Cell permeable PKCβII peptide inhibition reduced cell viability in MCF-7 breast cancer cells: Implications for ER+ breast cancer treatment

Philadelphia, PA

Introduction

MCF-7 human breast cancer cells have long been used as a model for hormone receptor-positive breast cancer, which is characterized by estrogen receptor-positive (ER+), progesterone receptor-positive (PR+), and human epidermal growth factor receptor 2-negative (HER2-) status. In ER+ breast cancer, estrogen binding stimulates the transcription of genes crucial to unchecked cell proliferation, such as cyclin D1. Protein Kinase C beta II (PKCβII) has been shown to enhance estrogen-driven growth in ER+ breast cancer through phosphorylation of estrogen receptors (ERs), leading to increased transcriptional activity and faster tumor growth. Additionally, PKCβII reduces apoptosis through upregulation of Bcl-2 expression and suppression of p53. By simultaneously enhancing proliferation and inhibiting apoptosis, PKCβ-II contributes to therapeutic resistance in hormone-positive breast cancer, limiting the efficacy of standard treatments. We hypothesize that treatment of MCF-7 cells with PKCβII inhibitor (PKCβII-) in vitro will reduce breast cancer cell viability. By targeting key signaling pathways implicated in the pathogenesis of ER+ breast cancer, we aim to explore the potential of PKCβII inhibition as a therapeutic adjunct to current treatment modalities.

Methods

MCF-7 were cultured in a 96-well plate, with 8 wells serving as untreated controls and 32 wells allocated for treatment with varying concentrations of PKCβII- conjugated to cell-permeable N-terminus conjugation of myristic acid (Myr) and Transactivator of Transcription (Tat) peptide (YGRKKRRQRRR) along with a cysteine-cysteine (C-C) spacer conjugated to PKCβII- cargo sequence (SLNPEWNET) (Myr-Tat-PKCβII-). Each concentration was tested in 8 replicate wells, while the remaining wells were left unused. The concentrations of Myr-Tat-PKCβII- used were obtained through serial dilution to achieve concentrations of 0.5-20μM. MCF-7 cells were treated with Myr-Tat-PKCβII- (0.5-20μM) or untreated control for 37 minutes. Cell viability was assessed by light microscopy and spectrophotometric analysis using a cell counting kit. Lower absorbance (measured at 450nm) indicates reduced cell viability or increased cell death. Absorbance data were analyzed using student T-test.

Results

Myr-Tat-PKCβII- concentration-dependently promoted MCF-7 cell death at 20μM 0.5±0.06; n=5, p< 0.05 compared to controls (1.03±0.09, n=5). Notably, lower doses of Myr-Tat-PKCβII- (0.5-5μM) demonstrated higher absorbance values (1.16±0.05, 1.20±0.07, 1.17±0.06, 1.2±0.05, 1.13±0.04; n=5, p>0.05, respectively) than untreated control.

Discussion

The decrease in absorbance following Myr-Tat-PKCβII- treatment suggests a reduction in metabolic activity and cell survival in MCF-7 cells at 20μM of Myr-Tat-PKCβII- compared to untreated controls. These findings support the possibility of a potential benefit for the adjunctive use of Myr-Tat-PKCβII- in current ER+ breast cancer treatment at specific doses. Cancer cells often respond to partial inhibition of pro-survival signaling proteins, such as kinases, by compensatory activation of the same or parallel pathways. At lower doses of Myr-Tat-PKCβII-, MCF-7 cells may utilize these compensatory mechanisms to sustain cell viability, as indicated by increased absorbance. Compensation may become less effective once a higher threshold of inhibitory treatment is reached. Further testing with an increased sample size at an optimized concentration of Myr-Tat-PKCβII- could strengthen the statistical power of these findings and provide more insight into the molecular mechanisms underlying PKCβII inhibitor-mediated MCF-7 cell death.