Vadeikienė, Roberta

Laryngeal cancer is a relatively uncommon malignancy with predisposing genetic factors that remain unclear. Single-nucleotide polymorphisms (SNPs) in genes involved in innate immune signaling may contribute to the development and progression of laryngeal carcinoma. This study aimed to evaluate the association of TLR4 (rs7037225, rs11536889, rs7037117) and MYD88 (rs7744, rs6853) polymorphisms with the risk of laryngeal squamous cell carcinoma (LSCC), as well as its clinical and pathological characteristics and survival. A retrospective case–control study involving 172 LSCC patients and 220 healthy controls was conducted. Genotyping was performed using real-time PCR from venous blood samples. MYD88 rs7744 was significantly associated with tumor size and lymph node involvement. Survival analysis showed a significant association between rs7744 and recurrence-free survival (RFS), with the AG and GG genotypes linked to poorer outcomes. Conversely, carriers of the TLR4 rs7037225 CT genotype showed significantly improved RFS, with p ranging from 0.024 to 0.037 across models. Considering the significant roles of TLR4 and MYD88 in Toll-like receptor signaling, these findings may reflect the involvement of innate immune pathways in LSCC progression. In summary, MYD88 rs7744 was associated with clinicopathological features and RFS, while TLR4 rs7037225 appeared to have a potential protective effect on survival.

Background and Objectives Breast cancer remains the most commonly diagnosed cancer among women globally. The complicated tumor microenvironment, aggressive behavior, heterogeneous nature, elevated proliferation rate, and treatment resistance are among the most characterized hallmarks of breast cancer. Many of these processes relate to the capacity of cancer cells to avoid apoptosis. Overexpressing anti-apoptotic genes is one strategy by which cancer cells prevent apoptosis. Consequently, anti-apoptotic gene expression reduction can promote the apoptosis mechanism and enhance the cells' sensitivity to cancer therapy. Chemical therapies are effective in treating cancer, but their adverse effects and resistance frequently result in treatment failure. Consequently, plant-based substances have recently received a lot of attention for their ability to inhibit cancer cell survival and increase sensitivity to treatment. One of these phytochemicals is sulforaphane (SFN), which can be found in cruciferous vegetables like broccoli and cauliflower. SFN possesses various attributes, including anti-inflammatory, cardioprotective, antioxidative, cytoprotective, and antimicrobial properties; however, its anticancer property is the most significant. Several studies demonstrated that SFN might induce apoptosis and inhibit the expression of cancer-specific genes. Thus, this study aimed to investigate the in vitro effects of SFN on breast cancer cells by evaluating BCL2 and BCL2L1 expression at both the gene and protein levels. Material and Method In this study, MCF-7 and MDA-MB-231 breast cancer cell lines were used. Cells were seeded in plates and incubated overnight. The next day, cells were treated with 25 µM and 50 µM concentrations of SFN or with DMSO as a control (0 µM). After 48 hours of incubation time, the expression of BCL2 and BCL2L1 genes was determined using reverse transcription-quantitative PCR (RT-qPCR). The total RNA was extracted from cells using the RNeasy Mini Kit. One microgram of total RNA was converted into cDNA using the High-Capacity RNA-to-cDNA Kit. RT-qPCR analysis was performed on a QuantStudio3 Real-Time PCR System. Relative gene expression was normalized to β-actin. BCL-2 and BCL-XL protein levels were assessed using Western blot analysis, involving separation by gel electrophoresis and transfer onto a membrane utilizing the semi-wet transfer unit Mini Blot module. Target proteins were detected using specific primary and secondary antibodies. The visualization of chemiluminescent imaging was done utilizing the Azure 280 system. The target protein expression level was normalized using the GAPDH protein as an internal control. Statistical analysis was performed using IBM SPSS Statistics 30 (version 30.0.0.0), and statistical significance was defined as a p-value less than 0.05. Results In MCF-7 cells, after the treatment with 25 µM and 50 µM concentrations of SFN, the BCL2 gene expression was reduced to 0.44 and 0.33, and BCL2L1 gene expression was 0.83 and 0.73, respectively. Additionally, BCL-2 protein levels decreased to 0.56 and 0.33 after the same SFN concentrations. The level of the BCL-XL protein did not alter significantly. In MDA-MB-231 cells, the BCL2 gene expression reduced to 0.88 and 0.82, and BCL2L1 gene expression was 0.73 and 0.32 after the treatment with 25 µM and 50 µM concentrations of SFN, respectively. Also, the concentrations of SFN used statistically significantly reduced protein levels. BCL-2 protein levels decreased to 0.85 and 0.78, and BCL-XL was 0.84 and 0.63. Conclusions and Recommendations Our study results revealed that SFN reduce the gene expression and protein levels of BCL2 and BCL2L1 in breast cancer cells. This suggests that SFN may be a potential anticancer agent whose effects are associated with the regulation of BCL-2 family proteins in the treatment of breast cancer.

