thymalfasin and Breast-Neoplasms

The immunologic effects of chemotherapy-induced tumor cell death are not completely understood. Accumulating evidence suggests that phagocytic clearance of apoptotic tumor cells, also known as efferocytosis, is an immunologically silent process, thus maintaining an immunosuppressive tumor microenvironment (TME). Here we report that, in the breast tumor microenvironment, thymosin α-1 (Tα-1) significantly reverses M2 polarization of IL10-producing tumor-associated macrophages (TAM) during efferocytosis induced by apoptotic cells. Mechanistically, Tα-1, which bound to phosphatidylserine on the surface of apoptotic tumor cells and was internalized by macrophages, triggered the activation of SH2-containing inositol 5'-phosphatase 1 (SHIP1) through the lysosomal Toll-like receptor 7 (TLR7)/MyD88 pathway, subsequently resulting in dephosphorylation of efferocytosis-activated TBK1 and reduction of efferocytosis-induced IL10. Tα-1 combined with epirubicin chemotherapy markedly suppressed tumor growth in an in vivo breast cancer model by reducing macrophage-derived IL10 and enhancing the number and function of tumor-infiltrating CD4+ and CD8+ T cells. In conclusion, Tα-1 improved the curative effect of chemotherapy by reversing M2 polarization of efferocytosis-activated macrophages, suggesting that Tα-1 injection immediately after chemotherapy may contribute to highly synergistic antitumor effects in patients with breast cancer.. Thymosin α-1 improves the curative effect of chemotherapy by reversing efferocytosis-induced M2 polarization of macrophages via activation of a TLR7/SHIP1 axis.

Topics: Breast Neoplasms; Female; Humans; Interleukin-10; Thymalfasin; Toll-Like Receptor 7; Tumor Microenvironment; Tumor-Associated Macrophages

Thymosin alpha 1 (Tα1) is a biological response modifier that has been introduced into markets for treating several diseases. Given the short serum half-life of Tα1 and the rapid development of Fc fusion proteins, we used genetic engineering method to construct the recombinant plasmid to express Tα1-Fc (Fc domain of human IgG4) fusion protein. A single-factor experiment was performed with different inducers of varying concentrations for different times to get the optimal condition of induced expression. Pure proteins higher than 90.3% were obtained by using 5 mM lactose for 4 h with a final production about 160.4 mg/L. The in vivo serum half-life of Tα1-Fc is 25 h, almost 13 times longer than Tα1 in mice models. Also, the long-acting protein has a stronger activity in repairing immune injury through increasing number of lymphocytes. Tα1-Fc displayed a more effective antitumor activity in the 4T1 and B16F10 tumor xenograft models by upregulating CD86 expression, secreting IFN-γ and IL-2, and increasing the number of tumor-infiltrating CD4+ T and CD8+ T cells. Our study on the novel modified Tα1 with the Fc segment provides valuable information for the development of new immunotherapy in cancer.

Topics: Animals; Biomarkers, Tumor; Breast Neoplasms; Disease Models, Animal; Disease Progression; Female; Half-Life; Humans; Hydrocortisone; Immunocompromised Host; Immunoglobulin Fc Fragments; Immunomodulation; Male; Melanoma; Melanoma, Experimental; Mice; Prognosis; Rats; Recombinant Fusion Proteins; Thymalfasin; Xenograft Model Antitumor Assays

Thymosin alpha1 (Tα1) is a multifunctional polypeptide involved in immunoregulation, which universally exists in various organs. The clinical application, however, is limited because of its short half-life. The Fc domain of human IgG has functional properties, improving the affinity and serum half-life. In this study, we fused Tα1 to Fc domain of human IgG1 for generation of a novel long-acting fusion protein, termed Tα1-Fc. Tα1-Fc was expressed, purified, and identified. The results showed that Tα1-Fc was more potent than Tα1 in inhibiting the growth of 4T1 and MCF-7 breast cancer cells in vivo. Furthermore, in the 4T1 tumor model the mice treated with Tα1-Fc exhibited higher level of CD4 and CD8 cells compared with that of the mice Tα1 treated. The interferon-γ and interleukin-2 level in the Tα1-Fc-treated mice was higher than that in the Tα1-treated mice. Tα1-Fc could also alleviate immunosuppression induced by hydrocortisone. In summary, Tα1-Fc provides a novel potent strategy to recruit immune cells against tumors and enhance the antitumor activity of Tα1.

