Abstract
Background/Aim: The effect of G-protein-coupled estrogen receptor 1 (GPER1) on tumors depends on tumor entity, with its expression level influencing signal transduction and function. Recent research suggests that GPER1 promotes tumor suppression in cervical carcinoma (CC). In contrast, silencing GPER1 increases expression of serpin family E member 1 (SERPINE1) and its protein, plasminogen activator inhibitor-1 (PAI-1), and promotes tumor progression, raising the question of whether PAI-1 might be a suitable target for the treatment of CC. To explore this, we examined the impact of PAI-1 inhibition using Tiplaxtinin (PAI-039, TPX).
Materials and Methods: The effects of TPX treatment on viability, colony formation, migration, and invasion of SiHa cervical squamous cell carcinoma (CSCC) and HeLa cervical adenocarcinoma (CAC) cells were assessed using AlamarBlue, colony formation, gap closure, and Boyden chamber assays, respectively. Apoptosis was examined using the Annexin/PI assay, while the cell cycle was analyzed in more detail using the PI assay.
Results: With increasing TPX concentration, viability and colony formation of SiHa and HeLa cells decreased significantly. Cell migration was strongly reduced under PAI-1 inhibitor treatment, while invasion showed a slight decline. Apoptosis and cell cycle were only minimally affected by TPX.
Conclusion: PAI-1 inhibitor TPX showed a strong inhibitory effect on both SiHa CSCC and HeLa CAC cells, significantly reducing their viability, colony formation, and migratory capacity. The observed effects suggest that TPX could potentially be used to target and hinder the growth and spread of both CSCC and CAC cells.
- Cervical carcinoma (CC)
- G-protein-coupled estrogen receptor 1 (GPER1)
- serpin family E member 1 (SERPINE1)
- plasminogen activator inhibitor-1 (PAI-1)
- inhibitor
- Tiplaxtinin
Introduction
Cervical carcinoma (CC), with about 600,000 new cases and 340,000 deaths annually, is the fourth most common cancer in women and the most frequently diagnosed gynecological tumor (1, 2). CC mainly arises from the squamous epithelium (90%), with adenocarcinomas accounting for 5%. These tumors often develop in the transformation zone between the endometrium and squamous epithelium, occasionally originating from the mucosal cells of the cervix. The process is usually triggered by premalignant changes, such as cervical intraepithelial neoplasia (CIN), associated with high-risk HPV types 16 and 18 (3, 4).
The G-protein-coupled estrogen receptor 1 (GPER1) plays a critical role in the progression, migration, and therapy resistance of various tumor entities (5). However, the overall picture remains inconsistent. GPER1 also appears to be involved in tumor suppression. Current research provides a range of answers to the question of whether GPER1 is tumor-promoting or tumor-suppressive. In CC, GPER1 seems to play a tumor-suppressive role (6).
Analysis of various pro-metastatic factors has shown that GPER1 knockdown leads to a significantly increased gene expression of the oncogene serpin family E member 1 (SERPINE1) and its corresponding protein, plasminogen activator inhibitor-1 (PAI-1). PAI-1, a serine protease inhibitor, is a key regulator of the plasminogen activation system (7). Additionally, SERPINE1 induces angiogenesis and is involved in cell motility and extracellular matrix homeostasis. PAI-1 protects tumor cells from apoptosis and promotes cell survival. It also has pro-angiogenic effects.
In CC, a correlation has been observed between SERPINE1 and poorer overall or disease-free survival (8). SERPINE1 is expressed in many tumors and is associated with decreased overall survival, as seen in breast cancer. Enhanced tumor metastasis and a poorer response to chemotherapy in breast cancer also appear to be linked to SERPINE1 (7).
SERPINE1/PAI-1 belong to the serine proteinase inhibitors and are primarily known to regulate fibrinolysis (9). PAI-1 is ubiquitous in the body and is found in many cell types, such as megakaryocytes, platelets, hepatocytes, adipocytes, smooth muscle cells and endothelial cells. In the bloodstream, PAI-1 is found in the plasma and in platelet α-granules (9). The inhibition of PAI-1 activity also seems to attenuate lung fibrosis (10).
