Enhanced protein carbonylation is associated with prolonged fibrin clot lysis in patients with advanced lung cancer: impact on long-term mortality

Introduction

Protein carbonylation (PC) is a marker of irreversible and irreparable oxidative damage to proteins, which is commonly used in clinical studies (1,2). The oxidative damage to proteins has been reported in the literature in neurodegenerative diseases (3), diabetes (4), stroke (5), obesity (6), aging (7), venous thromboembolism (VTE) (8) and malignancy. The role of increased generation of free radicals in cancer progression is poorly understood (9). Oxidative stress (OS) has been reported to be involved in cancer initiation by inducing genome instability, altering gene expression, inducing resistance to apoptosis, and metastatic tumor invasion (10,11). PC accumulation can occur during both the development of cancer and its progression (12,13). It has been demonstrated that PC can be rapidly induced through cytotoxic drug treatment, potentially inhibiting glycolytic proteins (14). This suggests that carbonylation may contribute to drug resistance in cancer cells (14).

Lung cancer (LC) is the second most commonly diagnosed cancer in the world and is the leading cause of cancer-related deaths, accounting for approximately 1.8 million deaths annually worldwide. Approximately 3% of individuals with diagnosed LC experience VTE within 2 years following their diagnosis (15). Within the first years, the cumulative VTE incidence is reported to be 10.2% among patients with small cell lung carcinoma (SCLC) (16), while it rises to about 22% in those with non-small cell lung carcinoma (NSCLC) (17,18). Dysregulated balance between blood coagulation and fibrinolysis could contribute to thromboembolic events in LC (19,20). Enhanced PC can affect fibrinolysis by changing protein functions involved in this process, stability, and the overall hemostatic balance, which leads to reduced clot susceptibility to enzymatic degradation (8,21,22).

To our knowledge, there have been no studies on the role of PC in inoperable LC patients in the context of fibrin clot formation, structure, and lysis, and its potential value in long-term prognosis. The aim of the present study was to investigate PC in advanced inoperable LC to address the above issues. We present this article in accordance with the STROBE reporting checklist (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-625/rc).

Methods

Study population

In a prospective study conducted in 2014 to assess the characteristics of fibrin clots, we evaluated 147 consecutive white patients with confirmed advanced LC. All individuals were recruited at the John Paul II Hospital, Krakow, Poland. The study population used in the current secondary analysis has been described in detail previously (23). In brief, patients were classified into two groups: those with SCLC and those with NSCLC. The NSCLC group encompassed adenocarcinoma, squamous cell carcinoma, and not otherwise specified (NOS) carcinoma, which included cases other than adenocarcinoma, squamous cell carcinoma, large cell carcinoma, or mixed/other histological types. The American Joint Committee on Cancer 7th edition tumor-node-metastasis (TNM) staging system was used based on available clinical data (24). Initially, all patients met all the following eligibility criteria for chemotherapy: an Eastern Cooperative Oncology Group (ECOG) performance status of 0 or 1; adequate organ function [white blood cell count ≥3×10³/µL and ≤12×10³/µL, neutrophil count ≥1.5×10³/µL, platelet count ≥100×10³/µL, hemoglobin ≥9.0 g/dL, total bilirubin ≤1.5 mg/dL, aspartate aminotransferase and alanine aminotransferase ≤2.5× upper limit of the reference range; creatinine ≤1.5 mg/dL or creatinine clearance ≥60 mL/min; arterial partial pressure of oxygen (PaO₂) ≥60 mmHg]. All patients initiated standard chemotherapy tailored to the histological type of cancer and any underlying any co-morbidities (23).

