Min Xiong

Legumain protease-activated TAT-liposome cargo for targeting tumours and their microenvironment

Specific targeting and cellular internalization are key properties for carriers of antitumor therapeutic agents. Here, we develop a drug carrier through the attachment of substrate of endoprotease legumain, alanine-alanine-asparagine (AAN), to cell-penetrating peptides (TAT, trans-activating factor). The addition of the AAN moiety to the fourth lysine in the TAT creates a branched peptide moiety, which leads to a decrease in the transmembrane transport capacity of TAT by 72.65%. Legumain efficiently catalyses the release of TAT-liposome from the AAN-TAT-liposome and thereby recovers the penetrating capacity of TAT. Doxorubicin carried by the AAN-TAT-liposome led to an increase in the tumoricidal effect of doxorubicin and a reduction in its systemic adverse effects in comparison with doxorubicin carried by a control delivery system. Thus, the specific targeting and high efficiency of this delivery platform offers a novel approach to limit the toxicity of anticancer agents as well as increasing their efficacy in cancer therapy.

Alginic acid-coated chitosan nanoparticles loaded with legumain DNA vaccine: effect against breast cancer in mice.

Legumain-based DNA vaccines have potential to protect against breast cancer. However, the lack of a safe and efficient oral delivery system restricts its clinical application. Here, we constructed alginic acid-coated chitosan nanoparticles (A.C.NPs) as an oral delivery carrier for a legumain DNA vaccine. First, we tested its characteristic in acidic environments in vitro. DNA agarose electrophoresis data show that A.C.NPs protected DNA better from degradation in acidic solution (pH 1.5) than did chitosan nanoparticles (C.NPs). Furthermore, size distribution analysis showed that A.C.NPs tended to aggregate and form micrometer scale complexes in pH<2.7, while dispersing into nanoparticles with an increase in pH. Mice were intragastrically administrated A.C.NPs carrying EGFP plasmids and EGFP expression was detected in the intestinal Peyer’s patches. Full-length legumain plasmids were loaded into different delivery carriers, including C.NPs, attenuated Salmonella typhimurium and A.C.NPs. A.C.NPs loaded with empty plasmids served as a control. Oral vaccination was performed in the murine orthotopic 4T1 breast cancer model. Our data indicate that tumor volume was significantly smaller in groups using A.C.NPs or attenuated Salmonella typhimurium as carriers. Furthermore, splenocytes co-cultured them with 4T1 cells pre-stimulated with CoCl2, which influenced the translocation of legumain from cytoplasm to plasma membrane, showed a 4.7 and 2.3 folds increase in active cytotoxic T lymphocytes (CD3+/CD8+/CD25+) when treated with A.C.NPs carriers compared with PBS C.NPs. Our study suggests that C.NPs coated with alginic acid may be a safe and efficient tool for oral delivery of a DNA vaccine. Moreover, a legumain DNA vaccine delivered orally with A.C.NPs can effectively improve autoimmune response and protect against breast cancer in mice.

Loss of vitamin D receptor in chronic kidney disease: a potential mechanism linking inflammation to epithelial-to-mesenchymal transition

Both peritubular inflammation and tubular epithelial-to-mesenchymal transition (EMT) are critical events during the pathogenesis of renal fibrosis. However, the relationship between these two processes is unclear. Here, we investigated the potential role of the vitamin D receptor (VDR) in coupling peritubular inflammation and EMT. In a mouse model of unilateral ureteral obstruction (UUO), loss of VDR was observed as early as 1 day after surgery. In cultured proximal tubular epithelial HK-2 cells, proinflammatory TNF-α inhibited the expression of VDR in a dose- and time-dependant manner. Treatment with TNF-α sensitized HK-2 cells to EMT stimulated by transforming growth factor (TGF)-β1. Ectopic expression of VDR counteracted the synergistic effect of TNF-α and TGF-β1 on EMT. Furthermore, knockdown of VDR using a small interfering RNA strategy mimicked the effect of TNF-α on facilitating EMT. Either TNF-α treatment or a loss of VDR induced β-catenin activation and its nuclear translocation. The VDR ligand calcitriol reversed the VDR loss and inhibited EMT in the mouse UUO model, and late administration of active vitamin D was effective in restoring VDR expression as well, and reduced collagen accumulation and deposition compared with the vehicle control. β-Catenin expression induced by UUO was also significantly inhibited after the late administration of vitamin D. These results indicate that the early loss of VDR in chronic kidney diseases was likely mediated by proinflammatory TNF-α, which renders tubular cells susceptible to EMT. Our data suggest that loss of VDR couples peritubular inflammation and EMT, two key events in renal fibrogenesis.

