cDNA cloning of a novel lectin that induce cell apoptosis from Artocarpus hypargyreus.

We isolated a novel lectin (AHL) from Artocarpus hypargyreusHance and showed its immunomodulatory activities. In this study, the amino acid sequence of AHL was determined by cDNA sequencing. AHL cDNA (875bp) contains a 456-bp open reading frame (ORF), which encodes a protein with 151 amino acids. AHL is a new member of jacalin-related lectin family (JRLs), which share high sequence similarities to KM+ and Morniga M, and contain the conserved carbohydrate binding domains. The antitumor activity of AHL was also explored using Jurkat T cell lines. AHL exhibits a strong binding affinity to cell membrane, which can be effectively inhibited by methyl-α-D-galactose. AHL inhibits cell proliferation in a time- and dose-dependent manner through apoptosis, evidenced by morphological changes, phosphatidylserine externalization, poly ADP-ribose polymerase (PARP) cleavage, Bad and Bax up-regulation, and caspase-3 activation. We further showed that the activation of ERK and p38 signaling pathways is involved for the pro-apoptotic effect of AHL.

Purification and characterization of a novel immunomodulatory lectin from Artocarpus hypargyreus Hance.

Lectins are proteins/glycoproteins of non-immune origin, which interact specifically and non-covalently with the carbohydrate moieties of respective receptors on the cell surface. In this study, a novel 65.2-kDa tetrameric lectin (AHL) was purified from Artocarpus hypargyreus Hance (A. hypargyreus) by affinity chromatography on a galactose-sepharose column. It is a glycoprotein with carbohydrate content of 6.91%. Its maximum haemagglutinating activity was maintained after incubation at a temperature range of 20-40 °C and pH range of 5.0-9.0. AHL-induced haemagglutination of erythrocytes was inhibited strongly by carbohydrates, such as methyl-galactose, methyl-mannose, and N-acetyl-d-galactosamine, indicating the existence of more than one carbohydrate binding sites in the AHL molecule. The AHL activity was gradually lost in the presence of urea and completely lost when being treated with ethylenediaminetetraacetic acid (EDTA). The immunomodulatory activity of AHL was assessed using human peripheral lymphocytes and rat peritoneal macrophages. AHL triggered proliferation and activation of human T lymphocytes and induced the release of Th1 cytokines, including IFN-γ, TNF-α and IL-6. Furthermore, AHL significantly stimulated the production of nitric oxide (NO) and pro-inflammatory cytokines, including TNF-α and IL-12, in rat peritoneal macrophages. However, AHL did not enhance proliferation of B cell-enriched rat splenocytes. Taken together, in this study, a novel immunomodulatory lectin was purified from A. hypargyreus and was found to be capable of inducing a Th1-type immune response, and thus, it may have potential immunoregulatory application in response to infections, immune diseases and cancer.

Mitogenic activity of Artocarpus lingnanensis lectin and its apoptosis induction in Jurkat T cells.

Lectins are a class of carbohydrate-binding proteins or glycoproteins and used in the purification and characterization of glycoproteins according to their specificity to carbohydrates. In the present study, the mitogenic activity of Artocarpus lingnanensis lectin (ALL) and its apoptosis induction in Jurkat T cells were explored. MTT assay revealed strong mitogenic potential of ALL. Meanwhile, the anti-cancer activity of ALL was also explored using the human leukemic Jurkat T cell line. ALL exhibited strong binding affinity (97%) to the cell membrane, which could be effectively inhibited by N-acetyl-D-galactosaminide (NAD). ALL induced time- and dose-dependent growth inhibition in Jurkat T cells. ALL could induce morphologic change and increase the hypodiploid cell population with the decreased population of S and G2/M phases. The induction of phosphatidylserine externalization and PARP cleavage further confirmed its apoptosis-inducing activity due to the activation of caspase-8 and -9. The inhibition of caspase-9 but not caspase-8 could rescue ALL-induced apoptotic cells. Further studies showed that ALL enhanced the cleavage of Bid, the release of cytochrome C, the depolarization of mitochondria and the activation of caspase-3. ALL downregulated the expression of Bcl-xl and Bcl-2 without impact on Bax and Bad. In addition, the activation of p38/JNK MAPK signaling pathways was observed to be a requisite for ALL apoptotic activity. In contrast, ALL could not induce apoptosis of normal T cells. These findings present the differential effect of ALL on Jurkat and normal T lymphocytes, suggesting its therapeutic value in leukemia.

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

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

Sensitization of cervical carcinoma cells to paclitaxel by an IPP5 active mutant.