The exclusion criteria were: active infections, the rate of glomerular filtration <60 mL/min, impaired thyroid function, recent thromboembolism, and current anticoagulation. However, patients receiving prophylactic treatment with low-molecular-weight heparins (LMWH) were considered eligible. Heart failure (HF) was defined by the presence of characteristic symptoms and signs, along with a left ventricular ejection fraction of ≤45%. Arterial hypertension was identified based on a documented history of high blood pressure (≥140/90 mmHg) or ongoing antihypertensive therapy. Type 2 diabetes was diagnosed based on blood glucose ≥7.0 mM at two different time points, or by the documented use of insulin or oral hypoglycemic agents. Coronary artery disease (CAD) was identified by a history of angina, previous myocardial infarction, or coronary revascularization. Ischemic stroke was diagnosed according to World Health Organization criteria. Chronic obstructive pulmonary disease was characterized by irreversible expiratory airflow limitation confirmed through spirometry. Current smoking status was assigned to those who have smoked at least 100 cigarettes and currently smoke one or more cigarettes per day, while a former smoker was referred to an individual who has smoked a minimum of 100 cigarettes in their lifetime but has abstained from smoking for at least the past month. The study was approved by the Local Ethics Committee (Okregowa Izba Lekarska in Krakow; No. 31/KBL/OIL/2013), and all patients gave informed consent. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Laboratory investigations

Blood samples were collected after overnight fasting from the antecubital vein, applying minimal stasis. Blood was drawn at enrollment, prior to the initiation of chemotherapy, and again before the 3^rd or 4^th chemotherapy cycle. For patients receiving LMWH, blood samples were collected >12 hours after the last dose. Citrate plasma (vol/vol, 9:1 of 3.2% sodium citrate) was used to determine fibrin clot characteristics. Blood cell count, glucose, and creatinine levels were measured using standard laboratory methods. Fibrinogen was assessed using the Clauss method. Plasma D-dimer levels were determined with the Innovance assay (Siemens, Marburg, Germany).

Carbonylation measurement

PC in plasma was evaluated by measuring carbonyl content using 2,4-dinitrophenylhydrazine, following the assay by Becatti et al. (25). In brief, plasma (100 µL) was mixed with dinitrophenylhydrazine (400 µL) and precipitated with trichloroacetic acid. The resulting pellet was well washed with an ethanol/ethyl acetate mixture (1:1) and then resuspended in guanidine hydrochloride (500 µL). PC content was measured at 370 nm using a spectrophotometer (Tecan, Sunrise) and expressed as nmol/mL per 1 mg of protein. The reference range for healthy subjects, established in our laboratory, was 0.54 to 2.03 nmol/mg (5).

Fibrin clot characteristics

Plasma fibrin clot properties were assessed in duplicate by technicians who were blinded to the sample origin (intra-assay and inter-assay coefficients of variation, below 7%).

Clot permeability

A permeation coefficient (Darcy’s constant; K_s), indicating the pore size within, was assessed using the pressure-driven system, as previously (26). Briefly, CaCl₂ (20 mM) and human thrombin (1 U/mL; Sigma-Aldrich, St. Louis, MO, USA) were mixed with citrated plasma (1:1). The tubes with the clots were attached to a reservoir filled with Tris-buffered saline (TBS; 0.01 M Tris, 0.1 M NaCl, pH 7.4), and the flow through the gel was measured. K_s was calculated based on the formula: QxLxη/txAxΔp, where Q represents the flow rate over time t, L is the length of the fibrin gel, η is the liquid viscosity (in poise), t is the percolation time, A is the cross-sectional area (in cm²), and Δp is the differential pressure (in dyne/cm²).

Turbidity measurements

Plasma was added to the activation mixture (2:1), containing TBS, human thrombin (0.6 U/mL; Sigma-Aldrich) and CaCl₂ (50 mM). Absorbance was read in a spectrophotometer (405 nm, Perkin-Elmer Lambda 4B, Molecular Devices, San Jose, CA, USA). The lag phase of the turbidity curve, reflecting the time needed for initial protofibril formation, and the maximum absorbance at the plateau phase (ΔAb_max), reflecting fibrin density, were noted (26).

Clot lysis assay

Clot lysis time (CLT) was determined as the time from the midpoint of the clear-to-maximum-turbid transition to the midpoint of the maximum-turbid-to-clear transition, as previously (26). Briefly, tissue factor (TF) at a final concentration of 0.6 pM (Innovin, Dade Behring, Deerfield, IL, USA), CaCl₂ at a final concentration of 17 mM), tissue plasminogen activator (tPA) at a final concentration of 30 U/mL (Boehringer Ingelheim, Ingelheim, Germany), and phospholipid vesicles at a final concentration of 10 mM in the 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer were mixed with citrated plasma in a total volume of 150 µL (26).

