The lncRNA SNHG5/miR-32 axis regulates gastric cancer cell proliferation and migration by targeting KLF4.

Long noncoding RNAs (lncRNAs) are emerging as important regulators in cellular processes, including the development, proliferation, and migration of cancer cells. We have demonstrated in a prior study that small nucleolar RNA host gene 5 (SNHG5) is dysregulated in gastric cancer (GC). To further explore the underlying mechanisms of SNGH5 function in the development of GC, in this study, we screened the microRNAs interacting with SNHG5 and elucidated their roles in GC. We showed that SNHG5 contains a putative miR-32-binding site and that deletion of this site abolishes the responsiveness to miR-32. Suppression of SNHG5 expression by miR-32 was found to be Argonaute (Ago)2-dependent. Immunoprecipitation showed that SNHG5 could be pulled down from the Ago-2 complex with miR-32. Furthermore, it was reported that Kruppel-like factor 4 (KLF4) is a target gene of miR-32. In agreement with SNHG5 being a decoy for miR-32, we showed that KLF4 suppression by miR-32 could be partially rescued by SNHG5 overexpression, whereas miR-32 mimic rescued SNHG5 overexpression-mediated suppression of GC cell migration. In addition, we identified a negative correlation between the expression of SNHG5 and miR-32 in GC tissues. Furthermore, KLF4 expression was significantly downregulated in GC specimens, and a negative correlation between miR-32 and KLF4 expression and a positive correlation between KLF4 and SNHG5 expression levels were detected. Overall, this study demonstrated, for the first time, that the SNHG5/miR-32/KLF4 axis functions as an important player in GC cell migration and potentially contributes to the improvement of GC diagnosis and therapy.-Zhao, L., Han, T., Li, Y., Sun, J., Zhang, S., Liu, Y., Shan, B., Zheng D., Shi, J. The lncRNA SNHG5/miR-32 axis regulates gastric cancer cell proliferation and migration by targeting KLF4.

A novel humanized anti-tumor necrosis factor-related apoptosis-inducing ligand-R2 monoclonal antibody induces apoptotic and autophagic cell death.

It is well known that the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL/TNFSF10) is specifically expressed in various tumor cells, but less or no expression in most normal tissues and cells. While TRAIL engages with its native death receptors, TRAIL receptor 1 (TRAIL-R1) or 2 (TRAIL-R2), usually elicits the tumor cell death by apoptosis. In this study, we report that a novel humanized monoclonal antibody against TRAIL-R2 (named as zaptuzumab) well remain the biological activity of the parental mouse antibody AD5-10 inducing cell death in various cancer cells, but little effect on normal cells. Zaptuzumab also markedly inhibited the tumor growth in the mouse xenograft of NCI-H460 without toxicity to the liver and kidney, and the efficacy of tumor suppression was increased significantly while it combined with cis-dichlorodiamineplatinum. Especially, 131 I-labeled zaptuzumab injected into mouse tail vein specifically targeted to the xenograft of the lung cancer cells. Confocal analysis showed that zaptuzumab bound with TRAIL-R2 on cell surface could be quickly internalized and transferred into the lysosome. Furthermore, zaptuzumab possessed a high level of antibody-dependent cytotoxicity as well as complement-dependent cytotoxicity. Study on the mechanisms of cell death induced by zaptuzumab showed that it efficiently induced both caspase-dependent apoptosis and autophagic cell death. These data suggest that the humanized anti-TRAIL-R2 monoclonal antibody or the second generation of the antibody may have an important clinical usage for cancer immunotherapy. © 2017 IUBMB Life, 69(9):735-744, 2017.

Mmu-miR-125b overexpression suppresses NO production in activated macrophages by targeting eEF2K and CCNA2.

