HPLC method for the determination of thymoquinone in growth cell medium.

BACKGROUND: Preclinical drug testing requires in vitro and in vivo assessments that are vital for studying drug pharmacokinetics and toxicity. Distinct factors that play an important role in drug screening, such as hydrophobicity, solubility of the substance and serum protein binding can be challenging by inducing result inconsistencies. Hence, establishing accurate methods to quantify drug concentrations in cell cultures becomes pivotal for reliable and reproducible results important for in vivo dosing predictions. OBJECTIVE: This research focuses on developing an optimized analytical approach via high-pressure liquid chromatography (HPLC) to determine thymoquinone (TQ) levels in monolayer cell cultures. METHODS: The method's validation adheres to the International Council for Harmonisation (ICH) guideline M10, ensuring its acceptance and applicability. Using an HPLC system with a Diode Array Detector (DAD), the study fine-tuned various parameters to achieve an efficient separation of TQ. Validation covered specificity, sensitivity, matrix effects, linearity, precision, and accuracy, alongside assessing TQ stability in RPMI-1640 medium. RESULTS: The HPLC method exhibited remarkable TQ specificity, free from interfering peaks at the analyte retention. Sensitivity analysis at the lower limit of quantification (LLOQ) revealed 5.68% %CV and 98.37% % mean accuracy. Matrix effect evaluation showcased accuracy within 85-115%. Linearity spanned in the concentration range of 2-10 µM with a correlation coefficient (r2) of 0.9993. Precision and accuracy were aligned with acceptance criteria. The proposed method was found to be greener in terms of usage of persistent, bioaccumulative, and toxic chemicals and solvents, corrosive samples, and waste production. CONCLUSION: The developed HPLC-DAD method emerges as specific, accurate, sensitive, and reliable for TQ determination in cell cultures. It ensures robust TQ quantification, enhancing precise in vitro assessments and dependable dosing predictions for in vivo studies. Further research is advocated to investigate TQ's stability across diverse environmental conditions.

In silico study of polyphenols as potential inhibitors of MALT1 protein in non-Hodgkin lymphoma.

Non-Hodgkin lymphoma (NHL) is one of the most common cancer types. Deregulated signaling pathways can trigger certain NHL subtypes, including Diffuse Large B-cell lymphoma. NF-ĸB signaling pathway, which is responsible for the proliferation, growth, and survival of cells, has an essential role in lymphoma development. Although different signals control NF-ĸB activation in various lymphoid malignancies, the characteristic one is the CARD11-BCL10-MALT1 (CBM) complex. The CBM complex is responsible for the initiation of adaptive immune response. Our study is focused on the molecular docking of ten polyphenols as potential CARD11-BCL10-MALT1 complex inhibitors, essentially through MALT1 inhibition. Molecular docking was performed by Auto Dock Tools and AutoDock Vina tool, while SwissADME was used for drug-likeness and absorption, distribution, metabolism, excretion, and toxicity (ADMET) analysis of the ligands. Out of 66 ligands that were used in this study, we selected and visualized five. Selection criteria were based on the binding energy score and position of the ligands on the used protein. 2D and 3D visualizations showed interactions of ligands with the protein. Five ligands are considered potential inhibitors of MALT1, thus affecting NF-ĸB signaling pathway. However, additional in vivo and in vitro studies are required to confirm their mechanism of inhibition.

Targeted NGS on sequential bone marrow biopsies aids in the evaluation of cytopenias and monocytosis and documents clonal evolution-a proof of principle study.

Differential diagnosis of clonal versus reactive cytopenia and monocytosis, respectively, frequently presents a diagnostic challenge. With the two recent classifications of myeloid disorders, mutational analysis has gained importance as a diagnostic tool. However, reports on its utility on trephine bone marrow biopsies (BMB) are sparse. The aim of our proof of principle study was to determine the suitability of targeted sequencing for the longitudinal evaluation of cytopenia and monocytosis and demonstration of clonal evolution on sequential BMB. Seventy-seven EDTA-decalcified BMB of 33 patients with peripheral cytopenia and/or monocytosis, including at least one follow-up biopsy/patient, were included. Initial morphological diagnoses were idiopathic cytopenia of undetermined significance (ICUS, 8 cases), MDS (without blast increase, 7 cases), MDS with increased blasts/excess blasts (MDS-IB/EB) (11 cases), and CMML (7 cases). Thirty-one genes relevant for myeloid disorders were examined using two custom AmpliSeq NGS panels. Mutations were found in the initial BMB of 5/8 cases of ICUS, thus changing the diagnosis to clonal cytopenia of unknown significance (CCUS), 5/7 MDS, 10/11 MDS-IB/EB, and 7/7 CMML. Clonal evolution was observed in 14/33 (42%) cases, mostly associated with disease progression. None of the wild-type patients acquired mutations during follow-up. NGS-based mutation profiling is a robust diagnostic tool for BMB and provides valuable additional information, especially for cases with no/minimal dysplasia, and for better risk stratification of MDS. Tracking variant allele frequency and appearance of mutations over time allows for observing clonal evolution or relapse.

