Anticancer Activity of HER2-targeting CPP-PTEN-THP Chimeric Proteins.

BACKGROUND/AIM: Protein phosphatase and tensin homolog (PTEN) is a tumor suppressor protein with potential to be a new biotechnological drug for PTEN-deficient cancer treatment. This study aimed to develop PTEN-based chimeric proteins (CPP-PTEN-THP) for human epidermal growth factor receptor 2 (HER2)-positive breast cancer treatment, addressing current limitations like inadequate delivery, poor tumor penetration, and low selectivity, while assessing their potential HER2-specific anticancer effects. MATERIALS AND METHODS: pCEFL-EGFP vector was used for both TAT-PTEN-LTV and KLA-PTEN-LTV construction. Non-contact co-cultures were employed using HEK-293T cells for protein expression, and HCC-1954 and MCF-7 cell lines for cytotoxicity testing. Protein detection was analyzed by western blotting and a docking prediction analysis was performed to infer the interactions. RESULTS: Endogenous and recombinant PTEN protein expression was confirmed in cell lysates. A 54-kDa signal matching the theoretical size of PTEN was detected, showing a greater level in TAT-PTEN-LTV (215.1±26.45%) and KLA-PTEN-LTV (129.2±1.44%) compared to endogenous PTEN. After the noncontact co-culture method, cytotoxic studies showed HCC-1954 preferential cell inhibition growth, with 25.95±0.9% and 12.25±1.29% inhibition by KLA-PTEN-LTV and TAT-PTEN-LTV respectively, compared to MCF-7 cells. An LTV-HER2 interaction model was proposed, inferring that LTV interactions are mainly due to the Pro, Trp, and Tyr residues that target HER2. CONCLUSION: The developed PTEN-based chimeric proteins have HER2-specific anticancer activity against HCC-1954 cells.

Design and Evaluation of a Humanized Anti-EGFR huscFv Antibody Fragment in Combination With Metformin in A549 Cells In Vitro.

BACKGROUND/AIM: The epidermal growth factor receptor (EGFR) is over-expressed in several types of cancer, and monoclonal antibody therapy has been the strategy that has shown the best results. This study focused on the construction of a humanized single chain antibody (huscFv) directed against EGFR (HER1). MATERIALS AND METHODS: The CDR grafting method was used to incorporate murine complementarity determining regions (CDRs) of cetuximab into human sequences. A dot blot assay was used to examine the affinity of the huscFv secreted by HEK293T for EGFR. The inhibitory effect on the viability of A549 cells was evaluated using the WST-1 assay. RESULTS: The incorporation of murine CDRs of cetuximab into human sequences increased the degree of humanness by 16.4%. The increase in the humanization of scFv did not affect the affinity for EGFR. Metformin had a dose-dependent effect, with an IC50 of 46 mM, and in combination with huscFv, the cell viability decreased by 45% compared to the 15% demonstrated by huscFv alone. CONCLUSION: The CDR grafting technique is efficient for the humanization of scFv, maintaining its affinity for EGFR and demonstrating its inhibitory effect when combined with metformin in A549 cells.

Molecular Cloning and Transient Expression of Recombinant Human PPARγ in HEK293T Cells Under an Inducible Tet-on System.

Peroxisome proliferator-activated receptor gamma (PPARγ) is involved in the regulation of lipid and glucose homeostasis and inflammation. PPARγ expression level has been widely studied in multiple tissues; however, there are few reports of preceding attempts to produce full-length human PPARγ (hPPARγ) in cellular models, and generally, expression level is not known or measurable. We propose an alternative strategy to express recombinant hPPARγ1, using a transient transfection with an inducible Tet-On 3G system where target and reporter gene were cloned in the same open reading frame. We transiently co-transfected human embryonic kidney 293T (HEK293T) cells with pTRE-ZsGreen1-IRES2-hPPARγ1 and pCMV-TET3G for inducible expression of hPPARγ1. Relative expression of the transcript was evaluated by RT-qPCR 48 h after transfection, obtaining a high expression level of hPPARγ (530-fold change, p < 0.002) in co-transfected HEK293T cells in the presence of doxycycline (1 µg/mL); also a significantly increased production of the reporter protein ZsGreen1 (3.6-fold change, p < 0.05) was determined by fluorescence analysis. These data indicated that HEK293T cells were successfully co-transfected and it could be an alternative model for hPPARγ expression in vitro. Additionally, this model will help to validate the quantification of inducible hPPARγ expression in vivo models for future research.