Pharmacological p38 MAPK inhibitor SB203580 enhances AML stem cell line KG1a chemosensitivity to daunorubicin by promoting late apoptosis, cell growth arrest in S-phase, and miR-328-3p upregulation.

Acute myeloid leukaemia (AML) is characterized by uncontrolled proliferation of myeloid progenitor cells and impaired maturation, leading to immature cell accumulation in the bone marrow and bloodstream, resulting in hematopoietic dysfunction. Chemoresistance, hyperactivity of survival pathways, and miRNA alteration are major factors contributing to treatment failure and poor outcomes in AML patients. This study aimed to investigate the impact of the pharmacological p38 mitogen-activated protein kinase (MAPK) inhibitor SB203580 on the chemoresistance potential of AML stem cell line KG1a to the therapeutic drug daunorubicin (DNR). KG1a and chemosensitive leukemic HL60 cells were treated with increasing concentrations of DNR. Cell Titer-Glo®, flow cytometry, phosphokinase and protein arrays, Western blot technology, and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were employed for assessment of cell viability, half-maximal inhibitory concentration (IC50) determination, apoptotic status detection, cell cycle analysis, apoptosis-related protein and gene expression monitoring. Confocal microscopy was used to visualize caspase and mitochondrial permeability transition pore (mPTP) activities. Exposed at various incubation times, higher DNR IC50 values were determined for KG1a cells than for HL60 cells, confirming KG1a cell chemoresistance potential. Exposed to DNR, late apoptosis induction in KG1a cells was enhanced after SB203580 pretreatment, defined as the combination treatment. This enhancement was confirmed by increased cleavage of poly(ADP-ribose) polymerase, caspase-9, caspase-3, and augmented caspase-3/-7 and mPTP activities in KG1a cells upon combination treatment, compared to DNR. Using phosphokinase and apoptosis protein arrays, the combination treatment decreased survival Akt phosphorylation and anti-apoptotic Bcl-2 expression levels in KG1a cells while increasing the expression levels of the tumor suppressor p53 and cyclin-dependent kinase inhibitor p21, compared to DNR. Cell cycle analysis revealed KG1a cell growth arrest in G2/M-phase caused by DNR, while combined treatment led to cell growth arrest in S-phase, mainly associated with cyclin B1 expression levels. Remarkably, the enhanced KG1a cell sensitivity to DNR after SB203580 pretreatment was associated with an increased upregulation of miR-328-3p and slight downregulation of miR-26b-5p, compared to DNR effect. Altogether, these findings could contribute to the development of a new therapeutic strategy by targeting the p38 MAPK pathway to improve treatment outcomes in patients with refractory or relapsed AML.

Exploring in vitro and in silico Biological Activities of Calligonum Comosum and Rumex Vesicarius: Implications on Anticancer and Antibacterial Therapeutics.

Introduction: The adverse effects of clinically used anti-cancer medication and the rise in resistive micro-organisms have limited therapeutic options. Multiple anti-cancer drugs are derived from medicinal herbs which also have shown anti-bacterial effects. This study aimed to identify the optimal extraction solvent for detecting the cytotoxic and anti-bacterial effects of Calligonum comosum (C. Comosum) and Rumex vesicarius (R. Vesicarius) extracts. Additionally, the study aimed to identify active metabolites and assess their potential as future drug candidates for anti-cancer and anti-bacterial therapeutics. Methods: Leaves from both plants were extracted using ethanol, ethyl acetate, chloroform, and water. The cytotoxic effects of the extracts were tested on liver, colon, and breast cancer cell lines. Apoptosis was assessed using High Content Imaging (HCI) and the ApoTox triplex Glo assay. The anti-bacterial effects were determined using agar-well diffusion. Liquid chromatography-mass spectrometry (LC-MS) was used to tentatively identify the secondary metabolites. In silico computational studies were conducted to determine the metabolites' mode of action, safety, and pharmacokinetic properties. Results: The ethanolic extract of C. Comosum exhibited potent cytotoxicity on breast cancer cell lines, with IC50 values of 54.97 µg/mL and 58 µg/mL for KAIMRC2 and MDA-MB-231, respectively. It also induced apoptosis in colon and breast cancer cell lines. All tested extracts of C. Comosum and R. Vesicarius demonstrated anti-bacterial activity against Staphylococcus aureus and Escherichia coli. Seven active metabolites were identified, one of which is Kaempferol 3-O-Glucoside-7-O-Rhamnoside, which showed strong (predicted) anti-cancer activity. Kaempferol 3-O-Glucoside-7-O-Rhamnoside and Quercetin-3-O-Glucuronide also exhibited potential anti-bacterial effects on gram-positive and negative bacteria. Conclusion: Ethanol extraction of C. Comosum solubilizes active metabolites with potential therapeutic applications in cancer treatment and bacterial infections. Kaempferol 3-O-Glucoside-7-O-Rhamnoside, in particular, shows promise as a dual therapeutic drug candidate for further research and development to improve its efficacy, safety, and pharmacokinetic profile.

