Applications To Health Promotion And Disease Prevention

The in vitro growth inhibition potential of Abrus lectins has been studied, and results show that the lectins inhibit the major protein synthesis pathway, which leads to cell death. Lectins (AGG and ABR) bind to the terminal galactose residue of cell surface receptors through the B subunit, enter the cells by receptor mediated endocytosis, and are finally transported to the endoplasmic reticulum (ER) by the retrograde pathway. The reduction of the A—B inter subunit disulfide bond, essential for the cytotoxicity that takes place in the ER, followed by translocation of the A chain to the cytosol A chain cleaves a specific adenine residue from the a-sarcin—ricin loop of the 28S rRNA in the cytosol of eukaryotes. The depurination prevents the binding of ribosome to elongation factors, thereby arresting the protein synthesis that causes cell death. The protein synthesis inhibitory activity of AGG (IC50 = 3.5 nM) is weaker than that of ABR (0.05 nM). As a consequence ABR is found to be extremely toxic, with an LD50 of 20 mg/kg of body weight, whereas the LD50 of the AGG is comparatively higher (5 mg/kg of body weight), implying lesser toxicity. Both the lectins trigger apoptosis by caspase-3 activation, and follow the intrinsic mitochondrial pathway involving potential damage to the the mitochondrial membrane, and reactive oxygen species production (Narayanan et al., 2004). Moreover, in vitro growth inhibitory properties of Abrus lectins have been evaluated in several tumor cells. For instance, ABR could bring about cell death of Dalton's lymphoma (DL), as is evident from typical morphological changes associated with apoptosis. However, necrotic cell death is dominant when a higher dose of ABR is used. ABR induces apoptosis by stimulating the expression of pro-apoptotic caspase-3, at the same time blocking the expression of Bcl-2 (Ramnath et al., 2007). Interestingly, AGG in a heat-denatured condition (prepared by maintaining it at 50° C for 30 minutes and then immediately at 100° C for 2 minutes in a water bath) exerts similar types of cellular growth inhibition potential as demonstrated in DL cells (Ghosh & Maiti, 2007).

Antitumor properties

An agent that can selectively induce cell death in tumor cells without affecting the normal cell population is considered to be an ideal candidate for cancer therapy. The selective

tumor-targeting nature of Abrus lectins was investigated in normal and tumor cells, and it was observed that ABR showed greater cytoagglutination against human cultured cell lines derived from acute lymphoblastic leukemia and adult T cell leukemia, and weak agglutination against normal lymphocytes (Kaufman & McPherson, 1975). Likewise, AGG-mediated human lymphoproliferative activity in vitro was also observed (Closs et al., 1975). Though the exact mechanisms are not known, this tumor-specificity of Abrus lectins may be investigated to develop a novel anticancer agent (Figure 49.2).

The first reports on the anticancer properties of Abrus lectins appeared before much was known about their mechanism of action. In the late 1960s and early 1980s, the effect of Abrus protein extract on growth inhibition of Yoshida sarcoma and Ehrlich ascites tumors in mice were investigated (Reddy & Sirsi, 1969; Lin et al., 1982). In the following years, the anticancer effect of the ABR was investigated in different experimental models, including human tumors grown in nude mice (Fodstad et al., 1977). Similarly, in another study, ABR showed a potent antitumor potential in a sarcoma mouse model, which is thought to be mediated by inhibition of DNA biosynthesis. These results were sufficiently promising to initiate a phase I clinical study of ABR. Though clinical study was found to be promising, unexpected observation in a few patients became an obstacle for further clinical investigation.

For the next couple of years, with improved protein separation techniques, a few papers appeared on Abrus lectin purification and crystallization, and the mechanism of intoxication. After the discovery of the detailed mechanism of cell growth inhibition, lectins again attracted the attention of the scientific community. Meanwhile, investigators came up with the idea that the toxins might be useful in treating cancer. ABR exhibits antitumor activity in mice at a sub-lethal dose and is able to decrease solid tumor mass development, as is evident from the study related to murine DL and the Ehrlich's ascites carcinoma (EAC) model (Ramnath et al., 2002). ABR with interperitonial administration increased the lifespan of ascites tumor-bearing mice, and showed maximum antitumor effect when used simultaneously with tumor cells. Such a study showed that prophylactic administration of ABR was ineffective. Similarly, the antitumor activity of AGG was evaluated in a murine DL ascites tumorogenic model. It was observed that both pre- and post-treatment of agglutinin at

FIGURE 49.2

Mechanism of action of Abrus lectins on tumor regression. Abrus lectins contribute to tumor inhibition by direct killing of tumor cells through mitochondrial-dependent pathways. Further, lectins are able to stimulate tumor associated macrophages and proliferation of splenocytes, leading to Th1 response and NK cell activation in tumor-bearing mice, which involves an antitumor response.

FIGURE 49.2

Mechanism of action of Abrus lectins on tumor regression. Abrus lectins contribute to tumor inhibition by direct killing of tumor cells through mitochondrial-dependent pathways. Further, lectins are able to stimulate tumor associated macrophages and proliferation of splenocytes, leading to Th1 response and NK cell activation in tumor-bearing mice, which involves an antitumor response.

a non-toxic dose significantly decreased the lymphoma cell numbers in DL-bearing mice. Moreover, heat-denatured AGG, like native AGG, was able to decrease the DL tumor cell number in vivo, and significantly increased the median survival time of DL bearing mice (Ghosh & Maiti, 2007). The in vivo tumoricidal property of lectins against DL and EAC-induced tumors was thought to be mediated through apoptosis (Ghosh & Maiti, 2007; Ramnath et al., 2007).

