Huizhong Li

Keap1: one stone kills three birds Nrf2, IKKβ and Bcl-2/Bcl-xL.

Oxidative stress, implicated in the etiology of cancer, results from an imbalance in the production of Reactive Oxygen Species (ROS) and cells own antioxidant defenses. As a oxidative stress sensor, Keap1 functions as both an adaptor for Cul3⋅Rbx1 E3 ligase complex mediated degradation of the transcription factor Nrf2, and a master regulator of cytoprotective gene expression. Although Nrf2 is a well known substrate for Keap1, the DGR domain of Keap1 has been reported also to bind other proteins directly or indirectly. IKKβ as positive regulator of NF-κB is also destabilized by Keap1, which resulted in inhibiting NF-κB-derived tumor promotion. In addition, anti-apoptotic Bcl-2/Bcl-xL protein was identified as another substrate for the Keap1-Cul3-E3 ligase complex. Keap1 led to the repression and destabilization of Bcl-2, decreased Bcl-2:Bax heterodimers and facilitated cancer cells apoptosis. Given that Keap1 might function as a tumor suppressor protein to mitigate tumor progression, the different kinds of Keap1 somatic mutations were detected in numerous cancer cells. Therefore, it is important to understand the Keap1-involved signaling cascades. This review primarily focuses on the prevention of tumorigenesis role of Keap1 through negative regulation of three substrates Nrf2, IKKβ and Bcl-2/Bcl-xL, with emphasis on the recent findings indicating the cancer guarder function of Keap1.

Immunoreceptor TIGIT inhibits the cytotoxicity of human cytokine-induced killer cells by interacting with CD155

T cell Ig and ITIM domain (TIGIT) is a newly identified inhibitory receptor expressed on T and natural killer (NK) cells. Cytokine-induced killer (CIK) cells express CD3 and CD56 molecules, and share functional properties with both NK and T cells. However, it remains unknown whether TIGIT is expressed in CIK cells. Here, we show that TIGIT is expressed by CIK cells and interacts with CD155. By blocking TIGIT using an anti-TIGIT functional antibody, we demonstrate that CIK cells display increased proliferation; higher cytotoxic targeting of tumor cells expressing CD155; and higher expression of interferon-γ (IFN-γ), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Furthermore, increases in IFN-γ and cytotoxicity by blockade of TIGIT were reduced by blocking DNAX accessory molecule-1 (DNAM-1) signaling, implying that TIGIT exerts immunosuppressive effects by competing with DNAM-1 for the same ligand, CD155. Our results provide evidence that blockade of TIGIT may be a novel strategy to improve the cytotoxic activity of CIK cells.

A Novel Strategy to Improve the Therapeutic Efficacy of Gemcitabine for Non-Small Cell Lung Cancer by the Tumor-Penetrating Peptide iRGD.

Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, comprising approximately 75–80% of all lung cancers. Gemcitabine is an approved chemotherapy drug for NSCLC. The objective of this study was to develop a novel strategy to improve the therapeutic efficacy of Gemcitabine for NSCLC by the co-administered iRGD peptide. We showed that the rates of positive expression of αvβ3, αvβ5 and NRP-1 in the A549 cell line were 68.5%, 35.3% and 94.5%, respectively. The amount of Evans Blue accumulated in the tumor of Evans Blue+iRGD group was 2.5 times that of Evans Blue group. The rates of growth inhibition of the tumors of the iRGD group, the Gemcitabine group and the Gemcitabine+iRGD group were 8%, 59.8% and 86.9%, respectively. The results of mechanism studies showed that PCNA expression in the Gemcitabine+iRGD group decreased 71.5% compared with that in Gemcitabine group. The rate of apoptosis in the Gemcitabine+iRGD group was 2.2 time that of the Gemcitabine group. Therefore, the tumor-penetrating Peptide iRGD can enhance the tumor-penetrating ability and therapeutic efficacy of Gemcitabine in the A549 xenograft. The combined application of Gemcitabine with iRGD may be a novel strategy to enhance the clinical therapeutic efficacy of Gemcitabine in patients with NSCLC.