Viral Agents as Potential Drivers of Diffuse Large B-Cell Lymphoma Tumorigenesis.

Among numerous causative agents recognized as oncogenic drivers, 13% of total cancer cases occur as a result of viral infections. The intricacy and diversity of carcinogenic processes, however, raise significant concerns about the mechanistic function of viruses in cancer. All tumor-associated viruses have been shown to encode viral oncogenes with a potential for cell transformation and the development of malignancies, including diffuse large B-cell lymphoma (DLBCL). Given the difficulties in identifying single mechanistic explanations, it is necessary to combine ideas from systems biology and viral evolution to comprehend the processes driving viral cancer. The potential for more efficient and acceptable therapies lies in targeted medicines that aim at viral proteins or trigger immune responses to either avoid infection or eliminate infected or cancerous cells. In this review, we aim to describe the role of viral infections and their mechanistic approaches in DLBCL tumorigenesis. To the best of our knowledge, this is the first review summarizing the oncogenic potential of numerous viral agents in DLBCL development.

Metformin and Thymoquinone Synergistically Inhibit Proliferation of Imatinib-Resistant Human Leukemic Cells.

Chemotherapy resistance is one of the major challenges in cancer treatment, including leukemia. A massive array of research is evaluating combinations of drugs directed against different intracellular signaling molecules to overcome cancer resistance, increase therapy effectiveness, and decrease its adverse effects. Combining chemicals with proven safety profiles, such as drugs already used in therapy and active substances isolated from natural sources, could potentially have superior effects compared to monotherapies. In this study, we evaluated the effects of metformin and thymoquinone (TQ) as monotherapy and combinatorial treatments in chronic myeloid leukemia (CML) cell lines sensitive and resistant to imatinib therapy. The effects were also evaluated in primary monocytic acute myeloid leukemia (AML) and chronic lymphocytic leukemia (CLL) cells. Both compounds induced a dose- and time-dependent decrease of viability and proliferation in tested cells. Metformin had similar IC50 values in imatinib-sensitive and imatinib-resistant cell lines. IC50 values of TQ were significantly higher in imatinib-resistant cells, but with a limited resistance index (2.4). Synergistic effects of combinatorial treatments were observed in all tested cell lines, as well as in primary cells. The strongest synergistic effects were observed in the inhibition of imatinib-resistant cell line proliferation. Metformin and TQ inhibited the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling and induced apoptosis in tested cell lines and primary cells. The enhanced effects of combinatorial treatments on the induction of apoptosis were more dominant in imatinib-resistant compared to imatinib-sensitive CML cells. Primary cells were more sensitive to combinatorial treatments compared to cell lines. A combination of 1.25 mM metformin and 0.625 µM TQ increased the levels of cleaved poly (ADP-ribose) polymerase (PARP), decreased the levels of proliferation regulatory proteins, and inhibited protein kinase B (Akt) and NF-κB signaling in primary CLL cells. This study demonstrates that combinatorial treatments of imatinib-resistant malignant clones with metformin and TQ by complementary intracellular multi-targeting represents a promising approach in future studies.

Curcumin Decreases Viability and Inhibits Proliferation of Imatinib-Sensitive and Imatinib-Resistant Chronic Myeloid Leukemia Cell Lines.

Chronic myeloid leukemia (CML) is a myeloproliferative haematological malignancy characterized by constitutive activation of BCR-ABL1 tyrosine kinase in the majority of patients. BCR-ABL1 expression activates signaling pathways involved in cell proliferation and survival. Current treatment options for CML include tyrosine kinase inhibitors (TKI) with resistance as a major issue. Various treatment options for overcoming resistance are being investigated. Among them, phytochemical curcumin could play an important role. Curcumin has been found to exhibit anti-cancerous effects in various models, including CML, through regulation of multiple molecular signaling pathways contributing to tumorigenesis. We have evaluated curcumin's effects on imatinib-sensitive LAMA84S and K562, as well as imatinib-resistant LAMA84R cell lines. Our results indicate a significant dose-dependent decrease in cell viability and proliferation of imatinib-sensitive and imatinib-resistant cell lines after curcumin treatment. Suppression of key signaling molecules regulating metabolic and proliferative events, such as Akt, P70S6K and NF-kB, was observed. Increased expression of caspase-3 suggests the potential pro-apoptotic effect of curcumin in the imatinib-resistant CML model. Additional in silico molecular docking studies revealed binding modes and affinities of curcumin with different targets and the results are in accordance with in vitro findings. Altogether, these results indicate the potential role of curcumin in the treatment of CML.

