Plumbagin as a preferential lead molecule to combat EGFR-driven matrix abundance and migration of cervical carcinoma cells.

The handshake between the complex networks of matrix components in the tumor micro-environment (TME) is considered as a crucial event in the progression of several cancers including cervical carcinoma (CC). A number of studies report a connection between epidermal growth factor (EGF) and matrix component production. Studies demonstrate that the mechano-transduction trigger by collagen, influences the tumor cells to undergo epithelial-mesenchymal transition (EMT) and block the entry of drugs. We hypothesize that the intervention to prevent EGF triggered deposition of matrix components could sensitize several therapies for CC cells. We utilized morphological assessment, MTT assay, mitored tracking, acridine orange (AO)/ ethidium bromide (EtBr) staining and bromodeoxyuridine (BrdU) assay to measure the cell viability, mitochondrial activity, cellular apoptosis, and DNA synthesis. Clonogenic assay and scratch healing assay were executed to address the stemness and migratory potential. Detection of glycosaminoglycan's (GAGs), collagen, matrix metalloproteinase (MMP)-2/9 secretion and calcium (Ca2+) ions were performed to assess the production of matrix components. Finally, the interaction between EGFR and plumbagin was evaluated by employing molecular dynamics (MD) simulation. Pre-treating the cells with plumbagin inhibited the EGF-induced EMT along with reduction in cell proliferation, migration, clonogenesis and depletion of matrix components. The actions of EGF and plumbagin were more pronounced in HPV-positive CC cells than HPV-negative CC cells. This study identified that increased matrix production triggered by EGF-rich milieu is inhibited by plumbagin in human papilloma viral (HPV) 68 positive ME180, HPV 16 positive SiHa and HPV-negative C33A cell lines. Delivery of plumbagin directly to TME would effectively accelerate the clearance of CC cells, reduce metastasis and matrix abundance by employing targeted delivery to minimize the undesired effects of plumbagin.

A comparative analysis of phyto-components on EGFR binding, viability, and migration in HPV positive ME180 and HPV negative C33A cervical cancer cells.

A need for effective implementation of cervical cancer (CC) even in developed countries insist the urge for developing an effective drug molecule to treat CC. Previously, we showed an inverse correlation between survival of CC patients and epidermal growth factor (EGF) receptor (EGFR) levels. Newer tyrosine kinase inhibitors to treat CC are being constantly pursued. In this context, the proposed study is an attempt to perform a comparative analysis using 20 phyto-components to determine the effective lead molecule. Molecular docking was utilized to determine the comparative efficacy of 20 phyto-components in binding to EGFR. It was then validated by cell viability, mitochondrial membrane potential, apoptosis, migration, and matrix metalloproteinase (MMP-2) in human papilloma virus (HPV) positive and HPV negative CC cells using top nine phyto-components based on computational screening. Computational analysis identified nine phyto-components out of which five compounds were effective in reducing the survival, mitochondrial membrane potential, apoptosis, migration, and MMP-2 secretion. EGCG, plumbagin, quercetin, emodin, and naringenin were identified as effective molecules in attenuating CC survival, proliferation, and migration.

Pharmacophore based virtual screening for identification of effective inhibitors to combat HPV 16 E6 driven cervical cancer.

Targeting HPV16 E6 has emerged as an effective drug target for the treatment/management of cervical cancer. We utilized pharmacophore-based virtual screening, molecular docking, absorption, distribution, metabolism and excretion (ADME) prediction, and molecular dynamics simulation approach for identifying potential inhibitors of HPV16 E6. Initially, we generated a ligand-based pharmacophore model based on the features of four known HPV16 E6 inhibitors (CA24, CA25, CA26, and CA27) via the PHASE module implanted in the Schrödinger suite. We constructed four-point pharmacophore features viz., three hydrogen bond acceptors (A) and one aromatic ring (R). The common pharmacophore feature further employed as a query for virtual screening against the ASINEX database via Schrödinger suite. The pharmacophore-based virtual screening filtered out top 2000 hits, based on the fitness score. We then applied the high throughput virtual screening (HTVS), standard precision (SP) and extra precision (XP). 1000 compounds were obtained from HTVS docking. Based on the glide score, they were further filtered to 500 hits by employing docking in standard precision mode. Finally, the best four hits and a negative molecule were identified using docking in XP mode. The four lead compounds and a negative molecule were then further subjected to ADME profile prediction by engaging Qikprop module. The ADME properties of the four lead molecules indicate good pharmacokinetic (PK) properties rather than the negative molecule. The binding stability of the HPV16 E6-hit complexes were investigated at a different time scale (100 ns) by using the desmond package and the results were examined using Root Mean Square Deviation (RMSD) and Root Mean Square Fluctuation (RMSF) and it revealed the stability of the protein-ligand complex throughout the simulation. Key residues, CYS 51 and GLN 107, also play a crucial role in enhancing the stability of the protein-ligand complex during the simulation. Furthermore, the binding free energy of the HPV16 E6-leads complexes was analyzed by prime which revealed that the ΔGbind coulomb and ΔGbind vdW interactions are crucially contributes to the binding affinity. In order to validate the computational findings, the efficacy of benzoimidazole and benzotriazole were ascertained for regulating ME180 cervical cancer cell survival, migration and ability to release MMP-2.

