KI Vaistų taikinių histopatologijos laboratorija (10.11)

Introduction: Hypoxic and ischemia-associated tissues are often characterized by acidic microenvironments, highlighting the need for drug delivery systems that enable enhanced drug release under acidic conditions. Curcumin (CUR) has antioxidant and cytoprotective properties, but its therapeutic application is limited by poor aqueous solubility and low bioavailability. This study aimed to optimize hybrid lipid-polymeric nanoparticles (HLPNs) as a CUR delivery platform and evaluate their physicochemical properties, colloidal stability, CUR release at pH 7.4 and pH 5.5, and cellular effects under normoxic and hypoxia-mimetic conditions.

Cytokinesis requires tightly coordinated membrane trafficking and cytoskeletal remodelling to ensure accurate physical separation of daughter cells. Rab11a, a recycling endosome-associated small GTPase, and KIF13A, a kinesin-3 family microtubule motor protein, have each been implicated in late mitotic events. However, the mechanistic relationship between Rab11a-mediated endosomal trafficking and KIF13A-driven transport during abscission remains insufficiently defined. This study aimed to elucidate the spatial and functional interplay between Rab11a and KIF13A during cytokinesis and to determine the consequences of disrupting this axis on mitotic progression and genomic stability. Rab11a and KIF13A were depleted in HeLa cells using shRNA- and siRNA-mediated knockdown strategies. Subcellular localisation and colocalisation at the intercellular bridge were examined by immunofluorescence microscopy and quantified using Pearson's correlation coefficient. Functional outcomes were assessed through multinucleation, micronuclei formation, telophase accumulation, and LAP2-positive chromatin bridge assays. Mitotic timing from nuclear envelope breakdown to abscission was quantified by live-cell spinning-disk confocal microscopy. Rab11a and KIF13A exhibited pronounced colocalisation at the ICB during cytokinesis. Depletion of either protein reduced its spatial association with the other. It resulted in marked cytokinetic defects, including increased multinucleation, micronuclei formation, telophase delay, and elevated chromatin bridge frequency. Live-cell imaging demonstrated a significant prolongation of the cytokinesis-to-abscission interval, accompanied by an extended overall mitotic M-phase. Mechanistically, loss of Rab11a or KIF13A impaired CHMP4B recruitment to the ICB, reduced PI3P accumulation, and enhanced Aurora B signalling, consistent with activation of the abscission checkpoint. These findings establish the Rab11a-KIF13A axis as a critical regulator of late mitosis, integrating endosomal trafficking with ESCRT-III-dependent abscission. Disruption of this pathway compromises abscission accuracy and promotes genomic instability, highlighting its essential role in maintaining mitotic integrity and its potential relevance in tumorigenesis. Continued exploration of the KIF13A-Rab11 interaction will yield a deeper understanding of mitotic progression and cellular organisation.

This study investigated the potential of high-pressure homogenization as a post-production modification to reduce particle size and enhance swallow-related properties of agar-based fluid gels. The effects of pressures ranging from 10 to 60 MPa on the stability, structural changes, and swallow-related rheological characteristics of dysphagia-oriented pre-prepared agar (1.5%) and agar-collagen (20%) fluid gels were examined. High-pressure homogenization reduced agar particle size, enhancing interactions among new agar domains. Droplet size and microstructural analyses showed that higher pressures produced smaller particles, which then re-aggregated after reaching a critical concentration. However, adding collagen prevented this re-aggregation, with particle size decreasing from 182.76 to 65.82 µm as pressure increased from 0 to 60 MPa. All treated fluid gels exhibited viscoelastic behavior, with pressure increasing viscosity—up to 4.5 in agar and twice as much in agar-collagen fluid gels—and samples transitioned from Level 2 to Level 3, according to the International Dysphagia Diet Standardization Initiative (IDDSI) testing. Furthermore, pressure-induced structural changes enhanced fluid gel stability under dynamic conditions, increasing it from 74.11% to 84.98% in agar and from 48.85% to 68.08% in agar-collagen fluid gels. These findings offer valuable insights into how post-processing high-pressure homogenization can reduce droplet size, enhance palatability, and preserve swallowing safety, influencing the properties of formulated fluid gels without requiring additional thickeners.

