an anti-cancer reagent in various cancer types. However, the anti-cancer activities of β-Asarone in lymphoma have not been shown.MethodsCell counting assay was used to evaluate Raji cell proliferation. CCK8 assay was used to evaluate the cell viability. Annexin-V/PI staining and flow cytometry analysis were used to evaluate apoptosis. ALDEFLUOR assay was used to evaluate the stem-like population. Luciferase reporter assay was used to examine the activation of NF-κB signaling. Western blot and polymerase chain reaction (PCR) were used to determine the expression of interested genes.ResultsWe showed that β-Asarone inhibited proliferation and induced apoptosis in Raji lymphoma cells in a dose-dependent manner. Additionally, β-Asarone functioned as a sensitizer of doxorubicin and resulted in synergistic effects on inhibition of proliferation and induction of apoptosis when combined with doxorubicin treatment. Interestingly, we found that β-Asarone also reduced the stem-like population of Raji lymphoma cells in a dose-dependent manner, and suppressed the expression of c-Myc and Bmi1. Importantly, β-Asarone abolished doxorubicin-induced enrichment of the stem-like population. In the mechanism study, we revealed that β-Asarone suppressed not only basal NF-κB activity but also Tumor necrosis factor α (TNF-α) induced NF-κB activity. Moreover, blocking NF-κB signaling inactivation was critical for β-Asarone induced apoptosis and inhibition of proliferation, but not for the effect on β-Asarone reduced stem-like population. In fact, β-Asarone suppressed stem-like population by destabilizing Bmi1 via a proteasome-mediated mechanism.ConclusionsOur data suggested the application of β-Asarone to lower the toxic effect of doxorubicin and increase the sensitivity of doxorubicin in clinical treatment. More importantly, our data revealed a novel role of β-Asarone which could be used to eliminate stem-like population in lymphoma, implying that β-Asarone might reduce relapse and drug resistance.

MiR-370 Sensitizes TMZ Response Dependent of MGMT Status in Primary Central Nervous System Lymphoma

Primary central nervous system lymphoma (PCNSL) is an aggressive and rare subtype of non-Hodgkin lymphoma, arising exclusively in the CNS with a poor prognosis. Previous evidence has proved that MGMT was a promising target involving in TMZ resistance of PCNSL. Our study described a new miR-370-mediated mechanism of MGMT regulation in PCNSL. We first showed that miR-370 was downregulated in PCNSL tissues, while MGMT was inversely overexpressed. It was also observed that miR-370 suppressed the expression of MGMT. Additionally, upregulation of miR-370 significantly increased TMZ sensitivity dependent of MGMT, thus suppressed Raji cell proliferation and induced apoptosis in vitro. In conclusion, these results suggest that miR-370 is a potential target in PCNSL treatment.

Optimization of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives as dual inhibitors of BTK and PI3Kδ

BTK and PI3Kδ play crucial roles in the progression of leukemia, and studies confirmed that the dual inhibition against BTK and PI3Kδ could provide superior anticancer agents to single targeted therapies. Herein, a new series of novel benzofuro[3,2-b]pyridin-2(1H)-one derivatives were optimized based on a BTK/PI3Kδ inhibitor 2 designed by our group. Biological studies clarified that compound 6f exhibited the most potent inhibitory activity (BTK: IC50 = 74 nM; PI3Kδ: IC50 = 170 nM) and better selectivity than 2. Moreover, 6f significantly inhibited the proliferation of Raji and Ramos cells with IC50 values of 2.1 μM and 2.65 μM respectively by blocking BTK and PI3K signaling pathways. In brief, 6f possessed of the potency for further optimization as an anti-leukemic drug by inhibiting BTK and PI3Kδ kinase.

Amphotericin B suppresses M2 phenotypes and B7-H1 expression in macrophages to prevent Raji cell proliferation

