Non-Small Cell Lung Cancer (NSCLC) constitutes over 85% of lung cancer (LC) cases [1]. Most NSCLC patients present with advanced disease at diagnosis, leading to a high incidence of distant metastasis post-curative surgery and poor prognosis. Advances in chemotherapy and the application of targeted therapies have markedly improved response rates and 5 years survival rates (SRs) for NSCLC. However, factors such as treatment resistance pose limitations to further research in various targeted therapies [2, 3].

The Topotecan (TPT) is derived from camptothecin and inhibits DNA topoisomerase I. It binds with both the enzyme and DNA, blocking the repair of single-strand DNA breaks [4]. DNA topoisomerase I is a crucial enzyme involved in DNA replication and repair, responsible for unwinding DNA and inducing single-strand breaks while forming a single-strand DNA sheath between DNA strands. As a DNA topoisomerase I inhibitor, TPT binds to the enzyme and disrupts DNA replication and repair processes, preventing cancer cells from continuing to grow and divide [5]. In addition to direct inhibition of DNA topoisomerase I, TPT also induces DNA breaks, further exacerbating damage to cancer cells [6]. Moreover, TPT induces cell apoptosis (programmed cell death) and arrests cells from progressing from the G2 phase to the M phase (mitosis), thereby further suppressing tumor cell proliferation [7]. The TPT is primarily used clinically to treat diseases such as SCLC and metastatic ovarian cancer. As a chemotherapy agent, TPT demonstrates significant efficacy and can synergize with taxanes or platinum-based drugs to enhance anticancer activity. Takahashi et al. [8] confirmed in clinical treatment of SCLC patients that employing TPT alone or in combination with Berzosertib suggested negligible differences in progression-free survival and adverse reactions, but combination therapy notably extended overall survival. Edelman et al. [9], discovered that combining TPT with Irinotecan drastically extended SR in patients with recurrent/refractory SCLC. Baize et al. [10], also found that combining TPT with cisplatin chemotherapy increased SR in SCLC patients. Common severe adverse effects include neutropenia, thrombocytopenia and anemia. Currently, TPT is approved for NSCLC. Nevertheless, more research is needed to explore the efficacy and mechanisms of TPT in treating NSCLC.

This work established an NSCLC nude mouse model by subcutaneously injecting H1993 human NSCLC cells into the right axillary fossa. Effects of cisplatin (DDP) and different concentrations of TPT on SRs, immune function, tumor growth and tumor cell apoptosis in the nude mouse model were compared. The goal was to provide reference data for investigating the potential mechanisms of TPT in treating NSCLC.

The TPT was observed to drastically enhance SRs and suppress tumor volume growth in nude mice carrying NSCLC tumors, showing a dose-dependent response. The NSCLC arises from the bronchial mucosal epithelium. Most patients have grim prognosis and numerous complications [11]. The DDP is standard first-line therapy for advanced NSCLC, often combined with radiotherapy to benefit patients drastically [12, 13]. Nevertheless, resistance to treatment can develop in some patients during therapy, which compromises its effectiveness. The mechanism of action of TPT in cancer treatment primarily involves inhibiting the activity of DNA topoisomerase I, thereby disrupting DNA replication and repair processes and inducing DNA breakage and cell apoptosis [14]. Zhang et al. [15], found that folate-conjugated TPT liposomes effectively reduced tumor volume and prolonged circulation time in rats bearing A549 LC xenografts. However, the precise mechanism by which TPT treats NSCLC requires further exploration.

The thymus and spleen are crucial immune organs, integral to both central and peripheral immune systems [16]. It was observed that after treatment with DDP, the thymus and spleen indices decreased in nude mice bearing NSCLC tumors, indicating immune suppression in the host organism. Conversely, following administration of TPT, an increase in thymus and spleen indices was noted, showing a dose-dependent effect. This suggests that TPT can enhance thymus and spleen indices in nude mice bearing NSCLC tumors, thereby boosting both humoral and cellular immune functions. During the growth of LC cells, drastic immune evasion mechanisms exist, which are crucial factors in inducing LC development [17]. Changes in T lymphocyte subset levels can directly reflect the body’s ability to inhibit tumor growth, including the major subsets CD4⁺ and CD8⁺ [18, 19, 20]. The CD4⁺ T cells facilitate immune cell proliferation and differentiation and coordinate interactions among immune cells [21]. As key effector cells in anti-tumor immunity, CD8⁺ cells typically inhibit tumor growth by directly killing tumor cells and secreting cytokines [22]. Conversely, an imbalance in the CD4⁺/CD8⁺ T-cell subset ratio or functional exhaustion of CD8⁺ T cells may impair antitumor immunity, thereby facilitating tumor immune escape. It has been observed that after treatment with DDP and TPT, the peripheral blood CD4⁺ cells and CD4⁺/CD8⁺ ratio markedly increased in nude mice bearing NSCLC tumors, while the proportion of CD8⁺ cells decreased markedly. Particularly, treatment with high doses of TPT led to more pronounced changes in T lymphocyte subset levels. This suggests that TPT can correct abnormal T lymphocyte subset levels in the body, improve immune suppression and thereby drastically enhance immune function in nude mice bearing NSCLC tumors.