In this study, we evaluated the impact of different in vitro 3D culture modelling methods on the activity of doxorubicin (DOX) and 5-fluorouracil (5-FU) in human melanoma spheroids. Human melanoma A375 and IGR39 spheroids were generated using the hanging drop and non-adhesive surface methods. Spheroid growth dynamics were assessed by measuring changes in spheroid diameter. To compare the effects of anticancer drugs in spheroids of different sizes, spheroids of approximately 200 and 400 µm were formed. Drug activity was evaluated based on spheroid growth and cell viability using the MTT assay. A375 spheroids formed using the non-adhesive surface method were more sensitive to DOX than spheroids formed using the hanging drop method. In smaller A375 spheroids, 10 µM 5-FU reduced cell viability more effectively in spheroids formed using the hanging drop method. In contrast, IGR39 spheroids formed by the hanging drop method were more resistant than those formed on a non-adhesive surface. However, in IGR39 spheroids, the effects of DOX and 5-FU on growth and viability did not significantly differ between formation methods. In conclusion, A375 spheroid growth was not significantly influenced by the formation method, whereas IGR39 spheroid growth depended on the method used. A375 spheroids formed on non-adhesive surfaces were more sensitive to DOX, whereas 5-FU activity depended on drug concentration and spheroid size. In IGR39 spheroids, the effects of DOX and 5-FU on growth and viability were largely independent of the spheroid formation method.

In this study, a series of novel S-alkylated imidazole derivatives 1-17, bearing a sulfonamide moiety, were synthesized. The ester group of starting material was utilized to produce compounds 2,3 bearing hydrazide and hydroxamic acid moieties. Furthermore, hydrazide 3 was successfully exploited in synthesis of various aromatic and heterocyclic hydrazones 4a10b, semicarbazides 13, 14 and azoles 11, 12, 15-17 (Fig. 1.). Compounds were tested against human prostate carcinoma PC3, triple-negative breast cancer MDA-MB-231, and glioblastoma U-87 cell lines, under normoxia and hypoxia. The effect on cell viability was determined by the MTT assay at 20 M of compounds after 72 hours of incubation. Compounds 1, 4a, 4b, 4c, 7a, 7b and 12 reduced the viability of all cell lines by up to appr. 20- 25% and were identified as the most active ones. The greatest difference in activity under normoxia and hypoxia conditions was determined in the MDA-MB-231 cell line, especially for compounds 4a, 4b, and 7b. Compound 1 showed the highest selectivity against cancer cell lines compared to human fibroblasts. The most active compound against all tested cancer cell lines was 7b (half maximum effective concentration EC50 values were in the range from 8.2 to 24.9 M).

1,2,4-Triazole-3-thiones bearing 2-amino-5-chloropyridine or 2-amino-3,5-dibromopyridine moieties were S-alkylated with substituted bromoacetamides to obtain target 1,2,4-triazole-3-thiols. Anticancer effects of the synthesized compounds were evaluated in vitro using cell monolayer (MTT and ‘wound healing’ assays) and 3D spheroid models against human lung adenocarcinoma (A549), triple-negative breast cancer (MDA-MB-231), and melanoma (IGR39) cell lines. In the initial screening, compounds incorporating the 3,5-dibromopyridyl fragment were generally the most cytotoxic and compound 34 (5-chloropyridinyl derivative) was the most potent, with EC50 values below 5 µM for all tested cancer cell lines. Although cytotoxic, they did not markedly inhibit cancer cell migration. In 3D spheroid cultures, 34 significantly inhibited the growth of MDA-MB-231 and IGR39 spheroids, while compounds 24 (phenyl) and 26 (p-tolyl) decreased spheroid viability without altering their size. Compound 26 also demonstrated notable antimicrobial properties, exhibiting stronger antifungal activity against C. tenuis (7.8 µg/mL) than nystatin (15.6 µg/mL) and matching the antibacterial potency of vancomycin against M. luteum (7.8 µg/mL). Molecular docking suggested possible MEK/BRAF-related interactions within the mitogen-activated protein kinase pathway. Compound 34 showed the most favourable predicted binding profile for MEK (-11.134 kcal/mol) and BRAF (–10.336 kcal/mol). However, these in silico findings require to be confirmed experimentally.

