Network pharmacology, molecular docking and experimental approaches of the anti-proliferative effects of Rhamnus prinoides ethyl-acetate extract in cervical cancer cells.

Background: Cervical cancer, one of the lethal cancers among women, is a challenging disease to treat. The current therapies often come with severe side effects and the risk of resistance development. Traditional herbal medicine, with its potential to offer effective and less toxic options, is a promising avenue. This study was undertaken to investigate the potential of Rhamnus prinoides (R. prinoides) root bark extracts in selectively inhibiting the proliferation of cervical cancer cells, using the HeLa cell line as an in vitro model. Methods: R. prinoides plant extracts were first screened at a fixed concentration of 200 µg/ml to determine the active extract. The selective anti-proliferative activity of the active extract was evaluated in a concentration dilution assay using the (3-(4,5-dimethylthiazol- 2-yl)-2,5-diphenyltetrazolium bromide) MTT assay on cancerous (HeLa) cells and non-cancerous (Vero) cells to determine the half-maximal inhibitory (IC50) and half-cytotoxic concentrations (CC50), respectively. Functional assays on cell morphology (by microscopy), cell migration (wound healing assay) and cell cycle (by flow cytometry) were also conducted. The active extract was analyzed using Gas Chromatography/Mass Spectrometry (GC/MS) to determine any compounds it contained. Following identification of possible gene targets by network pharmacology, the genes were validated by molecular docking and Real-Time Quantitative Polymerase Chain Reaction (RT-qPCR). Results: The ethyl acetate extract of R. prinoides (EARP), the most active extract, selectively inhibited the growth of cervical cancer cells, their migration and induced cell cycle arrest at the S phase. In silico analysis revealed that squalene, 3,3a,6,6-tetramethyl-4,5,5a,7,8,9-hexahydro-1H-cyclopenta[i]indene and Olean-12-en-3.beta.-ol, acetate showed acceptable drug-like characteristics and may be partly attributed to the bioactivity demonstrated and the deregulation of the mRNA expression of AKT1, NF-κB, p53, Bax, Bcl-2, and Er-b-B2. Conclusion: This study, for the first time, demonstrates the anti-proliferation effects of EARP and forms a firm foundation for further drug development studies.

Network pharmacology, molecular docking, and in vitro study on Aspilia pluriseta against prostate cancer.

BACKGROUND: Current prostate cancer treatments are associated with life-threatening side effects, prompting the search for effective and safer alternatives. Aspilia pluriseta Schweinf. ex Engl. has previously shown anticancer activity in lung and liver cancer cell lines. This study investigated its potential for prostate cancer. METHODS: A crude extract of A. pluriseta root was prepared using dichloromethane/methanol (1:1 v/v) and partitioned into hexane, ethyl acetate, and water fractions. The MTT assay was used to assess the antiproliferative activity of the fractions. The active fractions were tested at 6.25-200 µg/ml on human prostate cancer DU-145 cells and non-cancerous Vero E6 cells. Qualitative phytochemical and gas chromatography-mass spectrometry (GC-MS) analyses were conducted to identify chemical compounds. Network pharmacology was employed to predict molecular targets and modes of action of the identified chemical compounds, with subsequent validation through molecular docking and real-time PCR. RESULTS: Active extracts included crude dichloromethane/methanol, hexane, and ethyl acetate fractions, inhibiting DU-145 cell proliferation with IC50 values of 16.94, 20.06, and 24.14 µg/ml, respectively. Selectivity indices were determined to be 6.04 (crude), 3.62 (hexane), and 6.68 (ethyl acetate). Identified phytochemicals comprised phenols, terpenoids, flavonoids, tannins, sterols, and saponins. GC-MS analysis revealed seventy-nine (79) compounds, with seven (7) meeting ideal drug candidate parameters; their hub gene targets included MAPK3, MAPK1, IL6, TP53, ESR1, PTGS2, MMP9, MDM2, AR, and MAP2K1, implicating regulation of PI3K/Akt, MAPK, and p53 signaling pathways as potential modes of action. Core compounds such as 1-heneicosanol, lanosterol, andrographolide, and retinoic acid exhibited strong binding activities, particularly lanosterol with MAPK21 (-9.7 kcal/mol), ESR1 (-8.9 kcal/mol), and MAPK3 (-8.8 kcal/mol). Treatment with A. pluriseta downregulated AR expression and upregulated p53, while also downregulating CDK1 and BCL-2 and upregulating caspase-3. CONCLUSIONS: A. pluriseta extracts inhibited DU-145 cell growth without causing cellular toxicity, suggesting great potential for development as an anti-prostate cancer agent. However, further in vitro and in vivo experiments are recommended.

