Main Research Findings

1) High doses of Tα1 were found to exhibit anti-proliferative effects against cancer cells as well as antioxidant properties that decreased the production of reactive oxygen species and increased the activity of antioxidant enzymes.

2) Tα1 may have the potential to be used to develop novel cancer therapies due to its immunomodulatory effects that enhance curative effects and decrease immune-related adverse events

 

Selected Data

1) This study performed by Kharazmu-Khorassani et al investigated the biochemical effects of Tα1 on human lung cancer cells (A549), aiming to understand its influence on cellular proliferation, migration, reactive oxygen species (ROS) levels, and antioxidant enzyme activities, as well as its potential to induce apoptosis.

The core biological material for the study consisted of human lung epithelial adenocarcinoma cells, A549. These cells were maintained in a standard culture environment of RPMI medium supplemented with 10% heat-inactivated Fetal Bovine Serum (FBS) and a combination of antibiotics (100 units/mL penicillin and 100 mg/mL streptomycin). Cultures were sustained in 25-cm cell culture flasks and incubated at 37°C in a humidified atmosphere containing 5% carbon dioxide. To maintain optimal cell health and growth, the culture medium was refreshed every 1-2 times per week, and cells were sub-cultured when they reached approximately 80% confluence [1].

The Tα1 peptide was the primary experimental agent. For cytotoxicity assessments, A549 cells were treated with a broad range of Tα1 concentrations including 3, 6, 12, 24, and 48 µg/mL. For other biochemical assays, including those for antioxidant enzymes, ROS measurement, and cell migration, Tα1 was typically applied at concentrations of 3, 6, and 12 µg/mL. The standard incubation period with Tα1 for most experiments was 24 hours, though some assays extended to 48 hours.

Cytotoxicity was evaluated using the MTT assay. A549 cells were seeded at 10^4 cells per well in 96-well plates and allowed to adhere overnight at 37°C. Following this, they were treated with the specified Tα1 concentrations for 24 hours. After the treatment period, 20 µL of MTT solution was added to each well, and plates were incubated for an additional 4 hours. The supernatant was then discarded, and the formazan crystals, indicative of viable cells, were dissolved in 150 µL of DMSO per well. The absorbance was measured at 570 nm using an ELISA reader [1].

For antioxidant enzyme activity measurements (catalase, glutathione peroxidase, and superoxide dismutase) and total thiol content, cells were first prepared for lysis. Approximately 7 x 10^6 A549 cells were seeded in T75 flasks and incubated for 24 hours. After Tα1 treatment, cells were washed, scraped from the flasks, and centrifuged at 4000 rpm for 10 minutes. The resulting cell pellet was resuspended in 50 mM phosphate buffer and sonicated on ice for a minimum of 1 x 30 seconds using a Vibra Cell cup horn sonicator at 40% power. Protein content in the cell extract was quantified using the Bradford assay with bovine serum albumin as a standard. The cell lysates were stored at -20°C for up to 6 months.

Catalase (CAT) activity was determined by adding 50 µL of cell lysate containing 1.5 mg/mL protein, to 4 mL of phosphate buffer, and 1 mL of 30 mM H2O2 was added to initiate the reaction. Absorbance changes were measured every 30 seconds for 2 minutes at 240 nm to determine H2O2 decomposition. Glutathione Peroxidase (GPx) activity was assessed using the Ransel enzyme kit. The assay involved adding 10 µL of cell lysate to the kit reagents, with absorbance read at 340 nm. The method relies on GPx catalyzing the reduction of glutathione to its oxidized form, followed by regeneration with glutathione reductase using NADPH as an electron donor. Superoxide Dismutase (SOD) activity was also measured using the RANDOX enzyme kit. 10 µL of cell lysate containing 0.233 mg/mL protein was used, with absorbance read at 505 nm. SOD activity, expressed in U/mL, is based on its ability to catalyze the dismutation of superoxide radicals to hydrogen peroxide and molecular oxygen. Finally, total thiol content was determined using the Elman method. 20 µL cell lysate, 75 µL DTNB reagent, and 400 µL methanol were combined, and absorbance was evaluated at 412 nm using an ELISA reader [1].

