STAT3, a transcription factor with important roles in oncogenesis and cancer progression, is activated, phosphorylated, dimerized, and subsequently translocated into the nucleus in response to cytokines, growth factors, and other extracellular signals [39, 40]. Upon binding to sequence-specific DNA elements known as STAT3 response elements (SREs), p-STAT3 acts as a transcription factor, modulating the expression of numerous downstream target genes crucial for tumor growth and progression, such as cyclin B1, c-Myc, Bcl-xl, and Bcl-2 [6, 41, 42]. The significant involvement of STAT3 in cancer progression and tumorigenesis makes it a compelling molecular target for cancer therapy. A plethora of studies has shown demonstrated abnormal upregulation of STAT3 in various tumors, including cervical cancer [10, 43]. Until now, numerous small-molecule inhibitors have been developed to specifically target the SH2 domain of STAT3, crucial for STAT3 dimerization, resulting in reduced tumor cell viability, proliferation, and induction of apoptosis [44,45,46,47].

TTI-101 (c188-9) was discovered as a specific inhibitor targeting the phosphotyrosine peptide-binding site within the STAT3 SH2 domain with high affinity. Recent studies have increasingly focused on the potential of TTI-101 as an anti-tumor agent [48]. Experimental studies have revealed that TTI-101 enhances the therapeutic effectiveness of 5-Aza-2′-deoxycytidine (DAC) against pancreatic cancer through its regulation of demethylation [49]. Additionally, TTI-101 treatment demonstrated inhibition of hepatocellular carcinoma growth and protection of liver function in mice with hepatocyte-specific Pten deficiency [50]. Currently, several phase I/II clinical trial (e.g., NCT05440708, NCT05671835 and NCT05384119) have been launched to assess the safety and efficacy of TTI-101 in cancer patients. In a phase I clinical study (NCT03195699), TTI-101 demonstrated good tolerability in patients with solid tumors, with no severe side effects observed. Encouragingly, this treatment elicited an antitumor response, with 13% of patients showing partial responses and 41% experiencing stable disease. Particularly positive outcomes were noted in hepatocellular carcinoma (HCC), where 20% of patients achieved a partial response. Building on these promising results, Phase 2 studies are currently underway to further investigate the efficacy of TTI-101 in patients with HCC and metastatic breast cancer [51]. Subsequently, we conducted a comprehensive investigation into the anticancer effects of TTI-101 on cervical cancer through a multi-faceted approach encompassing in vitro and in vivo experiments, alongside molecular docking analysis.

Initially, utilizing molecular docking techniques, we elucidated a plausible binding mode of TTI-101 within the STAT3 SH2 domain, involving key interactions such as hydrogen bonds and pi-alkyl contacts. Molecular dynamics simulations validated the structural integrity of the STAT3-TTI-101 complex, exhibiting appropriate internal dynamics. Analysis of protein–ligand interactions provided insights into the intermittent nature of hydrogen bonding interactions between TTI-101 and STAT3 residues.

Subsequent in vitro experiments confirmed the inhibitory effects of TTI-101 on the viability, proliferation, and clonogenic ability of cervical cancer cells. MTT assay results revealed a dose- and time-dependent reduction in cell viability of HeLa cells following treatment with TTI-101. The IC50 value of TTI-101 decreased from 32.4 μM after 24 h of treatment to 18.7 μM after 48 h. Moreover, clonogenic assays demonstrated a notable decrease in the colony formation capacity of TTI-101-treated HeLa cells compared to the untreated control group.

In vivo studies have highlighted that STAT3 knockdown diminishes tumor growth and metastatic potential, underscoring the essential role of STAT3 activation in cancer progression and metastasis [52, 53]. Cervical cancer, known for its invasive and metastatic properties, poses challenges, making it imperative to explore compounds that suppress migration. Thus, we evaluated the effect of TTI-101 on cervical cancer cell migration through wound healing assays.

Our results revealed that TTI-101, along with its anti‐ proliferative properties, exhibited substantial anti-migratory activity in HeLa cells in a concentration-dependent fashion. The percentage of wound healing significantly decreased at TTI-101 concentrations of 20 μM and 40 μM, illustrating its efficacy in curtailing migration compared to the control group.

To gain insights into the mechanisms underlying the antiproliferative activity of TTI-101, we performed apoptosis analysis and cell cycle distribution assays. Our results indicated that TTI-101 treatment significantly increased the apoptosis rate in HeLa cells compared to the control group.