A connection has been established between PAI-1 and various diseases, including cardiovascular diseases (CVD), metabolic disorders, aging, cancer, tissue fibrosis, inflammation, and neurodegenerative diseases (11, 12). Analysis of various pro-metastatic factors revealed that GPER1 knockdown leads to a significantly increased gene expression of the oncogene SERPINE1 and its corresponding protein, plasminogen activator inhibitor-1 (PAI-1) (13).
Since the mechanisms of GPER1, which is associated with PAI-1, appear to be quite different in cervical adenocarcinoma (CAC) and in the more common cervical squamous cell carcinoma (CSCC), it should be examined whether inhibition of PAI-1 shows comparable inhibitory effects in CAC and CSCC cells. This study investigates the effect of treatment with the PAI-1 inhibitor Tiplaxtinin (PAI-039, TPX) on the CAC cell line HeLa and the CSCC cell line SiHa. Recent findings from our group suggest that GPER1 plays a role in tumor suppression in CC. It has been observed that a knockdown of GPER1 leads to increased expression of the oncogene SERPINE1 and its associated protein PAI-1. This raises the question of whether PAI-1 could be a suitable target for the treatment of both CC subtypes.
Materials and Methods
Cell culture. The human cervical carcinoma (CC) cell lines HeLa (CAC; HPV18+) and SiHa (CSCC; HPV16+) were obtained from the American Type Cell Collection (ATCC, Manassas, VA, USA) and cultured in minimum essential medium (MEM; L0416-500, Biowest, Nuaillé, France) supplemented with 10% fetal bovine serum (FBS; S181B-500, Biochrom, Berlin, Germany) and 1% Penicillin/Streptomycin (P/S; L0022-100, Biowest). To retain their identity, purchased cells were expanded and aliquots were frozen in liquid nitrogen. A new frozen stock was used every half year and mycoplasma testing of cultured cell lines was performed routinely using the polymerase chain reaction (PCR) Mycoplasma Test Kit I/C (D101-02, Vazyme, Düsseldorf, Germany). All cells were cultured in a humidified atmosphere with 5% CO2 at 37°C.
Treatments. The cells were treated with the PAI-1 inhibitor Tiplaxtinin (PAI-039, TPX) (MedChemExpress, Monmouth Junction, NJ, USA) dissolved in 2% v/v ethanol (Th. Geyer, Höxter, Germany), MEM with 2% v/v ethanol (ethanol control), or plain MEM (control). Initially, the chosen concentrations were 20 μM, 40 μM, 60 μM, 80 μM, and 100 μM. Once the E50 value of approximately 50 μM had been determined, all further experiments were carried out at this concentration.
Viability. For cell viability analysis via the Resazurin assay, 10,000 cells per ml were seeded in 96-well plates (Corning, Kennebunk, ME, USA) using Dulbecco’s Minimum Essential Medium without phenol red (DMEM; Thermo Fisher Scientific, Waltham, MA, USA). Treatments with TPX concentration series were initiated 24 h later. After a 72-h incubation, 20 μl of Resazurin (Thermo Fisher Scientific) was added to each well. Ten hours post-incubation, the fluorescence reduction to Resorufin was measured at wavelengths of 570 nm and 630 nm using a microplate reader (BioTek Instruments, Bad Friedrichshall, Germany). The data were analyzed with GEN5 1.08 software (BioTek Instruments).
Colony formation. To assess colony formation, 1,000 cells per well were seeded in a 6-well plate (Corning). Following a 24-h attachment period, cells were treated with TPX concentration series and incubated for 72 h. Once visible colonies had formed, plates were fixed and then stained with crystal violet (Sigma Aldrich, St. Louis, MO, USA). The plates were scanned using Epson Scan 2 software (Epson, Suwa, Japan), and analyzed for colony size and number using ImageJ 1.52a software (Wayne Rasband, National Institutes of Health, Bethesda, MD, USA).