The IL-4 is an anti-inflammatory cytokine secreted by Th2 cells that promotes differentiation of Th0 cells into Th2 cells [23]. The TNF-$\alpha$ is a cytokine secreted by immune cells that markedly inhibits tumor cell growth and induces tumor regression [24]. The IFN-$\gamma$, produced by activated NK cells, is a Th1-type cytokine that suppresses the expression of pro-cancerous diseases and can hinder the transformation of tumor cells [25]. It has been observed that treatment with DDP and TPT increases IL-4, TNF-$\alpha$ and IFN-$\gamma$ in the serum of nude mice bearing NSCLC, with TPT treatment exhibiting dose-dependent characteristics. The TNF-$\alpha$ can induce apoptosis and eliminate tumor cells, thereby inhibiting tumor growth [26]. The IFN-$\gamma$ can activate various immune cells in the body, thereby directly killing tumor cells or indirectly inhibiting the expression of pro-cancerous diseases [27, 28]. This indicates that TPT can inhibit T cell activation and proliferation and negatively regulate immune escape of tumor cells after immune response by affecting IL-4, TNF-$\alpha$ and IFN-$\gamma$. Ultimately, it exerts cytotoxic effects on tumor cells through the body’s immune mechanisms.

During tumor initiation and progression, dysregulated tumor suppressor gene phosphatase and tensin homolog deleted on PTEN can disrupt its normal control over the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway. This disruption leads to unchecked cell proliferation, contributing to the development of cancer [29]. Abnormal PI3K/AKT pathway activation promotes the proliferation, migration and invasion of LC cells, facilitating disease progression [30]. Glycogen synthase kinase-3$\beta$ (GSK-3$\beta$), as a downstream key effector molecule, not only participates in PI3K/AKT signaling, but also regulates tumor related pathways such as WNT/$\beta$-catenin [31, 32]. When PTEN function is impaired or PI3K is continuously activated, GSK-3$\beta$ is prone to phosphorylation inactivation (p-GSK-3$\beta$$\uparrow$), thereby weakening its induction of apoptosis and anticancer effects, promoting the survival and spread of tumor cells [33]. Conversely, upregulation of GSK-3$\beta$ expression can inhibit the WNT/$\beta$-catenin pathway and promote apoptosis of LC cells [34]. This study indicates that both DDP and TPT can upregulate the expression of PTEN and GSK-3$\beta$ in tumor tissues of NSCLC-bearing nude mice, significantly inhibit p-PI3K, p-PI3K/PI3K ratio, and p-GSK-3$\beta$ and its ratio, suggesting that they can effectively inhibit the PI3K/AKT/GSK-3$\beta$ pathway. Among them, TPT is dose-dependent, and the high-dose group has a better effect than DDP, showing stronger anti-tumor activity. Morscher et al. [35], noted the same, who claimed that TPT in combination with Temozolomide can mediate the PI3K/AKT/mTOR pathway to treat pediatric cancers. These results indicate that TPT can inhibit the growth of NSCLC tumors by affecting the activity of the PTEN/PI3K/GSK-3$\beta$ pathway.

The TPT, as a topoisomerase I inhibitor, holds significant importance in the study of NSCLC. This research demonstrates that TPT can correct the imbalance of T lymphocyte subsets by mediating the PTEN/PI3K/GSK-3$\beta$ signaling pathway. This finding offers new perspectives and strategies for immunotherapy in lung cancer. The study not only enhances our understanding of the mechanisms underlying TPT’s effects but also provides a theoretical foundation for developing more effective treatment regimens for lung cancer. Despite significant advances in the study of TPT in NSCLC, several limitations remain: (1) This study only compared the mechanisms of action of TPT monotherapy in Non-Small Cell Lung Cancer xenograft models; the potential enhanced efficacy of TPT in combination with other chemotherapeutic agents, such as DDP, requires further investigation. (2) Current research primarily focuses on xenograft models in mice and the applicability of these findings to human patients needs further validation and (3) The mechanisms of action of TPT warrant deeper investigation to understand its efficacy and safety in lung cancer treatment comprehensively. In summary, while the study of TPT in NSCLC is of significant importance and holds considerable promise for clinical application, it also faces limitations and challenges.

The TPT demonstrated dose-dependent characteristics in enhancing SRs and inhibiting tumor growth in nude mice with NSCLC xenografts. Its mechanism of action involves inducing tumor cell apoptosis, correcting imbalances in T lymphocyte subsets in nude mice, enhancing immune function and influencing the expression of PTEN, PI3K and GSK-3$\beta$ proteins. Future research should continue to explore its mechanisms of action and optimize treatment protocols to improve outcomes and quality of life for lung cancer patients.

This study investigated the efficacy of Topotecan in the treatment of Non-Small Cell Lung Cancer (NSCLC) via the PTEN/PI3K/GSK-3$\beta$ Pathway, a critical area for advancing and developing novel approaches to enhance the therapeutic outcomes in NSCLC. Its unique and significant contribution lies in elucidating how topotecan modulates the immune system through specific intracellular signaling pathways, thereby effectively improving patient survival rates. This finding not only provides new insights into the complex interplay between cancer and the immune system but also paves the way for the development of innovative immunotherapeutic strategies in cancer treatment, potentially driving future advancements in the field.

The datasets used and analyzed during the current study are available from the corresponding author on reasonable request.

CT and HPY designed the research study. CT and HPY performed the research. CT, ML and WL analyzed the data. CT and HPY drafted and revised the manuscript. All authors contributed to editorial changes in the manuscript. All authors read and approved the final manuscript. All authors have participated sufficiently in the work and agreed to be accountable for all aspects of the work.

The experimental protocol was approved by the Animal Ethical Committee of Tianjin Medical University Institute and Hospital (No. WDRY2022-K032). All the experimental protocols involved in the current investigation followed the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and the ARRIVE (Animal Research: Reporting of in vivo experiments) guidelines.

This research received no external funding.

The authors declare no conflict of interest.