BACKGROUND: MicroRNAs have been shown to be important regulators of the immune response and the development of the immune system. It was reported that microRNA-125b (miR-125b) was down-regulated in macrophages challenged with endotoxin. However, little is known about the function and mechanism of action of miR-125b in macrophage activation. Macrophages use L-arginine to synthesize nitric oxide (NO) through inducible NO synthase (iNOS), and the released NO contributes to the tumoricidal activity of macrophages. METHODS: Luciferase reporter assays were employed to validate regulation of a putative target of miR-125b. The effect of miR-125b on endogenous levels of this target were subsequently confirmed via Western blot. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was performed to determine the expression level of miR-125b in macrophage. MTS assays were conducted to explore the impact of miR-125b overexpression on the cell viability of 4T1 cells. RESULTS: Here, we demonstrate that mmu-miR-125b overexpression suppresses NO production in activated macrophages and that LPS-activated macrophages with overexpressed mmu-miR-125b promote 4T1 tumor cell proliferation in vitro and 4T1 tumor growth in vivo. CCNA2 and eEF2K are the direct and functional targets of mmu-miR-125b in macrophages; CCNA2 and eEF2K expression was knocked down, which mimicked the mmu-miR-125b overexpression phenotype. CONCLUSIONS: These data suggest that mmu-miR-125b decreases NO production in activated macrophages at least partially by suppressing eEF2K and CCNA2 expression.

Human T-cell leukemia virus type 1 Tax-deregulated autophagy pathway and c-FLIP expression contribute to resistance against death receptor-mediated apoptosis.

UNLABELLED: The human T-cell leukemia virus type 1 (HTLV-1) Tax protein is considered to play a central role in the process that leads to adult T-cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax-expressing cells show resistance to apoptosis induced by Fas ligand (FasL) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL). The regulation of Tax on the autophagy pathway in HeLa cells and peripheral T cells was recently reported, but the function and underlying molecular mechanism of the Tax-regulated autophagy are not yet well defined. Here, we report that HTLV-1 Tax deregulates the autophagy pathway, which plays a protective role during the death receptor (DR)-mediated apoptosis of human U251 astroglioma cells. The cellular FLICE-inhibitory protein (c-FLIP), which is upregulated by Tax, also contributes to the resistance against DR-mediated apoptosis. Both Tax-induced autophagy and Tax-induced c-FLIP expression require Tax-induced activation of IκB kinases (IKK). Furthermore, Tax-induced c-FLIP expression is regulated through the Tax-IKK-NF-κB signaling pathway, whereas Tax-triggered autophagy depends on the activation of IKK but not the activation of NF-κB. In addition, DR-mediated apoptosis is correlated with the degradation of Tax, which can be facilitated by the inhibitors of autophagy. IMPORTANCE: Our study reveals that Tax-deregulated autophagy is a protective mechanism for DR-mediated apoptosis. The molecular mechanism of Tax-induced autophagy is also illuminated, which is different from Tax-increased c-FLIP. Tax can be degraded via manipulation of autophagy and TRAIL-induced apoptosis. These results outline a complex regulatory network between and among apoptosis, autophagy, and Tax and also present evidence that autophagy represents a new possible target for therapeutic intervention for the HTVL-1 related diseases.

TRAIL receptor deficiency sensitizes mice to dextran sodium sulphate-induced colitis and colitis-associated carcinogenesis.

Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) and its receptor (TRAIL-R) play important roles in immune regulation and cancer cell death. Although TRAIL has been shown to induce chemokine release in various tumour cells, the function of TRAIL-R in the development of colitis and colitis-associated carcinogenesis has not been explored. In this study, we found that TRAIL-R-deficient mice exhibited a higher incidence of colitis and colitis-associated cancer than that of wild-type (WT) mice, and TRAIL-R expression was down-regulated in WT mice that were fed dextran sulphate sodium. Chemokines, including CCL2 and CXCL1, were highly expressed in the serum and inflammatory colon tissues of TRAIL-R(-/-) mice compared with WT mice, and TRAIL-R(-/-) mice showed a marked infiltration of immune cells during colitis. Hyperactivation of Janus kinase and nuclear factor-κB in colon epithelial cells was also observed, which correlated with the severity of colonic inflammation in TRAIL-R(-/-) mice. These data suggest that TRAIL-R plays a protective role in chemical-induced colon injury and negatively regulates mucosal immune responses.

