Obesity and Pro-Inflammatory Cytokines: Gene Expression Patterns Within Cervical Cancer Progression

Abstract

Background/Aim

Cervical cancer (CC) remains a significant global health concern, characterized by high incidence rates. Concurrently, the obesity epidemic continues to expand, affecting millions worldwide. Dysregulation of pro- and anti-inflammatory cytokines can promote chronic inflammation, which contributes to the pathogenesis of various diseases, including cancer. This study aimed to evaluate the gene expression levels of TNF-α, IL-6, IL-8, IL-10, and VEGF during CC progression in patients across different weight categories: normal weight, overweight, and obese.

Materials and Methods

Cervical samples were classified according to cervical diagnosis and body mass index (BMI). Gene expression levels of TNF-α, IL-6, IL-8, IL-10, and VEGF were assessed through PCR, and the Relative Expression Index was calculated using the 18S gene as a housekeeping reference.

Results

Eighty-one women from the Colposcopy Clinic of Health Jurisdiction II in Ciudad Juarez were evaluated and classified into four groups: no intraepithelial lesion (NIL) (n=18), low-grade squamous intraepithelial lesion (LSIL) (n=22), high-grade squamous intraepithelial lesion (HSIL) (n=24), and CC (n=17). These groups were also classified based on BMI. The overexpression of TNF-α (p=0.036), IL-6, and IL-10 increased with the severity of cervical lesions, whereas IL-8 expression was suppressed (p=0.53) as cervical pathology progressed. According to BMI, a trend toward overexpression of IL-6, IL-10, and TNF-α was observed with increasing adipose tissue in HSIL and CC. On the other hand, IL-8 and VEGF expression appeared to be repressed regardless of intraepithelial lesion grade or obesity status.

Conclusion

Obesity is a risk factor for the development of CC by promoting a proinflammatory environment combined with a VPH infection.

Introduction

Cytokines are low-molecular-weight proteins produced by various immune cells, such as macrophages, B and T lymphocytes, neutrophils, etc. They serve as mediators of intracellular signaling and are integral to immune modulation and inflammatory processes, encompassing both proinflammatory and anti-inflammatory responses (1, 2). Chronic inflammation has been implicated in the pathogenesis of numerous high-mortality diseases such as cardiovascular disease, diabetes mellitus, and cancer (3-5). Proinflammatory cytokines like IL-6, TNF-α, IL-8, IL-10, and vascular endothelial growth factor (VEGF) are commonly active in chronic inflammation, altering diverse signaling pathways including hypoxia, oxidative stress, fibrosis, insulin resistance, etc., leading to metabolic dysregulation (6, 7).

In cancer, these cytokines play a pivotal role in remodeling the tumor microenvironment, thus promoting angiogenesis, proliferation, invasion, and cell migration (8). Cervical cancer (CC) ranks fourth globally in incidence (9). In Mexico, the mortality rate attributable to cervical cancer is 10.92 per 100,000 population (10), positioning it as the second leading cause of death among women aged 30-59 years (11). On the other hand, it is estimated that by 2030, approximately 643 million women worldwide will be classified as obese, highlighting a significant global health concern (12). In Mexico, 41% of women exhibit some form of obesity, representing an alarming epidemiological condition (13). It is well established that obesity significantly elevates the risk of various cancers, with increases of up to 10% or even doubling the risk, particularly for endometrial and ovarian cancers (14, 15). Obesity promotes a chronic low-grade inflammatory state by the constant production of diverse proinflammatory cytokines and adipocytokines, such as TNF-α, IL-6, IL-10, leptin, etc. (16, 17). However, there is limited evidence examining concurrent cervical cancer progression, cytokines gene expression, and the influence of Body Mass Index (BMI). Therefore, this study aimed to evaluate the gene expression levels of IL-6, TNF-α, IL-8, IL-10, and VEGF in cervical samples at different stages of CC progression and to assess their variation relative to patient BMI within a population from Ciudad Juarez, Chihuahua, Mexico.

