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CURATOR
A pinboard by
Neha Arora

Ph.D Student, Indian Institute of Technology Guwahati

PINBOARD SUMMARY

Implication of Proteins in Cancer Therapeutics

Our body maintains the perfect balance between cell growth and cell death for its healthy survival. Breakdown of this mechanism leads to diseased condition. In cancer, there is a loss on the check of cell division leading to uncontrolled growth. This happens due to loss of tumor suppressor proteins whose main function is to regulate cell growth and division. One such tumor suppressor protein down regulated in cancer is PTEN. PTEN regulates a crucial signaling pathway inside our cells known as the AKT pathway. It is a dual specificity phosphatase that de-phosphorylates both lipid and protein substrates. One of the key substrate of PTEN is Phosphatidylinositol (3,4,5)-trisphosphate (PIP3). PTEN converts PIP3 to Phosphatidylinositol (4,5)-trisphosphate (PIP2). As a result PIP3 mediated activation of AKT is curbed. Once AKT level is reduced then the cycle of cell division is also under control. In case of most of the cancers, PTEN gene is mutated leading to low or no PTEN protein expression. Thus, the control over AKT pathway is lost. Our objective of study is to purify, characterize PTEN protein and deliver it exogenously to cancer cells and study its effect on regulation of AKT expression. To achieve our aim, we have cloned PTEN into bacterial expression system PGEX4T2 vector and obtained bacterially purified protein. The protein PTEN was characterized by biophysical techniques such as MALDI, circular dichroism and western blot. Once characterization was successful, the functional integrity of the purified protein was determined by Michaelis-Menten enzyme kinetics using para-nitrophenyl phosphate and PIP3 as substrates. To achieve delivery of the protein to cancer cells, the protein was bound to nanomaterial. The luminescent property of the nanomaterial was capitalized to achieve both delivery and tracking of the protein. Internalization studies were conducted using spectroscopic techniques. One crucial study is to determine the activity of the protein after binding to the nanomaterial. The activity of PTEN was determined by in vitro phosphates assay. Once the function of the protein was determined, we embarked on the study of cellular delivery and its subsequent implications in two cell lines namely; breast cancer cell line MCF7 and glioblastoma U-87 MG cells. It was interesting to observe cellular internalization and modulation of signaling, as determined by western blot studies, by exogenously delivered PTEN which paves the way for PTEN based therapy