TRAIL suppresses tumor growth in mice by inducing tumor-infiltrating CD4(+)CD25 (+) Treg apoptosis.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a promising and novel anticancer cytokine, specifically kills numerous tumor cells by apoptosis. However, some malignancies are resistant to TRAIL treatment in clinical trials, thus limiting its therapeutic potential. In the present study, the TRAIL-resistant murine hepatocellular carcinoma cell line Hepa1-6 was used to elucidate the physiological significance of TRAIL resistance, especially with respect to the immune regulatory function of TRAIL. Hepa1-6 cells were resistant to TRAIL-induced apoptosis in vitro; however, intratumoral injection of recombinant soluble TRAIL inhibited tumor growth and prolonged survival time in tumor-bearing mice. Local TRAIL treatment decreased the number of intratumoral CD4(+)CD25(+)Foxp3(+) regulatory T cells (Tregs) but did not affect CD4(+)CD25(+)Foxp3(+) Tregs in the draining lymph nodes and spleen. Further investigation showed that TRAIL induced apoptosis of tumor-activated CD4(+)CD25(+)Foxp3(+) Tregs, but not of CD4(+)CD25(-) T cells. Moreover, mouse TRAIL receptor DR5 expression was detected on the surface of the tumor-infiltrating CD4(+)CD25(+)Foxp3(+) Tregs, but not on naïve CD4(+)CD25(+)Foxp3(+) Tregs. Interestingly, intratumoral injection of TRAIL not only decreased the number of CD4(+)CD25(+)Foxp3(+) Tregs but also increased the number of tumor-specific CD8(+) CTL and augmented their cytotoxicity to the tumor cells. These data provide the novel evidence for an immune regulatory function of TRAIL and may shed light on the clinical application of TRAIL.

Epirubicin potentiates recombinant adeno-associated virus type 2/5-mediated TRAIL expression in fibroblast-like synoviocytes and augments the antiarthritic effects of rAAV2/5-TRAIL.

OBJECTIVE: Synovial cells in rheumatoid synovium display abnormal proliferation, which leads to joint destruction. TRAIL has been described as a proapoptotic factor in fibroblast-like synoviocytes (FLS). This study was undertaken to investigate the functions of rAAV2/5-TRAIL in human FLS and in arthritic mice. METHODS: Primary human FLS were infected with rAAV2/5-TRAIL in the presence or absence of epirubicin. Transgene expression was monitored by both enzyme-linked immunosorbent assay and flow cytometry. The disease-modulating activity of epirubicin plus rAAV2/5-TRAIL was investigated in mice with collagen-induced arthritis (CIA). RESULTS: Subtoxic doses of epirubicin potentiated rAAV2/5-mediated TRAIL expression in FLS and simultaneously enhanced the sensitivity of FLS to TRAIL. Epirubicin treatment up-regulated death receptor 4 (DR-4) and DR-5 expression and down-regulated FLIP expression, thereby enhancing the activation of procaspase 3, procaspase 8, and procaspase 9. An in vivo study showed that the combination of rAAV2/5-TRAIL gene therapy and epirubicin chemotherapy provided augmented antiarthritic effects in a mouse model of CIA. The intraarticular injection of rAAV2/5-TRAIL combined with epirubicin treatment significantly reduced the severity and incidence of CIA and joint swelling in the animals. Histologic evaluations revealed that inflammatory cell infiltration, cartilage destruction, and bone erosion were significantly reduced in the joints of the mice receiving the synthetic treatment. Results of a viral genome copy number assay indicated that epirubicin dramatically augmented the expression of rAAV2/5-TRAIL without altering its tissue distribution. CONCLUSION: These results suggest that epirubicin enhances the antiarthritic effect of rAAV2/5-TRAIL and that combination treatment might be an important therapeutic alternative, with practical significance for rheumatoid arthritis.