BackgroundMacrophages in the tumor microenvironment play a critical role in tumorigenesis and anti-cancer drug resistance. Burkitt’s lymphoma (BL) is a B-cell non-Hodgkin’s lymphoma with dense macrophage infiltration. However, the role for macrophages in BL remains largely unknown.MethodsB7-H1, a transmembrane glycoprotein in the B7 family, suppresses T cell activation and proliferation and induces the apoptosis of activated T cells. The expression of B7-H1 in BL clinical tissues was determined by streptavidin-peroxidase immunohistochemistry. The mutual regulation between macrophages and BL Raji cells was investigated in a co-culture system. The cell proliferation and cell cycle distribution of Raji cells were determined using BrdU staining coupled with flow cytometry. CD163, CD204 and B7-H1 expression was assessed by flow cytometry and Western blot. Cell invasion was analyzed by Transwell assay. The expression of cytokines was detected by quantitative RT-PCR. Immunofluorescence and allogeneic T-cell proliferation assays were used to compare the expression of B7-H1, p-STAT6, or p-STAT3 and CD3+ T cell proliferation treated with or without amphotericin B.ResultsB7-H1 was highly expressed in tumor infiltration macrophages in most clinical BL tissues. In vitro, Raji cells synthesized IL-4, IL-6, IL-10 and IL-13 to induce CD163, CD204 and B7-H1 expression in co-cultured macrophages, which in turn promoted Raji cell proliferation and invasion. Interestingly, antifungal agent amphotericin B not only inhibited STAT6 phosphorylation to suppress the M2 polarization of macrophages, but also promoted CD3+ T cell proliferation by regulating B7-H1 protein expression in macrophages.ConclusionAmphotericin B might represent a novel immunotherapeutic approach to treat patients with BL.

Effects of human umbilical cord-derived mesenchymal stem cells on hematologic malignancies.

Mesenchymal stem cells (MSCs) have been used in hematopoietic stem cell transplantation for years. However, the safety of MSCs applied in various types of hematologic malignancy has not been comprehensively explored. In the present study, the effects of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) on six representative hematologic malignancy cell lines were explored, including leukemia, multiple myeloma and lymphoma cells. Direct and indirect co-culture models were established, and cell proliferation was assessed by carboxyfluorescein diacetate succinimidyl ester staining. A cytometric bead array cytokine kit was used to quantify cytokines. The expression of interleukin (IL)-6 receptor elements on tumor cells was detected by reverse transcription-polymerase chain reaction and flow cytometry, and the effects of exogenous IL-6 on cell proliferation were determined using a Cell Counting kit-8 assay. The results demonstrated that hUC-MSCs inhibited the proliferation of most of the cell lines examined (THP-1, HL-60, K562 and RPMI-8226), but promoted the proliferation of Raji cells. In addition, hUC-MSCs secreted abundant IL-6, promoted the secretion of IL-10 by RPMI-8226 and Raji cells, and inhibited the secretion of tumor necrosis factor-α by THP-1 cells. These data indicate a varied effect of hUC-MSCs on various types of hematologic malignancy, including distinct mechanisms of cell-to-cell contact and cytokines. Researchers applying hUC-MSCs in lymphoma should be aware of a potential tumor growth-promoting effect.

Effect of rapamycin on the biological behaviors of human non-Hodgkin lymphoma Raji cells and its mechanism

Objective To investigate the effects of rapamycin on the biological behaviors of human non-Hodgkin lymphoma Raji cells with different concentrations and time, and to explore its mechanism. Methods Different concentrations (0, 10, 50, 100, 250, 500 nmol/L) of rapamycin were used in Raji cells for 24, 48, 72 h respectively. The apoptosis of Raji cells was detected by using CCK-8 method, and flow cytometry was used to detect the cell apoptosis and cycle of Raji cells. The enzymatic activity of Caspase-3 and Caspase-9 in Raji cells was detected by Caspase-3 and Caspase-9 activity testing kit. The expressions of bcl-2, p53 protein and mRNA were detected by Western blot method and reverse transcription-polymerase chain reaction (RT-PCR). Results The proliferative inhibition rate of Raji cells was increased from (23.7±4.2)% to (51.7±3.7)%, the cell apoptosis rate was increased from (4.9±1.9)% to (20.5±1.5)%, the proportion of G0/G1 was increased from (40.8±1.4)% to (63.6±1.7)%, the Caspase-3 enzyme activity of Raji cell in 24 h was increased from 0.16±0.05 to 1.08±0.04, Caspase-9 enzyme activity was increased from 0.19±0.04 to 1.34±0.06 after 24 h with the increasing concentration of rapamycin from 0 nmol/L to 500 nmol/L (P < 0.01). The mRNA of bcl-2 was decreased from 0.90±0.03 to 0.46±0.03, and mRNA of p53 was increased from 2.51±0.41 to 5.85±0.21. The protein expression of bcl-2 was reduced and the protein expression of p53 was increased. The experimental results of Raji cells in 48 h and 72 h were consistent with the experimental results of 24 h. Conclusion Rapamycin may inhibit the proliferation of Raji cells through Caspase-3, Caspase-9, bcl-2, p53 and induce its cell apoptosis. Key words: Lymphoma, non-Hodgkin; Sirolimus; Apoptosis; Caspase 3; Caspase 9; Genes, bcl-2; Genes, p53