Background and Objectives Breast cancer (BC) is a biologically heterogeneous disease with diverse molecular subtypes and clinical outcomes, strongly influenced by genetic factors. ATP-binding cassette (ABC) transporters play an important role in transmembrane processes, including drug efflux, cellular detoxification, and the maintenance of intracellular homeostasis. Variants in genes such as ABCB1 and ABCB8 may contribute to interindividual differences in tumor behavior, influencing clinicopathological characteristics and potentially affecting disease progression and treatment response. This study evaluated the association of two single nucleotide polymorphisms (SNPs), ABCB1 rs1128503 and ABCB8 rs56198402, with BC clinicopathological features. The findings aim to clarify their role in tumor biology and assess their potential as prognostic biomarkers, contributing to a better understanding of the genetic background of BC heterogeneity. Material and Method The study population comprised 170 female patients with histopathologically confirmed early-stage primary BC, aged 30 to 74 years (median age: 46 years). Clinical and tumor pathomorphological data, including tumor size, lymph node involvement, presence of metastases, degree of differentiation, estrogen (ER) and progesterone (PR) receptor status, human epidermal growth factor receptor 2 (HER2) status, disease progression, and mortality, were obtained from oncologists and the follow-up period extended until November 30, 2024. Genomic DNA was extracted from peripheral blood leukocytes using a column-based commercial DNA isolation kit in accordance with the manufacturer’s instructions. Genotyping of ABCB1 rs1128503 and ABCB8 rs56198402 was performed using TaqMan probe-based assays on the QuantStudio™ 3 Real-Time PCR System. The strength of associations between SNPs and BC clinicopathological characteristics was estimated using crude odds ratios (ORs) and corresponding 95% confidence intervals (CIs). Survival outcomes were assessed using Kaplan-Meier analysis. Statistical analysis was conducted using IBM SPSS Statistics, version 30.0.0.0, and a p-value < 0.05 was considered statistically significant. The study protocol was approved by the Kaunas Regional Biomedical Research Ethics Committee (approval numbers BE-2-10 and P1-BE-2-10/2014). Results Significant associations were identified between ABCB1 rs1128503 and PR status, metastatic disease, disease progression, and mortality. Specifically, GA genotype carriers had higher odds of progesterone receptor positivity than GG (OR=2.038; 95% CI 1.016–4.091; p=0.045), which was corroborated by allele-based analysis, showing similarly increased odds among A allele carriers (OR=1.986; 95% CI 1.034–3.816; p=0.039). Conversely, GA carriers demonstrated reduced odds of metastasis (OR=0.301; 95% CI 0.122–0.742; p=0.009), disease progression (OR=0.387; 95% CI 0.171–0.877; p=0.023), and mortality (OR=0.361; 95% CI 0.144–0.907; p=0.030). Furthermore, tumor size and lymph node involvement were also significantly associated (p-value < 0.05) with the studied genotype, indicating a protective effect against more advanced disease features. A significant difference in progression-free survival was observed across ABCB1 rs1128503 genotypes, with the GA genotype associated with more favorable outcomes (p-value < 0.05). No significant associations were found between the ABCB8 rs56198402 and clinicopathological characteristics of BC. Conclusions and Recommendations The ABCB1 rs1128503 was significantly associated with several breast cancer clinicopathological features, including progesterone receptor status, metastasis, disease progression, and progression-free survival, suggesting a potential protective effect of the GA genotype. In contrast, no significant associations were found for the ABCB8 rs56198402 variant. Further studies with larger cohorts are needed to confirm these findings and clarify the biological role of ABCB1 variants in breast cancer progression and prognosis.