Topics: Animals; Antineoplastic Agents; Breast Neoplasms; CD4-Positive T-Lymphocytes; CD8-Positive T-Lymphocytes; Cell Line, Tumor; Female; Humans; Immunoglobulin Fc Fragments; Immunoglobulin G; Interferon-gamma; Interleukin-2; MCF-7 Cells; Mice; Mice, Inbred BALB C; Mice, Inbred ICR; Mice, Nude; Rats; Rats, Wistar; Thymalfasin

Thymosin alpha 1 (Tα1), an immunoactive peptide, has been shown to inhibit cell proliferation and induce apoptosis in human leukemia, non-small cell lung cancer, melanoma, and other human cancers. However, the response and molecular mechanism of breast cancer cells exposed to Tα1 remain unclear. PTEN, a tumor suppressor gene, is frequently mutated in a variety of human cancers. In the present study, we aimed to investigate the biological roles of PTEN in the growth inhibition of human breast cancer cells exposed to Tα1. Using wild-type and mutant PTEN-expressing cells, we found a strong correlation between PTEN status and Tα1-mediated growth inhibition of breast cancer cells. The growth inhibition effect was more pronounced in breast cancer cells in which Tα1 enhanced PTEN expression, whereas endogenous PTEN knockdown reversed the growth inhibition effect of Tα1 in breast cancer cells. Further investigation revealed that PTEN up-regulation, which was induced by Tα1, can inhibit the activation of the PI3K/Akt/mTOR signaling pathway, leading to the growth inhibition of breast cancer cells. The addition of the synergy between Tα1 and the inhibition of PI3K/Akt/mTOR activation could strongly block cell viability in PTEN down-regulated breast cancer cells. PTEN-overexpressing cells not only up-regulated Bax and cleaved caspase-3/9 and PARP expression but also down-regulated Bcl-2 compared to the treatment with Tα1 alone. Together these findings suggest that PTEN mediates Tα1-induced apoptosis through the mitochondrial death cascade and inhibition of the PI3K/Akt/mTOR signaling pathway in breast cancer cells.

Topics: Apoptosis; Breast Neoplasms; Cell Line, Tumor; Cell Proliferation; Female; Humans; Mitochondria; Phosphatidylinositol 3-Kinases; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; Signal Transduction; Thymalfasin; Thymosin; TOR Serine-Threonine Kinases; Up-Regulation

Thymosin alpha 1 (Tα1) is commonly used for treating several diseases; however its usage has been limited because of poor penetration of the target tissue, such as tumor cells. In the present study, Tα1-iRGD, a peptide by conjugating Tα1 with the iRGD fragment, was evaluated its performance in MCF-7 and MDA-MB-231 human breast cancer cells. Compared with the wild-type peptide, Tα1-iRGD was more selective in binding tumor cells in the cell attachment assay. Furthermore, the MTT assay confirmed that Tα1-iRGD proved more effective in significantly inhibiting the growth of MCF-7 cells in contrast to the general inhibition displayed by Tα1. Further, conjugation of Tα1 with iRGD preserved the immunomodulatory activity of the drug by increasing the proliferation of mouse spleen lymphocytes. Further, compared with Tα1 treatment, Tα1-iRGD treatment of MCF-7 cells considerably increased the number of cells undergoing apoptosis, resulting in a dose-dependent inhibition of cancer cell growth, which was associated with a much better effect on up-regulation of the expression of BCL2-associated X protein (Bax), caspase 9, etc. More importantly, treatment with Ta1-iRGD was more efficacious than treatment with Ta1 in vivo. This study highlights the importance of iRGD on enhancement of cell penetration and tumor accumulation. In summary, our findings demonstrate that the novel modified Tα1 developed in this study has the potential to be used for treating breast cancer.

Topics: Animals; Antineoplastic Agents; Apoptosis; Breast Neoplasms; Cell Adhesion; Cell Line, Tumor; Cell Proliferation; Female; Heterografts; Humans; Lymphocytes; MCF-7 Cells; Mice, Inbred BALB C; Mice, Nude; Molecular Conformation; Molecular Targeted Therapy; Oligopeptides; Spleen; Thymalfasin; Thymosin

Clinical trails showed that thymosin alpha1 offers protection from toxicities (nausea, vomiting, fatigue) of chemotherapy. This study was designed to investigate the protection of thymosin alpha1 to nervous system.. Twenty-two patients with advanced lung cancer, or advanced breast cancer were treated with vinorelbine (25 mg/m(2), d(1), d(8)) combined with cisplatin (80 mg/m(2), d(1)), or gemcitabine (1.25 g/m(2), d(1), d(8)) combined with cisplatin (80 mg/m(2), d(1)),or paclitaxel (80 mg/m(2), d(1), d(8), d(15)) combined with carboplatin (AUC=6 d(1)),or paclitaxel (80 mg/m(2), d(1), d(8), d(15)) combined with epirubicin (80 mg/m(2), d(1)). They all experienced grade 2 to 4 of neurotoxicities according to common toxicity criteria of National Cancer Institute after chemotherapy. The same chemotherapy regimens were combined with thymosin alpha1 (1.6 mg/d for 4 days before chemotherapy, and 1.6 mg twice weekly for 1-3 weeks after chemotherapy began) in the next cycle. Clinical neurologic evaluation was performed at baseline every week.. In 10 patients (45.4%), neurotoxicities reduced from grade 2-4 before chemotherapy to less than grade 2 after chemotherapy.. Thymosin alpha1 may prevent patients from chemotherapy-induced neurotoxicities.

