Inhibition of NF-kB and COX-2 by andrographolide regulates the progression of cervical cancer by promoting PTEN expression and suppressing PI3K/AKT signalling pathway.

In the face of recent advances in Cervical cancer (CC) treatment, therapeutic and surgical procedures for CC management are still inadequate. In the current study for the first time Andrographolide (Andro) has been explored for its multitarget therapeutic efficacy on NF-kB, COX-2, and PI3K/AKT expressions together in CC. The expression levels of NF-kB, COX-2, PI3K and PTEN in the CC patient samples, both at mRNA and protein levels have shown significant association with poor survival and increased tumor aggressiveness. The binding efficacy of Andro was investigated using molecular docking and molecular dynamic simulations, and the protein and ligand complex for NF-kB and COX-2 has shown high binding energy. Andro displayed cytotoxicity by impeding the in-vitro proliferation of CC cells. Andro significantly supressed the NF-kB, COX-2, and PI3K expression and enhanced the expression levels of PTEN at protein levels in-vitro. Andro induced apoptosis in a dose dependent manner and significantly inhibited the migration and invasion of CC cells. Andro exhibited similar activity in-vivo and suppressed the CC tumor growth in xenograft C57BL/6 mice model. The anti-tumor activity of Andro, both in-vitro and in-vivo has shown considerable downregulation of NF-kB and COX-2 and induced apoptosis through impeding the PI3K/AKT signalling pathway. These findings from the above study projects, administration of Andro as an effective alternate safe compound to curtail and impede cervical cancer progression.

Virtual Screening, Molecular Docking, and Dynamic Simulations Revealed TGF-ß1 Potential Inhibitors to Curtail Cervical Cancer Progression.

Cervical cancer is one of the main causes of cancer death in women globally, and its epidemiology is similar to that of a low-infectious venereal illness. Many sexual partners and early age at first intercourse have been demonstrated to have a significant influence on risk. TGF-ß1 is a multifunctional cytokine that is required for cervical carcinoma metastasis, tumor development, progression, and invasion. The TGF-ß1 signaling system plays a paradoxical function in cancer formation, suppressing early-stage tumor growth while increasing tumor progression and metastasis. Importantly, TGF-ß1 and TGF-ß receptor 1 (TGF-ßR1), two components of the TGF-ß signaling system, are substantially expressed in a range of cancers, including breast cancer, colon cancer, gastric cancer, and hepatocellular carcinoma. The current study aims to investigate possible inhibitors targeting TGF-ß1 using molecular docking and dynamic simulations. To target TGF-ß1, we used anti-cancer drugs and small molecules. MVD was utilized for virtual screening, and the highest scoring compound was then subjected to MD simulations using Schrodinger software package v2017-1 (Maestro v11.1) to identify the most favorable lead interactions against TGF-ß1. The Nilotinib compound has shown the least XP Gscore of -2.581 kcal/mol, 30ns MD simulations revealing that the Nilotinib- TGF-ß1 complex possesses the lowest energy of -77784.917 kcal/mol. Multiple parameters, including Root Mean Square Deviation, Root Mean Square Fluctuation, and Intermolecular Interactions, were used to analyze the simulation trajectory. Based on the results; we conclude that the ligand nilotinib appears to be a promising prospective TGF-ß1inhibitor for reducing TGF-ß1 expression ad halting cervical cancer progression.

Entrectinib a Plausible Inhibitor for Osteopontin (SPP1) in Cervical Cancer-Integrated Bioinformatic Approach.