VDR Status Arbitrates the Prometastatic Effects of Tumor-Associated Macrophages

The relationship between tumor-associated macrophages (TAM) and epithelial-to-mesenchymal transition (EMT) during the initiation and progression of metastasis is still unclear. Here, a role for the vitamin D receptor (VDR) in metastasis was identified, as well as a role in the relationship between TAMs and EMT. First, the expression level of VDR was examined in clinical tissue from human patients with breast cancer or a mouse model of breast cancer with differential metastasis. These results revealed that VDR expression negatively correlates with metastasis in breast cancer. Second, coculture of VDR-overexpressing breast cancer cells with a macrophage cell line demonstrated that overexpression of VDR alleviated the prometastatic effect of cocultured macrophages on breast cancer cells. Furthermore, VDR overexpression abrogated the induction of EMT in breast cancer cells by cocultured macrophage cells, as measured by a loss of E-cadherin (CDH1) and induction of α-smooth muscle actin (α-SMA). TNFα in macrophage conditioned media inhibited VDR expression, whereas downregulation of VDR further mediated the promotion of TGFβ-induced EMT by TNFα. In addition, β-catenin expression was inhibited in VDR-overexpressing breast cancer cells and tumor xenografts. Finally, administration of calcitriol [1,25-(OH)2D3], an active vitamin D metabolite, exerted similar antimetastatic effects in breast cancer cells in vitro and a mouse model of breast cancer in vivo with preservation of VDR and suppression of β-catenin. Implications: VDR suppression by TNFα mediates the prometastatic effect of TAMs through enhancement of the β-catenin pathway. Mol Cancer Res; 12(8); 1181–91. ©2014 AACR.

Resveratrol induces apoptosis of human chronic myelogenous leukemia cells in vitro through p38 and JNK-regulated H2AX phosphorylation.

Aim:The phosphorylation of histone H2AX, a novel tumor suppressor protein, is involved in regulation of cancer cell apoptosis. The aim of this study was to examine whether H2AX phosphorylation was required for resveratrol-induced apoptosis of human chronic myelogenous leukemia (CML) cells in vitro.Methods:K562 cells were tested. Cell apoptosis was analyzed using flow cytometry, and the phosphorylation of H2AX and other signaling proteins was examined with Western blotting. To analyze the signaling pathways, the cells were transfected with lentiviral vectors encoding H2AX-wt or specific siRNAs.Results:Treatment of K562 cells with resveratrol (20–100 μmol/L) induced apoptosis and phosphorylation of H2AX at Ser139 in time- and dose-dependent manners, but reduced phosphorylation of histone H3 at Ser10. Resveratrol treatment activated two MAPK family members p38 and JNK, and blocked the activation of another MAPK family member ERK. Pretreatment with the p38 inhibitor SB202190 or the JNK inhibitor SP600125 dose-dependently reduced resveratrol-induced phosphorylation of H2AX, which were also observed when the cells were transfected with p38- or JNK-specific siRNAs. Overexpression of H2AX in K562 cells markedly increased resveratrol-induced apoptosis, whereas overexpression of H2AX-139m (Ser139 was mutated to block phosphorylation) inhibited resveratrol-induced apoptosis. K562 cells transfected with H2AX-specific siRNAs were resistant to resveratrol-induced apoptosis.Conclusion:H2AX phosphorylation at Ser139 in human CML cells, which is regulated by p38 and JNK, is essential for resveratrol-induced apoptosis.

Genome-wide transcriptional analysis of apoptosis-related genes and pathways regulated by H2AX in lung cancer A549 cells

Histone H2AX is a novel tumor suppressor protein and plays an important role in apoptosis of cancer cells. However, the role of H2AX in lung cancer cells is unclear. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. We showed that H2AX was involved in apoptosis of lung cancer A549 cells as in other tumor cells. Knockdown of H2AX strongly suppressed apoptosis of A549 cells. We clarified the molecular mechanisms of apoptosis regulated by H2AX based on genome-wide transcriptional analysis. Microarray data analysis demonstrated that H2AX knockdown in A549 cells affected expression of 3,461 genes, including upregulation of 1,435 and downregulation of 2,026. These differentially expressed genes were subjected to bioinformatic analysis for exploring biological processes regulated by H2AX in lung cancer cells. Gene ontology analysis showed that H2AX affected expression of many genes, through which, many important functions including response to stimuli, gene expression, and apoptosis were involved in apoptotic regulation of lung cancer cells. Pathway analysis identified the mitogen-activated protein kinase signaling pathway and apoptosis as the most important pathways targeted by H2AX. Signal transduction pathway networks analysis and chromatin immunoprecipitation assay showed that two core genes, NFKB1 and JUN, were involved in apoptosis regulated by H2AX in lung cancer cells. Taken together, these data provide compelling clues for further exploration of H2AX function in cancer cells.

