Effects Of Immunomodulatory Peptides On The Proliferation, Apoptosis And Differentiation In Human Leukemia Cells

Thymosinα₁(Tα₁), a 28-amino acid peptide, is a well-known immune system enhancer for the treatment of various diseases. In the present investigation, the effects of Tα₁ on the proliferation and apoptosis of human leukemia cell lines (HL-60, K562, and K562/ADM) were studied. Methods: The inhibition effects on proliferation induced by Tα₁ were analyzed by MTT assays and the induction of apoptosis was tested by cell morphology, nuclei condensation and annexin V binding. The expression of CD95, bcl-2 anti-apoptic gene and P-glycoprotein (P-gp) were analyzed by flow cytometry (FCM). The roles of nicotinic acetylcholine receptors (nAChRs) and muscaric acetylcholine receptors were also investigated. Results: The proliferation was significantly depressed after 96h of treatment with Tα₁, and obvious signs of apoptosis, i.e., cell morphology, nuclei condensation and annexin V binding, were observed thereafter. Moreover, the up-regulation of CD95 (Fas/Apol) and decrease in bcl-2 anti-apoptic gene expression were observed in apoptotic cells. The expression and the function of P-glycoprotein (P-gp) can be slightly inhibited by Tα₁. It is noteworthy that K562 and K562/ADM were more sensitive than HL-60 cells when subjected to Tα₁. Furthermore, HepG-2, the human hepatoma cell line, displayed significant less sensitivity to Tα₁ than all the human leukemia cell lines. D-tubocurarine (TUB), a nicotinic acetylcholine receptor (nAchRs) antagonist, significantly antagonized the inhibition effects induced by Tα₁, whereas atropine, a muscaric acetylcholine receptor antagonist, did not exhibit such effects. All the results indicated that Tα₁ was able to significantly suppress proliferation and induce apoptosis in human leukemia cell lines via nAchRs activation.Thymopentin (Arg-Lys-Asp-Val-Tyr, TP5) has shown immuno-regulatory activities in humans. In the present study, we investigated the effects of TP5 on the proliferation and differentiation of a human leukemia cell lines (HL-60, K562, and K562/ADM). Methods: The anti-proliferation and differentiation effects induced by TP5 were determined by cell viability, colony formation in soft agar, silver staining, NBT-reduction activity, benzidine staining and ALP activity. The cell cycle phase distribution, CD11b and CD14 cell surface antigen (markers of differentiation) expression and P-glycoprotein (P-gp) were analyzed by flow cytometry (FCM). The roles of nicotinic acetylcholine receptors (nAChRs) and muscaric acetylcholine receptors were also investigated. Results: It is noteworthy that TP5 displayed concentration-dependent inhibitory effects on the proliferation and colony formation of HL-60 and K562 cells. Furthermore, the decrease or even disappearance of AgNORs from nucleoli was observed in HL-60 cells after the treatment with TP5. The suppression induced by TP5 was accompanied by an accumulation of cell cycle in the G0/G1 phase. Moreover, TP5 significantly increased the NBT-reduction activity of HL-60 cells. Cytofluorometric and morphologic analysis indicated that TP5 had induced differentiation along the granulocytes lineage in HL-60 cells. D-tubocurarine (TUB) significantly antagonized the inhibitory effects induced by TP5, whereas atropine did not exhibit such effect. TP5 induced the apoptosis of K562 cells. TP5 could induce differentiation of K562 cells by benzidine-staining assay and decrease the ALP activity in K562 cells. Furthermore, the decrease of TNF secretion was observed in .HL-60 and K562 cells after the treatment with TP5. TP5 could slightly inhibit the proliferation of K562/ADM cells. The expression and the function of P-glycoprotein (P-gp) in K562/ADM cells could be significantly inhibited by TP5. All the results indicated that TP5 was able to significantly inhibit proliferation and induce apoptosis and differentiation in human leukemia cells. Our observations also implied that TP5 not only acted as an immunomodulatory factor in cancer chemotherapy, but is also a potential chemotherapeutic agent in the human leukemia therapy.