Interleukin-24 Research Articles

Effect of adenovirus-mediated interleukin 24 on glioma cells and its mechanism

Objective To investigate the effect of adenovirus-mediated interleukin 24 (Ad-IL-24) on proliferation and apoptosis of glioma cells and its mechanism. Methods We set up Ad-IL-24 group, Ad-GFP group and PBS group. The expression of IL-24 in each group was detected by real-time quantitative PCR (qRT-PCR) and Western blot, and the proliferation activity of 3 groups of U251 cells was detected by MTT assay. Apoptosis of U251 cells was detected by flow cytometry. The expression of Survivin and Bax in U251 cells was detected by qRT-PCR. Results (1) In group PBS and group Ad-GFP, U251 cells adhered well and grew well. The number of adherent growth in group Ad-IL-24 was significantly reduced and the infection efficiency in group Ad-IL-24 and Ad-GFP was above 95%. (2) The results of qRT-PCR and Western blot showed that the expression levels of IL-24 in PBS group and Ad-GFP group were significantly lower than those in Ad-IL-24 group (all P<0.05). (3) U251 cells infected with 100 MOI Ad-IL-24 for 48 h significantly inhibited the growth of U251 cells and the inhibitory effect was significantly higher than that in PBS and Ad-GFP groups (both P<0.05) and presented certain time dependence (P<0.05). (4) The U251 cells were infected with 100 MOI Ad-IL-24 for 48 h. Flow cytometry results showed that the rate of cell apoptosis in Ad-IL-24 group was 39.2%±4.2%, which was significantly higher than that of PBS group and Ad-GFP group (2.5%±1.0% and 4.9%±2.3%, respectively, both P<0.05). (5) The expression level of Survivin in Ad-IL-24 group was significantly lower than that in PBS group and Ad-GFP group (both P<0.05). The expression level of Bax was significantly higher in Ad-IL-24 group than in PBS group and Ad-GFP group (both P<0.05). Conclusions Ad-IL-24 expression could significantly inhibit the growth of U251 cells and induce apoptosis. It is speculated that the mechanism of action may be down-regulating the expression of apoptosis-inhibiting gene Survivin and up-regulating the expression of apoptosis-promoting gene Bax. Key words: Glioma; Cell proliferation; Apoptosis; Interleukin-24; Survivin; Bax

Eukaryotic Translation Initiation Factor 4A Down-Regulation Mediates Interleukin-24-Induced Apoptosis through Inhibition of Translation.

Dysregulated activity of helicase eIF4A drives transformation to and maintenance of cancer cell phenotype by reprogramming cellular translation. Interleukin 24 (IL-24) is a tumor-suppressing protein, which has the ability to inhibit angiogenesis, sensitize cancer cells to chemotherapy, and induce cancer cell-specific apoptosis. In this study, we found that eIF4A is inhibited by IL-24. Consequently, selective reduction of translation was observed for mRNAs harboring strong secondary structures in their 5′-untranslated regions (5′UTRs). These mRNAs encode proteins, which function in cell survival and proliferation. Consistently, overexpression of eIF4A conferred cancer cells with resistance to IL-24-induced cell death. It has been established that inhibition of eIF4A triggers mitochondrial-mediated apoptosis. We showed that IL-24 induces eIF4A-dependent mitochondrial depolarization. We also showed that IL-24 induces Sigma 1 Receptor-dependent eIF4A down-regulation and mitochondrial depolarization. Thus, the progress of apoptosis triggered by IL-24 is characterized by a complex program of changes in regulation of several initiation factors, including the eIF4A.

Oct4 suppresses IR‑induced premature senescence in breast cancer cells through STAT3- and NF‑κB-mediated IL‑24 production.