H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in chronic myelogenous leukemia cells induced by imatinib

Increasing evidence suggests that histone H2AX plays a critical role in regulation of tumor cell apoptosis and acts as a novel human tumor suppressor protein. However, the action of H2AX in chronic myelogenous leukemia (CML) cells is unknown. The detailed mechanism and epigenetic regulation by H2AX remain elusive in cancer cells. Here, we report that H2AX was involved in apoptosis of CML cells. Overexpression of H2AX increased apoptotic sensitivity of CML cells (K562) induced by imatinib. However, overexpression of Ser139-mutated H2AX (blocking phosphorylation) decreased sensitivity of K562 cells to apoptosis. Similarly, knockdown of H2AX made K562 cells resistant to apoptotic induction. These results revealed that the function of H2AX involved in apoptosis is strictly related to its phosphorylation (Ser139). Our data further indicated that imatinib may stimulate mitogen-activated protein kinase (MAPK) family member p38, and H2AX phosphorylation followed a similar time course, suggesting a parallel response. H2AX phosphorylation can be blocked by p38 siRNA or its inhibitor. These data demonstrated that H2AX phosphorylation was regulated by p38 MAPK pathway in K562 cells. However, the p38 MAPK downstream, mitogen- and stress-activated protein kinase-1 and -2, which phosphorylated histone H3, were not required for H2AX phosphorylation during apoptosis. Finally, we provided epigenetic evidence that H2AX phosphorylation regulated apoptosis-related gene Bim expression. Blocking of H2AX phosphorylation inhibited Bim gene expression. Taken together, these data demonstrated that H2AX phosphorylation regulated by p38 is involved in Bim expression and apoptosis in CML cells induced by imatinib.

Abstract 3535: Lymphoma-associated macrophages benefit for tumor progress via overexpressing legumain to degrade extracellular matrix in diffuse large B-cell lymphoma

Crosstalk between macrophages in the tumor microenvironment and malignant cells has been implicated during the pathogenesis of many kinds of tumors. However, role of lymphoma-associated macrophages in the progress of lymphoma as well as its underlying mechanism is far from completely clear yet. Here we evaluated the effect of lymphoma-associated macrophages on the characteristics of diffuse large B-cell lymphoma (DLBCL). Immunohistological staining demonstrated that level of CD163 positive macrophages, so called M2 type macrophage, was negatively related with the prognosis of DLBCL. In addition, M2 macrophage overexpressed asparaginyl endopeptidase legumain, as shown by double-immunofluoresence staining. Then we set up human DLBCL xenograft model. Mice were sacrificed at the different stages of tumor progress (average volume are 0.8 or 1.4 cm 3 . respectively. n=4). Real-time PCR results showed that mRNA expression of CD206 (M2 macrophage marker in mouse model) and legumain significantly increased with the progress of tumor. However, contents of matrix significantly decreased as indicated by sirus red and Masson9s staining. Overlapping existed in immunofluorescence staining of legumain and fibronectin/ collagen I. In vitro, we cultured monocytes U937 with supernatant from primary DLBCL cells. Real-time PCR and western blot demonstrated that mRNA and protein expression of M2 markers as well as legumain significantly increased in U937 after incubation of 48 hours. Further, we cultured the legumain overexpressed stable cell line in the wells coated with fibronectin or collagen I. Western blot showed that compared to control, legumain overexpression promoted the degradation of fibronectin and collagen I. Mice in human DLBCL xenograft model were randomly received clodronate liposome; PBS liposome or legumain inhibitor via i.v. injection (n=5). The results revealed that tumor growth was significantly delayed in the clodronate liposome group compared with PBS liposome control. Similarly, injection of legumain inhibitor mimicked the effect of clodronate liposome on the tumor progress. Our data suggests that lymphoma-associated macrophages polarized by lymophoma cells promote tumor progress and this effect might be mediated by degrading the matrix via overexpressed legumain. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3535. doi:1538-7445.AM2012-3535