Materials and Methods

Patients and samples. The study involved 81 patients from the Colposcopy clinic of the Sanitary Jurisdiction II at Ciudad Juarez, Chihuahua, Mexico. Cervical scraping samples without lesions, biopsies, and cervical scrapings with lesions/CC were collected.

Participants, women aged 18 years or older, signed an informed consent. Clinical and nutritional histories were recorded to assess risk factors. Samples were categorized based on lesion severity: 18 with no intraepithelial lesion (NIL), 22 low-grade squamous intraepithelial lesion (LSIL), 24 high-grade squamous intraepithelial lesion (HSIL), and 17 CC.

The study adhered to the Declaration of Helsinki and received approval from the Universidad Autonoma de Ciudad Juarez bioethics committee (CBE-ICB/004-01-14). As a risk factor, the presence of HPV in cervical samples was identified by amplifying a region of the HPV/L1 gene from sample DNA using the general primers GP5+/GP6+ (18).

DNA/RNA extraction and cDNA synthesis. DNA was extracted using the phenol/chloroform method and stored at -20°C until use. Total RNA was isolated with TRIzol reagent according to the manufacturer’s instructions and quantified using a Nanodrop 2000 (Thermo Fisher Scientific, Waltham, MA, USA). cDNA synthesis was performed with the ImProm-II Reverse Transcription System as described by the manufacturer (Promega Co., Madison, WI, USA).

Evaluation of gene expression. Gene expression of proinflammatory cytokines was evaluated by semiquantitative PCR. Primer sets for the IL-10, IL-8, VEGF, and 18S genes were designed based on sequences from the NCBI database. To amplify the IL-6 and TNF-α genes, primer sets previously reported in the literature were used (19, 20). Table I lists the primer sets and amplification conditions. PCR products were electrophoresed at 100 V for 45 min on agarose gels. The gel was visualized for 3.5 seconds under UV light using the EDAS 290 Kodak program. Densitometric analysis was performed. To normalize the data, the constitutive 18S gene was used as a reference.

Table I. Primers set and amplification conditions.

Statistical analysis. All data were analyzed using SPSS version 20.0 for Windows. Nonparametric tests were performed: Kruskal-Wallis and Mann-Whitney, with a significance level of p<0.05, and a chi-square (c²) test to evaluate the significance of risk factors, also at p<0.05.

Results

Clinical data. To identify risk factors in the study population, the clinical history of each patient was obtained. Table II presents the results.According to the literature, it is well established that HPV presence is correlated with carcinogenesis (21, 22). Consistent with these findings, our results indicate that nearly all samples with cervical cancer tested positive for HPV infection. Once the two variables of interest (BMI and lesion level) were obtained, the patient distribution is summarized in Figure 1. Notably, among normal-weight individuals, only one patient presented with cancer.

Table II. Percentage of distribution of risk factors according to type of lesion.

NIL: No intraepithelial lesion; LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; CC: cervical cancer.

Figure 1.

Distribution of patients by BMI and lesion level. NIL: No intraepithelial lesion; LSIL: low-grade squamous intraepithelial lesion; HSIL: highgrade squamous intraepithelial lesion; CC: cervical cancer; BMI: body mass index.

Cytokine expression according to progression of CC. To evaluate cytokine expression levels relative to CC progression, the NIL group served as the control and reference, with 18S rRNA as the housekeeping gene (expression level set at 100%). Table III presents the quantitative values and percentages of cytokine expression across the different lesion stages. Figure 2 shows cytokine expression during CC progression, with statistical significance in TNF-α and IL-8 between the normal and CC groups. Also, a statistical difference in IL-8 expression was observed between LSIL and HSIL.

Table III. Differences in cytokine expression according to the progression of CC.

NIL: No intraepithelial lesion; LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; CC: cervical cancer.

Figure 2.

Cytokines expression in the progression of CC. *Statistical significance at p<0.05. NIL: No intraepithelial lesion; LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; CC: cervical cancer.

