Anti-tumor activities and apoptotic mechanism of ribosome-inactivating proteins

Effects of RIPs on breast cancer

The type I RIPs TCS, momordica anti-human immunodeficiency virus (HIV) protein of 30 kDa (MAP30), gelonium anti-HIV protein of 31 kDa (GAP31), gelonin, marmorin, and α-momorcharin (α-MMC) have been shown to negatively affect the growth of breast tumor cells in vitro and in vivo [5–7]. TCS inhibits cell viability, causes cell cycle arrest, and significantly reduces tumor volume and weight by inducing apoptosis through caspase-8 and caspase-9 in breast tumor cells [5]. MAP30, which shares 59% sequence similarity with TCS, effectively inhibits human breast cancer MDA-MB-231 cells through down-regulating the expression of human epidermal growth factor receptor-2 (HER2), similarly to GAP31 [6]. HER2 overexpression is observed in approximately 30% of all human breast cancers, and HER2-overexpressing tumor cells may be less sensitive to chemotherapy; in this case, the combination of MAP30 and GAP31 could represent a therapeutic strategy [7]. HER2 and fibroblast growth factor-inducible 14-kDa protein (Fn14) are frequently co-expressed in human breast tumors, and HER2 directly induces increase in Fn14 expression, therefore sensitizing tumor cells to an immunotoxin generated by fusing Fn14 antibodies to recombinant gelonin (designated hSGZ) [8]. Indeed, hSGZ can rapidly internalize and deliver recombinant gelonin (rGel) to the cytosol of tumor cells, where it enzymatically blocks protein synthesis. Because Fn14 enhances breast cancer cell migration and invasion, a question of whether there is a way to damage tumor cells while reducing Fn14 expression was raised. We assume that MAP30 and hSGZ used together might achieve a better outcome, and breast tumor cells can be sequentially treated with MAP30 and hSGZ; MAP30 would decrease HER2 expression and lead to reduced Fn14 expression, then hSGZ would target Fn14-positive cells and exert its function without increasing the invasive capacity of tumor cells. However, this hypothesis remains to be verified by appropriate experiments.

Many cell membrane receptors are expressed at low levels in normal cells but are highly expressed in tumor cells. Estrogen receptor α (ERα) is expressed in approximately 75% of breast cancer tissues at higher levels compared with those in normal breast tissues (P = 0.001) [9]. In ERα-positive breast cancer cells, the ERα-mediated signaling pathway is involved in the inhibitory action of marmorin on proliferation; many drugs target ERα. Marmorin inhibits angiogenesis by lowering the viability of human umbilical vein endothelial cells in vitro; therefore, it was suggested that marmorin might starve tumors to death by reducing the amount of blood vessels in vivo [10]. Marmorin also induces DNA damage and endoplasmic reticulum stress, resulting in the induction of apoptosis in mice bearing MDA-MB-231 tumor xenografts [10].

Ribosome-inactivating proteins have the potential to become innovative anti-tumor agents, but they also possess toxic adverse effects, including severe systemic anaphylaxis, immunogenicity, and toxicity. To reduce the undesirable effects and achieve better therapeutic efficacy, Deng et al. [11] modified α-MMC with polyethylene glycol (PEG) to explore the anti-tumor efficacy on breast carcinoma; they demonstrated that α-MMC PEGylation extends the half-life of α-MMC and mitigates non-specific toxicity. Indeed, α-MMC-PEG exhibited improved anti-tumor efficacy with tolerable toxic reactions.