Breast cancer stem cells (BCSCs) are a small subpopulation of breast cancer cells that have been proposed to be a primary cause of failure of therapies, including ionizing radiation (IR). Their embryonic stem-like signature is associated with poor clinical outcome. In the present study, the function of octamer-binding transcription factor 4 (Oct4), an embryonic stem cell factor, in the resistance of BCSCs to IR was investigated. Mammosphere cells exhibited increased expression of stemness-associated genes, including Oct4 and sex-determining region Y-box 2 (Sox2), and were more resistant to IR compared with serum-cultured monolayer cells. IR-resistant MCF7 cells also exhibited significantly increased expression of Oct4. To investigate the possible involvement of Oct4 in IR resistance of breast cancer cells, cells were transfected with Oct4. Ectopic expression of Oct4 increased the clonogenic survival of MCF7 cells following IR, which was reversed by treatment with small interfering RNA (siRNA) targeting Oct4. Oct4 expression decreased phosphorylated histone H2AX (γ-H2AX) focus formation and suppressed IR-induced premature senescence in these cells. Mammosphere, IR-resistant and Oct4-overexpressing MCF7 cells exhibited enhanced phosphorylation of signal transducer and activation of transcription 3 (STAT3) (Tyr705) and inhibitor of nuclear factor κB (NF-κB), and blockade of these pathways with siRNA against STAT3 and/or specific inhibitors of STAT3 and NF-κB significantly increased IR-induced senescence. Secretome analysis revealed that Oct4 upregulated interleukin 24 (IL-24) expression through STAT3 and NF-κB signaling, and siRNA against IL-24 increased IR-induced senescence, whereas recombinant human IL-24 suppressed it. The results of the present study indicated that Oct4 confers IR resistance on breast cancer cells by suppressing IR-induced premature senescence through STAT3- and NF-κB-mediated IL-24 production.

Immunogenicity moderation effect of interleukin-24 on myelogenous leukemia cells.

Previous studies have shown that interleukin-24 (IL-24) has tumor-suppressing activity by multiple pathways. However, the immunogenicity moderation effect of IL-24 on malignant cells has not been explored extensively. In this study, we investigated the role of IL-24 in immunogenicity modulation of the myelogenous leukemia cells. Data show that myelogenous leukemia cells express low levels of immunogenicity molecules. Treatment with IL-24 could enhance leukemia cell immunogenicity, predominantly regulate leukemia cells to produce immune-associated cytokines, and improve the cytotoxic sensitivity of these cells to immune effector cells. IL-24 expression could retard transplanted leukemia cell tumor growth in vivo in athymic nude mice. Moreover, IL-24 had marked effects on downregulating the expression of angiogenesis-related proteins vascular endothelial growth factor, cluster of differentiation (CD) 31, CD34, collagen IV and metastasis-related factors CD147, membrane type-1 matrix metalloproteinase (MMP), and MMP-2 and MMP-9 in transplanted tumors. These findings indicated novel functions of this antitumor gene and characterized IL-24 as a promising agent for further clinical trial for hematologic malignancy immunotherapy.

Expression of interleukin-24 and its influence on bioactivity of non-small cell lung cancer cells