Cervical cancer is one of the major causes of death in women, especially in developing countries bearing more than a quarter of the global burden. Secreted phosphoprotein-1, also known as OPN (osteopontin), is an integrin-binding glycophosphoprotein that is overexpressed in a variety of tumors. OPN is a chemokine-like calcified ECM-associated protein that plays a crucial role in evaluating the metastatic potential of various cancers. However, the role of SPP1 in the tumor microenvironment and associated signaling pathways in CC is still unclear. In our study, three CC microarray datasets (GSE9750, GSE46857, and GSE67522) were obtained from the GEO database to identify the differentially expressed genes. Enrichment analysis was carried out by Enrichr and ShinyGO and the PPI interaction network was created by using String and Cytoscape. GEPIA datasets were used to validate the top 10 hub genes, and virtual screening, docking, and dynamic simulation studies were used to identify a suitable inhibitor against the OPN protein using MVD, PyRx, and GROMACS respectively. Our results show that a total of 11 DEGs were common for three datasets and gene ontology pathway enrichment analysis revealed that 2 biological processes i.e. programmed cell death and animal organ development commonly affected mechanisms in all three datasets. Docking and dynamic studies revealed that Entrectinib showed excellent binding affinity against OPN protein. Based on the results, we conclude that OPN is one of the most upregulated genes in cervical cancer and Entrectinib emerges to be a promising potential OPN inhibitor to curtail cervical cancer progression. Schematic representation: The schematic representation of methodology steps is illustrated in the graphical abstract. Schematic representation of methodology.

Molecular docking, synthesis, and biological evaluation of 7-azaindole-derivative (7AID) as novel anti-cancer agent and potent DDX3 inhibitor:-an in silico and in vitro approach.

The DEAD-box helicase family member DDX3 is involved in many diseases, such as viral infection, inflammation, and cancer. Many studies in the last decade have revealed the role of DDX3 in tumorigenesis and metastasis. DDX3 has both tumour suppressor and oncogenic effect, in the present study we have evaluated the expression levels of DDX3 in cervical squamous cell carcinoma at mRNA level via real-time PCR and protein level via Immunohistochemistry. DDX3 has become a molecule of interest in cancer biology that promotes drug resistance by adaptive response inevitably leading to treatment failure. One approach to avoid the development of resistant to disease is to create novel drugs that target the overexpressed proteins, we designed and synthesized a novel 7-azaindole derivative (7-AID) compound, {5-[1H-pyrrolo (2, 3-b) pyridin-5-yl] pyridin-2-ol]} that could lodge within the adenosine-binding pocket of the DDX3 (PDB ID: 2I4I). The binding efficacy of 7-AID compound with DDX3 was analysed by molecular docking studies. 7-AID was found to interact with the key residues Tyr200 and Arg202 from the Q-motif rendered by π-interactions and hydrogen bonds within the binding pocket with good docking score - 7.99 kcal/mol. The cytotoxicity effect of 7-AID compound was evaluated using MTT assay on human cervical carcinoma cells (HeLa) and breast cancer cells (MCF-7 and MDA MB-231) and the compound shown effective inhibitory concentration (IC50) on Hela cells 16.96 µM/ml and 14.12 and 12.69 µM/ml on MCF-7 and MDA MB-231, respectively. Further, the in-vitro, in-vivo anti-cancer and anti-angiogenic assessment of 7-AID compound was evaluated on Hela cells using scratch wound-healing assay, DAPI staining, cell cycle analysis, immunoblotting, and chorioallontoic membrane assay. Furthermore, the inhibitory effect of derivative compound on DDX3 was investigated in HeLa, MCF-7, and MDA MB-231 cells at the mRNA and protein levels. The results showed that the 7-AID compound effectively inhibited DDX3 in a dose-dependent manner, and the findings suggest that the compound could be used as a potential DDX3 inhibitor.

Inhibition of DDX3 and COX-2 by forskolin and evaluation of anti-proliferative, pro-apoptotic effects on cervical cancer cells: molecular modelling and in vitro approaches.

Several studies have reported up-regulation of both cyclooxygenase-2 (COX-2) and DEAD-box RNA helicase3 (DDX3) and have validated their oncogenic role in many cancers. Inhibition of COX-2 and DDX3 offers a potential pharmacological strategy for prevention of cancer progression. The COX-2 isoform is expressed in response to pro-inflammatory stimuli in premalignant lesions, including cervical tissues. This study elucidates the potential role of plant derived compound Forskolin (FSK) in plummeting the expression of COX-2 and DDX3 in cervical cancer. To establish this, the cervical cancer cells were treated with the FSK compound which induced a dose dependent significant inhibition of COX-2 and DDX3 expression. The FSK treatment also significantly induced apoptosis in cancer cells by modulating the expression of apoptotic markers like caspase-3, cleaved caspase-3, caspase-9, cleaved caspase-9, full length-poly ADP ribose polymerase (PARP), cleaved-poly ADP ribose polymerase (C-PARP) and Bcl2 in dose dependent manner. Further FSK significantly modulated the cell survival pathway Phosphatidylinositol 3-kinase (PI3-K)/Akt signalling pathway upon 24 h of incubation in cervical cancer cells. The molecular docking studies revealed that the FSK engaged the active sites of both the targets by interacting with key residues.