MDA-7/IL-24 inhibits Nrf2-mediated antioxidant response through activation of p38 pathway and inhibition of ERK pathway involved in cancer cell apoptosis.

Reactive oxygen species (ROS) have a crucial role in melanoma differentiation-associated gene-7 (MDA-7)/interleukin-24 (IL-24)-induced cancer cell apoptosis. However, cancer cell has a series of protective mechanisms to resist ROS damage. Nuclear factor erythroid 2-related factor 2 (Nrf2) activates antioxidant response element (ARE)-mediated gene expression involved in cellular protection against oxidative stress. As the Nrf2 repressor, Kelch-like ECH-associated protein-1 (Keap1) sequesters Nrf2 in cytoplasm to block Nrf2 nuclear translocation. In the present study, administration of MDA-7/IL-24 by means of tumor-selective replicating adenovirus (ZD55-IL-24) was used to investigate whether ZD55-IL-24 could attenuate Nrf2-mediated oxidative stress response in cancer cell. We found that ZD55-IL-24 effectively strengthened the association between Nrf2 and Keap1 to restrict Nrf2 nuclear translocation, thereby inhibiting ARE-dependent transcriptional response. To evaluate the detailed mechanism underlying the suppression of ZD55-IL-24 on Nrf2-mediated oxidative stress response, we further tested three different mitogen-activated protein kinase (MAPK) signaling pathways in A549 and HeLa cells transfected by ZD55-IL-24. Our data showed that ZD55-IL-24 inhibited extracellular signal-regulated kinase (ERK) signal pathway but activated p38 and c-Jun-NH2-kinase (JNK) signal pathways to exert the tumor-specific apoptosis. Moreover, ERK pathway inhibitor U0126 prevented Nrf2 phosphorylation at Ser40 to retard Nrf2 nuclear translocation, thus decreasing antioxidant gene transcription. In contrast, p38 pathway inhibitor SB203580 obviously promoted the dissociation of Nrf2 from Keap1 to promote antioxidant gene transcription. However, JNK pathway had no effect on Nrf2 subcellular localization or the association of Nrf2 with Keap1. Conclusively, our results indicate that ZD55-IL-24 inhibits Nrf2-mediated oxidative stress response not only by activating p38 signal pathway to potentiate the association of Nrf2 and Keap1 but also by suppressing ERK signal pathway to postpone Nrf2 nuclear translocation. Given the ‘dark’ side of Nrf2 on carcinoma cell survival and chemoresistance, our study provides a novel explanation about MDA-7/IL-24-induced cancer-specific apoptosis and therapeutic sensitization through suppression of the cytoprotective system.

Potent Antitumor Effect Elicited by RGD-mda-7, an mda-7/IL-24 Mutant, via Targeting the Integrin Receptor of Tumor Cells

The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, an mda-7/IL-24 mutant, RGD-mda-7, which includes the cell adhesive sequence 164Arg-165Gly-166Asp (RGD motif), was constructed and evaluated for bioactivity. RGD peptide binds to integrins α(V)β(3) and α(V)β(5), which are selectively expressed in tumor neovasculature and in the surface of some tumor cells. The wtmda-7/IL-24 and RGD-mda-7 were expressed in Escherichia coli and then purified and renatured. The immunostimulatory activity of RGD-mda-7 was assayed by stimulating peripheral blood mononuclear cells. The results suggested that the abilities of RGD-mda-7 to induce IL-6, TNF-α, and IFN-γ production were higher than wtmda-7/IL-24. Tumor targeting of RGD-mda-7 was assayed using cell adhesion experiments. The antitumor effect of the purified RGD-mda-7 on cell proliferation in vitro was evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake, cell apoptosis by staining with fluorescent probes of FITC-annexin V and DAPI, and caspase-3 expression and activity. The in vitro results showed that RGD-mda-7 inhibited the proliferation of multiple tumor cell lines (Hela, ACHN, HepG2, and A549). Staining with fluorescent probes of FITC-annexin V and DAPI indicated that RGD-mda-7 could induce apoptosis more effectively in four tumor cell lines than wtmda-7/IL-24, but has no effect on normal cell line NHLF. Western blotting showed that treatment of tumor cells with RGD-mda-7 could activate apoptotic pathway by cleavage of caspase-3 as same as wtmda-7/IL-24. Further, RGD-mda-7 group showed a higher cleaved level of caspase-3, but not in NHLF cells. These results demonstrate that RGD-MDA-7 possesses more potent antitumor effects than wtmda-7/IL-24 and therefore merits further investigation in preclinical and clinical studies.