Objective To investigate the expression of IL-24 in patients with non-small cell lung cancer (NSCLC), and to evaluate its influence on the bioactivity of NSCLC cells. Methods Thirty-nine patient with NSCLC (23 patients with adenocarcinoma and 16 patients with squamous carcinoma) and 17 healthy subjects were enrolled in this study. Serum samples and lung cancer tissues were collected. IL-24 expression in the serum samples was measured using enzyme-linked immunosorbent assay. Its expression at mRNA level in the lung cancer tissues was measured using reverse transcriptional real-time PCR. Adenocarcinoma cell line A549 and squamous carcinoma cell line NCI-H520 were stimulated with recombinant human IL-24 (10 ng/ml and 100 ng/ml) for 24 hours. Cell proliferation was measured using CCK-8 method. Apoptosis and cell cycle were measured using flow cytometry. Cell invasion was measured using Transwell assay. Results Serum IL-24 was significantly elevated in patients with NSCLC in comparison with that in healthy subjects [(144.10±64.43) vs (48.47±18.00) pg/ml]. No significant difference in IL-24 expression was found between patients with adenocarcinoma and squamous carcinoma. IL-24 expression at mRNA level in lung cancer tissues of patients with NSCLC was also significantly increased with an approximately 5-fold enhancement in comparison with that in normal lung tissues. Stimulation with low concentration of recombinant IL-24 (10 ng/ml) promoted the proliferation and suppressed the apoptosis of A549 and NCI-H520 cells. In contrast, high concentration of recombinant IL-24 (100 ng/ml) stimulation notably inhibited the proliferation and enhanced the apoptosis of lung cancer cell lines. No remarkable changes in cell cycle of the two kinds of lung cancer cells in response to IL-24 stimulation were observed. Moreover, low concentration of recombinant IL-24 (10 ng/ml) did not affect the invasion of A549 and NCI-H520 cells, while high concentration of recombinant IL-24 (100 ng/ml) significantly inhibited the invasion of lung cancer cells. Conclusion IL-24 might influence the bioactivity of NSCLC cells in a concentration-dependent manner. High concentration of IL-24 might counteract the invasion and metastasis of NSCLC, which is important to prevent disease promotion. Key words: IL-24; Non-small cell lung cancer; Cell proliferation; Apoptosis; Invasion

Iron-induced calcification in human aortic vascular smooth muscle cells through interleukin-24 (IL-24), with/without TNF-alpha

In CKD patients, arteriosclerotic lesions, including calcification, can occur in vascular smooth muscle cells in a process called Moenckeberg’s medial arteriosclerosis. Iron overload induces several complications, including the acceleration of arteriosclerosis. However, the relationship between Moenckeberg’s arteriosclerosis in vascular smooth muscle cells and iron accumulation has remained unknown. We tested the accelerated effect of iron on calcification in cultured human aortic vascular smooth muscle cells (HASMCs). After establishment of this model, we performed a microarray analysis using mRNA from early stage culture HASMCs after iron stimulation with or without TNF-alpha stimulation. The role of interleukin-24 (IL-24) was confirmed from candidate genes that might contribute to calcification. HASMCs demonstrated calcification induced by iron and TNF-alpha. Calcification of HASMCs was synergistically enhanced by stimulation with both iron and TNF-alpha. In the early phase of calcification, microarray analysis revealed up-regulation of IL-24. Stimulation of HASMCs by IL-24 instead of iron induced calcification. The anti-IL-24 antibody reversed the effect of IL-24, supporting the important role of IL-24 in HASMCs calcification. In conclusion, iron-induced calcification in vascular smooth muscle cells occurred via IL-24, IL-24 was increased during the calcification process induced by iron, and IL-24 itself caused calcification in the absence of iron.

Correlations between CXCL13, IL-24 genes and wrist arthritis.