Topics: Adult; Aged; Antineoplastic Combined Chemotherapy Protocols; Breast Neoplasms; Constipation; Female; Humans; Hypesthesia; Hypokinesia; Lung Neoplasms; Lymphatic Metastasis; Male; Middle Aged; Neoplasm Staging; Neuroprotective Agents; Thymalfasin; Thymosin

Synaptophysin and/or chromogranin A may be expressed in epithelial cells of normal and dysplastic mammary gland and in some cancer of the breast. This work indicates that some stromal cells form thin walled vascular channels and from their perivascular mesenchyma arise myoid-like cells, some with cross-like striations. These myoid-like cells have been stained with antibody to smooth muscle actin, rarely to desmin and sarcomeric actin as well as are strongly positive for synaptophysin and/or chromogranin A. From those cells arise cancer cells. Some cancer cells also expressed desmin, sarcomeric actin or smooth muscle actin. Because synaptophysin positive are neuromuscular endplates, it is a question whether the presence of synaptophysin in the progenitor of neoplastic cells of myogenic origin is due to the synaptic dysfunction in molecular mechanism of the epithelial-parenchyma and mesenchymal stroma interaction. The location of S100-protein positive dendritic cells distributed in regular pattern in suprabasal layer of the mammary gland indicate that these cells may arise from preadipocytes which take part in morphogenesis of the breast. All cases of the breast cancer demonstrated thymosin alpha 1, some of them also showed Hassall's-like bodies, mucin secretion and minute calcification. Whether the interaction of epithelial cells and S100-protein positive dendritic cells in tissue other than the thymus has similar dynamic activity to that of Hassall's bodies of the thymus remains to be determined. Both, epithelial cells of Hassall's bodies and epithelial cells of the investigated breast are of myogenic origin. The above consideration suggests that the cells forming vascular channels are with a differentiation defect as they are created in altered stromal mesenchyma and the presence of thymic growth factors might induce tumor growth.

Topics: Adjuvants, Immunologic; Biomarkers, Tumor; Breast Neoplasms; Carcinoma; Chromogranin A; Chromogranins; Disease Progression; Humans; Immunohistochemistry; Prognosis; S100 Proteins; Synaptophysin; Thymalfasin; Thymosin

A radioimmunoassay specific for the C-terminus of human prothymosin alpha was developed using the synthetic peptide [Cys-Aca degrees]-human prothymosin alpha (90-109)-OH coupled to KLH as antigen and the analogue [Tyr-Aca degrees]-human prothymosin alpha (90-109)-OH labelled with 125I as tracer. The radioimmunoassay measured intact prothymosin alpha, in the range of 2-100 pmol and does not cross-react with the partly homologous polypeptide parathymosin alpha. A major epitope was located in the segment 95-107. A radioimmunoassay specific for the N-terminus of human parathymosin alpha, also measuring intact parathymosin alpha in the range of 1-20 pmol and not cross-reacting with prothymosin alpha, was developed using the synthetic peptide [Cys-Aca degrees]-human parathymosin alpha (1-30)-OH as antigen coupled to KLH and the analogue [Tyr-Aca degrees]-human parathymosin alpha (1-10)-OH labelled with 125I as tracer. A major epitope was located in the segment 1-10. These radioimmunoassays, together with a previously established radioimmunoassay for the N-terminus of prothymosin alpha, permitted the identification of the molecular forms of the cross-reactive materials in both normal and neoplastic breast tissue extracts as intact prothymosin alpha and parathymosin alpha. It was also possible to reveal significantly higher levels of both alpha-thymosins in breast cancer tissue compared to the nearby healthy tissue--the mean of 14 samples was over 14-fold higher--suggesting a role of both prothymosin alpha and parathymosin alpha in cell proliferation. The reported radioimmunoassays are expected to facilitate the search for prognostic and/or diagnostic applications of these polypeptides in human cancer.

Topics: Aged; Aged, 80 and over; Amino Acid Sequence; Animals; Breast Neoplasms; Chromatography, High Pressure Liquid; Cross Reactions; Epitopes; Female; Humans; Middle Aged; Molecular Sequence Data; Peptides; Protein Precursors; Rabbits; Radioimmunoassay; Thymalfasin; Thymosin

Growth of normal and malignant cells is controlled by the interplay of several hormones and growth factors present in the body. The growth of the human breast cancer cell line MCF-7 is stimulated in vitro by estradiol which has been shown to induce the release of numerous polypeptide growth factors into the culture media. In a search to identify polypeptide growth factors for MCF-7 breast cancer cells we have detected the thymic hormone, thymosin alpha one (TA1) in culture supernatants and cytosol preparations of MCF-7 cells grown in TA1 free media. TA1 was identified by reverse phase-high performance liquid chromatography (RP-HPLC) followed by a specific radioimmunoassay for TA1 (TA1-RIA). Indirect immune fluorescence localized TA1 on, or within, the cytoplasm of MCF-7 cells grown in TA1 free media. The results of this data along with our previously published findings fulfills three of the four criteria needed to establish thymosin alpha-1 as an autocrine growth factor for MCF-7 cells in culture.

Topics: Breast Neoplasms; Cell Line; Chromatography, High Pressure Liquid; Female; Fluorescent Antibody Technique; Humans; Radioimmunoassay; Thymalfasin; Thymosin; Tumor Cells, Cultured