Migration. For gap closure assay, 100,000 cells per well (equivalent to 1,000,000 cells/ml) were plated in a 24-well plate (Corning) using a 2-well culture-insert (ibidi, Gräfelfing, Germany) to create a gap. After 24 h of incubation, the cells were washed with Dulbecco’s Phosphate Buffered Saline (DPBS; Pan Biotech, Aidenbach, Germany) and treated with 50 μM TPX. Images of the gap closure process were captured at every 8 h till the gaps were closed using uEye Cockpit 2.0 software (IDS Imaging Development Systems, Obersulm, Germany). The closure rate was analyzed using ImageJ 1.52a software (Wayne Rasband).
Invasion. Matrigel (Corning) was mixed with DMEM (Thermo Fisher Scientific) in equal parts (150 μl each) and distributed on the bottom of inserts in a 24-well plate, incubated for 1 h. Cells (25,000 cells/ml) were resuspended in medium and added to the inserts, which were incubated for 24 h. To attract carcinoma cells, MG63 cells (ATCC) (33,33 cells/ml) were seeded in a second 24-well plate. After 24 h, inserts were transferred into the wells with attractor cells. After 96 h of co-culture, the inserts were stained using PBS (PAN-Biotech), dH2O, methanol (Th. Geyer), hematoxylin (Merck, Darmstadt, Germany), and tap water. The stained inserts were transferred to microscope slides, covered with Aquatex (Merck), and sealed with a coverslip. After drying, the slides were imaged and analyzed with Cell Sens Dimension software (Olympus, Tokyo, Japan) and ImageJ software (Wayne Rasband).
Apoptosis. SiHa and HeLa cells (200,000 cells/ml) were seeded into a 6-well plate (Corning) and treated after 24 h with BSA (Sigma Aldrich) in MEM, ethanol solution, or TPX. On day 5, the cells were prepared for FACS analysis by transferring the medium into Eppendorf tubes (Greiner Holdings, Kremsmünster, Austria), centrifuging, and adding trypsin (Thermo Fisher Scientific) to detach the cells. After counting, the samples were centrifuged again, and Binding Buffer, Annexin (Southern Biotech, Birmingham, AL, USA), and Propidium Iodide (Sigma Aldrich) were added. The samples were incubated in the dark, filtered, and transferred into polystyrene tubes for analysis on the Cytoflex S flow cytometer (Beckman Coulter, Brea, CA, USA). The data was processed using FlowJo software v10.10 (BD Bioscience, Bedford, MA, USA).
Cell cycle. Cells were seeded in culture dishes to achieve a count of 0.5 to 1×106 cells per condition for PI staining. On day one, 2 ml of SiHa and HeLa cells were added to a 6-well plate (Corning Life Sciences). After 24 h, the medium was removed, and cells were treated with BSA (Sigma Aldrich), ethanol (Th. Geyer Ingredients), or TPX solution. The media were collected to capture dead cells, and the remaining cells were detached with trypsin (GibcoTM, Thermo Fisher Scientific). Cell counts were adjusted, and the cells were fixed in 70% ethanol. For RNA digestion, FACS buffer (BD Bioscience) with RNase (Thermo Fisher Scientific) was used. After washing and resuspending, Propidium Iodide (Sigma Aldrich) was added for staining.
The cells were analyzed with the Cytoflex SX flow cytometer (Beckman Coulter), measuring 10,000 to 30,000 events per sample. Data were processed using FlowJo software (BD Bioscience).
Statistical analysis. All experiments were conducted with at least three biological replicates and three technical replicates. The data are presented as mean±SEM. Statistical analyses, including one-way ANOVA, two-way ANOVA, and unpaired t-tests, were carried out using GraphPad Prism (v. 8.0.1, GraphPad Software Inc., San Diego, CA, USA). Following one-way ANOVA, Dunnett’s or Tukey’s multiple comparisons tests were applied. Unpaired, two-tailed parametric t-tests were performed, assuming equal standard deviations between the groups being compared. Statistical significance was determined for p-values <0.05.