Background and Objectives Hypopharyngeal squamous cell carcinoma (HSCC) is a relatively rare but aggressive head and neck malignancy, characterized by poor prognosis and low survival rates. Despite advances in treatment, the molecular mechanisms underlying HSCC development and progression remain misunderstood. The MYD88 gene, which encodes a crucial adaptor protein in innate immune signaling pathways, and its dysregulation have been linked to oncogenesis across many tumor types. However, the role of MYD88 genetic variants in HSCC has not been thoroughly investigated. Material and Method This retrospective case–control study was conducted in the Lithuanian University of Health Sciences, Department of Otorhinolaryngology, between 2017 and 2024, including 79 male patients with histologically confirmed HSCC and 220 healthy male controls. Clinical and pathological data were collected from medical records. Genomic DNA was extracted from peripheral blood samples, and selected MYD88 polymorphisms (rs7744 and rs6853) were genotyped using real-time PCR with TaqMan probes in the Oncology Research Laboratory, Institute of Oncology. Statistical analyses were performed using SPSS (version 30.0). Results The MYD88 rs7744 polymorphism was significantly associated with distant metastasis. Carriers of the GG genotype had a markedly increased risk compared to AA genotype carriers (OR = 28.46, 95% CI: 1.85–438.82, p = 0.016). No significant associations were observed between MYD88 polymorphisms and HSCC susceptibility, relapse-free survival, or overall survival. Additionally, rs6853 showed no association with clinicopathological features. Conclusions and Recommendations The MYD88 rs7744 variant might play a role in tumor progression, specifically regarding distant metastasis, rather than in susceptibility to HSCC.

Introduction Radiotherapy is a vital therapeutic option in the treatment of breast cancer nowadays. However, a major obstacle to the full effectiveness of radiation therapy is still the radioresistance of cancer cells. Various studies have proven sulforaphane's (SFN) beneficial effects against cancer and its possible utilization as a radiosensitizer in radiotherapy. This study aimed to investigate whether SFN has a radiosensitizing effect on breast cancer cells.

Material and methods The anticancer efficiency of SFN and radiosensitizing effect in MCF-7 and MDA-MB-231 cell lines were assessed by the MTT assay. Using a flow cytometric assay, the apoptosis level and changes in the cell cycle were measured. RT-qPCR and Western blot analysis were used to determine BCL-2 and BCL-XL genes expresion and proteins level.

Results According to our results, SFN reduced the viability of cells, and combining SFN with radiation therapy (IR) caused much greater anticancer effects on cells. SFN+IR was shown to enhance the number of cells in the G2/M phase and the percentage of cells going through apoptosis. SFN reduced the expression of apoptosis-relative genes BCL-2 and BCL-XL. Consistent with this data, Western blot analysis revealed that BCL-2 and BCL-XL protein levels were decreased in tested cells. As a result of the combination treatment, the downregulation of the BCL-2 protein was observed only in MDA-MB-231 cells.

Conclusions These results indicate that SFN acts as a radiosensitizer by enhancing apoptotic cell death and reducing anti-apoptotic genes in breast cancer cells.

Myeloproliferative neoplasms (MPNs) are clonal hematopoietic disorders characterized by excessive proliferation of one or more myeloid lineages, frequently accompanied by an elevated risk of thrombotic events. Matrix metalloproteinases (MMPs), a family of zinc-dependent endopeptidases, are implicated in numerous inflammatory and vascular pathophysiological processes. In this study, we analyzed the association between selected MMP polymorphisms, rs1799750, rs243865, rs3025058, rs3918242, and rs17576, and thrombotic risk as well as clinical characteristics in patients with MPNs. Genotyping was performed using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Among the polymorphisms analyzed, a statistically significant association was identified between the MMP-9 rs3918242 CT genotype and an increased risk of arterial thrombosis (OR = 4.206, CI 1.337–13.234, p = 0.014). Moreover, rs3918242 CT was associated with thrombotic events (both arterial and venous thrombosis combined), suggesting a potential contributory role in the prothrombotic phenotype observed in MPNs (OR = 3.200, CI 1.110–9.258, p = 0.031). These findings indicate that genetic variation in MMP-9, particularly rs3918242, may serve as a predictive marker for vascular complications in MPN patients. Further studies with larger cohorts are warranted to confirm these associations and to elucidate the molecular mechanisms underlying the contribution of MMP polymorphisms to thrombosis in MPNs.