Phytochemical analysis, in-vitro and in-silico study of antiproliferative activity of ethyl acetate fraction of Launaea cornuta (Hochst. ex Oliv. & Hiern) C. Jeffrey against human cervical cancer cell line.

Introduction: Cervical cancer is one of the leading causes of death among women globally due to the limitation of current treatment methods and their associated adverse side effects. Launaea cornuta is used as traditional medicine for the treatment of a variety of diseases including cancer. However, there is no scientific validation on the antiproliferative activity of L. cornuta against cervical cancer. Objective: This study aimed to evaluate the selective antiproliferative, cytotoxic and antimigratory effects of L. cornuta and to explore its therapeutical mechanisms in human cervical cancer cell lines (HeLa-229) through a network analysis approach. Materials and methods: The cytotoxic effect of L. cornuta ethyl acetate fraction on the proliferation of cervical cancer cells was evaluated by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) bioassay and the antimigratory effect was assessed by wound healing assays. Compounds were analysed using the qualitative colour method and gas chromatography-mass spectroscopy (GC-MS). Subsequently, bioinformatic analyses, including the protein-protein interaction (PPI) network analysis, Gene Ontology (GO), and Kyoto Encyclopaedia of Genes and Genomes (KEGG) analysis, were performed to screen for potential anticervical cancer therapeutic target genes of L. cornuta. Molecular docking (MD) was performed to predict and understand the molecular interactions of the ligands against cervical cancer. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) was performed to validate the network analysis results. Results: L. cornuta ethyl acetate fraction exhibited a remarkable cytotoxic effect on HeLa-229 proliferation (IC50 of 20.56 ± 2.83 µg/mL) with a selectivity index (SI) of 2.36 with minimal cytotoxicity on non-cancerous cells (Vero-CCL 81 (IC50 of 48.83 ± 23.02). The preliminary screening revealed the presence of glycosides, phenols, saponins, terpenoids, quinones, and tannins. Thirteen compounds were also identified by GC-MS analysis. 124 potential target genes associated with the effect of L. cornuta ethyl acetate fraction on human cervical cancer were obtained, including AKT1, MDM2, CDK2, MCL1 and MTOR were identified among the top hub genes and PI3K/Akt1, Ras/MAPK, FoxO and EGFR signalling pathways were identified as the significantly enriched pathways. Molecular docking results showed that stigmasteryl methyl ether had a good binding affinity against CDK2, ATK1, BCL2, MDM2, and Casp9, with binding energy ranging from -7.0 to -12.6 kcal/mol. Tremulone showed a good binding affinity against TP53 and P21 with -7.0 and -8.0 kcal/mol, respectively. This suggests a stable molecular interaction of the ethyl acetate fraction of L. cornuta compounds with the selected target genes for cervical cancer. Furthermore, RT-qPCR analysis revealed that CDK2, MDM2 and BCL2 were downregulated, and Casp9 and P21 were upregulated in HeLa-229 cells treated with L. cornuta compared to the negative control (DMSO 0.2%). Conclusion: The findings indicate that L. cornuta ethyl acetate fraction phytochemicals modulates various molecular targets and pathways to exhibit selective antiproliferative and cytotoxic effects against HeLa-229 cells. This study lays a foundation for further research to develop innovative clinical anticervical cancer agents.