Meet the Insidious Players: Review of Viral Infections in Head and Neck Cancer Etiology with an Update on Clinical Trials.

Head and neck cancers (HNC) occur in the upper aerodigestive tract and are among the most common cancers. The etiology of HNC is complex, involving many factors, including excessive tobacco and alcohol consumption; over the last two decades, oncogenic viruses have also been recognized as an important cause of HNC. Major etiological agents of nasopharynx carcinoma and oropharyngeal carcinoma include Epstein-Barr virus (EBV) and human papillomaviruses (HPVs), both of which are able to interfere with cell cycle control. Additionally, the association of hepatitis C and hepatitis B infection was observed in oral cavity, oropharyngeal, laryngeal, and nasopharyngeal cancers. Overall prognoses depend on anatomic site, stage, and viral status. Current treatment options, including radiotherapy, chemotherapy, targeted therapies and immunotherapies, are distributed in order to improve overall patient prognosis and survival rates. However, the interplay between viral genome sequences and the health, disease, geography, and ethnicity of the host are crucial for understanding the role of viruses and for development of potential personalized treatment and prevention strategies. This review provides the most comprehensive analysis to date of a vast field, including HNC risk factors, as well as viral mechanisms of infection and their role in HNC development. Additionally, currently available treatment options investigated through clinical practice are emphasized in the paper.

Association of IRS1 genetic variants with glucose control and insulin resistance in type 2 diabetic patients from Bosnia and Herzegovina.

Background Previous studies reported conflicting results regarding association of insulin receptor substrate 1 (IRS1) gene variation with type 2 diabetes (T2D) and insulin resistance (IR) in different ethnic groups. We examined the association of rs7578326, rs2943641, and rs4675095 in the IRS1 gene with T2D and related traits in a population from Bosnia and Herzegovina, which is one of the European countries with the highest T2D prevalence of 12.5%. Methods Our study included 390 T2D patients and 252 control subjects. Biochemical parameters, including fasting glucose (FG), fasting insulin (FI), homeostasis model assessment insulin resistance index (HOMA-IR), and HbA1c were measured in all participants. Genotyping analysis was performed by Mass Array Sequenom iPlex platform. Results Our results demonstrated that rs7578326 and rs4675095 variants were associated with increased FG levels. The rs7578326 was also associated with higher FI, HOMA-IR (B = 0.08, 95% CI [0.01, 0.15], padd = 0.025; B = 0.079, 95% CI [0.006, 0.150], padd = 0.033, respectively) in T2D, and with HbA1c (B = 0.034, 95% CI [0.003, 0.065], pdom = 0.035) in non-drug-treated T2D. In contrast, rs2943641 C allele was associated with lower FG levels in control subjects (B = -0.17, 95% CI [-0.03, -0.002], padd = 0.030) and HbA1c (B = 0.03, 95% CI [0.002, 0.06], pdom = 0.040) in non-drug-treated T2D. Conclusions We report the association between common variants in IRS1 gene with insulin resistance, glucose, and HbA1c levels in Bosnia and Herzegovina's population.

Perceptions of students in health and molecular life sciences regarding pharmacogenomics and personalized medicine.

BACKGROUND: Increasing evidence is demonstrating that a patient's unique genetic profile can be used to detect the disease's onset, prevent its progression, and optimize its treatment. This led to the increased global efforts to implement personalized medicine (PM) and pharmacogenomics (PG) in clinical practice. Here we investigated the perceptions of students from different universities in Bosnia and Herzegovina (BH) towards PG/PM as well as related ethical, legal, and social implications (ELSI). This descriptive, cross-sectional study is based on the survey of 559 students from the Faculties of Medicine, Pharmacy, Health Studies, Genetics, and Bioengineering and other study programs. RESULTS: Our results showed that 50% of students heard about personal genome testing companies and 69% consider having a genetic test done. A majority of students (57%) agreed that PM represents a promising healthcare model, and 40% of students agreed that their study program is well designed for understanding PG/PM. This latter opinion seems to be particularly influenced by the field of study (7.23, CI 1.99-26.2, p = 0.003). Students with this opinion are also more willing to continue their postgraduate education in the PM (OR = 4.68, CI 2.59-8.47, p < 0.001). Furthermore, 45% of students are aware of different ethical aspects of genetic testing, with most of them (46%) being concerned about the patient's privacy. CONCLUSIONS: Our results indicate a positive attitude of biomedical students in Bosnia and Herzegovina towards genetic testing and personalized medicine. Importantly, our results emphasize the key importance of pharmacogenomic education for more efficient translation of precision medicine into clinical practice.