PAX5 haploinsufficiency induce cancer cell dormancy in Raji cells

PAX5 mutations have important role in leukemogenesis and leukemia relapse, cancer cell dormancy participates in cancer relapse, but there was no report about PAX5 mutation inducing cancer cell dormancy. we constructed the PAX5 deletion Raji cell lines using gene editing technology, evaluated dormancy biological characteristics of cell lines. Our results showed PAX5 haploinsufficiency restrained the proliferation of Raji cells, induced G0/G1 arrest of Raji cells, reduced chemotherapy sensitivity. The tumor formation rate reduced in PAX5 mutation Raji cells. Our results showed PAX5 insufficiency induced cancer cell dormancy in Raji cell.

Cloning of a novel lectin from Artocarpus lingnanensis that induces apoptosis in human B-lymphoma cells.

We isolated a novel lectin (Artocarpus nitidus subsp. lingnanensis lectin, ALL) from Artocarpus nitidus subsp. lingnanensis and showed its mitogenic activities. In this study, we determined the amino acid sequence of ALL by cDNA sequencing. ALL cDNA (933bp) contains a 657-bp open reading frame (ORF), which encodes a protein with 218 amino acids. ALL shares high sequence similarities with Jacalin and Morniga G and belongs to jacalin-related lectin family. We also examined the antitumor activity of ALL using Raji, a human B-lymphoma cell line. ALL exhibits a strong binding affinity to cell membrane, which can be effectively inhibited by N-acetyl-D-galactosamine (GalNAc). ALL inhibits Raji cell proliferation in a time- and dose-dependent manner through apoptosis, evidenced by morphological changes, phosphatidylserine externalization, poly ADP-ribose polymerase (PARP) cleavage, Bcl-2 down-regulation, and caspase-3 activation. We further showed that the activation of p38 mitogen-activated protein kinase (MAPK) signaling pathways is required for the pro-apoptotic activity of ALL.

High expression of PRPS1 induces an anti-apoptotic effect in B-ALL cell lines and predicts an adverse prognosis in Chinese children with B‑ALL

Phosphoribosyl pyrophosphate synthetase 1 (PRPS1) is closely associated with a number of diseases; however, its influence in B-cell acute lymphoblastic leukemia (B-ALL) and the potential molecular mechanisms involved remain unclear. The present study aimed to evaluate the expression of PRPS1 in Chinese children with B-ALL and to investigate the mechanism of action of PRPS1 in this disease. A Cell Counting Kit-8 (CCK-8) assay was performed to examine the proliferation of B-ALL Sup-B15 and Raji cells, and flow cytometric analysis was conducted to determine the cell cycle distribution and rate of apoptosis. The mRNA and protein expression levels of PRPS1, MYC proto-oncogene, bHLH transcription factor, cyclin E1, B-cell lymphoma-2 (Bcl-2), cyclin dependent kinase 2 and caspase-3 were detected by reverse transcription-quantitative polymerase chain reaction and western blot analysis, respectively. Elevated PRPS1 expression was associated with a high-risk stratification and poor prognosis in patients with B-ALL. Furthermore, overexpression of PRPS1 accelerated the growth of and inhibited apoptosis in Sup-B15 and Raji cells as well as increasing the expression of Bcl-2 to induce an anti-apoptotic effect in B-ALL cell lines. The results of the present study indicate that PRPS1 regulates multiple processes in B-ALL and may be an attractive therapeutic target.