To investigate the relationship between B lymphocyte chemokine 1 (CXCL13) and interleukin-24 (IL-24) gene and wrist arthritis. A total of 122 cases of patients with wrist arthritis treated in our hospital from May 2013 to April 2016 were randomly selected as wrist arthritis group, while 120 normal subjects were selected as normal control group. Venous blood was collected from all patients in normal control group and wrist arthritis group, respectively. Rheumatoid factor (RF), human C-reactive protein (CRP), and erythrocyte sedimentation rate (ESR) in venous blood were measured. The visual analogue scale (VAS) score was used to statistically analyze the pain of subjects in normal control and wrist arthritis groups; the wrist flexion and extension activities of subjects in normal control group and wrist arthritis group were measured. The expressions of CXCL13 and IL-24 mRNA in synovial tissue of normal control group and wrist arthritis group were detected by reverse transcription-polymerase chain reaction (RT-PCR). Western blotting was used to detect the expressions of CXCL13 and IL-24 in normal control group and wrist arthritis group. The levels of CRP, RF, and ESR in the normal control group were within the normal range, but the levels of CRP, RF, and ESR in the wrist arthritis group were significantly higher than those in the normal control group. VAS scores and joint flexion extension activities in the normal control group were at normal levels. The VAS score of wrist arthritis group was significantly higher than that of the normal control group, and the joint flexion extension activities were significantly lower than that in the normal control group. The results of RT-PCR showed that the expression of CXCL13 mRNA in synovial tissue of wrist arthritis was significantly higher than that in the normal control group, while the expression of IL-24 mRNA in synovial tissue of wrist arthritis was significantly lower than that in normal control tissues. Western blotting showed that the expression of CXCL13 in synovial tissue of wrist arthritis was significantly higher than that in the normal control group, while the expression of IL-24 in synovial tissue of wrist arthritis was significantly lower than that in normal control groups. Analysis of variance showed that the expressions of CXCL13 and IL-24 in the normal control group and wrist arthritis group had statistically significant differences (p<0.01). The abnormal expressions of CXCL13 and IL-24 are closely related to the occurrence and development of wrist arthritis. This study shows that CXCL13 and IL-24 have important research values in wrist arthritis. CXCL13 and IL-24 expressions can be used as new indicators of the diagnosis and treatment of wrist arthritis.

Anti-tumor effects of oncolytic adenovirus expressing human interleukin-24 gene on neuroblastoma implanted in nude mice

Objective To evaluate the anti-tumor effect of oncolytic adenovirus expressing human interleukin-24 (IL-24) gene on neuroblastoma implanted in nude mice. Methods BALB/c nude mice were subcutaneously inoculated with LA1-55N cells to establish a model of neuroblastoma. Murine models were randomly divided into four groups to be treated with an oncolytic adenovirus carrying interleukin-24 (ZD55-IL-24), adenovirus carrying interleukin-24 (Ad-IL-24), oncolytic adenovirus fusing with enhanced green fluorescent protein (ZD55-EGFP), phosphate buffer saline (PBS), respectively, when the volume of implanted tumor reached 100-150 mm3. The tumor size was measured at all interval of 4 days for 6 weeks. The expression of Caspase-3, E1 A and IL-24 was detected by Western blotting analysis. The apoptosis was determined by TdT mediated dUTP nick end labeling (TUNEL). Results The group treated with ZD55-IL-24 significantly inhibited the growth of tumor. The mean tumor volume was (91.20±39.31) mm3 in the ZD55-IL-24 group, significantly smaller than that in Ad-IL-24 group (997.89±51.65) mm3, ZD55-EGFP group (943.46±69.62) mm3, and PBS group (1 980.24±98.78) mm3 s after 6 week. Western blotting analysis confirmed that ZD55-IL-24 could express IL-24 and E1 A protein in LA1-55N cell line with high efficiency, and obviously activate of Caspase-3. TUNEL showed ZD55-IL-24 could induce obviously apoptotic effects. The percentage of the apoptotic cells was(52.10±6.21)%, (21.30±3.62)%, (20.60±2.01)% (P=0.003), respectively, after treatment with ZD55-IL-24, Ad-IL-24 and ZD55-EGFP. Conclusion ZD55-IL-24 has a significant inhibitory effect on the growth of neuroblastoma implanted in nude mice, which maybe related with activation of Caspase-3. Key words: Neuroblastoma; Oncolytic adenovirus; Intedeukin-24

CCL2 Enhances the Viability of Human Chorionic Trophoblast Cell Line HTR-8/SVneo Cells by Inhibiting Interleukin-24 Expression