Follow-up

The primary endpoint was death or LC progression. Follow-up was carried out either during a clinic visit or through a phone call. We requested that patients or their families provide medical records to verify the information regarding the causes of death. Patient status was finally evaluated in January 2024 in the National Drug Program Monitoring System Registry. The definition of LC death included death due to the following International Statistical Classification of Diseases 10 (ICD-10) codes: C34.0-3, C34.8-9.

Statistical analysis

We estimated that with the sample size of 29 subjects, the study would have 90% power to detect a 10% difference in PC as compared to the mean value of PC based on the previous study (4) at a two-sided alpha level of 0.05. The Shapiro-Wilk test was used to assess the distribution of continuous variables. Continuous data were presented as mean ± standard deviation (SD) and medians and interquartile range (IQR) for data with normal and non-normal distribution, respectively. The comparison of continuous data between 4 quartiles of carbonyl was verified by the Kruskal-Wallis test. The post-hoc analysis to Kruskal-Wallis test was also performed. For comparison of continuous data between patients in stage IV and stage III the Mann-Whitney U-test was used. The differences of qualitative variables between two or more groups were assessed by the chi-square test for independence. Due to departures from normal distribution, the correlations between continuous variables were assessed using the Spearman test. The survival time in studied group was assessed by the Kaplan-Meier method. The proportional hazard Cox regression was applied to determine the impact of stage IV, age, PC, statins and smoking on survival time. The proportional hazard assumption was verified by the Schoenfeld Residuals test and the C-index [c-statistics, area under the curve (AUC)] was used to estimate the predictive accuracy of the presented models. The goodness of fit for the Cox regression models was assessed by the Grønnesby and Borgan test and the deviance residuals. The Cox regression results were presented as hazard ratios (HRs) with 95% confidence intervals (CIs).

A P value <0.05 was considered statistically significant for two-sided tests. All statistical analyses were carried out in R (27) v. 4.1.0 and Statistica 13 softwares (StatSoft Inc., Tulsa, Oklahoma, USA). The power analysis was performed in G*Power 3.1.9.4 (28).

Results

At baseline

As shown in Table 1, patients were mostly elderly and females predominated (69.4%). There were 57 patients (38.8%) diagnosed with SCLC and 90 patients (61.2%) diagnosed with NSCLC. Metastatic LC was identified in 90 patients (61.2%), while locally advanced NSCLC or SCLC with limited disease (LD) was recognized in 57 patients (38.8%).

Plasma PC levels [median 3.01 (IQR, 2.53–3.87) nmol/mg protein] were 58% higher than the upper limit of the reference range in 99% of patients (Figure 1). PC concentrations were positively associated with age (R=0.39, P<0.001) but not with sex or BMI. There was a notable association between PC levels and smoking status. Former smokers (n=54, 36.7%) exhibited higher PC levels than non-smokers [median, 3.71 (IQR, 2.78–4.04) vs. 2.87 (IQR, 2.41–3.6) nmol/mg protein, P<0.001, respectively], whereas active smokers (n=80, 54.4%) had lower PC levels than non-smokers [median, 2.77 (IQR, 2.42–3.36) vs. 3.67 (IQR, 2.76–4.04) nmol/mg protein, P=0.005 respectively]. The differences remained significant after adjustment for age. Patients with stage IV (n=90, 61.2%) had a 14.4% higher PC content compared to those with stage III (n=57) [3.26 (IQR, 2.64–3.92) vs. 2.85 (IQR, 2.4–3.62) nmol/mg protein; Figure 2]. The differences remained significant after adjustment for age. No similar associations were observed for metastasis or histologic type (all P>0.05).

Figure 1 Carbonyl concentration depending on the carbonyl quartile in lung cancer patients. The line represents the upper limit of the reference range observed in healthy controls.