2A peptide-based, lentivirus-mediated anti-death receptor 5 chimeric antibody expression prevents tumor growth in nude mice.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a member of the TNF superfamily, induces tumor cell death via death receptors on target cells, without adverse effects on most normal cells. Its receptors are therefore an attractive target for antibody-mediated tumor therapy. Here, we report the creation of a lentivirus vector constructed by linking the heavy chain and the light chain of the antibody with a 2A/furin self-processing peptide in a single open reading frame that expresses a novel chimeric antibody (named as zaptuximab) with tumoricidal activity, which is consisted of the variable region of a mouse anti-human DR5 monoclonal antibody, AD5-10, and the constant region of human immunoglobulin G1. Lentivirus-expressed zaptuximab bound specifically to its antigen, DR5, and exhibited significant apoptosis-inducing activity in various tumor cell lines. The packaged recombinant virus lenti-HF2AL showed strong apoptosis-inducing activity in vitro. Meanwhile, inoculated subcutaneous human colon HCT116 tumor formation in nude mice were inhibited significantly. Moreover, there was a synergistic effect of mitomycin C (MMC) on the observed tumoricidal efficacy, prolonging the life span of nude mice with orthotopic human lung tumor cancers. These data suggest that lentivirus-mediated, 2A peptide-based anti-DR5 chimeric antibody expression may have clinical utility as an anticancer treatment and may represent a rational adjuvant therapy in combination with chemotherapy.

Therapeutic efficacy of a MMAE-based anti-DR5 drug conjugate Oba01 in preclinical models of pancreatic cancer.

Pancreatic cancer (PC) is among the most aggressive malignancies associated with a 5-year survival rate of <9%, and the treatment options remain limited. Antibody-drug conjugates (ADCs) are a new class of anticancer agents with superior efficacy and safety profiles. We studied the antitumor activity of Oba01 ADC and the mechanism underlying the targeting of death receptor 5 (DR5) in preclinical PC models. Our data revealed that DR5 was highly expressed on the plasma membrane of PC cells and Oba01 showed potent in vitro antitumor activity in a panel of human DR5-positive PC cell lines. DR5 was readily cleaved by lysosomal proteases after receptor-mediated internalization. Monomethyl auristatin E (MMAE) was then released into the cytosol to induce G2/M-phase growth arrest, cell death via apoptosis induction, and the bystander effect. Furthermore, Oba01 mediated cell death via antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity. For improved potency, we investigated the synergetic effect of Oba01 in combination with approved drugs. Oba01 combined with gemcitabine showed better antiproliferative activity than either standalone treatment. In cell- and patient-derived xenografts, Oba01 showed excellent tumoricidal activity in mono- or combinational therapy. Thus, Oba01 may provide a novel biotherapeutic approach and a scientific basis for clinical trials in DR5-expressing patients with PC.

A novel anti-DR5 chimeric antibody and epirubicin synergistically suppress tumor growth.

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of tumor cells. TRAIL receptor 2 (DR5) expression is high in tumor cells, transformed cells, and clinical tumor specimens and is low in most normal cells and tissues; therefore, DR5 is considered an attractive target for cancer therapy. In this study, HMCAZ5, a novel mouse-human chimeric antibody based on AD5-10, was generated and stably expressed in CHO-dhfr(-) cells. Highly purified HMCAZ5 exhibits a high affinity for the receptor that is equal to the parental mouse antibody, induces apoptosis in various cancer cells but not in normal hepatocytes, and elicits both antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity in various human cancer cells. The anthracycline anticancer drug epirubicin (EPB) synergizes the cytotoxicity of HMCAZ5 in cancer cells by upregulating DR5 expression on the cell surfaces, enhancing p53 expression, Bid cleavage, and JNK phosphorylation and downregulating c-FLIP expression and Akt phosphorylation. Moreover, HMCAZ5 alone suppresses tumor growth, and EPB augments the tumoricidal activity in human colorectal and hepatocellular tumor xenografts in athymic nude mice. These data suggest that the anti-DR5 chimeric antibody HMCAZ5 may have a clinical use and represents a useful immunological strategy, in combination with chemotherapy, for the treatment of cancer.