Effects of mTOR Inhibitor Rapamycin on Burkitt's Lymphoma Cells

To explore the effects of mTOR inhibitor rapamycin on proliferation, cell cycle and apoptosis of Burkitt's lymphoma cell line Raji and CA46 cells and its mechanism, so as to provide the experimental evidence for a therapeutic target of Burkitt's lymphoma. 3-(4,5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide(MTT) assay was performed to assess the inhibitory effect of rapamycin on proliferation of Burkitt's lymphoma cell line Raji and CA46 cells. The cell cycle distribution of Raji and CA46 cells was analyzed by flow cytometry with propidium iodide(PI) single staining. The cell apoptosis of Raji and CA46 cells was analyzed by flow cytometry with FITC Annexin V+PI double staining. The expressions of RPS6, p-RPS6, survivin and caspase-3 proteins were detected by Western blot after treating with rapamycin. Rapamycin markedly inhibited the proliferation of both Raji and CA46 cells in a time- and concentration-dependent manners, showing good biological activity, the cell proliferation inhibition rate reached about 20% after treatment with 1 nmol/L rapamycin. After treatment with different concentrations of rapamycin for 24 and 48 hours, the proportion of both cells in G1/G0 phase in the treated groups was significantly increased in a time- and concentration-dependent manners in comparison with the solvent control group. With regard to the cells in S and G2/M phase, the decreased population was accompanied by the increase of G1/G0 phase cells. After treatment with 100 nmol/L rapamycin for 48 hours, both Raji and CA46 cells demonstrated an apparent apoptosis,especially late apoptosis by flow cytometry with Annexin V+PI staining. After treatment with rapamycin, the expression of p-RPS6 and survivin of Raji and CA46 cells was obviously down-regulated, the expression of caspase-3 was obviously up-regulated in a time- and dose-dependent manners. However, rapamycin did not obviously affect the expression of RPS6. The rapamycin can effectively inhibit cell proliferation, arrest Raji and CA46 cells in G1/G0 phase, and this effect associates with inhibiting the activation of mTOR/RPS6 signal pathway through down-regulating the expression of phosphorylated RPS6, i.e. mTOR downstream signal pathway. It also can induce apoptosis by down-regulating the expression of anti-apoptotic protein survivin and activating the intrinsic pro-apoptotic protein caspase-3.

Mda‑7/IL‑24 induces the differentiation of Bcell lymphoma via activation of the P38 mitogen activated protein kinase signaling pathway.

Interleukin 24 (IL‑24) is a unique cytokine encoded by the melanoma differentiation associated gene‑7 (Mda‑7), and was first discovered inhuman melanoma cells. Exogenous Mda‑7/IL‑24 has been shown to inhibit the proliferation and invasion of a broad spectrum of human cancer cells, but its effect on the differentiation of B cell lymphoma is not yet clear. To the best of our knowledge, the present study demonstrated for the first time that overexpressing Mda‑7/IL‑24 can induce differentiation in human B cell lymphomacells, and the underlying mechanism was investigated. The proliferation of stable Mda‑7/IL‑24 overexpressing Raji and Daudi cells was assessed by the MTS method. The immunophenotype, apoptosis level and cell cycle distribution of Raji and Daudi cells were analyzed by flow cytometry. The expression of PR domain zinc finger protein 1 (Blimp1) and B‑cell lymphoma 6 (Bcl6) were analyzed by western blotting. Additionally, western blotting assay was also performed to study the effect of Mda‑7/IL‑24 on the activity of the P38 mitogen activated protein kinase (MAPK) signaling pathway in Raji and Daudi cells. Proliferation of Raji and Daudi cells overexpressing Mda‑7/IL‑24 was inhibited significantly, compared with those of parent cells and cells transfected with the empty vector alone. Apoptosis was not involved in the proliferation inhibition, while the cell cycle was arrested in G1 phase in the Raji and Daudi cells overexpressing Mda‑7/IL‑24. Overexpressing Mda‑7/IL‑24 resulted in a significantly decreased expression of cluster of differentiation (CD)10, and increased expression of CD45 and CD138 in the cell surface of Raji and Daudi cells. The expression of Blimp1 was upregulated, while the levels of Bcl6 protein was downregulated, in Raji and Daudi cells overexpressing Mda‑7/IL‑24. Furthermore, the activities of the P38 MAPK signaling pathway in lymphoma cells were upregulated. These results indicated that Mda‑7/IL‑24 could induce terminal differentiation of B lymphoma cells by regulating the expression of Blimp1 and Bcl6 via altering the P38 MAPK signaling pathway, suggesting that Mda‑7/IL‑24 may therefore be a potential differentiation therapeutic agent to be applied in clinical treatment of B cell lymphoma.