The generation of ROS was measured using 2,7-dichlorodihydrofluorescein-diacetate (DCFH-DA). A549 cells were plated in six-well plates and treated with 3, 6, and 12 µg/mL Tα1 for 24 hours. 50 µL DCFH-DA was then added, and after 30 minutes, cells were trypsinized, centrifuged, and resuspended in PBS/FBS. Fluorescent signals, indicative of ROS production, were detected using flow cytometry and analyzed with Flow-Jo software. Cell migration was assessed via a scratch assay. A549 cells were plated in 24-well plates and incubated for 48 hours to form a monolayer. A scratch was then made using a sterilized pin. After washing to remove suspended cells, new medium containing 3, 6, or 12 µg/mL Tα1 was added. Images were captured at 0, 24, and 48 hours using an inverted microscope, and migration distance was calculated using ImageJ software.

To investigate apoptosis and cell cycle perturbation, two methods were employed:
Hoechst 33342 staining that included plating A549 cells in 24-well plates, treating with Tα1 for 24 hours, washing, and permeabilizing with ethanol. After washing, 1 µg/mL Hoechst 33342 was added and incubated for 30 minutes in the dark. Cells were observed under a fluorescence-inverted microscope with a blue filter. The second method was cell cycle analysis by flow cytometry that included plating A549 cells in 24-well plates and treating with 3, 6, or 12 µg/mL Tα1 for 24 hours. After trypsinization, centrifugation, and washing, cells were fixed with ethanol. Propidium iodide (PI) was added for DNA staining. Fluorescent signals were detected using flow cytometry (BD Accuri C6) and analyzed with Flow-Jo software to assess cell cycle phases (G1, S, G2/M) and sub-G1 population as an indicator of apoptosis [1].

2) Tα1 is a naturally occurring polypeptide, comprising 28 amino acids, that holds a central role in immune system regulation. Researchers Wei et al completed a comprehensive review regarding the compound’s therapeutic potentials. First isolated from thymic tissue,Tα1 is primarily produced by thymic epithelial cells. These cells are integral to providing the microenvironment necessary for thymocyte maturation and secreting active peptides that contribute to T-cell differentiation and proliferation. Physiologically, Tα1 is essential for maintaining normal immune homeostasis throughout life, with circulating levels acting as a prognostic indicator for various pathological conditions, including hepatitis, systemic erythematosus lupus, rheumatoid arthritis, sepsis, and cystic fibrosis. The administration of exogenous Tα1 aims to restore these levels, thereby modifying, enhancing, and restoring compromised immune functions across a spectrum of diseases, encompassing viral infections, immunodeficiencies, and malignancies [2].

Tα1 exerts a profound influence on both adaptive and innate immune responses, showcasing a pleiotropic regulatory capacity that adapts to different disease contexts. In the adaptive immune system, Tα1 is crucial for T cell maturation, promoting the differentiation of CD34+ stem cells into CD3+CD4+ cells. It actively stimulates the production of vital Th1-type cytokines, such as interleukin (IL)-2, interferon-gamma (IFN-γ), and IL-3, which are critical for cell-mediated immunity.

Furthermore, Tα1 induces a shift towards the Th1 subtype of T cells, which is instrumental in driving neurogenesis, enhancing cognitive functions, and bolstering anti-tumor responses. Its regulatory effects extend to reducing the number of regulatory T cells (Tregs) through increased apoptosis and improving T cell counts in conditions characterized by severe lymphocytopenia, such as in certain COVID-19 patients, while also decreasing the expression of exhaustion markers like PD-1 and TIM3 on CD8+ T cells. Beyond T cells, Tα1 indirectly stimulates B cells to differentiate into antibody-producing plasma cells, leading to an improved response to vaccines and enhancing deficient IL-10-producing regulatory B cell subsets in patients with multiple sclerosis [2].