Immunostimulatory properties

Along with direct antitumor potential, these lectins have a functional role in tumor defense by immunomodulation. The mitogenic properties of ABR and AGG were studied in human as well as mice immune cells, and both lectins showed significant adjuvant activities in oil emulsion and aqueous solution in animal models. It has been demonstrated that heat-denatured AGG in oil emulsion produces a humoral immune response comparable with that of traditional complete Freund's adjuvant against ovalbumin in a rat model (Suryakala et al., 2000). Immunoadjuvant activity of AGG and ABR were investigated in aqueous solution with ovalbumin, diphtheria toxoid, and lysozyme as test antigens in mouse model, and induction of humoral immunity by native and heat-denatured AGG were comparable with that of Freund's adjuvant (Tripathi & Maiti, 2003). All these findings indicate that Abrus lectins maybe used as immunoadjuvants in aqueous solution for raising a high antibody titer with a high avidity for weak antigens, thus potentiating the systemic immune response.

Lectins are known as polyclonal activators of lymphocytes, and work through the induction of a battery of cytokines. Treatment of AGG increases the expression of activation markers (CD25, CD71) in B and T cells, which implies that the mitogenic stimulus of agglutinin influences not only proliferation but also the activation status of splenocytes (Ghosh et al., 2009). Both native and heat-denatured AGG activate splenocytes and induce the production of cytokines like interleukin-2 (IL-2), interferon-g (IFN-g), and tumor necrosis factor-a (TNF-a) indicating a Th1 type of immune response (Tripathi & Maiti, 2005). The natural killer (NK) cells, innate effector cells, are also activated in the presence of AGG. Furthermore, AGG stimulates the innate effector arms, like macrophages, by up-regulating pro-inflammatory cytokine expression. Increased production of nitric oxide and hydrogen peroxide, and a high phagocytic and bactericidal activity of macrophages, are potentiated by native as well as heat-denatured AGG. These results suggest that both forms of AGG can act as immunostimulants in vitro. In vivo administration of native and heat-denatured AGG showed macrophage and NK cell activation (Ghosh & Maiti, 2007). Similarly, ABR also augments the humoral and cell-mediated immune response of the host. A non-toxic dose of ABR, (1.25 mg/kg body weight) for 5 consecutive days of treatment in normal mice stimulates specific humoral responses, and a significant increase is observed in the total leukocyte count, lymphocytosis, the weights of the spleen and thymus, circulating antibody titer, antibody forming cells, bone marrow cellularity, and a-esterase positive bone marrow cell.

The host response to cancer therapies might be potentiated by the simultaneous administration of these type-1 immunoadjuvant Abrus lectins, along with cancer drugs. Splenocytes from native and heat-denatured AGG-treated DL-bearing mice exhibited the Th1 type of cytokine (IL-2 and IFN-g) response, which could generate a robust antitumor immune response (Ghosh & Maiti, 2007). AGG, in its native and heat-denatured forms, modulates tumor associated macrophage (TAM) by increasing in vitro cytotoxicity towards tumor cells, and production of nitric oxide. Similarly, ABR augments antitumor immunity in tumor-bearing hosts by boosting the cell-mediated immune effector arms, such as NK cells of tumor-bearing hosts. Antibody-dependent cellular cytotoxicity (ADCC), which links humoral and cellmediated immunity, was enhanced in the ABR-treated tumor-bearing mice. Early antibody-dependent complement-mediated cytotoxicity was also observed in the ABR-treated mice. Immunization with ABR-treated Meth-A tumor cells induces a strong antitumor immunity in

syngeneic BALB/c mice, and such an immunizing effect is stronger than that produced by an irradiated Meth-A tumor cell vaccine (Shionoya et al., 1982). Studies on the adjuvant effects of ABR in a tumor condition indicate its potential use as an immunoadjuvant.

Many proteins are degraded or processed into smaller peptide products through natural mechanisms, giving rise to a newly recognized natural "peptidome," which potentially contains a valuable source of anticancer agents. Abrus lectin derived peptides represent a prime example of anticancer peptide segments encrypted within lectins. In an initial study, tryptic digestion of Abrus agglutinin stimulates macrophages, as is evident from the increasing phagocytic and bactericidal activity, as well as hydrogen peroxide production. It also proliferates splenocytes, leading to Th1 response and NK cell activation. Interestingly, tryptic-digested AGG and ABR derived peptides, named AGP and ABP respectively (obtained from 10-kDa molecular weight cut-off membrane permeate), molecular weight in the range of 500 Da to 1500 Da, induce mitochondria-dependent cell death. Sub-lethal doses of peptide fractions showed significant growth inhibitory properties in an in vivo DL model, which was found to be mediated through apoptosis (Bhutia et al., 2009a). Along with direct antitumor potential, these peptides activate in vitro immune cells such as macrophages, NK cells, and B and T cells in the tumor microenvironment, which, in a concerted way, inhibits tumor progression (Bhutia et al., 2009b).

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