In breast cancer, over-expression of EGFR and HER2 is often correlated with poor clinical prognosis and decreased overall survival. Lapatinib and Gefitinib are EGFR antagonists used to act on breast cancer cells with high levels of EGFR due to their ability to activate apoptosis and inhibit cell proliferation. However, the resistance of tumor cells to drug-induced apoptosis has hindered effective treatment. We propose that this resistance to treatment is a result of dys-regulation of inhibitor of apoptosis proteins (IAPs), which plays a key role in caspase inhibition. Hence, we hypothesized that IAP-mediated apoptosis resistance may contribute to insensitivity towards cancer treatment. We suggest that inhibition of IAP, namely Survivin and cIAP2, can overcome the intrinsic resistance of mouse mammary gland cells to targeted therapies against EGFR receptors, namely Lapatinib and Gefitinib, and increase their efficiency.
2. Materials and methodology
Cells were plated in 6-well plates at a density of 1 × 105 and grown in DMEM medium. Following cell adherence, siRNA transfection was performed to reduce the levels of Survivin, cIAP2 or a combination of both. Following transfection, the control and IAPs- inhibited cells were either incubated for 24 hours and harvested for Western Blotting to determine reduction in IAP levels, or incubated for another 2 hours in preparation for drug treatments of Lapatinib and Gefitinib. After exposure to treatment, untreated and treated samples were further incubated for another 96 h and harvested to examine effects on apoptosis and cell proliferation. Cells were collected for apoptotic studies using ApoDirect TUNEL-staining kit. The samples were prepared and stained with FITC and PI. The samples were then analyzed by flow cytometry and immunofluorescence (IF). Cell proliferation was measured using MTT Cell Proliferation.
3. Results and discussion
It is observed from the Western Blotting results that levels of both Survivin and cIAP2 have been successfully inhibited through transfection with siRNA.
To assess if the reduction in levels of IAPs would increase apoptosis, flow cytometry and IF were used to measure apoptosis qualitatively and quantitatively. Results from analysis of the TUNEL assay by IF and flow cytometry revealed that reduction in levels of IAPs increased the apoptosis of these cells when exposed to Lapatinib and Gefitinib. This indicates that the combination of IAP antagonists with drugs that target EGFR receptors promotes apoptosis and increases efficiency of drug treatments.
The results from cell proliferation show that the inhibition of IAPs significantly reduced number of viable cells. The knockdown of IAPs, even in the absence of any treatment is sufficient to cause a consistent decrease in cell proliferation of about 20% compared to control cells. However, we also found that the combined effect of inhibiting both IAPs did not result in any greater decrease in rate of cell proliferation as compared to inhibiting either one IAP.
From our findings, we suggest that the use of IAP antagonists in combination with clinically relevant EGFR antagonists decrease cell proliferation and promote apoptosis. We therefore propose that, together with appropriate biomarkers, treating certain patients with IAP inhibitors and EGFR antagonists together could be of clinical value.
The inhibition of IAPs increases the efficiency of treatments with Lapatinib and Gefitinib
The combination of IAP antagonists with drugs that target EGFR receptors promotes apoptosis
The combination of IAP antagonists with drugs that target EGFR receptors leads to reduction of the cell proliferation of non-malignant mouse mammary gland cells.