A crucial role of angiotensin converting enzyme 2 (ACE2) in SARS coronavirus-induced lung injury.

During several months of 2003, a newly identified illness termed severe acute respiratory syndrome (SARS) spread rapidly through the world. A new coronavirus (SARS-CoV) was identified as the SARS pathogen, which triggered severe pneumonia and acute, often lethal, lung failure. Moreover, among infected individuals influenza such as the Spanish flu and the emergence of new respiratory disease viruses have caused high lethality resulting from acute lung failure. In cell lines, angiotensin-converting enzyme 2 (ACE2) has been identified as a potential SARS-CoV receptor. The high lethality of SARS-CoV infections, its enormous economic and social impact, fears of renewed outbreaks as well as the potential misuse of such viruses as biologic weapons make it paramount to understand the pathogenesis of SARS-CoV. Here we provide the first genetic proof that ACE2 is a crucial SARS-CoV receptor in vivo. SARS-CoV infections and the Spike protein of the SARS-CoV reduce ACE2 expression. Notably, injection of SARS-CoV Spike into mice worsens acute lung failure in vivo that can be attenuated by blocking the renin-angiotensin pathway. These results provide a molecular explanation why SARS-CoV infections cause severe and often lethal lung failure and suggest a rational therapy for SARS and possibly other respiratory disease viruses.

[Molecular mechanism of hydroxyurea enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand].

OBJECTIVE: To explore the molecular mechanism via which the chemotherapeutic drug hydroxyurea (HU) enhances K562 cell apoptosis induced by tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). METHODS: Chronic myelogenous leukemia-derived K562 and SVT-35 cells were treated with recombinant soluble TRAIL (rsTRAIL) alone or combined with HU for a time course, and the cell viability was determined by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-4-sulfophenyl-2H-tetrazolium-phenazine methosulphate assay. Western blot was performed to analyze the activation of apoptosis-related protein kinases and the expression of apoptosis inhibitor molecules. RESULTS: The survival rates of SVT-35 and K562 cells treated with 1 µg/ml rsTRAIL for 24 hours were 32% and 93%, respectively. HU significantly increased the sensitivity of K562 cells to rsTRAIL cytotoxicity. Combination of rsTRAIL and HU resulted in the phosphorylation of rat sarcoma (RAS), mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase and in the significant reduction of apoptosis-inhibited molecule Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 in K562 cells. CONCLUSIONS: HU enhanced K562 cell sensitivity to rsTRAIL is mediated by Ras-MEK-ERK signaling pathway. Expression of antiapoptotic proteins cellular Fas associated death domain protein-like interleukin-1 beta-convening enzyme inhibitory protein and cellular inhibitor of apoptosis protein-1 is also down-regulated during this process. These results may through light on the therapeutic study of human chronic myelogenous leukemia.

Targeting a novel N-terminal epitope of death receptor 5 triggers tumor cell death.