The TCR assigns naive T cells to a preferred lymph node.

Naive T cells recirculate between the spleen and lymph nodes where they mount immune responses when meeting dendritic cells presenting foreign antigen. As this may happen anywhere, naive T cells ought to visit all lymph nodes. Here, deep sequencing almost-complete TCR repertoires led to a comparison of different lymph nodes within and between individual mice. We find strong evidence for a deterministic CD4/CD8 lineage choice and a consistent spatial structure. Specifically, some T cells show a preference for one or multiple lymph nodes, suggesting that their TCR interacts with locally presented (self-)peptides. These findings are mirrored in TCR-transgenic mice showing localized CD69 expression, retention, and cell division. Thus, naive T cells intermittently sense antigenically dissimilar niches, which is expected to affect their homeostatic competition.

Evaluation of the antimalarial properties of Solanum incanum L. leaf extract fractions and its ability to downregulate delta aminolevulinate dehydratase to prevent the establishment of malaria infection.

ETHNOPHARMACOLOGICAL RELEVANCE: Solanum incanum L. is commonly used in traditional herbal medicine (THM) in Kenya for treating various ailments. Recent developments in disease treatment have introduced the concept of host-directed therapy (HDT). This approach involves targeting factors within the host cell that can impede the growth or replication of a pathogen. One such host factor is delta aminolevulinate dehydratase (δ-ALAD), the second enzyme in the heme biosynthesis pathway utilized by Plasmodium for growth. Studies using mice models have shown an increase in δ-ALAD expression during Plasmodium berghei infection. Another plant in the Solanum genus, S. guaranticum, has been found to inhibit δ-ALAD in red blood cells in vitro and in the brain in vivo. Is it possible that the bioactive compounds in S. incanum extracts could also be effective in HDT for malaria treatment? AIM OF STUDY: To better assess the effectiveness of S. incanum leaf extracts as a curative and prophylaxis in malaria parasite infection, and to test the plant's ability to decrease δ-ALAD expression. MATERIALS AND METHODS: The leaves of S. incanum were collected, dried, and pulverized before being subjected to a successive extraction protocol to obtain crude, hexane, ethyl acetate, and aqueous extract fractions. Phytochemical analysis was conducted on all extract fractions, followed by GC-MS analysis of the fraction with the most potent antimalarial activity. An acute toxicity study was also performed on the extracted fractions. The potency of the extract fractions as curative and prophylactic antimalarial was then evaluated in THM using Plasmodium berghei-infected mice at a dose of 100 mg/kg. The extract fraction with the highest activity was further evaluated at varying doses and its effect on δ-ALAD was measured using RT-qPCR. The percentage of parasitemia and chemosuppression, and mean survival time were used as indices of activity. RESULTS: Phytochemical analysis revealed that the ethyl acetate and aqueous extract fractions contained high terpenoids, flavonoids, and phenols levels. However, alkaloids were only present in moderate quantities in the aqueous extract, and quinones were found in high levels only in the crude extract. Additionally, all extract fractions contained saponins in high levels but lacked tannins. While the plant extracts were found to be non-toxic, they did not exhibit curative antimalarial activity. However, all extract fractions showed prophylactic antimalarial activity, with the ethyl acetate extract having the highest percentage of chemosuppression even at doses of 250 and 1000 mg/kg. In the negative control, the expression of δ-ALAD was 5.4-fold, but this was significantly reduced to 2.3-fold when mice were treated with 250 mg/kg of the ethyl acetate fraction. GC-MS analysis of the ethyl acetate fraction revealed high percentages of 2-methyloctacosane, tetracosane, and decane. CONCLUSION: The fractions extracted from S. incanum leaves have been found to possess only antimalarial prophylactic properties, with the ethyl acetate extract fraction showing the most effective results. The activity of this fraction may be attributed to its ability to decrease the expression of δ-ALAD, as it contains an alkane compound implicated with enzyme-inhibitory activity.