Cytokine expression according to BMI. The cytokine expression profile across different stages of cervical CC progression and BMI categories is depicted in Figure 3. Results indicate that obese patients exhibit a progressive upregulation of IL-6 and TNF-α correlating with the severity of cervical lesions. In contrast, IL-10 expression appears to be more closely associated with BMI than lesion type, with overexpression (relative expression >1.5) observed in obesity across LSIL, HSIL, and CC. In overweight individuals, IL-10 expression patterns are more influenced by lesion type. Additionally, IL-8 expression is suppressed in HSIL and CC independently of BMI status. VEGF levels are consistently downregulated in obesity in conjunction with cancer progression.

Figure 3.

Cytokine expression according to BMI and type of lesion. (A) IL-6, (B) TNF-α, (C) IL-10, (D) IL-8, (E) VEGF. All graphs compare expression patterns of normoweight, overweight and obesity groups in the different stage of lesion. NIL: No intraepithelial lesion; LSIL: low-grade squamous intraepithelial lesion; HSIL: high-grade squamous intraepithelial lesion; CC: cervical cancer.

Discussion

The association between reproductive factors such as parity, age at menarche, sexual activity, age, number of sexual partners, hormonal contraception usage, and HPV as risk determinants is well established. Specifically, a high parity, defined as four or more pregnancies, shows a positive correlation with cervical cancer, which is supported by our data demonstrating a statistically significant link between parity of four or more and CC (23, 24). Additionally, our findings indicate that early initiation of sexual activity is significantly associated with an increased risk of cervical cancer, consistent with established literature (23, 25, 26).

Analysis of cytokine expression during CC progression (Figure 2) reveals a 49.27% increase in IL-6 levels, though not reaching statistical significance. These findings align with existing literature demonstrating significant IL-6 upregulation during cancer progression, particularly in HPV-positive samples (27, 28). This elevated IL-6 may facilitate oncogenesis via the JAK/STAT pathway, which is known to be modulated by HPV oncogenes E5, E6, and E7 to promote cellular proliferation and immune evasion (29-31).

The analysis of TNF-α gene expression demonstrates a progressive increase correlated with lesion type and CC. A notable overexpression of 204% was observed in the CC group, reaching statistical significance (p=0.039). TNF-α contributes to chronic inflammation, facilitating CC progression by inducing epithelial-mesenchymal transition (EMT) via the NF-κB/Twist signaling axis (32). Additionally, TNF-α has been implicated in tumorigenesis and lymphatic metastasis through VEGFC-mediated activation of AKT and ERK pathways (33). Chronic inflammation driven by TNF-α is also associated with oral carcinogenesis linked to HPV infection (34). Moreover, TNF-α expression patterns in CC may serve as prognostic biomarkers for response to immunotherapy (35).

IL-10 gene expression exhibits a linear increase correlating with lesion progression. Relative expression levels increase from 0.9 in normal tissue to 1.33 in affected tissue, indicating a 48% increase in CC, although this difference is not statistically significant. Data suggest that IL-10 upregulation is associated with HPV infection (27, 36, 37). IL-10 functions dually as an anti-inflammatory and proinflammatory cytokine. Its overexpression is known to facilitate chronic infection by reducing T lymphocyte populations (38), inhibiting antigen presentation, and inducing apoptosis within immune cells (39).

In this study, we observed a significant reduction in IL-8 expression associated with lesion progression, decreasing from a relative expression value of 1.03 in normal tissue to 0.38 in CC, reflecting a 63.4% decline (p=0.05). These results contrast with existing literature (40, 41), which indicates that IL-8 is associated with cellular migration and proliferation, often overexpressed in malignancies (42). The suppression of the proinflammatory cytokine IL-8 may be attributed to HPV oncoproteins E6/E7, which inhibit transcription of the IL-8 promoter (43). On the other hand, decreased IL-8 levels have been linked to improved survival outcomes in HPV-associated oropharyngeal squamous cell carcinoma (44). Regarding VEGF, it was downregulated in 47.9% of CC cases. VEGF’s role in angiogenesis and its contribution to tumor cell proliferation and migration are well established (45). Its expression is more prevalent in cervical adenocarcinomas and correlates with disease progression (46). Notably, VEGF expression is often detectable at low levels in early-stage tumors, with approximately 55% of samples showing expression (47). Consistent with our results, which show that only 9 CC samples expressed VEGF (data not shown), it has been proposed that the histological grade of cancer can influence the progressive increase in expression (48).

