Margarita Skopeliti

The immunologically active site of prothymosin α is located at the carboxy-terminus of the polypeptide. Evaluation of its in vitro effects in cancer patients

Prothymosin α (proTα) is a 109 amino acid long polypeptide presenting distinct immunoenhancing activity in vitro and in vivo. Recent reports suggest that in apoptotic cells, proTα is cleaved by caspases at its carboxy(C)-terminus generating potentially bioactive fragments. In this study, we identified the peptide segment of proTα presenting maximum immunomodulatory activity. Calf thymus proTα was trypsinised, and the five fragments produced (spanning residues 1–14, 21–30, 31–87, 89–102 and 103–109) were tested for their ability to stimulate healthy donor- and cancer patient-derived peripheral blood mononuclear cell (PBMC) proliferation in autologous mixed lymphocyte reaction (AMLR), natural killer and lymphokine-activated killer cell activity, intracellular production of perforin, upregulation of adhesion molecules and CD25 expression. ProTα(89–102) and proTα(103–109) significantly fortified healthy donor-lymphocytes’ immune responses to levels comparable to those induced by intact proTα. These effects were more pronounced in cancer patients, where peptides proTα(89–102) and proTα(103–109) partly, however significantly, restored the depressed AMLR and cytolytic ability of PBMC, by simulating the biological activity exerted by intact proTα. ProTα(1–14), proTα(21–30) and proTα(31–87) marginally upregulated lymphocyte activation. This is the first report showing that proTα’s immunomodulating activity can be substituted by its C-terminal peptide(s). Whether generation and externalization of such immunoactive proTα fragments occurs in vivo, needs further investigation. However, if these peptides can trigger immune responses, they may eventually be used therapeutically to improve some PBMC functions of cancer patients.

Prothymosin α immunoactive carboxyl-terminal peptide TKKQKTDEDD stimulates lymphocyte reactions, induces dendritic cell maturation and adopts a β-sheet conformation in a sequence-specific manner

Prothymosin alpha (ProTalpha) is a small acidic polypeptide with important immunostimulatory properties, which we have previously shown to be exerted by its carboxyl (C)-terminus. It exerts immunoenhancing effects through stimulation of monocytes via toll-like receptor (TLR) triggering. Here, we assayed the activity of synthetic peptides homologous to ProTalphas C-terminus to stimulate lymphocyte functions, in particular natural killer cell cytotoxicity of peripheral blood mononuclear cells isolated from healthy donors. A synthetic decapeptide TKKQKTDEDD was identified as the most potent lymphocyte stimulator. The activity of this peptide was sequence-specific and comparable to that of the intact molecule, suggesting that ProTalphas immunoactive segment encompasses the nuclear localization signal sequence of the polypeptide. Because ProTalpha stimulates immune responses in a monocyte-dependent manner, we further investigated whether the entire molecule and its peptide TKKQKTDEDD specifically act on monocytes and show that both can promote maturation of monocyte-derived dendritic cells (DC). Finally, knowing that, under specific conditions, ProTalpha forms amyloid fibrils, we studied the amyloidogenic properties of its C-terminal peptide segments, utilizing ATR FT-IR spectroscopy and transmission electron microscopy (negative staining). Although the peptide TKKQKTDEDD adopts an antiparallel beta-sheet conformation under various conditions, it does not form amyloid fibrils; rather it aggregates in globular particles. These data, in conjunction with reports showing that the peptide TKKQKTDEDD is generated in vivo upon caspase-cleavage of ProTalpha during apoptosis, strengthen our hypothesis that immune response stimulation by ProTalpha is in principle exerted via its bioactive C-terminal decapaptide, which can acquire a sequence-specific beta-sheet conformation and induce DC maturation.