Overexpression of Secreted Phosphoprotein 1 (SPP1) predicts poor survival in HPV positive cervical cancer.

Cervical cancer (CC) is the most prevalent malignant gynecological tumor with limited treatments. The present study describes the role of SPP1 in cancer progression, SPP1 emerged as one of the most overexpressed genes identified through clariom D transcriptome microarray. This investigation aims towards identifying a potential gene with significant prognostic value for detection and early diagnosis of cervical cancer. The elevated expression of SPP1 in cervical squamous cell carcinoma tissue was validated across GEO (Gene Expression Omnibus) microarray data sets, TCGA (The Cancer Genome Atlas), and Oncomine databases. SPP1 expression was found to be prognostically significant, showing association with poor survival rate of the patients. Our study intended to assess the expression of secreted phosphoprotein (SPP1) gene at mRNA and protein levels, and to explore the association of single nucleotide polymorphisms of SPP1 with risk of CC. Further, receiver operating characteristics (ROC) curve was plotted to determine the levels of SPP1 to differentiate CC against control. Results revealed significant (p < 0.01) stage-wise upregulation of SPP1 in CC compared to the normal cervical tissue and this was further confirmed using Immunohistochemistry and real-time PCR. The ROC for SPP1 demonstrated good selective power to differentiate malignant CC and non-malignant cervical tissues. The SPP1 gene -443 T > C promoter polymorphisms are found to be significantly predominant in the disease group and Insilico analysis by the TRANSFAC software confirms its association with loss of STAT6 transcription factor binding site leading to overexpression of the SPP1.

Screening and Identification of Potential iNOS Inhibitors to Curtail Cervical Cancer Progression: an In Silico Drug Repurposing Approach.

Cervical cancer is the second most common cause of cancer deaths in women worldwide and remains the main reason of mortality among women of reproductive age in developing countries. Nitric oxide is involved in several physiological functions inclusive of inflammatory and immune responses. However, the function of NO in tumor biology is debatable. The inducible NOS (iNOS/NOS2) isoform is the one responsible to maintain the levels of NO, and it exhibits pleotropic effects in various cancers with concentration-dependent pro- and anti-tumor effects. iNOS triggers angiogenesis and endothelial cell migration in tumors by regulating the levels of vascular endothelial growth factor (VEGF). In drug discovery, drug repurposing involves investigations of approved drug candidates to treat various other diseases. In this study, we used anti-cancer drugs and small molecules to target iNOS and identify a potential selective iNOS inhibitor. The structures of ligands were geometrically optimized and energy minimized using Hyperchem software. Molecular docking was performed using Molegro virtual docker, and ligands were selected based on MolDock score, Rerank score, and H-bonding energy. In the study shown, venetoclax compound demonstrated excellent binding affinity to iNOS protein. This compound exhibited the lowest MolDock score and Rerank score with better H-bonding energy to iNOS. The binding efficacy of venetoclax was analyzed by performing molecular docking and molecular dynamic simulations. Multiple parameters were used to analyze the simulation trajectory, like root mean square deviation (RMSD), radius of gyration (Rg), and hydrogen bond interactions. Based on the results, venetoclax emerges to be a promising potential iNOS inhibitor to curtail cervical cancer progression.

Inhibition of Inducible Nitric Oxide Synthase (iNOS) by Andrographolide and In Vitro Evaluation of Its Antiproliferative and Proapoptotic Effects on Cervical Cancer.