Background and Objectives Head and neck cancer is the 6th most common cancer worldwide. This is a large group of cancers that are different in their representation, aggressiveness and outcomes. Laryngeal squamous cell carcinoma (LSCC) accounts for 30-40 % and hypopharyngeal squamous cell carcinoma (HSCC) for around 3-5 % of head and neck cancers. Though there are a few known risk factors of these diseases, most notably alcohol and tobacco consumption, only a small number of patients that are exposed are diagnosed with these cancers. This suggest that there is individual genetic predisposition to the disease, although the genetic biomarkers are yet to be discovered. One of the target genes of Notch signaling pathway, a process which is linked with tumor formation, is HESR family genes, including HEYL gene. Some studies suggest HEYL gene and its single nucleotide polymorphisms (SNPs) to have association with risk of various cancers, though its impact on head and neck cancers remains unclear. The objective of this study was to evaluate the association between selected single nucleotide polymorphisms HEYL rs41264499 and rs11206478 and risk of LSCC and HSCC; secondly, the objective was to assess if there is a link between HEYL selected SNPs and LSCC as well as HSCC pathomorphological characteristics. Material (patients) and research method used Two groups, consisting of 251 male patients were included in this retrospective casecontrol study study: 79 male patients with diagnosed hypopharyngeal squamous cell carcinoma and 172 male patients with diagnosed laryngeal squamous cell carcinoma. There was a control group of 220 healthy male individuals included during their routine annual health checkup at the family doctor in Kaunas Clinics. The study was approved by the Kaunas Regional Biomedical Research Ethics Committee (Protocols No. BE-2- 37, No. BE-2-10, and No. P1-BE-2-10/2014.). Data was collected on tumor size, lymph node involvement, extranodular extention, distant metastasis, stage, histological grade and association with HPV infection. DNA was extracted from peripheral venous blood samples of all individuals using a commercial DNA extraction kit (Thermo Fisher Scientific Baltics) as indicated on the manufacturer's guidelines. Genotyping of the selected polymorphisms HEYL rs41264499 and rs11206478 was performed using TaqMan® SNP Genotyping Assays on the QuantStudio™ 3 Real-Time PCR System. Statistical analysis was performed using IBM SPSS Statistics for Windows, v. 25.0. Findings/results in sufficient details to support conclusions In this study we determined that the HEYL rs41264499 CG genotype carriers had a 1.874-fold increased risk of LSCC (OR = 1.874, CI 95% (1.057-3.323), p = 0.031). Additionally, we found that the distribution of HEYL rs41264499 G allele carriers and non-carriers is statistically significantly different in patients with LSCC compared to the control group (p = 0.030). The binary logistic regression analysis showed that the G allele carriers of HEYL rs41264499 had an increased risk of LSCC (OR = 1.838, CI 95% (1.055-3.203), p = 0.032). No clear link was found between HEYL rs11206478 and risk of LSCC. Both of the selected polymorphisms showed no association on risk of HSCC. In addition, there was no significant association between selected HEYL SNPs and tumor size, lymph node involvement, metastasis, stage, histological grade, HPV infection and extranodular extention. Conclusions and recommendations HEYL rs41264499 CG genotype carriers as well as G allele carriers in the allelic model had an increased risk of developing laryngeal squamous cell carcinoma. There was no association between selected SNPs and laryngeal and hypopharyngeal cancer pathomorphological features. Study results suggest that HEYL rs41264499 may have a value in determining risk of LSCC.

Background: Radiotherapy is frequently used in the treatment of breast cancer. However, radioresistance remains the primary disadvantage of this therapeutic approach. Therefore, the search of chemicals, which could induce radiosensitivity is of great importance. One of these chemicals is resveratrol (RSV). Several investigations revealed RSV’s ability to inhibit the expression of cancer-specific genes, induce changes in the cell cycle, and activate apoptosis. Our study aimed to investigate the effects of RSV on radiosensitivity and the expression of BCL2 gene in the breast cancer. Material and Methods: An X-ray linear accelerator was used to irradiate cells with 2 or 4 Gy doses. The anti-proliferative effect of RSV in MCF-7 cells was determined by colony formation assay. The apoptosis level and changes in the cell cycle were measured using the Muse Cell Analyzer. Real-time PCR was used to quantitatively determine BCL2 gene expression. Results: Our results indicated, that RSV decreased MCF-7 cell viability. When a combination of RSV and radiation therapy (IR) was used, the anticancer effects on cells were noticeably stronger. It was demonstrated that RSV+IR increased the proportion of cells undergoing apoptosis and the number of cells in the G2/M phase. Based on RT-PCR results, RSV+IR combinations statistically significantly reduced BCL2 gene expression compared to RSV-alone or IR-alone treatment. Conclusion: According to the findings of our study, resveratrol is a potential radiosensitizer of MCF-7 breast cancer cells. RSV and IR combinations decreased cell proliferation, which was associated with the induction of apoptosis and reduced expression of the anti-apoptotic BCL2 gene.