Cohesin-mediated NF-κB signaling limits hematopoietic stem cell self-renewal in aging and inflammation.

Organism aging is characterized by increased inflammation and decreased stem cell function, yet the relationship between these factors remains incompletely understood. This study shows that aged hematopoietic stem and progenitor cells (HSPCs) exhibit increased ground-stage NF-κB activity, which enhances their responsiveness to undergo differentiation and loss of self-renewal in response to inflammation. The study identifies Rad21/cohesin as a critical mediator of NF-κB signaling, which increases chromatin accessibility in the vicinity of NF-κB target genes in response to inflammation. Rad21 is required for normal differentiation, but limits self-renewal of hematopoietic stem cells (HSCs) during aging and inflammation in an NF-κB-dependent manner. HSCs from aged mice fail to down-regulate Rad21/cohesin and inflammation/differentiation signals in the resolution phase of inflammation. Inhibition of cohesin/NF-κB reverts hypersensitivity of aged HSPCs to inflammation-induced differentiation and myeloid-biased HSCs with disrupted/reduced expression of Rad21/cohesin are increasingly selected during aging. Together, Rad21/cohesin-mediated NF-κB signaling limits HSPC function during aging and selects for cohesin-deficient HSCs with myeloid-skewed differentiation.

Methanolic extract of Agerantum conyzoides exhibited toxicity and growth disruption activities against Anopheles gambiae sensu stricto and Anopheles arabiensis larvae.

BACKGROUND: Vector control remains the mainstay to effective malaria management. The negative implications following persistent application of synthetic insecticides geared towards regulation of mosquito populations have necessitated prospection for ecofriendly effective chemistries. Plant-derived compounds have the potential to control malaria-transmitting mosquito populations. Previously, Agerantum conyzoides extracts have demonstrated toxicity effects on disease-transmitting mosquitoes. However, their efficacy in controlling Afrotropical malaria vectors remains unclear. Herein, the toxicity and growth disruption activities of crude methanolic leaf extract of A. conyzoides on Anopheles gambiae sensu stricto and An. arabiensis larvae were assessed. METHODS: Late third (L3) instars of An. gambiae s.s and An. arabiensis larvae were challenged with increasing doses of crude methanolic extract of A. conyzoides. The larval mortality rates were recorded every 24 h and the LC50 values determined at their associated 95% confidence levels. ANOVA followed by Post-hoc Student-Newman-Keuls (SNK) test was used to compare results between treatment and control groups. Phytochemical profiling of the extract was performed using standard chemical procedures. RESULTS: Treatment of larvae with the methanolic extract depicted dose-dependent effects with highest mortality percentages of ≥ 69% observed when exposed with 250 ppm and 500 ppm for 48 h while growth disruption effects were induced by sublethal doses of between 50-100 ppm for both species. Relative to experimental controls, the extract significantly reduced larval survival in both mosquito species (ANOVA, F(8,126) = 43.16776, P < 0.001). The LC50 values of the extract against An. gambiae s.s ranged between 84.71-232.70 ppm (95% CI 81.17-239.20), while against An. arabiensis the values ranged between 133.46-406.35 ppm (95% CI 131.51-411.25). The development of the juvenile stages was arrested at pupal-larval intermediates and adult emergence. The presence of alkaloids, aglycone flavonoids, triterpenoids, tannins and coumarins can partly be associated with the observed effects. CONCLUSION: The extract displayed considerable larvicidal activity and inhibited emergence of adult mosquitoes relative to experimental controls, a phenomenon probably associated with induced developmental hormone imbalance. Optimization of the bioactive compounds could open pathways into vector control programmes for improved mosquito control and reduced malaria transmission rates.

Antimicrobial and cytotoxicity properties of the organic solvent fractions of Clerodendrum myricoides (Hochst.) R. Br. ex Vatke: Kenyan traditional medicinal plant.