p53 regulates Ki-67 promoter activity through p53- and Sp1-dependent manner in HeLa cells

The expression of the human Ki-67 protein, which is strictly associated with cell proliferation, is regulated by a variety of cellular mediators. In this study, we studied the effects of p53 on Ki-67 promoter in HeLa cells using luciferase reporter assay. The results showed that: (1) p53 inhibited Ki-67 promoter activity in a dose-dependent manner, (2) the p53-binding motifs mediated part of the transcriptional repression of Ki-67 promoter through a sequence-specific interaction with p53, (3) p53 was able to repress the Sp1-stimulated Ki-67 promoter activity, and (4) the Sp1-binding sites were responsible for the p53-mediated transcriptional repression of Ki-67 promoter. In conclusion, p53 inhibited Ki-67 promoter activity via p53- and Sp1-dependent pathways, and the interaction between p53 and Sp1 might be involved in the transcriptional regulatory mechanisms.

Enhanced apoptosis-inducing function of MDA-7/IL-24 RGD mutant via the increased adhesion to tumor cells.

Melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has shown potent tumor cell apoptosis inducing capacity in multiple cancers. However, the apoptosis induction capacity of mda-7/IL-24 was low and directly correlated with the adhesion to tumor cells.Cell adhesion molecule integrin α(v)β(3) expressed on the surface of several types of solid tumor cells, and they bind to arginine-glycine-aspartic acid (RGD) which enhanced the adhesion to tumor cells. This rout was exploited to construct a tumor-targeting gene RGD-IL-24 which can express RGD-MDA-7/IL-24 protein that includes the cell adhesive sequence (164)Arg-(165)Gly-(166)Asp (A Glycine residue was inserted into the recombinant MDA-7/IL-24 between Arg164 and Asp165 to form a RGD motif). We successfully got the MDA-7/IL-24 mutant by overlapping polymerase chain reaction (PCR) and evaluated its therapeutic efficacy for tumor cell lines MCF-7, HeLa, HepG2, and normal human lung fibroblast (NHLF) line. And we found that the expression of pCDNA3.1/RGD-IL-24 was same to the expression of pCDNA3.1/IL-24. The RGD-IL-24 enhanced the apoptosis-inducing function in tumor cells, but not in normal cells. In tumor cell lines, the apoptosis-inducing activities of RGD-IL-24 was significantly higher than IL-24 detecting by MTT assay, Annexin V, and Hoechst 33258 analysis. Further, pCDNA3.1/RGD-IL-24 showed a significant increase in the ratio of pro-apoptotic (bax) to anti-apoptotic (bcl-2) proteins in tumor cell lines, but not in NHLF cell line. Together, these results suggest that RGD-IL-24 can enhance the apoptosis of tumor cells and may provide a promising drug in tumor therapy.