Triptonide acts as a novel potent anti-lymphoma agent with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn signal pathway

Lyn is a proto-oncogene overexpressed and constitutively activated in lymphoma, and plays an important role in lymphoma initiation and malignant progression. Hence, the oncogenic Lyn has recently been targeted for novel anti-lymphoma drug discovery; however, the effective Lyn-targeted drug for lymphoma treatment with low toxicity is absent in the clinical setting. The goal of this study is to explore powerful and low toxic Lyn-targeted anti-lymphoma agent. Here we show that triptonide, a small molecule purified from the herb Tripterygium wilfordii Hook F, potently inhibits the proliferation of human B-lymphoma Raji and T-lymphoma Jurkat cells with IC50 of 5.7nM and 4.8nM, respectively. Strikingly, triptonide at a dose of 5mg/kg/day almost completely inhibited the lymphoma growth in human lymphoma cells-xenografted mice without obvious side effects, particularly; the tumors in 6 mice among the 8 xenografted mice were completely eradicated in vivo. Cell biological studies showed that triptonide at the doses of 2.5–10nM notably suppressed B-lymphoma cell colony-forming capability, and that triptonide at the dose of 20nM promoted apoptosis through activation of PARP and caspase 3, but reduction of BCL2 protein levels in the lymphoma cells. Molecular studies revealed that triptonide markedly inhibited oncogenic Lyn transcription through suppressing the promoter activity of the gene, and that it remarkably reduced both total and phosphorylated Lyn proteins, and diminished Lyn downstream ERK and ATK signal pathways. Additionally, triptonide significantly enhanced p38 phosphorylation. Together, triptonide exerts potent anti-lymphoma effect with low toxicity mainly through inhibition of proto-oncogene Lyn transcription and suppression of Lyn downstream ERK and ATK signal pathways, providing an attractive drug candidate for development of novel anti-lymphoma therapeutics.

SIL-24 peptide, a human interleukin-24 isoform, induces mitochondrial-mediated apoptosis in human cancer cells.

Interleukin-24 (IL-24) is a unique cytokine in the IL-10 family that reveals tumor-suppressive activity against a broad range of cancers. Alternative splicing of human IL-24 generates several isoforms with different pro-apoptotic activities. In the current study, we aimed to investigate the cytotoxic properties of a recombinant smallest isoform of IL-24 (sIL-24) and the underlying molecular mechanisms in PC-3, A549, U937, and Raji cancer cells as well as normal cell line MRC-5. Following treatment of the cells with recombinant sIL-24 peptide and full-length IL-24 protein, cytotoxicity was determined by MTT assay. Apoptosis induction was evaluated using annexin-V/PI double staining flow cytometry and Hoechst 33342 staining. The expression of Bax, Bcl-2, cytochrome c, and caspase-3 was analyzed by Western blotting. MTT assay exhibited that sIL-24 dose and time dependently inhibited the proliferation of IL-24 receptor-positive PC-3, U937, and Raji cells more effectively than full-length IL-24. In contrast, sIL-24 had little cytotoxic effect on A549 cells lacking the IL-24 receptor, or on MRC-5 normal cells. Flow cytometric analysis and morphological observation revealed an efficient apoptosis induction in the receptor-positive cells. Furthermore, Western blot assay demonstrated that cell death induced by sIL-24 was associated with upregulation of the Bax/Bcl-2 ratio, cytochrome c release, and the expression of cleaved caspase-3, suggesting that sIL-24 induced apoptosis mainly through the mitochondrial pathway. Notably, among the tested cells, induction of apoptosis was more significant in PC-3 cells. Our results suggest that the sIL-24 peptide is a promising candidate for potential treatment of human cancers.

Cytotoxic mechanism of Baccharis milleflora (Less.) DC. essential oil

The aim of this study was to investigate the cytotoxic activity of the essential oil from cladodes of Bacharis milleflora in relation to Jurkat, Raji and HL-60 cells, as well as exploring the cell mechanisms in order to elucidate how the cytotoxic process occurs. The presence of the following volatile compounds was detected by GC–MS: bicyclogermacrene (12.16%), germacrene D (11.18%), (E)-caryophyllene (9.28%), and α-humulene (8.05%). In general, IC50 values lower than 50μg/mL were obtained for all the tumor cells at 24, 48 and 72h by MTT assay. The decrease in cell DNA content was demonstrated due to the inhibition of the proliferation of Jurkat, Raji and HL-60 cells by B. milleflora essential oil. In particular, Raji cells presented the greatest inhibition of cell proliferation and they were subsequently used to investigate cell death mechanisms. B. milleflora essential oil promoted G0/G1 arrest and also induced cell fragmentation, which was represented by an increase in the sub-G0 population, indicating cell death induced by apoptosis. The selectivity index was 3.97. Necrotic cell death, coupled with low levels of apoptotic cell death, was observed by conventional EB/AO and Hoechst 33342 staining assays, demonstrating that this essential oil acts via both necrotic and apoptotic mechanisms.