Results
Impact of Tiplaxtinin (TPX) treatment on the viability of HeLa and SiHa CC cells. SiHa: At the tested concentrations of 20 μM (105.3±1.873%, p=0.9176, n=8) and 40 μM (103.6±2,784%, p=0.9868, n=6), a slight increase in cell viability was observed. Starting at a concentration of 60 μM (89.55±5.575%, p=0.4943, n=6), viability decreased, although no significant differences were noted compared to the ethanol control. However, a significant reduction in viability was observed at a TPX concentration of 80 μM (77.56±7,493%, p=0.0306, n=5). The lowest viability was recorded at a TPX concentration of 100 μM, with a value of 31.98±5,174% (Figure 1A). This value demonstrated high significance in the one-way ANOVA test (p<0.0001, n=6), indicating a pronounced effect of TPX on cell viability at this concentration. HeLa: In treated HeLa cells, a progressive decline in viability was observed with increasing TPX concentrations. A significant reduction in viability was already evident at a concentration of 40 μM (76.82±4,975%, p=0.0201, n=5). An even more pronounced significance was noted at 60 μM (60.72±6,115%, p=0.0001, n=5). The most substantial and highly significant reductions in viability compared to the ethanol control were observed at 80 μM (49.12±4.960%, p<0.0001, n=5) and 100 μM (27.65±4.716%, p<0.0001, n=6) (Figure 1B).
Effects of Tiplaxtinin (TPX) treatment on viability (A, B) and colony formation (C, D) of cervical cancer (CC) cells. Relative viability of SiHa (A) and HeLa (B) CC cells after treatment with increasing concentrations of TPX (20-100 μM) over three days versus ethanol control (=100%). Comparison of relative colony formation of SiHa (C) and HeLa (D) CC cells treated with increasing concentrations of TPX (20-100 μM) versus ethanol control (=100%). One-way ANOVA; *p<0.05; ***p<0.001; ****p<0.0001.
Impact of Tiplaxtinin (TPX) treatment on colony formation of HeLa and SiHa CC cells. SiHa: At TPX concentrations of 20 μM (96.43±3,606%, n=4), 40 μM (91.13±6,482%, n=4), and 60 μM (90.2±2,603%, n=5), a slight but not statistically significant reduction in colony formation of SiHa cells was observed. In contrast, a significant reduction in colony formation was evident at a concentration of 80 μM (49.17±2,450%, p<0.0001, n=5). The highest tested TPX concentration of 100 μM resulted in a substantial and statistically significant reduction in colony formation, reaching 16.02±2,071% (p<0.0001, n=5) (Figure 1C). HeLa: At a TPX concentration of 20 μM (100.4±4,494%, n=4), a marginal increase in the relative colony formation rate was observed. A non-significant reduction in colony formation was detected at a concentration of 40 μM (99.25±7.022%, p=0.9999, n=4). A significant decrease in colony formation was evident at a TPX concentration of 60 μM (70.53±11.977%, p=0.0095, n=5). The highest levels of significance were observed at concentrations of 80 μM (47.57±2.612%, p=0.0002, n=5) and 100 μM (14.62±2.693%, p<0.0001, n=5) (Figure 1D).
Impact of Tiplaxtinin (TPX) treatment on migration of HeLa and SiHa CC cells. SiHa: The closure of gaps was significantly slowed under treatment with TPX. Statistical analysis was performed using two-way ANOVA, comparing the means of the treatments to the ethanol control. All gaps were normalized at time 0 h. Significant differences became apparent at 24 h. At this time point, the gap area under ethanol control was 29.60% smaller than under TPX treatment (95.12±1.492%, p=0.0273, n=3). More pronounced differences emerged at 32 h, with the gap area under TPX treatment (93.22±1.483%, p =0.0274, n=3) being 29.60% larger than the ethanol control. By 40 h, the gap area under TPX treatment measured 93.32±3.20 % (p=0.0047, n=3), 46.43% larger than the control. At 48 h, TPX treatment (92.02±3.074%, p =0.0044, n=3) again showed a significant difference, with a 40.54% larger gap area compared to ethanol control (Figure 2A and C). HeLa: TPX treatment was observed to reduce the migration of HeLa cells. Statistical analysis was performed using two-way ANOVA, comparing the mean values of the treatment group to the ethanol control. The gap areas of all controls and treatments were normalized to their respective values at 0 h. Over time, the gap area under TPX treatment remained larger compared to the ethanol control at several time points: 16 h (EtOH: 76.64±6.289% vs. TPX: 85.14±5.831%, n=4), 32 h (EtOH: 48.67±11.846% vs. TPX: 74.25±11.603%, n=4), 40 h (EtOH: 39.26±12.013% vs. TPX: 68.16±13.431%, n=3), and 48 h (EtOH: 30.07±12.627% vs. TPX: 58.16±13.431%, n=3). However, these differences were not statistically significant compared to the control (Figure 2B and D).