This work is aimed at investigating the expression levels of inducible nitric oxide synthase (iNOS) in cervical cancer and identifying a potential iNOS inhibitor. The data mining studies performed advocated iNOS to be a promising biomarker for cancer prognosis, as it is highly overexpressed in several malignant cancers. The elevated iNOS was found to be associated with poor survival and increased tumor aggressiveness in cervical cancer. Immunohistochemical and RT-PCR investigations of iNOS showed significant upregulation of endogenous iNOS expression in the cervical tumor samples, thus making iNOS a potent target for decreasing tumor inflammation and aggressiveness. Andrographolide, a plant-derived diterpenoid lactone, is widely reported to be effective against infections and inflammation, causing no adverse side effects on humans. In the current study, we investigated the effect of andrographolide on the prognostic value of iNOS expression in cervical cancer, which has not been reported previously. The binding efficacy of andrographolide was analyzed by performing molecular docking and molecular dynamic simulations. Multiple parameters were used to analyze the simulation trajectory, like root mean square deviation (RMSD), torsional degree of freedom, protein-root mean square fluctuations (P-RMSF), ligand RMSF, total number of intramolecular hydrogen bonds, secondary structure elements (SSE) of the protein, and protein complex with the time-dependent functions of MDS. Ligand-protein interactions revealed binding efficacy of andrographolide with tryptophan amino acid of iNOS protein. Cancer cell proliferation, cell migration, cell cycle analysis, and apoptosis-mediated cell death were assessed in vitro, post iNOS inhibition induced by andrographolide treatment (demonstrated by Western blot). Results. Andrographolide exhibited cytotoxicity by inhibiting the in vitro proliferation of cervical cancer cells and also abrogated the cancer cell migration. A significant increase in apoptosis was observed with increasing andrographolide concentration, and it also induced cell cycle arrest at G1-S phase transition. Our results substantiate that andrographolide significantly inhibits iNOS expression and exhibits antiproliferative and proapoptotic effects on cervical cancer cells.

Identification of Differentially Expressed Genes in Cervical Cancer Patients by Comparative Transcriptome Analysis.

Cervical cancer is one of the most malignant reproductive diseases seen in women worldwide. The identification of dysregulated genes in clinical samples of cervical cancer may pave the way for development of better prognostic markers and therapeutic targets. To identify the dysregulated genes (DEGs), we have retrospectively collected 10 biopsies, seven from cervical cancer patients and three from normal subjects who underwent a hysterectomy. Total RNA isolated from biopsies was subjected to microarray analysis using the human Clariom D Affymetrix platform. Based on the results of principal component analysis (PCA), only eight samples are qualified for further studies; GO and KEGG were used to identify the key genes and were compared with TCGA and GEO datasets. Identified genes were further validated by quantitative real-time PCR and receiver operating characteristic (ROC) curves, and the highest Youden index was calculated in order to evaluate cutoff points (COPs) that allowed distinguishing of tissue samples of cervical squamous carcinoma patients from those of healthy individuals. By comparative microarray analysis, a total of 108 genes common across the six patients' samples were chosen; among these, 78 genes were upregulated and 26 genes were downregulated. The key genes identified were SPP1, LYN, ARRB2, COL6A3, FOXM1, CCL21, TTK, and MELK. Based on their relative expression, the genes were ordered as follows: TTK > ARRB2 > SPP1 > FOXM1 > LYN > MELK > CCL21 > COL6A3; this generated data is in sync with the TCGA datasets, except for ARRB2. Protein-protein interaction network analysis revealed that TTK and MELK are closely associated with SMC4, AURKA, PLK4, and KIF18A. The candidate genes SPP1, FOXM1, LYN, COL6A3, CCL21, TTK and MELK at mRNA level, emerge as promising candidate markers for cervical cancer prognosis and also emerge as potential therapeutic drug targets.

Role of Biosynthesized Ag-NPs Using Aspergillus niger (MK503444.1) in Antimicrobial, Anti-Cancer and Anti-Angiogenic Activities.