Abstract 341: Imatinib induces expression of Bim and apoptosis in chronic myelogenous leukemia cells via p38/H2AX pathway

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA H2AX is a novel human tumor suppressor protein and plays an important role in apoptosis of cancer cells. Increasing published data indicate that H2AX phosphorylation (Ser139) contributes to its regulation of cancer cell apoptosis. Thus, charactering the mechanisms and signaling pathways involved in H2AX phosphorylation (Ser139) will certainly provide better understanding on H2AX function in cancer cells. Our previous report showed that caspase-3/Mst1 pathway regulates phosphorylation of H2AX at Ser139 in chronic myelogenous leukemia (CML) cells. Recently we found another pathway which regulates H2AX phosphorylation (Ser139) in CML cells. When the CML cells were subjected to imatinib treatment, MAPK family member ERK1/2 activity was inhibited, but p38 activity was increased, another member JNK1/2 had no response. Then we used p38 inhibitor SB202190 and siRNA against p38 to block p38 activation and observed that H2AX phosphorylation and apoptosis of CML cells was concurrently inhibited. Our results also showed that inhibition of MSK1/MSK2 did not affect H2AX phosphorylation (Ser139), although the p38 and ERK1/2 downstream MSK1/MSK2 could be activated by imatinib in CML cells. Overall, these data confirmed that p38 signaling pathway is also required for H2AX phosphorylation (Ser139) in CML cells. Our further evidences indicated that inhibition of H2AX phosphorylation (Ser139) regulated by p38 during apoptotic induction with imatinib suppressed the expression of apoptosis-related gene Bim. Taken together, these data demonstrated that p38/H2AX pathway regulates Bim expression and subsequent apoptosis in CML cells. Citation Format: Min Xiong, Tianhui Niu, Yaqiong Dong, Chengrong Lu. Imatinib induces expression of Bim and apoptosis in chronic myelogenous leukemia cells via p38/H2AX pathway. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 341. doi:10.1158/1538-7445.AM2014-341

Abstract 5614: Legumain protease substrate modified TAT-liposome cargo: an efficient tool for targeting malignant diseases.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Tumor targeting specificity and efficacy of the internalization of macro-biomolecular drugs have emerged as two major obstacles hampering the application of liposome-based delivery system against tumor. Trans-activating transcriptional activator (TAT) is a member of the cell-penetrating peptides which enhance the penetration of various macro-biomolecular cargos. In this study, we developed a modified TAT-Liposome cargo in which the activation of TAT peptide was blocked by Legumain protease substrate Ala-Ala-Asn-(AAN). Firstly, we confirmed the extremely high level of Legumain expression in a variety of solid tumors, as well as its specific effect on recognition and digestion of AAN. Then we conjugated AAN with the TAT peptide and constructed the modified liposome (AAN-TAT-Lipo) cargo. Flow-cytometry assay showed the internalization of AAN-TAT-Lipo was significantly higher than that of AAN-Lipo in 4T1 cells which expressed active Legumain. In vivo, the concentration of Dox encapsulated in AAN-TAT-Lipo was significantly higher in the tumor sites and lower in normal tissues compared with Free-Dox, AAN-Lipo-Dox and TAT-Lipo-Dox groups after administration in mouse model of orthotopic breast cancer. Luciferase assay showed that the AAN-TAT-Lipo-Dox dramatically inhibited tumor growth compared with other groups. TUNEL staining demonstrated that the apoptotic cells in tumor sites were significantly increased in the group of AAN-TAT-Lipo-Dox, compared with other groups. Whereas, the apoptotic cells in the heart, liver, spleen and kidney were decreased in the group of AAN-TAT-Lipo-Dox. Taken together, the modified TAT-lipo cargo via blocking TAT peptide activation with a Legumain specific substrate, AAN, is proven effective for improving the efficacy of internalization, and reducing the systemic toxicity of the tumor targeting delivery system as well. Citation Format: Ze Liu, Min Xiong, Debbie Liao, Dan Lv, Ralph A. Reisfeld, Wolfgang Wrasidlo, Si Chen, Dwayne G. Stupack, Peiqing Sun, Xiaoyue Tan, Rong Xiang. Legumain protease substrate modified TAT-liposome cargo: an efficient tool for targeting malignant diseases. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5614. doi:10.1158/1538-7445.AM2013-5614