Strategies to Improve the Clinical Performance of Chimeric Antigen Receptor-Modified T Cells for Cancer

Clinical trials of chimeric antigen receptor (CAR)-modified T cells have shown promise in hematologic malignancies. However, in solid tumors, the clinical responses have been less impressive. It is important to determine how to further improve the clinical effects of CAR-modified T cells. In this review, we focus on recent clinical trials and analyze the factors that determine clinical responses, including the following: 1) the composition of the CAR; 2) the preparation of CAR-modified T Cells; 3) the clinical treatment schedule; 4) the patient characteristics. We also propose future Strategies that must be investigated before the technology can be used in a wider range of clinical applications.

MDA-7/IL-24 induces Bcl-2 denitrosylation and ubiquitin-degradation involved in cancer cell apoptosis.

MDA-7/IL-24 was involved in the specific cancer apoptosis through suppression of Bcl-2 expression, which is a key apoptosis regulatory protein of the mitochondrial death pathway. However, the underlying mechanisms of this regulation are unclear. We report here that tumor-selective replicating adenovirus ZD55-IL-24 leads to Bcl-2 S-denitrosylation and concomitant ubiquitination, which take part in the 26S proteasome degradation. IL-24-siRNA completely blocks Bcl-2 ubiquitination via reversion of Bcl-2 S-denitrosylation and protects it from proteasomal degradation which confirmed the significant role of MDA-7/IL-24 in regulating posttranslational modification of Bcl-2 in cancer cells. Nitric oxide (NO) is a key regulator of protein S-nitrosylation and denitrosylation. The NO donor, sodium nitroprusside (SNP), down-regulates Bcl-2 S-denitrosylation, attenuates Bcl-2 ubiquitination and subsequently counteracts MDA-7/IL-24 induced cancer cell apoptosis, whereas NO inhibitor 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxy-3-oxide (PTIO) shows the opposite effect. At the same time, these NO modulators fail to affect Bcl-2 phosphorylation, suggesting that NO regulates Bcl-2 stability in a phosphorylation-independent manner. In addition, Bcl-2 S-nitrosylation reduction induced by ZD55-IL-24 was attributed to both iNOS decrease and TrxR1 increase. iNOS-siRNA facilitates Bcl-2 S-denitrosylation and ubiquitin-degradation, whereas the TrxR1 inhibitor auranofin prevents Bcl-2 from denitrosylation and ubiquitination, thus restrains the caspase signal pathway activation and subsequent cancer cell apoptosis. Taken together, our studies reveal that MDA-7/IL-24 induces Bcl-2 S-denitrosylation via regulation of iNOS and TrxR1. Moreover, denitrosylation of Bcl-2 results in its ubiquitination and subsequent caspase protease family activation, as a consequence, apoptosis susceptibility. These findings provide a novel insight into MDA-7/IL-24 induced growth inhibition and carcinoma apoptosis.

EXPRESSION, PURIFICATION, AND CHARACTERIZATION OF RGD-mda-7, A HIS-TAGGED mda-7/IL-24 MUTANT PROTEIN

RGD peptide (Arg-Gly-Asp tripeptide) binds to integrin αVβ3 and αVβ5, which is selectively expressed in tumor neovasculature and on the surface of some tumor cells. Some studies showed that coupling the RGD peptides to anticancer drugs yielded compounds with increased efficiency against tumors and lowered toxicity to normal tissues. The melanoma differentiation-associated gene-7/interleukin-24 gene (mda-7/IL-24) is a novel tumor-suppressor/cytokine gene that exhibits potent tumor-suppressive activity without damaging normal cells. To enhance the antitumor effect, we inserted a glycine residue into the wild type (mda-7/IL-24) between 164Arg and 165Asp to form a RGD peptide, named RGD-mda-7, then expressed RGD-mda-7 in Escherichia coli. Herein, we describe the expression and purification of RGD-mda-7. We detected the characterizations of immunostimulatory activity, tumor targeting, potent cytopathic effect, and apoptosis inducing exploited by RGD-mda-7 in tumor cells, and also compared these characterizations with wtmda-7/IL-24. The data showed that RGD-mda-7 had more potent tumor targeting and apoptosis-inducing effects than wtmda-7/IL-24.