Laukaitienė, Danguolė

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.

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: 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 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.

Background and objectives The ABCB8, a mitochondrial ATP-binding cassette transporter, is critical in regulating iron homeostasis and protecting cells from oxidative stress. Polymorphisms in the ABCB8 gene, including rs17545756 and rs4148844, are likely to affect gene expression and protein functionality, thereby impacting cancer risk. In breast cancer (BC), these variants can affect tumor behavior by modifying mitochondrial function, metabolic pathways, or therapeutic resistance. This study aims to analyze the association of ABCB8 polymorphisms rs17545756 and rs4148844 with BC by evaluating their correlation with tumor phenotype and disease prognosis in BC patients. Understanding these associations may provide insights into the molecular mechanisms of BC progression and support the development of more targeted diagnostic and therapeutic strategies. Material and Method In this study, we analyzed ABCB8 rs4148844 and rs17545756 polymorphisms in 170 patients with BC. Medical information such as age at diagnosis, tumor size, grade, lymph node status, estrogen (ER), progesterone (PR), human epidermal growth factor 2 (HER2) receptor status, metastatic status, disease progression, and death was collected. Genomic DNA was isolated from peripheral blood leukocytes using a commercially available DNA extraction kit (Thermo Fisher Scientific Baltics, Vilnius, Lithuania). The polymorphisms mentioned were assessed using TaqMan (Applied Biosystems Europe BV, UK Branch, Warrington, Cheshire, UK) according to the manufacturer’s instructions on the QuantStudio 3 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The statistical analysis was done with Statistical Package for the Social Sciences (SPSS) 25.0 for Windows. The study was approved by the Kaunas Regional Biomedical Research Ethical Committee (protocol No. 2024- BEC2-137). Results The association between ABCB8 rs17545756 and lymph node involvement was determined (χ2 (2) = 5.479, p = 0.040). Logistic regression analysis revealed that individuals carrying the CT genotype had a significantly higher risk of lymph node metastasis compared to those with the CC genotype (OR = 2.909, 95% CI 1.003–8.439, p = 0.049). Moreover, T allele carriers (vs. non-carriers) had a 3.200-fold increased risk of lymph node metastasis (OR = 3.200, 95% CI 1.121-9.131, p = 0.030). The association between ABCB8 rs17545765 and lymph node metastasis remained statistically significant in a multivariate logistic regression analysis, where factors, such as age at diagnosis, primary tumor size, grade, and receptor status, were included as potential confounding variables. No statistically significant correlations were found between the ABCB8 rs4148844 polymorphism and BC phenotype or prognosis. Conclusions and Recommendations Our findings indicate a significant association between the ABCB8 rs17545756 and lymph node involvement in breast cancer patients. Patients carrying the CT genotype and T allele were at a higher risk of lymph node metastasis, suggesting a potential role of this variant in promoting tumor spread. These results suggest that rs17545756 may serve as a potential genetic marker for metastatic risk in breast cancer.

Myeloproliferative neoplasms (MPNs) are characterized by increased proliferation of myeloid lineages in the bone marrow. Calreticulin (CALR) 52 bp deletion and CALR 5 bp insertion have been identified in essential thrombocythemia (ET) and primary myelofibrosis (PMF). There is not much data on the crosstalk between mutated CALR and MPN-related signaling pathways, such as JAK/STAT, PI3K/Akt/mTOR, and Hedgehog. Calreticulin, a multifunctional protein, takes part in many cellular processes. Nevertheless, there is little data on how mutated CALR affects the oxidative stress response and oxidative stress-induced DNA damage, apoptosis, and cell cycle progression. We aimed to investigate the role of the CALR 52 bp deletion and 5 bp insertion in the pathogenesis of MPN, including signaling pathway activation and functional analysis in CALR-mutated cells. Our data indicate that the JAK/STAT and PI3K/Akt/mTOR pathways are activated in CALR-mutated cells, and this activation does not necessarily depend on the CALR and MPL interaction. Moreover, it was found that CALR mutations impair calreticulin function, leading to reduced responses to oxidative stress and DNA damage. It was revealed that the accumulation of G2/M-CALR-mutated cells indicates that oxidative stress-induced DNA damage is difficult to repair. Taken together, this study contributes to a deeper understanding of the specific molecular mechanisms underlying CALR-mutated MPNs.

Background and Objectives BCR/ABL1-negative myeloproliferative neoplasms (MPNs) such as primary myelofibrosis (PMF), essential thrombocythemia (ET), and polycythemia vera (PV) arise from the clonal proliferation of neoplastic stem cells in the bone marrow. Matrix metalloproteinases (MMPs), zinc-dependent endopeptidases, play a role in extracellular matrix and bone marrow remodeling. Moreover, it was suggested that MMPs modulate platelet function and thrombus formation. It is well known that MPN patients often experience thrombosis and other disease complications. However, assessing the thrombotic events risk is difficult because of the complexity of the interactions of multiple factors, e.g. age, history of previous thrombosis, cardiovascular comorbidities, and mutational status, that affect thrombosis. So, there is a need for research that will help to understand whether MMP genetic variants may be a potential early marker for thrombotic events in patients with MPNs. Here, we analyzed the effect of MMP1, MMP2, and MMP9 gene polymorphisms on the risk of thrombotic events and clinical characteristics in patients with PMF, ET, and PV. [...].

Background and Objectives Radiotherapy is an important treatment modality in modern cancer therapy besides surgery and systemic therapy. However, radioresistance of breast cancer cells remains the major limiting factor of this treatment option. Therefore, there is a need for research related to compounds that can improve the radiotherapy effect. One of these is luteolin (LUT). Luteolin, a flavonoid that is found in many fruits, vegetables, and herbs, exhibits numerous biological activities, including anti-inflammatory, antidiabetic, and anticancer properties. Through a variety of experimental cancer models, luteolin has been extensively researched in many cancer types and has been related to its ability to inhibit tumor growth by targeting cellular processes such as apoptosis, angiogenesis, migration, and cell cycle progression. However, a knowledge gap remains regarding the studies on luteolin efficacy and its the mechanism of radiosensitization. Thus, the aim of this work was to analyze the potential radiosensitizing effect of luteolin on MCF7 breast cancer cells. [...].

Introduction Radiation therapy is frequently applied in breast cancer treatment. However, the radioresistance and side effects remain the major limiting factors of this treatment option (1). Therefore, the studies of substances as modifiers of radiation treatment efficacy are very relevant. One of these is sulforaphane (SFN). Several studies have shown the proapoptotic and antiproliferative activity of SFN in multiple cell types (2). Aim The aim of the study was to analyze the potential radiosensitizing effect of SFN on MDA-MB231 breast cancer cells.