Background: To investigate the regulatory effect of interleukin-24 (IL-24) on cell viability of human chorionic trophoblast cell line (HTR-8/SVneo cells). Methods: Immunohistochemical staining was used to detect the expression of IL-24 and its receptors IL-20R1, IL-20R2, and IL-22R1 in villus tissue at early normal pregnancy. The effect of thymic stromal lymphopoietin (TSLP) and chemokine CCL2 on the expression of IL-24 in human chorionic trophoblast cell line HTR-8/SVneo cells was analyzed by In-cell Western. In addition, the effect of recombinant human IL-24 (rhIL-24) and CCL2 on the viability of HTR-8/SVneo cells was analyzed by MTT assay. Results: IL-24 and its receptors showed a strong positive staining in trophoblasts at early normal pregnancy. Compared with control group, expression of IL-24 in HTR-8/SVneo cells was significantly inhibited after in vitro stimulation of recombinant human CCL2 protein (rhCCL2) (P Conclusions: CCL2 enhances the viability of human trophoblast cell line HTR-8/SVneo cells in vitro by inhibiting the secretion of IL-24, which may be beneficial to blastocyst implantation and placental development.

Reversal effect of adenovirus-mediated human interleukin24 transfection on the cisplatin resistance of A549/DDP lung cancer cells.

Interleukin-24 (IL-24) is a tumor-suppressor gene that has been documented in human melanoma cells. IL-24 has marked antitumor activities on various types of human cancer, but its underlying mechanism remains unclear. In the present, we investigated the effects of human IL-24 (hIL-24) on the chemotherapy resistance of lung cancer cells. The cisplatin (DDP)-resistant lung carcinoma cell line A549/DDP was subjected to adenovirus-mediated transfection with the human IL-24 gene (Ad-hIL-24). The growth-inhibitory and apoptotic effects of Ad-hIL-24 on A549/DDP cells were observed, and the expression levels of AKT, phosphorylated-AKT (p-AKT) and P-glycoprotein (P-gp) were detected. Ad-hIL-24 significantly decreased the levels of p-AKT and P-gp, and effectively inhibited A549/DDP cell growth. Furthermore, A549/DDP cells exhibited a significantly increased rate of apoptosis, as well as G2/M-phase arrest, following transfection with Ad-hIL-24, and these effects were increased in cells treated with Ad-IL-24 combined with DDP when compared with those treated with Ad-hIL-24 or DDP alone. These results suggest that hIL-24 can reverse the DDP resistance of lung cancer cells, and that the associated mechanism involves the induction of apoptosis and G2/M-phase arrest through the phosphoinositide3-kinase (PI3K)/AKT signaling pathway, as well as a decrease in drug resistance through P-gp expression.

Coactosin-like protein CLP/Cotl1 suppresses breast cancer growth through activation of IL-24/PERP and inhibition of non-canonical TGFβ signaling.

Coactosin-like protein (CLP, or Cotl1), is an F-actin-binding protein, whose role in cancer is largely unknown. Here we show that CLP/Cotl1 is highly expressed in a rat epithelial breast cancer cell line (FE1.3) compared with its mesenchymal counterpart (FE1.2). Knockdown of CLP/Cotl1 in FE1.3 cells increased cell proliferation, whereas its overexpression in FE1.2 cells inhibited proliferation in culture and reduced tumor growth in xenograft assays in mice. Mechanistically, we identified two major pathways through which CLP/Cotl1 exerts its suppressive effects. First, CLP/Cotl1 re-expression in FE1.2 and in human MCF7 breast cancer cells induced expression of the growth-suppressor gene interleukin-24 (IL-24), which independently of p53 upregulates the tumor-suppressor genes p53 apoptosis effector related to PMP-22 (PERP) and p21cip1. Second, overexpression of CLP/Cotl1 potentiated the growth-suppressive effect of transforming growth factor-β1 (TGFβ1), leading to downregulation of TGFβ-responsive genes vascular growth factor A/B (VEGFA/VEGFB), hypoxia inducing factor 1α (HIF-1α) and trombospondin 1 (TSP1), which mediate various hallmarks of cancer progression including angiogenesis, invasion and metastasis. CLP/Cotl1 inhibited TGFβ signaling via a non-canonical signaling involving IL-24-instigated inhibition of mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) phosphorylation and subsequent post-transcriptional downregulation of SMAD2 and SMAD4. We also showed that CLP/COTL1 expression sensitizes breast cancer cells to chemotherapeutic drugs, and this was further enhanced by addition of exogenous TGFβ1. CLP/Cotl1 expression is lost in many human malignancies including prostate, uterine and breast cancers. Thus, our results uncover a novel tumor-suppressor role for CLP/Cotl1 and identify the downstream effectors interleukin 24 (IL-24)/PERP and IL-24/MAPK/ERK/TGFβ as potential targets for precision therapy.