Green synthesis of nanoparticles is regarded as a safe and non-toxic process over conventional synthesis. Owing to the medicinal value of biologically derived biomolecules and utilizing them in synergy with nanoscience to offer more accurate therapeutic options to various diseases is an emerging field. One such study we present here with highlights of the synthesis and efficacy of biogenic silver nanoparticles produced from the extract of Aspergillus niger SAP2211 (accession number: MK503444.1) as an antimicrobial, anti-cancerous and anti-angiogenic agent. The synthesized Ag-NPs were characterized following UV-vis, FTIR, XRD, SEM and TEM, and were found to possess bactericidal activity against the selected pathogenic microbes, such as Staphylococcus aureus, Escherichia coli, and Salmonella typhi. Further, we evaluated cytotoxicity effect of this biogenic Ag-NPs using MMT assay on normal cardio myoblast (H9C2) and cancerous human cervical carcinoma (HeLa) cells. Doxorubicin used as positive control. This Ag-NPs have shown trivial cytotoxicity at the IC50 concentration on normal cells (IC50 = 47.17 µg/ml) over the cancer cells (IC50 = 8.609 µg/ml) with nearly 7 fold difference, indicating it as a selective anti-cancerous agent in contrast to standard drug doxorubicin (IC50 = 6.338 µg/ml). Further in-vitro assessment of wound healing capability by scratch wound healing assay, invasion by transwell matrigel invasion assay, and apoptosis via DAPI and annexin V-FITC assays were studied in HeLa cells. Synthesized biogenic Ag-NPs have shown to be anti-angiogenic in nature, which was established by in-vivo chick chorioallantois membrane assay. Overall, in vitro studies revealed that biogenic Ag-NPs positively inhibited migration, invasion, and induced apoptosis, and in-vivo CAM assay revealed that intercapillary network was reduced and the angiogenesis was inhibited.

Crotalaria verrucosa Leaf Extract Mediated Synthesis of Zinc Oxide Nanoparticles: Assessment of Antimicrobial and Anticancer Activity.

In this work, we present an ecofriendly, non-hazardous, green synthesis of zinc oxide nanoparticles (ZnO NPs) by leaf extract of Crotalaria verrucosa (C. verrucosa). Total phenolic content, total flavonoid and total protein contents of C. verrucosa were determined. Further, synthesized ZnO NPs was characterized by UV-visible spectroscopy (UV-vis), X-ray diffractometer (XRD), Fourier transform infra-red (FTIR) Spectra, transmission electron microscope (TEM), and Dynamic light scattering (DLS) analysis. UV-vis shows peak at 375 nm which is unique to ZnO NPs. XRD analysis demonstrates the hexagonal phase structures of ZnO NPs. FTIR spectra demonstrates the molecules and bondings associated with the synthesized ZnO NPs and assures the role of phytochemical compounds of C. verrucosa in reduction and capping of ZnO NPs. TEM image exhibits that the prepared ZnO NPs is hexagonal shaped and in size ranged between 16 to 38 nm which is confirmed by DLS. Thermo-gravimetric analysis (TGA) was performed to determine the thermal stability of biosynthesized nanoparticles during calcination. The prepared ZnO NPs showed significant antibacterial potentiality against Gram-positive (S. aureus) and Gram-negative (Proteus vulgaris, Klebsiella pneumoniae, and Escherichia coli) pathogenic bacteria and SEM image shows the generalized mechanism of action in bacterial cell after NPs internalization. In addition, NPs are also found to be effective against the studied cancer cell lines for which cytotoxicity was assessed using MTT assay and results demonstrate highest growth of inhibition at the concentration of 100 µg/mL with IC50 value at 7.07 µg/mL for HeLa and 6.30 µg/mL for DU145 cell lines, in contrast to positive control (C. verrucosa leaf extract) with IC50 of 22.30 µg/mL on HeLa cells and 15.72 µg/mL on DU145 cells. Also, DAPI staining was performed in order to determine the effect on nuclear material due to ZnO NPs treatment in the studied cell lines taking leaf extract as positive control and untreated negative control for comparison. Cell migration assay was evaluated to determine the direct influence of NPs on metastasis that is potential suppression capacity of NPs to tumor cell migration. Outcome of the synthesized ZnO NPs using C. verrucosa shows antimicrobial activity against studied microbes, also cytotoxicity, apoptotic mediated DNA damage and antiproliferative potentiality in the studied carcinoma cells and hence, can be further used in biomedical, pharmaceutical and food processing industries as an effective antimicrobial and anti-cancerous agent.