BACKGROUND/AIM: Clerodendrum myricoides is a Kenyan herbal plant used in the management of respiratory diseases. In the current study, we investigated in vitro antimicrobial activity, cytotoxicity, and phytochemical screening of C. myricoides. MATERIALS AND METHODS: Antimicrobial activities of C. myricoides organic fractions against array of microorganisms including: (i) Mycobacterium tuberculosis (MTB) H37Rv, (ii) Staphylococcus aureus, (iii) Klebsiella pneumoniae, (iv) Escherichia coli, (v) Candida albicans, (vi) Pseudomonas aeruginosa, (vii) Cryptococcus neoformans, (viii) Salmonella typhi, (ix) Shigella sonnei, and (x) Methicillin-resistant S. aureus (MRSA) were investigated by disc diffusion and microdilution techniques. Antituberculous activity was investigated using BACTEC MGIT 960 system while cytotoxicity was analyzed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay on HEp-2 cells. Finally, phytochemicals were screened using standard procedures. RESULTS: Methanolic fractions exhibited a broad spectrum activity inhibiting 75% of test pathogens. It had the highest activity with minimal inhibition concentration (MIC) values of ≤62.5 µg/ml recorded against 62.5% tested microbes. It yielded the highest zone of inhibition of 20.3 mm (S. aureus), lowest MIC of <12.5 µg/ml (MTB), and the lowest minimal bactericidal concentration of 62.5 µg/ml (C. albicans), within the acceptable toxicity limit (CC50 >90 µg/ml). The phytochemicals largely believed to be responsible for the observed activity included: Alkaloid, phenols, anthraquinones, terpenoids, and flavonoids. CONCLUSION: Methanolic fraction had remarkable activity against MRSA, S. aureus, E. coli, S. sonnei, C. albicans, and MTB, which are of public health concerns due to drug resistance and as sources of community and nosocomial infections. To the best of our knowledge, this is the first report exploring the antituberculous activity of C. myricoides and thence a major output in search of novel, safe drug leads to mitigate the global tuberculosis threat.

Antimicrobial and cytotoxicity properties of the crude extracts and fractions of Premna resinosa (Hochst.) Schauer (Compositae): Kenyan traditional medicinal plant.

BACKGROUND: Premna resinosa (Hochst.) Schauer also called "mukarakara" in Mbeere community of Kenya is used in the management of respiratory illness. In this study we investigated antituberculous, antifungal, antibacterial activities including cytotoxicity and phytochemical constituents of this plant. METHODS: Antibacterial and antifungal activities were investigated by disc diffusion and micro dilution techniques. Antituberculous activity was investigated using BACTEC MGIT 960 system while cytotoxicity was analyzed by MTT assay on Vero cells (Methanolic crude extract) and HEp-2 cells (fractions). Finally, phytochemicals were profiled using standard procedures. RESULTS: P. resinosa had high antituberculous activity with a MIC of <6.25 µg/ml in ethyl acetate fraction. The antibacterial activity was high and broad spectrum, inhibiting both Gram positive and Gram negative bacteria. Dichloromethane fraction had the best antibacterial MIC of 31.25 µg/ml against Methicillin-resistant S. aureus while Ethyl acetate fraction had the highest zone of inhibition of 22.3±0.3 against S. aureus. Its effects on tested fungi were moderate with petro ether fraction giving an inhibition of 10.3±0.3 on C. albicans. The crude extract and two fractions (petro ether and methanol) were not within the acceptable toxicity limits, however dichloromethane and ethyl acetate fractions that exhibited higher activity were within the acceptable toxicity limit (CC50<90). The activity can to some extent be associated to alkaloids, flavonoids, terpenoids, anthraquinones and phenols detected in this plant extracts. CONCLUSION: Our findings demonstrate that P. resinosa has high selective potential as a source of novel lead for antituberculous, antibacterial and antifungal drugs. Of particular relevance is high activity against MRSA, S. aureus, C. albicans and MTB which are great public health challenge due to drug resistance development and as major sources of community and hospital based infections.