Effects of Tiplaxtinin (TPX) treatment on migration of CC cells. Comparison of relative size of the gap of SiHa (A) and HeLa (B) CC cells after treatment with 50 μM TPX over 48 h versus ethanol control (=100%). Representative bright-field images compare the gap sizes of SiHa (C) and HeLa (D) CC cells treated with 50 μM TPX versus ethanol control over 48 h. Images were taken every 8 h. Two-way ANOVA; *p<0.05; **p<0.01.
Impact of Tiplaxtinin (TPX) treatment on invasion of HeLa and SiHa CC cells. SiHa: Under TPX treatment, a reduction in cell invasion by 13.14±4.110% was observed compared to the control group (86.86±4.110%, p=0.0127, n=5). The unpaired t-test confirmed the significance of the results (Figure 3A). HeLa: A reduction in cell invasion with TPX treatment by 9761±1.543% was observed compared to the ethanol control (90.24±1.543%, p=0.0002, n=5), indicating a potential inhibitory effect of TPX on cell motility. The results were confirmed as significant using the unpaired t-test (Figure 3B).
Effects of Tiplaxtinin (TPX) treatment on invasion of CC cells. Comparison of relative number of migrated SiHa (A) and HeLa (B) CC cells treated with 50 μM TPX versus ethanol control (=100%). Unpaired t-test; *p<0.05; ***p<0.001.
Impact of Tiplaxtinin (TPX) treatment on apoptosis of HeLa and SiHa CC cells. SiHa: Using FACS analysis, the different apoptosis time points in SiHa cells were examined. The number of viable cells was lower in the EtOH control (80.14±4.487%, p=0.4615, n=5) compared to the TPX treatment group (84.70±1.511%, p=0.4615, n=5). A significant difference was observed in early apoptosis, with a higher percentage in the EtOH control (10.46±2.425%, p=0.0314, n=5) compared to the TPX-treated cells (1.162±0.216%, p=0.0314, n=5). However, no significant effect was detected in late apoptosis, as indicated by the PI-positive phase (EtOH: 8.496±1.974%, p =0.9175, n=5 vs. TPX: 6.538±1.217%, p=0.9175, n=5) (Figure 4A and C). HeLa: The number of living cells was comparable between the EtOH control (73.44±4.4779%, p =0.9952, n=5) and the TPX treatment (74.54±5.860%, p =0.9952, n=5). Similarly, no significant differences in cell count were observed during early apoptosis (EtOH: 4.170±1.849%, n=5 vs. TPX: 4.468±1.929%, n=5). In late apoptosis, a slight reduction in the number of treated cells was observed (EtOH: 14.46±2.337%, p=0.9978, n=5 vs. TPX: 13.60±2.997%, p=0.9978, n=5); however, also this difference was not significant (Figure 4B and D).
Effects of Tiplaxtinin (TPX) treatment on apoptosis of CC cells. Comparison of the cell count in SiHa (A) and HeLa (B) cells treated with 50 μM TPX versus the ethanol control, depicting live cells, early apoptosis, and late apoptosis. Analysis of flow cytometry results of different passages SiHa (C) (1: P21,2: P60, 3: P63, 4: P64) and HeLa (D) (1: P55, 2: P56, 3: P77, 4: P78) CC cells treated with TPX versus ethanol control. Q1 (upper left quadrant) shows the cells in early apoptosis, which are both Annexin and Propidium iodine (PI) positive. Q2 (upper right quadrant) represents late apoptosis, where only the PI-positive cells are visible. The live cells are located in Q4 (lower left quadrant). Two-way ANOVA; *p<0.05.