Background: Laryngeal squamous cell carcinoma (LSCC) is the most common cancer of the head and neck region. Despite the great research effort, the role of potential behavioral and genetic risk factors remains unclear. Therefore, it is essential to search for genetic factors that could contribute to earlier diagnostics and help improve patient survival. Material and Methods: 172 men with diagnosed LSCC were enrolled in this retrospective case-control study with the control group of 220 healthy men. Genomic DNA was extracted from EDTA-preserved peripheral venous blood samples from all individuals. Genotyping of selected polymorphisms HEYL rs41264499 and rs11206478, HEY1 rs2467789 and rs1046472 using real-time PCR was performed at the Oncology Research Laboratory of Oncology Institute at the Lithuanian University of Health Sciences. Statistical data analysis was performed using SPSS v29.0. Results: Our study results show that the HEYL rs41264499 CG genotype carriers had a 1.874-fold increased risk of LSCC (OR = 1.874, CI 95% (1.057-3.323), p = 0.031). Also, it was determined that the G allele carriers of HEYL rs41264499 had an increased risk of LSCC (OR = 1.838, CI 95% (1.055-3.203), p = 0.032). Further, HEY1 rs1046472 TT genotype carriers (compared to GG genotype) had an increased odds of LSCC by 2.527-fold (OR=2.527, 95% CI (1.028-6.212), p = 0.043). HEY1 rs1046472 G allele carriers had a reduced likelihood of LSCC compared to the control group (OR = 0.395, 95% CI (0.163-0.955, p = 0.039). Conclusion: HEYL rs41264499 and HEY1 rs1046472 polymorphisms have significant association with LSCC development.

Background and Objectives Breast cancer remains the most frequent malignancy among women worldwide. The complex tumor microenvironment, aggressive behavior, heterogenous nature, high rate of proliferation, and treatment resistance are among breast cancer's most well-known characteristics. Most of these processes are related to the ability of cancer cells to evade apoptosis. One way cancer cells prevent apoptosis is by overexpressing anti-apoptotic genes. Therefore, reducing the expression of anti-apoptotic genes can activate the apoptosis mechanism and thus increase the sensitivity of cells to cancer treatment. Although chemical treatments are successful in treating cancer, their side effects and resistance often lead to treatment failure. Therefore plant-derived chemicals have attracted a lot of attention due to their ability to efficiently inhibit cancer cell survival and induce sensitivity to cancer treatment. One of these phytochemicals is resveratrol (RSV) which can be found in common foods, such as pistachios, peanuts, bilberries, blueberries, and grapes. RSV is a phytoestrogen that possesses antioxidant, antiinflammatory, cardioprotective, and anti-cancer properties. Several investigations demonstrated that RSV could trigger apoptosis and suppress the expression of genes specific to malignancy. Thus, this study aimed to investigate the in vitro effects of RSV on the expression of anti-apoptotic genes BCL-2, MCL-1 and BCL-XL in breast cancer cells. Material and Method In this study, cell lines from different subtypes of breast cancer were used: the MCF-7 cell line is the luminal A and the MDA-MB-231 cells are the triple negative subtype. To determine the expression of the anti-apoptotic BCL-2, MCL-1, and BCL-XL genes, we used reverse transcription-quantitative PCR (RT-qPCR). Cells were seeded in 6- well plates and incubated overnight. The next day, cells were treated with 50 µM and 80 µM concentrations of RSV or with DMSO as a control (0 µM). After 48 hours of incubation time, the total RNA was extracted from cells using the RNeasy Mini Kit according to the manufacturer’s instructions. The High-Capacity RNA-to-cDNA Kit was used to produce cDNA from 1 µg of total RNA. RT-qPCR analysis was performed on a QuantStudio3 Real-Time PCR System. Relative gene expression was normalized to β-actin. Statistical significance was established using Student's t-tests. The result was considered statistically significant if p<0.05. Results RT-qPCR analysis showed that RSV significantly reduced the expression of BCL-2, MCL-1 and BCL-XL genes in the MDA-MB-231 cell line. Compared with the untreated control group, after the treatment with 50 µM and 80 µM concentrations of RSV, the BCL-2 gene expression was reduced to 0.76 and 0.73, MCL-1 gene expression decreased to 0.41 and 0.42, BCL-XL gene expression was 0.38 and 0.40, respectively. In MCF-7 cells, only the BCL-2 gene expression decreased to 0.79 and 0.85 after the treatment with 50 µM and 80 µM concentrations of RSV, respectively. The expression of the MCL-1 and BCL-XL genes did not alter significantly. Our findings demonstrate that RSV had a stronger impact on anti-apoptotic genes expression in the MDA-MB-231 cell line, than in MCF-7. Conclusion and recommendations Our study results revealed that RSV reduced the expression of anti-apoptotic BCL-2, MCL-1, and BCL-XL genes in breast cancer cells. However, the effectiveness of RSV depends on the subtype of breast cancer. The triple negative breast cancer cell line MDA-MB-231 was more sensitive to the effects of RSV and may be a potential target for RSV therapy.