Effects of RIPs on leukemia and lymphoma

Trichosanthin significantly inhibits the proliferation of various leukemia and lymphoma cell lines [12]. Notably, TCS can damage leukemia and lymphoma cells through different mechanisms according to the cell type. TCS induces apoptosis in T-lymphocyte cell lines, but inhibits growth of B-lymphocyte cell lines via S-phase cell cycle arrest [12]. It has been suggested that cucurmosin is more potent than TCS in killing the chronic myelogenous leukemia K562 cells; both cucurmosin and TCS down-regulate P210^Bcr-Ab1 and inhibit tyrosine kinase, resulting in cell growth suppression [13]. Cucurmosin also inhibits proliferation and induces apoptosis in tumor cells; interestingly, cucurmosin combined with trans-retinoic acid or arsenic trioxide was shown to synergistically increase these effects on the human acute promyelocytic leukemia NB4 cell line [14].

Articulatin-D, the first cytotoxic RIP with a B-chain lacking sugar-binding activity, has been shown to highly inhibit leukemia and lymphoma cells in vitro; the highest toxicity was obtained with Jurkat cells, followed by Molt-4, U-937, HL-60, and Raji cells [15]. With its special physical properties, articulatin-D is a good candidate for the synthesis of immunotoxins capable of efficiently and specifically killing tumor cells.

Immunotoxins are emerging targeted agents composed of a toxin fragment and an antibody/cytokine. Saporin and rGel have been widely used to construct immunotoxins, which have been reported to be useful in cancer treatment by multiple studies [16–18]; several such molecules have been evaluated clinically [19, 20]. It is feasible to locate cancer cells through membrane proteins CD22, CD7, CD19, and CD38, and corresponding antibody HB22.7, HB2, BU12, and OKT10 are used to construct immunotoxins. HB22.7-saporin was cytotoxic against a panel of non-Hodgkin’s lymphoma (NHL) cell lines and was shown to significantly prevent tumor development in a xenograft model of NHL [21]. HB2-saporin, BU12-saporin, and OKT10-saporin were shown to be selectively cytotoxic toward human acute lymphoblastic leukemia in vitro and in vivo [22–24].

Luster et al. [25] have reported that treatment with rGel-BLyS, rGel fused to a B-lymphocyte stimulator, rapidly reduced the tumor burden and markedly prolonged survival in xenograft mouse models of spread lymphoma or leukemia; in this setting, cell death was not induced by caspase activation but rather was partially mediated by the ribotoxic stress response. Furthermore, the rGel-BLyS fusion toxin combined with the proteasome inhibitor bortezomib restrained lymphoma growth and down-regulated nuclear factor kappa B (NF-κB) activity, which is critical for cellular proliferation and survival [26].

Effects of RIPs on hepatoma and other cancers

Entry mechanism

Ribosome-inactivating proteins must enter cells to inactivate the eukaryotic ribosome via their RNA N-glycosidase activity. First, type II RIPs bind to glycoproteins and/or glycolipids on the cell membrane and enter the cell via endocytosis; then, RIPs undergo retrograde transport from the Golgi apparatus to the endoplasmic reticulum via an intracellular pathway [30]. The enzymatic moieties will not be released to cytosol and reach the ribosomes to exert their function until they exploit the endoplasmic reticulum-associated degradation pathway [3].

It is difficult for type I RIPs to enter cells because of their sugar-binding activity deficiency. They can enter cells to some extent, probably due to their interaction with phospholipids in the cell membrane; however, the exact entry mechanism remains unclear. To facilitate the entry of type I RIPs into cells, they can be linked to proper carriers such as monoclonal antibodies and other molecules. The resulting conjugates can be specifically toxic to target cells. Several immunotoxins have been well studied in experiment therapies against hematologic and solid tumors. The entry pathways of type I RIPs, type II RIPs, and immunotoxins are shown in Figure 1.

Induction of apoptosis in tumor cells

Caspases play an important role in apoptosis. They are classified into three types: initiator, executioner, and cytokine processor caspases. Great progress has been made in studying the three signaling pathways related to caspase activation, including mitochondrial, death receptor, and endoplasmic reticulum stress signaling pathways. The connections among these pathways are shown in Figure 2 [31]. Apoptosis also occurs through apoptosis-inducing factor (AIF), which is caspase-independent [32].