Figure 2 Protein carbonyl levels in stage III and IV lung cancer patients.

Linear regression showed that active smoking only seemingly affects PC [beta =0.29, standard error (SE) =0.17], whose levels are mainly modulated by age (per year, beta =0.038, SE =0.009) and stage IV (beta =0.38, SE =0.13; R²=16.6%). Active smoking was a confounding variable, and its removal from the model did not change its goodness of fit (R²=15.6%).

No associations were observed between PC concentrations and comorbidities (Table 1).

PC levels increased with the prolongation of CLT and correlated positively with this measure (r=0.35, P<0.001) but not with the other fibrin clot variables measured, along with fibrinogen, D-dimer, or plasminogen (Table 2). CLT did not differ between LC patients with stage III or IV (P>0.05).

Follow-up

During a follow-up performed between 2014 and 2024, the primary endpoint occurred in 142 (96.6%) patients (0.43 per 100 patient-years). Almost all patients died due to progression of LC. In 12 cases, the cause of death remained unknown. Patients with PC level higher than the reference value had a greater risk of death (HR =1.85; 95% CI: 1.29–2.66, P=0.04). As expected, those with stage IV were almost twice as likely to die as those with stage III (HR =1.89; 95% CI: 1.32–2.70, P<0.001).

Baseline PC levels were inversely correlated with survival time (r=−0.18, P=0.03; Figure 3). Quartile analysis of PC concentrations showed that patients with high PC levels, defined as the top quartile (i.e., >3.87 nmol/mg protein), were older, more frequently used ACE-I or ARB and statins, exhibited longer CLT (Table 1), and had shorter survival times compared to those in the lower quartiles (Figure 4). In multivariable analysis, stage IV disease, statin therapy, and PC levels in the highest quartile were associated with shorter survival time (Table 3).

Figure 3 An association between PC and CLT content (A) and survival time (B). CLT, clot lysis time; PC, protein carbonylation.

Figure 4 Kaplan-Meier curves showing the probability of death with regard to protein carbonylation quartiles measured in patients with advanced lung cancer.

Discussion

To our knowledge, this is the first study to demonstrate that high PC levels are present in advanced LC and correlate with disease stage. Moreover, we showed that higher PC impaired fibrinolysis and, therefore, may, at least in part, exacerbate disease severity and negatively impact survival rates. Elevated PC levels have been reported in several types of cancer (29,30). A poor prognosis has been associated with cholangiocarcinoma linked to inflammation-induced PC (31). Altered antioxidant levels and higher serum PC have been observed in prostate cancer patients compared to controls (32). It has been confirmed that reactive oxygen species (ROS) are present in the neoplastic environment and play an essential role in cancer development, as demonstrated in breast and gastric cancers (33,34). Rossner et al. (35) suggested that increased PC may be associated with a higher risk of breast cancer. OS, which leads to PC generation, is known to contribute to endothelial dysfunction and increased inflammation (36). This process can result in increased fibrin production and subsequent fibrinolysis, as reflected by elevated D-dimer. As for fibrinogen, the presence of sulfur-containing amino acids makes it particularly susceptible to oxidative modification (37). Its carbonylation is associated with the altered architecture of the fibrin clot, disrupting its function (37). Proteins involved in fibrinolysis, such as plasminogen, plasmin, tPA, and plasminogen activator inhibitor (PAI-1), undergo carbonylation in OS environments, impairing fibrinolysis and potentially causing thrombus persistence (8). On the other hand, certain elements of OS process may correlate with programmed cell death 1 (PD-1) expression, inhibit tumor cell immune evasion, and improve the prognosis of lung adenocarcinoma (38). A key finding of our study is the marked increase in PC levels among LC patients and the positive correlation with CLT. This suggests a complex relationship between OS and fibrinolysis in LC patients. In recent studies, we also observed an association between higher PC levels and prolonged CLT in patients with advanced, stable CAD (39). Similar findings have been reported in patients with acute ischemic stroke (5). Hypoxic conditions are known to intensify oxidative processes (40). Notably, patients with an ECOG-1 health status in our study did not exhibit chronic respiratory failure, yet PC, linked to oxidative cellular damage, was elevated in nearly all cases. This suggests that advanced and diffused LC may predominantly drive OS.