EGCG attenuate EGF triggered matrix abundance and migration in HPV positive and HPV negative cervical cancer cells.

Our previous laboratory findings suggested the beneficial effects of epigallocatechin gallate (EGCG) against cervical cancer (CC) cells survival. The present study is aimed at identifying the effects of EGCG in preventing the actions of epidermal growth factor (EGF) in human papilloma virus (HPV) 68 positive ME180 and HPV negative C33A CC cells. An elevated level of EGF in tumor micro-environment (TME) is linked to the metastasis of several cancers including CC. We hypothesized that EGCG has the ability to block the actions of EGF. To test this, survival assay was performed in cells treated with or without EGF and EGCG. The mitochondrial activity of cells was ascertained using MTT assay and mitored staining. Protein and non-protein components in the extracellular matrix such as collagen and sulphated glycosaminoglycans (GAGs) were evaluated using sirius red and alcian blue staining, respectively. Matrix metalloproteinase-2 (MMP-2) gene expression and enzymatic activity were assessed using real time-reverse transcriptase-polymerase chain reaction (RT-PCR) and gelatin zymography. Wound healing assay was performed to assess the EGF induced migratory ability and its inhibition by EGCG pre-treatment. Clonogenic assay showed that EGCG pre-treatment blocked the EGF driven colony formation. In silico analysis performed identified the efficacy of EGCG in binding with different domains of EGF receptor (EGFR). EGCG pre-treatment prevented the epithelial-mesenchymal transition (EMT) and metabolic activity induced by EGF, this is associated with concomitant reduction in the gene expression and enzyme activity of MMP-2. Further, reduced migration and ability to form colonies were observed in EGCG pre-treated cells when stimulated with EGF. HPV positive ME180 cells showed increased migratory and clonogenic ability upon EGF stimulation, whose effects were not much significant in HPV negative C33A cells. EGCG effectively blocked the actions of EGF in both HPV positive and HPV negative conditions and can be advocated as supplementary therapy for the management of EGF driven CC. However, further studies using cell line-derived xenograft (CDX)/patient-derived xenograft (PDX) model system is warranted to validate the therapeutic utility of EGCG.

A review on the role of epidermal growth factor signaling in the development, progression and treatment of cervical cancer.

The sub-committee constituted by the Indian Council of Medical Research (ICMR) for the management of cervical cancer (CC) detailed in the consensus document (2016) reported CC as a significant cause of morbidity and mortality in women. The incidence of an increase in CC and associated mortality in women is a major cause of cancer. To date, human papilloma viral (HPV) infection accounts for more than 99% of CC. However, there are individuals infected with HPV do not develop CC. There is a greater correlation between HPV infection and upregulation of the epidermal growth factor receptor (EGFR) signaling cascade during the initiation, sustenance, and progression of CC. Therefore, EGFR is often targeted to treat CC using tyrosine kinase inhibitors (TKIs) and monoclonal antibodies (mAB). The current review analyzed the existing clinical/pre-clinical studies and the significance of EGFR abundance using the Kaplan-Meier (KM) survival plot analysis for disease-free survival (DFS) and overall survival (OS). We performed a series of bioinformatics analyses to screen the crucial role of the EGFR gene in CC. Further, different transcription factors that are dysregulated due to EGFR abundance and their relevance were determined using computational tools in this review. Endogenous microRNAs (miRNA) that undergo changes due to alterations in EGFR during CC were identified using computational database and consolidated the information obtained with the published in the area of miRNA and EGFR with special reference to the initiation, sustenance and progression of CC. The current review aims to consolidate contemporary approaches for targeting CC using EGFR and highlight the current role of miRNA and genes that are differently regulated during CC involving EGFR mutations. Potential resistance to the available EGFR therapies such as TKIs and mABs and the need for better therapies are also extensively reviewed for the development of newer therapeutic molecules with better efficacy.