In line with these findings, there was a noteworthy decrease in Bcl-2 expression at both the gene and protein levels, accompanied by an increase in Bax and Caspase-3 levels. These genes are pivotal in apoptosis, with Bax and Caspase-3 promoting cell death, while Bcl-2 acts as an inhibitor of apoptosis [54, 55].

In the present project, we verified that the cell cycle in TTI-101-treated HeLa cells was arrested at the G2/M phase (Fig. 4B). Proper cell cycle regulation is pivotal in preventing uncontrolled cell growth and proliferation [56]. The cell cycle checkpoint serves as a crucial regulatory point in controlling the progression of the cell cycle. It acts as a gatekeeper, allowing the cell to proceed to the next phase of the cycle only after successfully passing the checkpoint examination. Disrupted control of the cell cycle is a significant factor in the development of tumors. One significant regulatory factor in governing the checkpoint process is the CDK1/cyclin B complex [57]. At the end of the S phase, the activated CDK1/cyclin B complex triggers entry into mitosis [58]. Our findings indicate that following TTI-101 treatment, the expression levels of STAT3 and its target genes, including survivin, CDK1, cyclin B1, and c-Myc were notably reduced in HeLa cells.

Survivin is typically undetectable in most adult tissues, but its expression is significantly upregulated in a wide range of cancers [59]. It is cell cycle-regulated, with peak expression during the G2/M phase and lower levels during the G1 phase [60]. c-Myc is another STAT3 downstream gene that plays a crucial role in regulating CDK1/cyclin B1-dependent G2/M cell cycle progression [61, 62].

We speculated that inducing cell cycle arrest by targeting cell checkpoints and the STAT3 pathway may be another mechanism through which TTI-101 combats cervical cancer cells.

Finally, to validate the relevance of our findings in a physiologically relevant setting, we conducted in vivo experiments using a well‐established subcutaneous cervical cancer xenograft models. It is worth noting that the concentrations of TTI-101 utilized in this study were selected based on prior research [24, 49, 63]. For instance, Kong et al. reported that TTI-101 (100 mg/kg) can augment the anti-metastasis effect of DAC in mouse pancreatic cancer models by inhibiting EMT [49]. In addition, Bharadwaj et al. showed that there were no observed clinical, anatomical, histological, or laboratory abnormalities in rats when they administered daily doses of up to 200 mg/kg/day for a duration of 28 days [24]. Thus, in present study, TTI-101 was tested on cervical cancer model in 100 mg/kg concentration. Consistent with previous studies, our results demonstrated that TTI-101 noticeably hindered tumor growth compared to the vehicle control group, indicating its potent antitumor efficacy. Notably, the treatment with TTI-101 did not result in significant changes in body weight, showing its potential as a well-tolerated therapeutic option for cervical cancer.

We also found that STAT3 knockout significantly inhibited the tumor growth in xenograph animal model. These observations highlight the potential efficacy of targeting STAT3 as a therapeutic strategy for cervical tumors, warranting further investigation into the underlying mechanisms of STAT3 inhibition in tumor suppression.

The docking results, corroborating the direct binding of TTI-101 to STAT3, align with our experimental findings, highlighting the anticancer effects of TTI-101 in cervical cancer through STAT3/c-Myc signaling inhibition (Fig. 6). The results emphasized that TTI-101 has the ability to modulate multiple intracellular pathways involved in cellular proliferation, apoptosis, and migration. This underscores TTI-101 as a promising therapeutic candidate and emphasizes the significance of targeting STAT3 in cervical cancer treatment.

Schematic illustration of the anti-tumor activity of TTI-101 on human cervical cancer cells by inhibiting the activity of STAT3/c-Myc signaling. Persistent STAT3 activation resulting from the activation of upstream growth factor receptors (e.g., IL-6 receptor), leads to abnormal proliferation and tumorigenesis in these cells. TTI-101 inhibits cell proliferation and promotes apoptosis through STAT3 downstream target genes in human CC cells

The lack of direct comparison data between cancer and normal cells is a limitation of the current study. Ideally, future studies should include the evaluation of TTI-101's effects on normal cervical epithelial cells to further demonstrate the therapeutic window and selectivity of this compound. However, we can discuss the potential selectivity of TTI-101 based on the in vivo xenograft experiments performed. In these studies, we did not observe any significant adverse effects on the body weight of the mice treated with TTI-101. This suggests that the compound may have a preferential cytotoxic effect on the tumor cells without causing major systemic toxicity.