Pancreatic adenocarcinoma is one of the deadliest gastrointestinal cancers, accounting for over 90% of pancreatic malignancies and being frequently diagnosed at advanced stages due to late-onset symptoms and the lack of specific biomarkers [1,2]. Hypoxia within the tumor microenvironment plays a crucial role in pancreatic cancer progression by promoting aggressiveness and resistance to therapy [3]. This study aimed to evaluate the cytotoxic activity and selectivity of hydrazone derivatives synthesized at Kaunas University of Technology in human pancreatic cancer cells in vitro under normoxic and hypoxic conditions. The effects of 22 hydrazone derivatives were assessed in MIA PaCa-2 and PANC-1 cell lines using the MTT assay. Several compounds significantly reduced cell viability, and IT199, IT206, IT207, and IT227 were selected for further analysis. Their EC₅₀ values were determined in pancreatic cancer cells and human fibroblasts, with dasatinib used as a reference compound. Compared to dasatinib, the activity of the investigated hydrazone derivatives was 3–146-fold lower, depending on the cell line and oxygen conditions. Under normoxia, IT227 was the most active compound in MIA PaCa-2 (EC₅₀= 1.7 µM) and PANC-1 (EC₅₀ = 4.3 µM) cells, whereas under hypoxia, IT207 (EC₅₀= 27.3 µM) and IT199 (EC₅₀ = 21.7 µM) showed the highest activity. Under normoxic conditions, the compounds were largely non-selective toward cancer cells (SI ≤ 1), whereas under hypoxia, both potency and selectivity increased. Notably, IT227 demonstrated very high selectivity under hypoxia in both PANC-1 (SI = 15.9) and MIA PaCa-2 (SI = 30.3) cells, while IT207 (SI = 3.1) and IT199 (SI = 2.3) showed moderate hypoxia-associated selectivity. In conclusion, the hydrazone derivatives exhibited differential cytotoxic effects against pancreatic cancer cell lines, with compounds containing a double hydrazone fragment showing higher activity. The most active compounds (IT199, IT206, IT207, and IT227) were 3–146-fold less potent than dasatinib. IT206, bearing a double hydrazone fragment with methyl and naphthalene substituents, demonstrated increased cytotoxic activity under hypoxic conditions but limited selectivity toward cancer cells (SI ≈ 1). Further studies are required to elucidate the mechanisms of action and therapeutic potential of the investigated compounds

Triple-negative breast cancer accounts for less than 15% of all new cases of breast cancer, but due to its biologically aggressive and highly proliferative nature, it has a poor prognosis [1]. Melanoma is the most aggressive and severe form of skin cancer, which accounts for less than 2% of global cancer diagnoses and is the fifth most common cancer in the US [2]. The global incidence of glioblastoma is considered rare but one of the most aggressive and lethal brain tumors in adults and children, with a poor prognosis and a survival rate of 14-15 months [3]. Targeted drugs have led to major breakthroughs in cancer treatment. Therefore, hydrazone derivatives as potential kinase inhibitors are anticipated to contribute to cancer treatment. Our research aimed to evaluate the anticancer activity of hydrazone derivatives synthesized at Kaunas University of Technology (Lithuania) in human triple-negative breast, melanoma, and glioblastoma cell lines. In our previous study, we investigated the activity of 22 hydrazone derivatives bearing substituted aromatic and different heterocyclic moieties on triple-negative breast cancer cell line MDA-MB-231, melanoma cell line IGR39, and glioblastoma cell line U87. Compounds IT199, IT207, and IT227 showed selectivity and a significant reduction of cell viability by MTT assay. In this study, we determined the compound effect on cell spheroid growth and viability at 5 μM concentration after 10 days of incubation, compared with anticancer drug dasatinib. We also investigated the impact on cell migration using a single-cell migration method in the presence of 5 μM of the tested compound during 8 hour period. Compounds showed a lower viability-reducing effect on cancer cell 3D spheroids compared to their activity in a cell monolayer. Compound IT227 significantly reduced spheroid growth by up to 47% and viability by up to 86%, while another compound, IT207, inhibited only spheroid growth by up to 9%. IT199 reduced spheroid cell viability by up to 44% and showed little to no effect on spheroid growth. Dasatinib reduced MDA-MB-231 and U87 cell spheroid viability by approximately 49%, and IGR39 spheroid viability by less than 40%. Based on preliminary results from single-cell migration analysis, the average speed of cell migration was slightly reduced by compound IT227 on MDA-MB-231 cell line, while IT199 and IT207 had no significant effect on migration. A similar effect was observed with IGR39 and U87 cell lines. In conclusion, compounds IT199, IT207, and IT227 showed generally weaker inhibition of spheroid formation compared to 2D monolayers. Among the tested compounds, IT227 exhibited the most pronounced anticancer activity, significantly reducing both spheroid growth and cell viability, as well as inhibiting cell migration in multiple cancer cell lines, thus indicating the compound as a promising candidate for further investigation.