Tumor necrosis factor-related apoptosis-inducing ligand receptors death receptor (DR) 4 and DR5 are potential targets for antibody-based cancer therapy. Activation of the proapoptotic DR5 in various cancer cells triggers the extrinsic and/or intrinsic pathway of apoptosis. It has been shown that there are several functional domains in the DR5 extracellular domain. The cysteine-rich domains of DR5 have a conservative role in tumor necrosis factor-related apoptosis-inducing ligand-DR5-mediated apoptosis, and the pre-ligand assembly domain within the N1-cap contributes to the ligand-independent formation of receptor complexes. However, the role of the N-terminal region (NTR) preceding the N1-cap of DR5 remains unclear. In this study, we demonstrate that NTR could mediate DR5 activation that transmits an apoptotic signal when bound to a specific agonistic monoclonal antibody. A novel epitope in the NTR of DR5 was identified by peptide array. Antibodies against the antigenic determinant showed high affinities for DR5 and triggered caspase activation in a time-dependent manner, suggesting the NTR of DR5 might function as a potential death-inducing region. Moreover, permutation analysis showed that Leu(6) was pivotal for the interaction of DR5 and the agonistic antibody. Synthetic wild-type epitopes eliminated the cytotoxicity of all three agonistic monoclonal antibodies, AD5-10, Adie-1, and Adie-2. These results indicate that the NTR of DR5 could be a potential target site for the development of new strategies for cancer immunotherapy. Also, our findings expand the current knowledge about DR5 extracellular functional domains and provide insights into the mechanism of DR5-mediated cell death.

Synergistic antitumor effect of AAV-mediated TRAIL expression combined with cisplatin on head and neck squamous cell carcinoma.

BACKGROUND: Adeno-associated virus-2 (AAV-2)-mediated gene therapy is quite suitable for local or regional application in head and neck cancer squamous cell carcinoma (HNSCC). However, its low transduction efficiency has limited its further development as a therapeutic agent. DNA damaging agents have been shown to enhance AAV-mediated transgene expression. Cisplatin, one of the most effective chemotherapeutic agents, has been recognized to cause cancer cell death by apoptosis with a severe toxicity. This study aims to evaluate the role of cisplatin in AAV-mediated tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) expression and the effect on HNSCC both in vitro and in vivo. METHODS: Five human HNSCC cell lines were treated with recombinant soluble TRAIL (rsTRAIL) and infected with AAV/TRAIL to estimate the sensitivity of the cancer cells to TRAIL-induced cytotoxicity. KB cells were infected with AAV/EGFP with or without cisplatin pretreatment to evaluate the effect of cisplatin on AAV-mediated gene expression. TRAIL expression was detected by ELISA and Western blot. Cytotoxicity was measured by MTT assay and Western blot analysis for caspase-3 and -8 activations. Following the in vitro experiments, TRAIL expression and its tumoricidal activity were analyzed in nude mice with subcutaneous xenografts of HNSCC. RESULTS: HNSCC cell lines showed different sensitivities to rsTRAIL, and KB cells possessed both highest transduction efficacy of AAV and sensitivity to TRAIL among five cell lines. Preincubation of KB cells with subtherapeutic dosage of cisplatin significantly augmented AAV-mediated transgene expression in a heparin sulfate proteoglycan (HSPG)-dependent manner. Furthermore, cisplatin enhanced the killing efficacy of AAV/TRAIL by 3-fold on KB cell line. The AAV mediated TRAIL expression was observed in the xenografted tumors and significantly enhanced by cisplatin. AAV/TRAIL suppressed the tumors growth and cisplatin augmented the tumoricidal activity by two-fold. Furthermore, Combination treatment reduced cisplatin-caused body weight loss in nude mice. CONCLUSION: The combination of AAV-mediated TRAIL gene expression and cisplatin had synergistic therapeutic effects on head and neck cancers and reduced the potential toxicity of cisplatin. These findings suggest that the combination of AAV/TRAIL and cisplatin may be a promising strategy for HNSCC therapy.

[Expression and tumoricidal activity of a human-mouse chimeric anti-DR5 antibody through a Furin/2A peptide].