Paclitaxel is one of the best anticancer agents that has been isolated from plants, but its major disadvantage is its dose-limiting toxicity. In this study, we obtained evidence that the active mutant IPP5 (8-60hIPP5m), the latest member of the inhibitory molecules for protein phosphatase 1, sensitizes human cervix carcinoma cells HeLa more efficiently to the therapeutic effects of paclitaxel. The combination of 8-60hIPP5m with paclitaxel augmented anticancer effects as compared to paclitaxel alone as evidenced by reduced DNA synthesis and increased cytotoxicity in HeLa cells. Furthermore, our results revealed that 8-60hIPP5m enhances paclitaxel- induced G2/M arrest and apoptosis, and augments paclitaxel-induced activation of caspases and release of cytochrome C. Evaluation of signaling pathways indicated that this synergism was in part related to down- regulation of NF-?B activation and serine/threonine kinase Akt pathways. We noted that 8-60hIPP5m down- regulated the paclitaxel-induced NF-?B activation, I?Bα degradation, PI3-K activity and phosphorylation of the serine/threonine kinase Akt, a survival signal which in many instances is regulated by NF-?B. Together, our observations indicate that paclitaxel in combination with 8-60hIPP5m may provide a therapeutic advantage for the treatment of human cervical carcinoma.

8-60hIPP5(m)-induced G2/M cell cycle arrest involves activation of ATM/p53/p21(cip1/waf1) pathways and delayed cyclin B1 nuclear translocation.

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. The active mutant IPP5 (8-60hIPP5(m)), the latest member of the inhibitory molecules for PP1, has been shown to inhibit the growth of human cervix carcinoma cells (HeLa). In order to elucidate the underlying mechanisms, the present study assessed overexpression of 8-60hIPP5(m) in HeLa cells. Flow cytometric and biochemical analyses showed that overexpression of 8-60hIPP5(m) induced G2/M-phase arrest, which was accompanied by the upregulation of cyclin B1 and phosphorylation of G2/M-phase proteins ATM, p53, p21(cip1/waf1) and Cdc2, suggesting that 8-60hIPP5(m) induces G2/M arrest through activation of the ATM/p53/p21(cip1/waf1)/Cdc2/ cyclin B1 pathways. We further showed that overexpression of 8-60hIPP5(m) led to delayed nuclear translocation of cyclin B1. 8-60hIPP5(m) also could translocate to the nucleus in G2/M phase and interact with pp1α and Cdc2 as demonstrated by co-precipitation assay. Taken together, our data demonstrate a novel role for 8-60hIPP5(m) in regulation of cell cycle in HeLa cells, possibly contributing to the development of new therapeutic strategies for cervix carcinoma.

IPP5, a novel inhibitor of protein phosphatase 1, suppresses tumor growth and progression of cervical carcinoma cells by inducing G2/M arrest.

Protein phosphatase 1 (PP1) is a major serine/threonine phosphatase that controls gene expression and cell cycle progression. Here, we report the characterization of a novel human bone marrow stromal cell (BMSC)-derived protein called protein phosphatase 1 inhibitor 5 (IPP5), which was obtained by large-scale random sequencing of a human BMSC cDNA library. The human IPP5 cDNA encodes a protein of 116 amino acid residues, which shares high homology with human protein phosphatase 1 inhibitor-1 (PPI-1). The effect of IPP5 on tumor growth and the underlying molecular mechanisms were investigated by overexpression of IPP5 in HeLa cells, a human cervical carcinoma cell line. Our results demonstrated that overexpression of the active mutant IPP5 inhibited the growth of HeLa cells both in vitro and in vivo. Biochemical analysis demonstrated that active mutant IPP5-mediated G2/M arrest of HeLa cells involved regulation of cyclin A1, cyclin B1, CDK1, p21, and p53, as well as increased inhibition of ERK activation. Furthermore, overexpression of the active mutant IPP5 leads to the formation of dikaryons following the failure of cytokinesis. Therefore, IPP5 might be a potential growth inhibitor for human tumor cells, especially for cervical carcinoma cells, and it could contribute to the development of new therapeutic strategies for human cervical cancer treatment.

Effect of IPP5, a novel inhibitor of PP1, on apoptosis and the underlying mechanisms involved.

Genes encoding apoptosis-inducing proteins are postulated to be candidate tumour suppressors. The identification of such proteins may benefit the early diagnosis and therapy of tumours. In the present study, we characterized the function of a novel human BMSC (bone marrow stromal cell)-derived protein {IPP5 [inhibitor-5 of PP1 (protein phosphatase 1)]} by large-scale random sequencing of a human BMSC cDNA library. hIPP5 (human IPP5) cDNA encodes a protein of 116 amino acid residues, which shares high homology with human PPI-1 (inhibitor-1 of PP1). The effect of IPP5 on apoptosis and the underlying molecular mechanisms were investigated by overexpression of IPP5 in HeLa cells, a human cervical carcinoma cell line. Our results showed that overexpression of active mutant IPP5 inhibited anchorage-dependent growth and induced apoptosis in HeLa cells, which may be attributed to the up-regulation of p21(waf/cip1) (a 21 kDa cell-cycle regulatory protein), p53 and Bcl-2-antagonist/killer, and down-regulation of Bcl-2 and Bcl-X(L). We also showed that the expression of active mutant IPP5 in HeLa cells was further enhanced on TNF (tumour necrosis factor) treatment and overexpression of active mutant IPP5 sensitized HeLa cells to TNF-induced JNK (c-Jun N-terminal kinase) and p38 activation as well as TNF-mediated apoptosis. Thus overexpression of active mutant IPP5 may increase cell susceptibility to TNF-induced apoptosis by the activation of p38 and JNK pathways. In addition, IPP5 active mutant could interact with PP1alpha as demonstrated by the co-precipitation assay.