Distinct Mechanisms of Cytotoxicity in Novel Nitrogenous Heterocycles: Future Directions for a New Anti-Cancer Agent.

Electron-rich, nitrogenous heteroaromatic compounds interact more with biological/cellular components than their non-nitrogenous counterparts. The strong intermolecular interactions with proteins, enzymes, and receptors confer significant biological and therapeutic properties to the imidazole derivatives, giving rise to a well-known and extensively used range of therapeutic drugs used for infections, inflammation, and cancer, to name a few. The current study investigates the anti-cancer properties of fourteen previously synthesized nitrogenous heterocycles, derivatives of imidazole and oxazolone, on a panel of cancer cell lines and, in addition, predicts the molecular interactions, pharmacokinetic and safety profiles of these compounds. METHOD: The MTT and CellTiter-Glo® assays were used to screen the imidazole and oxazolone derivatives on six cancer cell lines: HL60, MDA-MB-321, KAIMRC1, KMIRC2, MCF-10A, and HCT8. Subsequently, in vitro tubulin staining and imaging were performed, and the level of apoptosis was measured using the Promega ApoTox-Glo® triplex assay. Furthermore, several computational tools were utilized to investigate the pharmacokinetics and safety profile, including PASS Online, SEA Search, the QikProp tool, SwissADME, ProTox-II, and an in silico molecular docking study on tubulin to identify the critical molecular interactions. RESULTS: In vitro analysis identified compounds 8 and 9 to possess the most significant potent cytotoxic activity on the HL60 and MDA-MB-231 cell lines, supported by PASS Online anti-cancer predictions with pa scores of 0.413 and 0.434, respectively. In addition, compound 9 induced caspase 3/7 dependent-apoptosis and interfered with tubulin polymerization in the MDA-MB-231 cell line, consistent with in silico docking results, identifying binding similarity to the native ligand colchicine. All the derivatives, including compounds 8 and 9, had acceptable pharmacokinetics; however, the safety profile was suboptimal for all the tested derivates except compound 4. CONCLUSION: The imidazole derivative compound 9 is a promising anti-cancer agent that switches on caspase-dependent apoptotic cell death and modulates microtubule function. Therefore, it could be a lead compound for further drug optimization and development.

The Role of Myrrh Metabolites in Cancer, Inflammation, and Wound Healing: Prospects for a Multi-Targeted Drug Therapy.

BACKGROUND: Myrrh extract is a well-known medicinal plant with significant therapeutic benefits attributed to the activity of its diverse metabolites. It has promising activity against cancer and inflammatory diseases, and could serve as a potential therapeutic alternative since most therapeutic agents have severe side effects that impair quality of life. METHOD: The current study identified the active metabolites from the myrrh resin methanolic extract. Then, the extracts were tested for in vitro anti-inflammatory and anti-cancer activity using cancer cell lines and Tamm-Horsfall Protein 1 (Thp-1)-like macrophage cell lines. Furthermore, using an in vivo rat model, the extracts' anti-inflammatory and wound-healing activity was investigated. In addition, in silico predictions of the myrrh constituents highlighted the pharmacokinetic properties, molecular targets, and safety profile, including cytochrome P 450 (CYP) inhibition and organ toxicity. RESULTS: Nine secondary metabolites were identified, and computational predictions suggested a good absorption profile, anticancer, anti-inflammatory, and wound-healing effects. The myrrh extract had moderate cytotoxic activity against both HL60 and K562 leukemia cell lines and the KAIMRC1 breast cancer cell line. Myrrh caused a dose-dependent effect on macrophages to increase the reactive oxygen species (ROS) levels, promote their polarization to classically activated macrophages (M1) and alternatively activated macrophages (M2) phenotypes, and consequently induce apoptosis, highlighting its ability to modulate macrophage function, which could potentially aid in several desired therapeutic processes, including the resolution of inflammation, and autophagy which is an important aspect to consider in cancer treatment. The topical application of myrrh improved wound healing, with no delayed inflammatory response, and promoted complete re-epithelization of the skin, similar to the positive control. In conclusion, we provide evidence for the methanolic extract of myrrh having cytotoxic activity against cancer cells and anti-inflammatory wound-healing properties, which may be attributed to its role in modulating macrophage function. Furthermore, we suggest the active constituents responsible for these properties, which warrants further studies focusing on the precise roles of the active metabolites.