Development and immunochemical evaluation of antibodies Y for the poorly immunogenic polypeptide prothymosin alpha

Since conserved mammalian polypeptides are believed to exhibit enhanced immunogenicity in avian species, hens were immunized against the poorly immunogenic, highly conserved mammalian polypeptide prothymosin alpha (ProTalpha), i.e. against either non-conjugated ProTalpha (isolated from bovine thymus) or ProTalpha conjugated to keyhole limpet hemocyanin (ProTalpha/KLH). The antibodies Y were isolated from the egg yolk and evaluated through suitable dot-blot and ELISA systems in parallel with antibodies G isolated from the antiserum of rabbits immunized against the same immunogens. As revealed, antibodies Y and G of low titer and/or affinity were obtained against non-conjugated ProTalpha, while antibodies Y against ProTalpha/KLH had a better apparent titer, could better discriminate between ProTalpha and the closely related bioactive peptide thymosin alpha 1, and were obtained at much larger quantities than the corresponding antibodies G.

Phenoxodiol, an anticancer isoflavene, induces immunomodulatory effects in vitro and in vivo

Phenoxodiol (PXD) is a synthetic analogue of the plant isoflavone genistein with improved anticancer efficacy. Various properties and mechanisms of action have been attributed to the drug, the most important being its ability to sensitize resistant tumour cells to chemotherapy, which led to its fast track FDA approval for phase II/III clinical trials. In this study, we examined the effects of PXD on human peripheral blood mononuclear cells (PBMC) and its potential role in regulating immune responses. We show that PXD, at concentrations ≥1 μg/ml (4 μM), inhibited proliferation and reduced the viability of healthy donor‐derived PBMC. In contrast, lower PXD concentrations (0.05–0.5 μg/ml) augmented, upon 3‐day incubation, PBMC cytotoxicity. Experiments with purified CD56+ lymphocytes revealed that PXD enhanced the lytic function of natural killer (NK) cells by directly stimulating this lymphocytic subpopulation. Furthermore, in an in vivo colon cancer model, Balb/C mice administered low‐dose PXD, exhibited significantly reduced tumour growth rates and prolonged survival (in 40% of the animals). Ex vivo results showed that PXD stimulated both NK and tumour‐specific cell lytic activity. We conclude that PXD, when administered at low concentrations, can act as an immunomodulator, enhancing impaired immune responses, often seen in cancer‐bearing individuals.

Ovarian malignant ascites-derived lymphocytes stimulated with prothymosin α or its immunoactive decapeptide lyse autologous tumour cells in vitro and retard tumour growth in SCID mice

BACKGROUND Tumour-associated lymphocytes (TALs) present in effusions of ovarian cancer patients exhibit impaired activities, due to the immunosuppressive environment of the ascites. Means to enhance their cytotoxicity against autologous tumour cells are of clinical importance. The immunomodulator prothymosin alpha (proTα) increases the specific lysis of tumour cells by activated CD8(+) T-lymphocytes and its immunoreactivity is exerted by the carboxy-terminal decapeptide, proTα(100-109). These two molecules were studied on TALs in vitro, and in SCID mice bearing human ovarian tumours. METHODS TALs and tumour cells were isolated from 41 ovarian cancer patients and co-cultured in the presence of proTα or proTα(100-109). The cytotoxicity of peptide-stimulated TALs was tested against autologous tumour cells and K562. Ex vivo peptide-stimulated TALs from three patients were adoptively transferred intraperitoneally in SCID mice, previously inoculated with each patients autologous tumour cells. RESULTS ProTα and its immunoreactive peptide proTα(100-109), enhanced the cytotoxic activity of TALs against autologous tumour cells in vitro, but marginally affected the lysis of K562. The effect of proTα and proTα(100-109) was higher after 7-14 days of stimulation, whereas TAL cytotoxicity was significantly decreased after 21 days. Mice administered TALs, ex vivo activated with proTα or proTα(100-109) for 7 days, showed a relatively lower tumour increase rate and a prolongation of their survival, compared to controls. CONCLUSION Our data demonstrate that, in the presence of tumour antigens, proTα and proTα(100-109) enhance the depressed cytotoxicity of TALs against autologous tumour cells in vitro and retard tumour growth in vivo.