The innate immune system also benefits significantly from Tα1’s immunomodulatory actions. Tα1 is known to potentiate the cytolytic activity of natural killer (NK) cells, an early line of defense against cancer and viral infections. Its effects on dendritic cells (DCs) are particularly well-documented; Tα1 acts as an adjuvant, activating DCs and inducing their functional maturation. In conventional DCs, it promotes IL-12 production, while in plasmacytoid DCs, it facilitates IL-10 secretion in response to fungal pathogens, thereby balancing inflammation and tolerance through a Th1/Treg anti-fungal response. Moreover, Tα1 demonstrates a dual regulatory effect on human monocyte-derived DCs during viral and bacterial infections. It increases the expression of maturation markers (CD40, CD80, MHC I/II) and proinflammatory cytokines (IL-6, IL-8, TNF-α, IFN-α/γ), enhancing antiviral immunity, while simultaneously suppressing excessive inflammation caused by bacterial infections [2].

Macrophages, another critical component of innate immunity, are also profoundly affected by Tα1. It enhances the efficiency of antigen presentation by macrophages and dramatically induces phagocytosis, as evidenced by its rapid clearance of Aspergillus niger conidia. Tα1 increases macrophage motility, chemotaxis, pinocytosis, and their anti-tumor cytotoxicity.

Critically, Tα1 plays a role in shaping the plasticity of M2 Tumor-Associated Macrophages (TAMs) towards an anti-tumoral M1 phenotype. This is achieved by downregulating M2 markers like IL-10 and CD206, and simultaneously upregulating M1 markers such as IL-6 and CD86, which culminates in a CD4+ and CD8+ T cell-dependent anti-tumor immune response. Furthermore, Tα1 exhibits an anti-tumor effect by promoting apoptosis in peripheral blood monocytic myeloid-derived suppressor cells (MDSCs) and inhibiting their migration into the tumor microenvironment [2].

A key mechanism underlying Tα1’s diverse effects is its modulation of Toll-like Receptors (TLRs), a family of transmembrane receptors crucial for recognizing pathogen-associated molecular patterns. Tα1’s pleiotropic regulatory functions involve various TLRs and their downstream signaling pathways, allowing it to fine-tune cellular immune responses. For instance, Tα1 acts as an agonist for TLR2 and TLR9, increasing their expression in murine DCs and enhancing IL-12 production through the MyD88-MAPK-NF-κB pathway. It also boosts TLR2 expression in human conventional DCs and increases TLR9 expression in plasmacytoid DCs, promoting IL-10 secretion for anti-fungal responses and enhancing IFN-α secretion via the TLR9-MyD88-IRF7 pathway for antiviral immunity.

Tα1 is also implicated in TLR7/8-mediated immunoregulation, activating the TNF receptor-associated factor 6-aPKC-IKK signaling pathway in macrophages, leading to cytokine gene expression. It enhances pro-inflammatory cytokine expression and MHC I/II in TLR7/8 agonist-treated human monocyte-derived DCs. Intriguingly, Tα1 influences the peripheral blood mononuclear cell response in multiple sclerosis patients, reducing proinflammatory cytokines while increasing anti-inflammatory ones.

At a molecular level, Tα1 binds to phosphatidylserine (PS) on apoptotic tumor cells, which is critical for its ability to reprogram immunosuppressive M2 TAMs towards an immunostimulatory M1 phenotype. This involves a complex interplay of signaling pathways, including activating TLR7-MyD88-SHIP1 to reduce TLR4-MyD88-TBK1-IRF3 driven IL-10, and promoting IL-6 secretion through the TLR8-MyD88-IRAK4-JNK/P38/IKKα/β-IκBα-p65 pathway. This intricate context-dependent modulation of TLRs underscores Tα1’s versatility in orchestrating immune responses [2]

 

Discussion

1) The investigation completed by the research team of Kharazmi-Khorassani et al into the biochemical effects of Tα1 on A549 human lung cancer cells yielded several key findings, primarily highlighting Tα1’s anti-proliferative, anti-migratory, and antioxidant properties, while showing no significant effect on apoptosis.