New Born Calf Serum Can Induce Spheroid Formation in Breast Cancer KAIMRC1 Cell Line.

Three-dimensional (3D) cell culture systems have become very popular in the field of drug screening and discovery. There is an immense demand for highly efficient and easy methods to produce 3D spheroids in any cell format. We have developed a novel and easy method to produce spheroids from the newly isolated KAIMRC1 cell line in vitro. It can be used as a 3D model to study proliferation, differentiation, cell death, and drug response of cancer cells. Our procedure requires growth media supplemented with 10% new born calf serum (NBCS) and regular cell culture plates to generate KAIMRC1 spheroids without the need for any specialized 3D cell culture system. This procedure generates multiple spheroids within a 12-24-h culture. KAIMRC1 spheroids are compact, homogeneous in size and morphology with a mean size of 55.8 µm (±3.5). High content imaging (HCI) of KAIMRC1 spheroids treated with a panel of 240 compounds resulted in the identification of several highly specific compounds towards spheroids. Immunophenotyping of KAIMRC1 spheroids revealed phosphorylation of FAK, cJUN, and E-cadherin, which suggests the involvement of JNK/JUN pathway in the KAIMRC1 spheroids formation. Gene expression analysis showed upregulation of cell junction genes, GJB3, DSC1, CLDN5, CLDN8, and PLAU. Furthermore, co-culture of KAIMRC1 cells with primary cancer-associated-fibroblasts (CAFs) showcased the potential of these cells in drug discovery application.

Metabolites Profiling, In Vitro, In Vivo, Computational Pharmacokinetics and Biological Predictions of Aloe perryi Resins Methanolic Extract.

BACKGROUND: Aloe perryi is a traditional herb that has various biological and pharmacological properties such as anti-inflammatory, laxative, antiviral, antidiabetic, and antitumor effects, which have not been deliberated before. The current investigation aims to evaluate in vitro cytotoxicity against several cancer cell lines in addition to in vivo anti-inflammatory activities of Aloe perryi extract using a rat animal model. Moreover, the pharmacokinetic properties of bioactive constituents and possible biological targets were assessed and evaluated. The methanolic extract of Aloe perryi was prepared by maceration, to tentatively identify the biomolecules of the Aloe perryi extract, analytical LC-QTOF-MS method was employed for Aloe perryi methanolic extract. The cytotoxic activity was examined in six cancer cell lines using Titer-Glo assay and the IC50s were calculated in addition to in silico target predictions and in vivo anti-inflammatory activity assessment. Subsequently, the pharmacokinetics of the identified active components of Aloe perryi were predicted using SwissADME, and target prediction using the Molinspiration webserver. The cytotoxic activity on HL60 and MDA-MB-231 was moderately affected by the Aloe perryi extract with IC50 of 63.81, and 89.85 µg/ml, respectively, with no activity on other cells lines. Moreover, the Aloe perryi extract exhibited a significant increase in wound contraction, hair growth, and complete re-epithelization when compared with the negative control. The pharmacokinetic properties of the bioactive constituents suggested a good pharmaceutical profile for the active compounds and nuclear receptors and enzymes were the two main possible targets for these active compounds. Our results demonstrated the promising activity of Aloe perryi extract with cytotoxic and anti-inflammatory properties, indicating a potential therapeutic utility of this plant in various disease conditions.