Prostate cancer makes up 13,5% of diagnosed cancer wordwide, marking it as the fourth most widespread and the second most commonly diagnosed cancer in men [1]. Triple-negative breast cancer accounts for 10–15% of all new breast cancer cases, as it is an aggressive subtype with unfavorable prognosis [2]. Sulfonamide derivatives are potential anticancer agents with the ability to inhibit carbonic anhydrase IX, that are more expressed in cancer cells[3]. Research for new effective substances creates progress in the treatment of cancer. Our research aimed to evaluate the anticancer activity of sulfonamide derivatives synthesized at Kaunas University of Technology (Lithuania) on human prostate and triple-negative breast cancer cell line viability. In our study, we investigated the activity of 23 sulfonamide derivatives bearing substituted aromatic and different heterocyclic moieties on prostate cancer cell line PC3 and triple-negative breast cancer cell line MDA-MB231. The effect on cell viability was determined by the MTT assay at 20 μM of compounds after 72 hours of incubation. The comparative compounds with significant cell viability-reducing capabilities were chosen for further studies, and the EC50 values were established. Compounds 1, 4a, 4b, 4c, 7a, 7b and 12 reduced the viability of all cell lines by up to appr. 20-25%. These compounds were selected for the determination of EC50 values. The greatest difference in normoxia and hypoxia conditions were to MDA-MB231 cell line, as in hypoxia, viability was reduced by up to appr 70-90%. Compounds 1 and 4c are selective to the MDA-MB231 cell line, but compound 1 was more active under normoxia, while 4c was more active under hypoxia. Also, compounds 4b, 7a and 7b are only selective to MDA-MB231 cell line in hypoxia, although 7b had similar EC50 values in normoxia for both cell lines. Compounds 4a and 12 are more selective to the PC3 cell line in normoxia, but under hypoxia they become selective to the MDA-MB231 cell line. In conclusion, compounds 1, 4a, 4b, 4c, 7a, 7b and 12 showed a significant reduction of cell viability by MTT assay. Compound 1, 7a and 7b are more effective in normoxia, while 4a, 4b, 4c and 12 reduce the viability higher in hypoxia. Especially effective in hypoxia are compounds 4a and 12 in the MDA-MB231 cell line.

Melanoma is the third most common form of skin cancer and is associated with a high risk of metastasis. Incidence and mortality rates are increasing each year, and it is predicted that the number of melanoma cases will continue to rise rapidly in the coming decades [1]. For these reasons, further research is required to improve our understanding of melanoma biology and to identify new effective anticancer drugs that could improve patient survival [2]. In this study, we evaluated the impact of in vitro 3D culture modelling of the human melanoma A375 on the activity of doxorubicin (DOX) and 5-fluorouracil (5-FU). These drugs were selected based on their differences in mechanism of action. DOX is a cytotoxic drug that inhibits topoisomerase II, causing DNA damage and cell death [3]. In contrast, 5-FU is a cytostatic drug that inhibits DNA and RNA synthesis, thereby suppressing cell proliferation [4]. Two of the most commonly used 3D culture formation methods were selected: the hanging drop method and the non-adhesive surface formation method [5]. Spheroid growth dynamics were assessed by phase-contrast microscopy through analysis of changes in spheroid diameter. To compare the effects of anticancer drugs in spheroids of different sizes, spheroids were generated using two initial cell numbers (250 and 2000 cells per spheroid). Following treatment with DOX and 5-FU,spheroids were incubated for 8 days. Drug activity was evaluated based on changes in spheroid diameter and cell viability, measured using the MTT assay. DOX transport into spheroids was assessed by measuring DOX fluorescence intensity over the total spheroid area using fluorescence microscopy. In general, A375 spheroids formed using the non-adhesive surface method were more sensitive to DOX than spheroids formed using the hanging drop method. In smaller spheroids, 10 µM 5-FU inhibited viability more efficiently when spheroids were formed using the hanging drop method. DOX transport into spheroids varied depending on the concentration of DOX used. When spheroids were incubated with 10 µM DOX, accumulation was higher compared to incubation with 2 µM DOX. No statistically significant differences in DOX transport were determined in spheroids formed by different methods, nor in spheroids of different diameters (200 µm and 400 µm). In conclusion, A375 spheroids formed using the non-adhesive surface method are more sensitive to DOX, while the effect of 5-FU varies depending on its concentration and the size of the spheroids. DOX transport into A375 spheroids does not depend on the formation method but is dependent on the DOX concentration used.