OBJECTIVE: To express a full-length human-mouse chimeric anti-DR5 antibody from a single open reading frame with tumoricidal activity to various cancer cells. METHODS: The heavy and light chains of chimeric antibody were joined by the Furin and 2A (F/2A) self-cleavage peptide and cloned into a lentiviral vector of pWPXL. Then the HEK293 cells were infected with the constructed expression vector pWPXL-HF2AL. Western blot, enzyme-linked immunosorbent assay (ELISA) and MTS assay were used to detect the chimeric antibody expression, cleavage, binding affinity to the antigen and tumoricidal activity to various tumor cells. RESULTS: The recombinant chimeric antibody was successfully expressed from a single open reading frame in pWPXL-HF2AL construct. And it possessed a similar binding affinity to the parental murine counterpoint and strong tumoricidal activity to various cancer cells. For example, on the concentration of 3 µg/ml, it made the relative cells viability of HCT116, SMMC7721, A549 and U251 down to 20.6% ± 2.6%, 35.1% ± 2.7%, 76.1% ± 6.1% and 15.6% ± 2.0% respectively. CONCLUSIONS: The human-mouse chimeric anti-DR5 antibody of F/2A peptide is successfully expressed. Possessing a strong tumoricidal activity in various cancer cells, it may provide a novel strategy for cancer biotherapy.

[Killing effects of controllable expression of tumor necrosis factor-related apoptosis-inducing ligand from mesenchymal stem cells on hepatocellular carcinoma cells].

OBJECTIVE: To study the controllable expression of soluble tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) in mesenchymal stem cells and evaluate its potential tumoricidal effects in cancer therapy. METHODS: The controllable TRAIL expression vector of Ad-Tet-TRE-TRAIL was established in an adenovirus vector for transfection into murine mesenchymal stem cells. The controllable expression and secretion of TRAIL were detected by Western blot and enzyme-linked immunosorbent assay. The viability of hepatocellular carcinoma cells was determined by MTT assay. The tumoricidal activity of TRAIL was determined by Annexin-V/PI staining and flow cytometry. RESULTS: The murine expression model of TRAIL was successfully established in the presence of doxycycline. The secreted TRAIL in cell culture medium could efficaciously suppress the growth of human hepatocellular carcinoma SMMC-7402 by induced apoptosis. The cell viability of SMMC-7402 was 66.5% ± 4.8% and 42.9% ± 6.5% at post-treatment versus 97.3% ± 2.2% and 99.4% ± 4.7% in the control group at 24 h and 48 h. CONCLUSION: The controllable TRAIL expression mediated by mesenchymal stem cells kills human hepatocellular carcinoma cells effectively. And it may provide a novel therapeutic strategy for hepatocellular carcinoma.

[Combination of AD5-10 and epirubicin in treating rheumatoid arthritis].

OBJECTIVE: To investigate the mechanism of anti-death receptor 5-10 (AD5-10) combined with epirubicin in treating rheumatoid arthritis (RA). METHODS: We detected the cell viability of the fibroblast-like synoviocytes (FLS) from RA patients with MTT. The expression level of apoptosis signaling pathways protein, p53, and p21 were evaluated with Western blot. RESULTS: We found that epirubicin, at different doses, could enhance the effect of AD5-10 on FLS, promoting the apoptosis of FLS. The expression levels of caspase-3, -8, -9, c-FLIP, Bcl-2, p53, and p21 in the FLS changed after epirubicin treatment. CONCLUSION: Epirubicin may coordinate with AD5-10 in inducing FLS apoptosis through affecting the levels of p53, p21, c-FLIP, and Bcl-2.

Preclinical evaluation of a novel antibody-drug conjugate targeting DR5 for lymphoblastic leukemia therapy.