Impact of Tiplaxtinin (TPX) treatment on cell cycle of HeLa and SiHa CC cells. SiHa: The FACS analysis allowed for the quantification of the different cell cycle phases. In the G1 phase, the cell count was almost identical between the EtOH control (69.70±0.819%, p=0.9010, n=3) and the TPX treatment (68.80±1.308%, p=0.9010, n=3). In the S phase, a slight reduction in cell number was observed with TPX (EtOH: 11,94±1.173%, p=0.5374, n=3 vs. TPX: 10.13±0.185%, p=0.5374, n=3). In contrast, a moderate increase in cell number was noted in the G2/M phase with TPX treatment (EtOH: 12.77±1.354%, p=0.1247, n=3 vs. TPX: 15.97±0.636%, p=0.1247, n=3). None of the results were found to be significant (Figure 5A and C). HeLa: The cell counts in the different cell cycle phases differed only slightly between the EtOH control and the TPX treatment. A minimal increase in cell number was also observed in the G2/M phase (EtOH: 10.52±1.614%, p =0.9853, n=3 vs. TPX: 11.60±1.190%, p=0.9853, n=3) (Figure 5B and D).
Effects of Tiplaxtinin (TPX) treatment on cell cycle of CC cells. Comparison of the cell count in SiHa (A) and HeLa (B) cells treated with 50 μM TPX versus the ethanol control, depicting the cell cycle in G1, S and G2/M phases. Analysis of flow cytometry results of different passages SiHa (C) (1: P21, 2: P63, 3: P64) and HeLa (D) (1: P78, 2: P77, 3: P65) CC cells treated with TPX versus ethanol control. Two-way ANOVA.
Discussion
Each year, several hundred thousand women worldwide are diagnosed with CC, and more than half succumb to the disease’s progression (14). These alarming statistics underscore the urgent need to expand the repertoire of available therapeutic options. This study aimed to investigate the effects of the PAI-1 inhibitor TPX on the HeLa and SiHa cell lines. Recent findings suggest that GPER1 plays a role in tumor suppression in CC. A GPER1 knockdown has been shown to increase the expression of the oncogene SERPINE1 and its corresponding protein, PAI-1. While the role of GPER1 as a tumor suppressor or promoter remains debated, mounting evidence suggests a tumor-suppressive function of GPER1 in CC (6). However, the role of GPER1 may also be different in the CC subtypes CAC and CSCC. Over-expression of GPER1 enhanced tumorigenic properties in CSCC cells, but showed tumor suppressive effects in CAC cells (15, 16). To evaluate the effects of TPX on CC cells, this study analyzed viability, colony formation, migration and invasion. Finally, apoptosis and the cell cycle were also investigated.
Increasing concentrations of TPX significantly reduced the viability of both HeLa CAC and SiHa CSCC cells. HeLa cells exhibited a marked reduction in viability starting at 40 μM TPX, suggesting heightened sensitivity to the inhibitor. In contrast, SiHa cells only showed a significant viability reduction at 80 μM, indicating a comparatively lower sensitivity. This differential response may be due to distinct genetic and molecular characteristics, particularly in the expression of signaling proteins involved in cell colony formation and survival. Further studies are necessary to elucidate the mechanisms through which TPX affects cell viability and to determine whether these differences are due to varying PAI-1 activity or other molecular factors driving tumor progression. Colony formation decreased significantly with rising TPX concentrations. In HeLa cells, a substantial reduction was observed at 60 μM TPX, while SiHa cells exhibited a similar response at 80 μM, demonstrating differing sensitivities between the two cell lines. Gomes-Giacoia et al. showed that the reduction of PAI-1 could inhibit the viability and proliferation of HeLa cells (17). TPX exhibited a pronounced inhibitory effect on the migration of SiHa cells. After 24 h, the gap size in TPX-treated cells was approximately 30% larger compared to ethanol-treated controls, a significant difference that persisted after 48 h, with the gap size being approximately 40% larger. This strongly suggests that TPX effectively impairs SiHa cell migration. While HeLa cells did not exhibit statistically significant differences, the gap sizes in treated cells were noticeably larger than in ethanol-treated controls, indicating a potential inhibitory effect. However, within the 24- to 48-h window, some observed effects may also be attributable to cell proliferation rather than migration alone. TPX treatment showed no significant reduction in cell invasion for either HeLa or SiHa cells, suggesting minimal effects on invasion. This finding aligns with prior research indicating that invasion assays can sometimes lack precision (18).