The cytotoxicity assay, performed using the MTT method, demonstrated that Tα1 exerted an anti-proliferative effect on A549 cells in a concentration-dependent manner. After 24 hours of treatment, Tα1 at concentrations of 24 and 48 µg/mL significantly reduced cell viability. Specifically, Tα1 at concentrations of 3, 6, and 12 µg/mL also reduced cell viability, though the difference was not statistically significant at these lower doses, contributing to a 20-36% reduction in cell viability across the 3-48 µg/mL range. This indicates that higher concentrations of Tα1 were required to achieve a statistically significant cytotoxic effect on A549 cells within a 24-hour period, with the most pronounced growth inhibition observed at 24 and 48 µg/mL [1].

A significant portion of the study focused on Tα1’s antioxidant properties, evaluated through its impact on key antioxidant enzymes and reactive oxygen species (ROS) levels. CAT activity in A549 cell extracts showed a concentration-dependent enhancement. The activity of catalase was significantly higher at 12 µg/mL of Tα1, measuring approximately 1.75 times that of the control group. While 3 and 6 µg/mL also increased CAT activity, these differences were not statistically significant compared to the control. GPx activity was also significantly increased in A549 cells treated with Tα1. At 3, 6, and 12 µg/mL, GPx activities were measured at 69.3, 96.7, and 90.4 unit/mL respectively, relative to a control value of 12.6 unit/mL. This indicated a statistically significant enhancement of GPx activity at all tested concentrations, with specific significant differences observed between 3 and 6 µg/mL, and 3 and 12 µg/mL [1].

SOD activity similarly demonstrated a positive association with Tα1 concentration. At 6 and 12 µg/mL, SOD activity was 1.36 and 1.56 times higher than the control, respectively, indicating a significant boost in this crucial antioxidant enzyme’s function. The cellular thiol assay indicated that Tα1 at concentrations of 3, 6, and 12 µg/mL enhanced the total cellular thiol content compared to the control. However, unlike the enzyme activities, the differences in thiol levels between the various Tα1 treatment groups were not statistically significant. This suggests a general supportive role for Tα1 in maintaining cellular redox balance, primarily through boosting enzyme activity rather than direct, dose-dependent changes in overall thiol content.

Figure 1: Changes in A) CAT activity, B) Gpx activity, C) SOD activity, and D) % Thiol in response to various treatment concentrations of Tα1.

Complementing the antioxidant enzyme findings, the ROS assay directly measured the generation of cellular ROS using DCFH-DA and flow cytometry. The results showed a significant reduction in cellular ROS levels in A549 cells treated with Tα1. At concentrations of 3, 6, and 12 µg/mL, ROS production was significantly lower by 2.31%, 3.71%, and 1.61% respectively compared to the control group at 31% after 24 hours. Notably, the lowest production of ROS was observed at the highest Tα1 concentration (12 µg/mL), confirming Tα1’s ability to suppress oxidative stress in cancer cells [1].

The scratch assay was employed to assess Tα1’s effect on cell migration. The results indicated that Tα1 significantly inhibited the migration of A549 lung cancer cells in a concentration-dependent manner. After 24 hours, the migration percentages at 3, 6, and 12 µg/mL Tα1 were 51%, 51%, and 47% respectively, compared to a control migration of 62.5%. This inhibitory effect became even more pronounced after 48 hours, with Tα1 reducing migration by 20%-26.5% at the same concentrations. These findings demonstrate Tα1’s capacity to suppress the invasive potential of A549 cells.