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.

Expression, Purification and Functional Assessment of Smallest Isoform of Human Interleukin-24 in Escherichia coli

ABSTRACT Interleukin-24 (IL-24) is a novel tumor-suppressor gene that has different alternative splice isoforms. It has been shown that new smallest isoform of human IL-24 gene, lacking three exons, induces higher levels of cytotoxicity than all the isoforms, indicating shortest isoform of IL-24 may be a new promising anti-cancer agent. In this study, we aimed to provide a reproducible method for recombinant production of the smallest isoform of IL-24 (sIL-24). The Structure of sIL-24 was analyzed using bioinformatics tools (I-TASSER, Prosa, RAMPAGE and SPDBV version 4.1). The DNA sequence encoding sIL-24 was chemically synthesized and sub-cloned into the pET-32a (+) vector for further protein expression in Escherichia coli BL21 (DE3) strain. Upon IPTG induction, sIL-24 peptide was expressed as a thioredoxin fusion protein. The recombinant sIL-24 was released from the fusion by TEV protease cleavage followed by nickel affinity chromatography. The yield of the purified sIL-24 was estimated about 380 μg/ml. MTT assay showed that sIL-24 peptide inhibited the proliferation of PC-3 cancer cells more effectively than full length IL-24 protein, while none affect the survival of MRC-5 normal cells. These results indicate that the presented expression system is an efficient system for the production of small functional recombinant sIL-24 peptide.This functional peptide may have cancer therapeutic application.

Oncolyic adenovirus SG600-IL24 expressing human melanoma differentiation associated gene-7/interleukin-24 selectively induces growth arrests and apoptosis in hepatocellular carcinoma cell lines

Objective To investigate the effect of oncolytic adenovirus vector SG600-IL24 expressing human melanoma differentiation associated gene-7/interleukin-24 (MDA-7/IL-24) on hepatocellular carcinoma cell lines with different metastatic potentials (HepG2, Hep3B, SMMC7721 and normal liver cell line L02). Methods The oncolytic adenovirus SG600-IL24 carrying MDA-7/IL-24 gene was transfected into hepatocellular carcinoma cell lines and normal liver cell line. The mRNA and protein expression of MDA-7/IL-24 in HepG2, Hep3B, SMMC7721 and L02 cells was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), enzyme linked immunosorbent assay (ELISA) and Western blotting respectively. Methyl thiazol tetrazolium (MTT) assay and flow cytometry were used to study tumor cell proliferation and cell cycle in vitro. Hoechst 33258 and flow cytometry were studied to indicate the apoptosis effects. Results It was confirmed by RT-PCR, ELISA and Western blotting that the exogenous MDA-7/IL-24 gene was highly expressed in HepG2, Hep3B, SMMC7721 and L02 cells. MTT and apoptosis detection indicated that MDA-7/IL-24 could induce the growth suppression [inhibition rate: (75.0±2.5)%, (85.0±2.0)%, and (86.0±3.5)% respectively. for HepG2: F=5.860; for Hep3B: F=7.210; for SMMC7721: F=7.980, P=0.000], promote apoptosis [apoptosis rate: (56.5±4.0)%, (34.4±2.0)% and (43.3±2.5)% respectively. For HepG2: F=203.400; for Hep3B: F=313.200; for SMMC7721: F=130.500, P=0.000], and block cancer cells in G2/M phase [the blocking rate: (35.4±4.2)%, (47.3±6.2)% and (40.5±5.0)%] but not L02 cells. Conclusion Oncolytic adenovirus vector SG600-IL24 can selectively induce growth suppression and promote apoptosis in hepatocellular carcinoma lines in vitro but not in L02 cells. Key words: Carcinoma, hepatocellular; Oncolytic viruses; Melanoma differentiation associated gene-7/interleukin-24 gene; Gene therapy

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.

