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

Abstract

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 thatEGCG 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.