Finally, the study investigated Tα1’s influence on apoptosis and cell cycle progression using Hoechst 33342 staining and cell cycle analysis by flow cytometry. Hoechst 33342 staining showed that in A549 cells treated with 3, 6, and 12 µg/mL of Tα1, the number of living cells was not significantly reduced compared to the control. This indicated that Tα1 did not induce apoptosis in these lung cancer cells under the tested conditions. Cell cycle analysis corroborated these findings. Although Tα1 treatment at various concentrations (3, 6, and 12 µg/mL) led to a reduction in the cell population in the G1 phase of the cell cycle, this change was not statistically significant. Furthermore, the sub-G1 profile analysis, which is a common indicator of apoptosis, showed no significant increase in apoptotic cells after Tα1 exposure. These results collectively suggest that Tα1, under the experimental conditions of this study, did not primarily induce cell cycle arrest or programmed cell death in A549 cells [1].

In conclusion, Tα1 at high concentrations exhibited anti-proliferative effects on A549 lung cancer cells, though its primary mechanism appeared to be unrelated to the induction of apoptosis or significant cell cycle arrest. Its most prominent and consistent effects were its antioxidant properties, demonstrated by the suppression of ROS production and the enhancement of key antioxidant enzyme activities, along with its ability to inhibit cancer cell migration. The study positions Tα1 as a potential therapeutic agent that can induce antioxidant effects and suppress migration in A549 cancer cells, warranting further investigation into its role in various cancer contexts [1].

2) The research team of Wei et al completed a comprehensive review regarding the therapeutic potential of Tα1. Tα1 has emerged as a significant immunomodulator, extensively utilized for treating various conditions, including viral infections, immunodeficiencies, and, most notably, malignancies. Its ability to stimulate both innate and adaptive immune responses, coupled with its pleiotropic regulatory mechanisms that adapt to specific disease contexts and immune microenvironments, positions it as a highly promising therapeutic agent. Preclinical studies consistently demonstrate Tα1’s potential to enhance the curative effects of conventional cancer therapies and mitigate immune-related adverse events, particularly those associated with immune checkpoint inhibitors, thus paving the way for novel cancer treatment strategies [2].

In terms of chemotherapy, Tα1 has shown strong synergistic effects by augmenting anti-tumor immune responses, thereby counteracting the immunosuppressive environment often induced by cytotoxic agents. While chemotherapy effectively eliminates tumor cells, it can also create an immunosuppressive tumor microenvironment that hinders effective immune surveillance. Tα1 acts as a crucial adjuvant, restoring immune balance and enhancing the efficacy of chemotherapy. For instance, its combination with IL-2 and 5-fluorouracil significantly reduces liver metastases in rat models of colorectal carcinoma. In experimental models of peritoneal metastases from colonic carcinoma, Tα1, when administered after hyperthermic intraperitoneal chemotherapy, increases overall survival rates and promotes a robust Th1 anti-tumor immune response, leading to increased CD8+ T cell infiltration.

Furthermore, combining Tα1 with gemcitabine has been shown to yield superior anti-tumor effects in nasal natural killer/T cell lymphoma, by attenuating Epstein-Barr virus (EBV) viral load, promoting the expression of proinflammatory factors, inhibiting epithelial-mesenchymal transition (EMT) and cell growth, and enhancing both autophagy and apoptosis within tumor cells. Similarly, in breast cancer models, Tα1 combined with epirubicin markedly increases the number and function of tumor-infiltrating T cells, contributing to suppressed tumor growth. These findings collectively highlight Tα1’s capacity to induce powerful synergistic anti-tumor effects across various cancer types when used in conjunction with conventional chemotherapy [2].