Tumor-suppressive effects of interferon-β through interleukin-24 in melanoma

Background: Interferon beta (IFN-β) is widespread used as a treatment against melanoma to improve its prognosis. Several literature has suggested that the sensitivity of melanoma cells to IFN-β shows wide spectrum through in vivo and in vitro examinations including our previous study that RPMI-7951 and A375 is relatively sensitive to IFN-β, while G361, SK-MEL-5 and SK-MEL-1 is relatively resistant. Interleukin24 (IL-24) has been reported to inhibit the tumor growth by inducing the tumor-selective apoptosis. This study was conducted to elucidate the relation with IL-24 in inhibition effects of IFN-β on melanoma cells.

A conditionally replicating adenovirus expressing IL-24 acts synergistically with temozolomide to enhance apoptosis in melanoma cells in vitro.

Malignant melanoma is characterized by suppressed apoptosis in tumor cells and high levels of invasion. Temozolomide (TMZ) is one of the most effective single chemotherapeutic agents for patients with malignant melanoma, but resistance develops quickly and frequently. Therapeutic cytokines such as interleukin-24 (IL-24) and conditionally replicating adenoviruses have exhibited promising results as complementary therapies. Thus, the present study hypothesized that a conditionally replicating adenovirus expressing IL-24 combined with TMZ may exhibit increased antitumor activity compared with either treatment alone in melanoma A375 and M14 cell lines in vitro. The present study constructed an E1B-55 gene-deleted conditionally replicating adenovirus expressing the IL-24 gene (ZD55-IL-24). IL-24 was expressed at high levels in melanoma cells infected with ZD55-IL-24 in the presence of TMZ. The combination of ZD55-IL-24 + TMZ induced higher protein expression levels of the proapoptotic proteins B-cell lymphoma-2 (Bcl-2)-like protein 4 and phosphorylated protein, γ-H2A histone family member X (γ-H2AX), and reduced the levels of the antiapoptotic proteins Bcl-2, myeloid cell leukemia-1and nuclear factor-κB compared with either treatment individually. A dose-dependent increase in the cytopathic effects for the combination of ZD55-IL-24 and TMZ was also observed. The data of the present study suggest that the ZD55-IL-24 + TMZ combination induced increased levels of apoptosis, possibly by triggering DNA damage, in melanoma cells in vitro compared with either treatment alone. These findings suggest that this strategy may be a promising approach for the treatment of patients with malignant melanoma.

Interleukin-24 inhibits osteosarcoma cell migration and invasion via the JNK/c-Jun signaling pathways.

Approximately 25% of osteosarcoma patients present with clinically detectable metastatic disease at the time of initial diagnosis. High-dose chemotherapy and/or surgery for the treatment of primary metastatic osteosarcoma is ineffective, and <20% of patients will survive 5 years from diagnosis. Therefore, the treatment of metastases is critical for the improvement of the prognosis of primary metastatic osteosarcoma patients. We have previously observed that overexpression of interleukin-24 (IL-24) inhibits neuroblastoma cell proliferation, migration and invasion in vitro. The present study investigated whether IL-24 may be a novel agent for osteosarcoma metastasis-suppressive treatment. It was observed that IL-24 is able to inhibit migration and invasion in spontaneously metastasizing human 143B osteosarcoma cells via the c-Jun N-terminal kinase (JNK)/c-Jun signaling pathway. IL-24 was effective in inhibiting JNK and c-Jun phosphorylation to downregulate matrix metalloproteinase (MMP)-2 and MMP-9, which contributed to the suppression of cell migration and invasion. It was concluded that IL-24 may be a potent agent in the inhibition of highly metastatic 143B osteosarcoma cells, and IL-24 may have translational potential as an effective therapeutic agent for the treatment of metastatic osteosarcoma.