As is well established, obesity induces a state of low-grade chronic inflammation (49). A recent study (50) has demonstrated a link between BMI and cervical cancer, suggesting that one pathway through which obesity influences carcinogenesis is the modulation of tumor-promoting inflammation via proliferative inflammatory signals (51). In this context, the study’s findings on gene expression related to inflammation showed that in CC, IL-6 expression was increased by 276% in patients with obesity compared to weight-matched controls without lesions. Similarly, TNF-α expression was elevated by 3,808.1% in patients with obesity. Conversely, IL-8 was downregulated by 68.6% in patients with cervical cancer and obesity relative to normal-weight individuals without lesions. VEGF expression was repressed by 2.9% in the obesity cohort. No statistically significant correlation was observed between lesion type and BMI, although a discernible pattern of gene expression variation across different BMI classes and lesion types was noted. It is established that inflamed adipocytes release proinflammatory cytokines such as IL-6, TNF-α, VEGF, and IL-10 through multiple mechanisms, which are implicated in promoting tumor progression via activation of various signaling pathways (52).

The correlation between obesity and IL-6 is well established; elevated serum IL-6 levels are positively associated with increased visceral adiposity (53). IL-6 is secreted by adipose tissue and adipose tissue macrophages (ATMs), contributing to ATM accumulation and an ensuing inflammatory state (54, 55). Obesity is characterized by a significant accumulation of visceral fat, distributed across the abdominal (anterior and posterior), gluteofemoral regions, and enveloping intraperitoneal and retroperitoneal organs (56, 57). Evidence indicates that individuals with increased visceral adiposity and a corresponding high inflammatory index, strongly linked to visceral fat, exhibit reduced cancer survival rates (58-60).  VEGF a key regulator of angiogenesis, supports healthy adipose tissue; diminished VEGF expression has been associated with inflammation (61) and proposed as a protective factor against obesity (62). Our findings suggest that VEGF repression may be linked to an inflammatory phenotype driven by visceral fat accumulation.

IL-10 cytokine expression was elevated by 124% in the obesity plus cancer cohort. Evidence suggests that IL-10 promotes tumorigenesis through activation of the STAT signaling pathway (38), which can affect the development of CC. Additionally, studies indicate IL-10 overexpression in obese women (63, 64). Our findings demonstrate that IL-10 levels are significantly modulated by both obesity and HPV infection, highlighting its potential utility as a diagnostic biomarker for CC.

Conclusion

The inflammatory process of obesity, which overexpresses proinflammatory cytokines such as IL-6, IL-10, and TNFα, is associated with the inflammatory environment in cervical intraepithelial lesions caused by HPV infection. Thus, obesity combined with HPV could favor the development of CC.

Conflicts of Interest

All Authors declare no competing interests.

Authors’ Contributions

Conceptualization, F.J.-V.; formal analysis, A.L.A-O.; investigation, L.P-P.; methodology, F.J.-V.; resources, F.J.-V.; writing – review and editing, I.A.G.-M., M.S.-V. J.C.S-E. All Authors have read and agreed to the published version of the manuscript.

Acknowledgements

All Authors acknowledge to UACJ for the support and infrastructure, SECIHTI for the scholarship granted to Livier Picazo Perez for her master’s degree studies, and MD Cecilia Diaz Hernández from the Colposcopy Clinic, Sanitary Jurisdiction II of Ciudad Juárez, for proving all the samples.

Funding

This research receives no external funding.

Artificial Intelligence (AI) Disclosure

During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning-based image enhancement tools.