High PC levels have been linked to poor outcomes in various cancers and increased mortality risks. For the first time, our analysis indicates that elevated PC levels in LC patients correlate with disease progression and may adversely affect survival rates. Our study demonstrated higher PC and D-dimer levels in LC groups at stages III and IV, while CLT values remained consistent across these stages. In our previous research, we found prolonged CLT in the LC group compared to controls, particularly in smokers (23). Thus, regardless of whether LC is in stage III or IV, the risk of VTE appears to be consistently high, underscoring the potential benefits of antithrombotic prophylaxis at all stages. In this context, PC could serve as a diagnostic marker and may have prognostic value in assessing LC severity. Moreover, PC has potential as a candidate for biochemical staging of LC, especially in cases where imaging studies provide ambiguous results, though further research is warranted. Elevated PC observed in LC patients in our study aligns with previous reports on advanced and metastatic cancers (20). This confirms that high OS is a pervasive condition in the LC tumor microenvironment. It is possible that OS in advanced LC is a consequence or a trigger for oncogenic mutations and cancer progression, but it is certainly associated with a prothrombotic state and protein structure modification.

Additionally, our findings demonstrate that oxidative damage to biomolecules strongly correlates with disease advancement. This underscores the role of OS in LC progression, suggesting the potential need for antioxidant-based interventions.

The current study has several limitations. First, we compared PC results in the study group to the reference value rather than to a control group adjusted for gender and age. On the other hand, selecting an appropriate control group with diseases coexisting with LC and a carcinogenic environment is practically impossible. The study included patients with advanced, inoperable LC, which is, however, a heterogeneous group due to variations in stages of progression and histopathological diagnoses. For example, stage IV includes all patients regardless of the number of distant metastases. The involvement of parenchymal organs varied significantly. Preliminary analyses, however, did not show any differences in carbonylation levels depending on the involvement of specific organs. A difference was observed between stage III and stage IV. The study group included patients with NSCLC and SCLC. These diagnoses differ significantly in tumor biology, treatment, and prognosis. Compared to NSCLC, SCLC is characterized by very rapid growth and early metastasis to distant organs such as the brain, liver, or bones, which may interfere with fibrinolysis and carbonylation processes in various ways. Moreover, SCLC is strongly associated with cigarette smoking, which is an independent factor influencing fibrinolysis, oxidative processes and carcinogenesis (41,42). Therefore, selecting homogeneous groups of LC patients appears necessary for future studies. Finally, associations reported in this study cannot directly mean the cause-and-effect relationship and further studies are highly warranted.

Conclusions

This study shows that increased PC is associated with LC stage and long-term mortality in patients with inoperable LC. Elevated PC levels may also exacerbate the prothrombotic state in LC, emphasizing the clinical relevance of targeting OS in these patients. Further research is needed to clarify whether PC is a consequence or a trigger of LC progression.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the STROBE reporting checklist. Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-625/rc

Data Sharing Statement: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-625/dss

Peer Review File: Available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-625/prf

Funding: This work was supported by the Polish National Science Centre (No. UMO-2013/09/B/NZ5/00254 to A.U.) and the Science Fund of the Saint John Paul II Hospital, Krakow, Poland and the Frycz-Modrzewski University in Krakow (No. FN/01/2025 to G.K.).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-625/coif). G.K. reports that this work was supported by the Polish National Science Centre (No. UMO-2013/09/B/NZ5/00254), and the Science Fund of the Saint John Paul II Hospital, Krakow, Poland and the Frycz-Modrzewski University in Krakow (No. FN/01/2025). A.U. reports that this work was supported by the Polish National Science Centre (No. UMO-2013/09/B/NZ5/00254). The other authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was approved by the Local Ethics Committee (Okregowa Izba Lekarska in Krakow; No. 31/KBL/OIL/2013), and all patients gave informed consent. This study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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