Long intergenic non-coding RNA 00152 promotes lung adenocarcinoma proliferation via interacting with EZH2 and repressing IL24 expression

BackgroundNumerous studies have shown that long non-coding RNAs (lncRNAs) behave as a novel class of transcript during multiple cancer processes, such as cell proliferation, apoptosis, migration, and invasion. LINC00152 is located on chromosome 2p11.2, and has a transcript length of 828 nucleotides. The biological role of LINC00152 in LAD(lung adenocarcinoma) remains unknown.MethodsQuantitative reverse transcription PCR(qRT-PCR) was used to detect LINC00152 expression in 60 human LAD tissues and paired normal tissues. In vitro and in vivo studies showed the biological function of LINC00152 in tumour progression. RNA transcriptome sequencing technology was performed to identify the downstream suppressor IL24(interleukin 24) which was further examined by qRT-PCR, western bolt and rescue experiments. RNA immunoprecipitation (RIP), RNA pulldown, and Chromatin immunoprecipitation (ChIP) assays were carried out to reveal the interaction between LINC00152, EZH2 and IL24.ResultsLINC00152 expression was upregulated in 60 human LAD tissues and paired normal tissues. High levels of LINC00152 expression were correlated with advanced TNM stage, larger tumor size, and lymph node metastasis, as well as shorter survival time. Silencing of LINC00152 suppressed cell growth and induced cell apoptosis. LINC00152 knockdown altered the expression of many downstream genes, including IL24. LINC00152 could interact with EZH2 and inhibit IL24 transcription. Moreover, the ectopic expression of IL24 repressed cell proliferation and partly reversed LINC00152 overexpression-induced promotion of cell growth in LAD.ConclusionsOur study reveals an oncogenic role for LINC00152 in LAD tumorigenesis, suggesting that it could be used as a therapeutic target in LAD treatment.

Interleukin-24 Concentrations Not Related with Age, but Affect Pro- and Anti-Inflammatory Cytokines Differently in Healthy Donors.

Interleukin 24 (IL-24) is expressed at different levels in a variety of tumor tissues and matched normal tissues and is regarded as a potential tumor biomarker as its expression levels in tumor tissues are associated with tumor patient prognosis. At present, the expression level of IL-24 in healthy human peripheral blood is unknown. In this study, 1940 blood samples were collected using different processing methods from healthy donors. ELISA was used to detect IL-24 concentrations. The results showed that processing methods had the greatest influence on test results, with the highest IL24 concentration in EDTA plasma and the lowest in sodium citrate plasma. Lengths of storage time at 4°C had no obvious effect on IL-24 test results, and IL-24 in peripheral blood was stable for 15 days. IL-24 concentration in the sera of healthy donors showed no associations with age, blood glucose, hemoglobin, total cholesterol, carcinoembryonic antigen, absolute lymphocyte counts, alpha fetoprotein, white blood cells, thyroid stimulating hormone, or cereal third transaminase. We also confirmed that IL-24 expression level in the blood of healthy subjects was positively correlated with pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF-α), and interleukin 6 (IL-6), but negatively correlated with anti-inflammatory cytokine, IL-10. We observed that sample processing methods influence the detection of IL-24 levels as EDTA plasma had the highest IL-24 concentration, and citric acid sodium, the lowest. Age, gender, and physical and chemical indicators were not related to IL-24 concentrations. We confirmed the IL-24 concentration was positively related to IL-6 and TNF-α and negatively to IL-10.