Acute lymphoblastic leukemia (ALL) is an aggressive hematological neoplasm resulting from immature lymphoid precursors. An antibody-drug conjugate (ADC), coupling a small molecule covalently with a targeting antibody, can specifically kill tumor cells. Death receptor 5 (DR5) is considered as a promising anti-tumor drug target. In this study, we describe the preclinical evaluation of a novel DR5-targeting ADC (Oba01) as a potential therapeutic against ALL. Oba01 utilizes anti-DR5 humanized monoclonal antibody (zaptuzumab) coupled via a cleavable linker to monomethyl auristatin E (MMAE). Oba01 can specifically bind to DR5 on the tumor cells and transfer into lysosome via DR5-mediated endocytosis. It then effectively releases the MMAE, which can bind to the tubulin and prevent its aggregation, thereby leading to a significant inhibition of proliferation and cell death in tumor cells. Additionally, Oba01 displays significant dose-dependent tumoricidal activity in cell-derived xenograft (CDX) and patient-derived xenograft (PDX) mouse models. More importantly, toxicity analysis of Oba01 showed a favorable safety profile, and pharmacokinetic analysis illustrated an excellent stability and tolerability in rats and cynomolgus monkeys. Taken together, our data conclusively demonstrate that Oba01 is an attractive candidate for further clinical trials in DR5-positive ALL patients.

Knockdown of c-FLIP(L) enhanced AD5-10 anti-death receptor 5 monoclonal antibody-induced apoptosis in human lung cancer cells.

It is reported that the agonistic antibodies against death receptors 4 and 5 (DR4, DR5) are cytotoxic to various cancer cells. In the present study, the sensitivity of five human lung cancer cell lines to previously reported AD5-10 agonistic antibody against DR5 were investigated. Of these cell lines, A549 and small cell lung cancer showed a moderate sensitivity to AD5-10 and three other cell lines were resistant. Cell line H460 is resistant to AD5-10 despite a high level of cell-surface DR5 expression. We demonstrated that the resistance of H460 cells to AD5-10 was not related to the expression level of DR5, but the expression and cleavage of c-FLIP(L) in the cells. Inhibition of endogenous c-FLIP(L) expression by siRNA significantly enhanced AD5-10-induced cell death in these lung cancer cells. We further showed that this sensitizing effect was associated with decreased expression of Bcl-2 family proteins Bid and Bcl-X(L), change of mitochondrial membrane potential, release of cytochrome c from mitochondria, and caspase activation. Therefore, these data provide evidence that c-FLIP(L) is involved in the resistance of lung cancer cells to AD5-10-induced apoptosis. Moreover, immunohistochemistry on paraffin-embedded tissue revealed that c-FLIP(L) was expressed in 87.9% (29 of 33) of lung carcinoma tissues from the patients, but little in tissues from normal controls. This suggests that inhibition of c-FLIP(L) expression might be a potential strategy for lung cancer therapy, especially for those lung cancers resistant to the agonistic antibody against death receptors.

Tax1 enhances cancer cell proliferation via Ras-Raf-MEK-ERK signaling pathway.

Erbin is an ErbB2 binding protein, which belongs to the LAP (leucine-rich repeat (LRR) and PDZ domain) protein family. We previously reported that Tax1, a protein of the human T-cell leukemia virus type I (HTLV-I), associated with Erbin by using Erbin PDZ domain as a bait to screen a human T lymphocyte cDNA library by a yeast two hybrid strategy. In the present study, we demonstrated that Tax1 enhances cancer cell proliferation via Ras-Raf-MEK-ERK signaling pathway by using molecular section strategy. The pull-down assay showed that the four amino acid domain, that is, Tax1 350-353, might specifically interact with Erbin, but not any other Tax1 deletion mutants. The coimmunoprecipitation assay confirmed that Tax1 350-353 domain bound with Erbin in vivo. Functional study demonstrated that overexpression of Tax1 in cancer cell lines of liver cancer SMMC-7721, colon cancer HCT-116, and breast cancer MCF-7 facilitated the cell proliferation. And the transfection of Tax1 353 in MCF-7 cells with endogenous Erbin expression markedly increased phosphorylation of Ras, Raf, MEK1/2, ERK1/2, PI3K, and IkappaBalpha, suggesting that Tax1-enhanced cell proliferation tracks Ras-Raf-MEK-ERK signaling pathway.