Beyond chemotherapy, Tα1 holds significant promise as an adjuvant in contemporary immunotherapy, particularly with immune checkpoint inhibitors (ICIs). While ICIs like anti-CTLA-4 and anti-PD-1/PD-L1 antibodies have revolutionized cancer treatment, their efficacy is not universal, and patient responses are often dictated by the tumor immune microenvironment (TME). Tα1 has been shown to synergize with anti-CTLA-4 therapy; pretreatment with Tα1 enhances long-term overall survival in metastatic melanoma patients treated with ipilimumab. For anti-PD-1/PD-L1 therapies, Tα1’s ability to modulate the TME is critical. It can potentially convert less responsive TME types into a Type II TME, which is characterized by the presence of both PD-L1 expression and tumor-infiltrating lymphocytes, making it amenable to anti-PD therapy. Tα1 achieves this by increasing the number of tumor-infiltrating lymphocytes (TILs) and inducing a stronger specific anti-tumor response, partly by enhancing MHC class I expression in tumor cells [2].

Moreover, Tα1 promotes the apoptosis of myeloid-derived suppressor cells (MDSCs) and re-polarizes tumor-associated macrophages (TAMs) from an immunosuppressive M2 phenotype to an immunostimulatory M1 phenotype. In NSCLC cells with high PD-L1 expression, Tα1 suppresses proliferation and migration, thereby enhancing the efficacy of anti-PD-L1 antibodies. The combination of low-doseTα1 and an anti-PD-L1 antibody has also been shown to increase efficacy in lung metastasis melanoma models.

A crucial aspect of Tα1’s role in immunotherapy involves its interaction with the efferocytosis process, particularly concerning TAMs. PD-1+ TAMs are typically M2-like macrophages with limited efferocytotic capabilities. Anti-PD-1/PD-L1 antibodies improve efferocytosis, and Tα1s ability to regulate the phenotype of efferocytosed TAMs suggests a powerful synergistic effect when combined with anti-CD47 immune checkpoint inhibitors. This combination could significantly promote tumor cell efferocytosis and further drive TAM polarization towards an anti-tumoral M1 phenotype, offering a novel treatment approach that integrates both innate and adaptive immune responses [2].

Significantly, Tα1 may also address a major challenge associated with ICIs: immune-related adverse events (irAEs). While ICIs cause dysimmune toxicities across various organ systems, Tα1 has been observed to potentiate the anti-tumor activity of anti-CTLA-4 therapy while simultaneously protecting against irAEs, such as anti-CTLA-4-induced colitis, through the IDO1 tolerogenic pathway. This dual capacity to enhance efficacy and mitigate side effects makes Tα1 an exceptionally promising candidate for combination immunotherapies.

In conclusion, Tα1’s established role as an immunomodulator, orchestrating both adaptive and innate immune responses primarily through the activation of Toll-like receptors and their downstream signaling pathways, underscores its therapeutic value. Its pleiotropic effects on immune cells make it an attractive candidate for combination therapies, especially in oncology. The promising preclinical findings regarding its synergy with chemotherapy and, more importantly, its potential to enhance the efficacy of immune checkpoint inhibitors (anti-CTLA4, anti-PD-1/PD-L1, and anti-CD47 antibodies) while mitigating their associated toxicities, highlight a significant avenue for future cancer treatment strategies. Further research will be crucial to fully elucidate its complex mechanisms and optimize its application in clinical settings to maximize patient benefit [2].

 

Disclaimer

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Kharazmi-Khorassani J, Asoodeh A. Thymosin alpha-1; a natural peptide inhibits cellular proliferation, cell migration, the level of reactive oxygen species and promotes the activity of antioxidant enzymes in human lung epithelial adenocarcinoma cell line (A549). Environ Toxicol. 2019;34(8):941-949. doi:10.1002/tox.22765

[2] Wei Y, Zhang Y, Li P, Yan C, Wang L. Thymosin α-1 in cancer therapy: Immunoregulation and potential applications. Int Immunopharmacol. 2023;117:109744. doi:10.1016/j.intimp.2023.109744

 

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