A challenge in the therapeutic application of PAI-1 inhibitors lies in their potential systemic effects on blood coagulation. By inhibiting PAI-1’s regulatory role in plasmin-mediated fibrinolysis, small molecule inhibitors might disrupt hemostasis, leading to unexpected bleeding events. While studies in non-human primates have shown no adverse effects on systemic coagulation after prolonged PAI-1 inhibitor administration (19), this aspect warrants further investigation.
The analysis of the effect of TPX on apoptosis of CC cells revealed that the number of SiHa cells was significantly reduced in early apoptosis and slightly reduced in late apoptosis. The significant reduction in cell count in early apoptosis of SiHa cells after TPX treatment could indicate an accelerated progression of cell death (20). It is also possible that the reduction in cell count in late apoptosis suggests that TPX promotes primary cell death through necrosis instead of apoptosis (21). In contrast, HeLa cells showed a slight reduction in cell count during early and late apoptosis. Previous studies have shown that treatment with TPX enhances or induces programmed cell death in HeLa cells (17). The cell cycle analysis regarding the influence of TPX revealed a slight, non-significant increase in the number of cells in the G2/M phase in both SiHa and HeLa cells. Such cell cycle arrest in the G2/M phase could indicate a potential inhibition of tumor cells or an enhanced induction of apoptosis (22).
Conclusion
This study underscores the therapeutic potential of the PAI-1 inhibitor TPX, particularly in reducing cell viability, colony formation, and migration in both CSCC and CAC cells. However, its limited effect on cell invasion and the necessity to evaluate long-term systemic risks highlight the complexity of developing PAI-1 inhibitors as anticancer agents (9).
Future experimental work should focus on several key aspects to further explore and enhance the utility of TPX. First, detailed investigations into the molecular pathways influenced by TPX are necessary. This includes its interactions with key signaling proteins involved in tumor progression, such as those regulating apoptosis, oxidative stress, and epithelial mesenchymal transition (EMT). Understanding these pathways could provide insight into the mechanistic basis of TPX’s observed effects.
Another promising avenue involves exploring combination therapies. Evaluating the efficacy of TPX in conjunction with existing chemotherapeutic agents or targeted therapies could reveal potential synergistic effects. Such combinations might amplify therapeutic outcomes while reducing systemic risks by lowering the required dosage of each agent. To generalize the results, the effects of TPX need to be tested on a broader range of CC cell lines, including those with different HPV status and genetic profiles. This step would help to identify any cell line-specific responses and refine the scope of its applicability.
Acknowledgements
The Authors thank Sonja Blume and Matthias Läsche for the excellent technical assistance.
Footnotes
Authors’ Contributions
Conceptualization, C.G.; investigation, S.W.; writing - original draft preparation, S.W. and C.G.; writing - review and editing, J.G.; project administration, C.G. All Authors have read and agreed to the published version of the manuscript.
Conflicts of Interest
The Authors declare no conflicts of interest in relation to this study.
Funding
This research received no external funding.
Artificial Intelligence (AI) Disclosure
No artificial intelligence (AI) tools, including large language models or machine learning software, were used in the preparation, analysis, or presentation of this manuscript.
- Received April 8, 2025.
- Revision received April 13, 2025.
- Accepted April 14, 2025.
- Copyright © 2025 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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