Dual targeting of HSP90 and BCL-2 in breast cancer cells using inhibitors BIIB021 and ABT-263

Abstract

Purpose: The incidence of breast cancer has been increasing in recent years, and monotherapy approaches are not sufficient alone in the treatment of breast cancer. In the combined therapy approach, combining two or three different agents in lower doses can mitigate the side effects on living cells and tissues caused by high doses of chemical agents used alone. ABT-263 (navitoclax), a clinically tested Bcl-2 family protein inhibitor, has shown limited success in clinical trials due to the development of resistance to monotherapy in breast cancer cells. This resistance shows that monotherapy approaches are inadequate and more effective treatment strategies are needed. It is the ability of HSP90 inhibitors to destabilize many oncoproteins that are critical for the survival of cancer cells. This study aimed to examine the anticancer activity of the combination of ABT-263 with BIIB021, a new generation HSP90 inhibitor, on two widely used breast cancer cell lines: MCF-7 (ER-positive) and MDA-MB-231 (triple-negative breast cancer, TNBC). These cell lines were selected to represent distinct breast cancer subtypes with different molecular characteristics and clinical behaviors.

Methods: Single and combined cytotoxic effects of this agents on MCF-7 and MDA-MB-231 breast cancer cell lines were determined using the MTT cell viability test. The combined use of these two agents showed a synergistic effect, and this effect was assigned using the Chou and Talalay method. mRNA and protein levels of apoptosis-related genes Bax, Bcl-2, Casp9, and Heat Shock Proteins HSP27, HSP70, and HSP90 were analyzed using Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) and Western Blotting, respectively.

Results: The cytotoxicity analysis, combined with the application of the Chou-Talalay method, demonstrated that the BIIB021 and ABT-263 combination exhibited significantly greater anticancer activity compared to the individual effects of either BIIB021 or ABT-263 in breast cancer cell lines. The analysis of mRNA and protein levels indicated that the BIIB021+ABT-263 combination may have triggered the intrinsic apoptotic pathway in breast cancer cells.

Conclusion: This study showed that co-administration of ABT-263 and BIIB021 agents exhibited synergistic cytotoxic effects and increased the expression of apoptosis-related genes in breast cancer cell lines.

Keywords: BIIB021; Breast cancer; Combination treatment; Hsp90 inhibition; Navitoclax.

Fig. 1

The dose- and time-dependent effects of ABT-263 and BIIB021 on the viability of MCF-7 and MDA-MB-231 cell lines, along with the calculated IC₅₀ values of ABT-263 and BIIB021. The cell viability of MCF-7 (A) and MDA-MB-231 (B) cells after exposure to various doses of ABT-263 after 24 and 48 h. The cell viability of MCF-7 (C) and MDA-MB-231 (D) cells after exposure to various doses of BIIB021 after 24 and 48 h

Fig. 2

The combined cytotoxic effects of ABT-263 and BIIB021 in MCF-7 (A) and MDA-MB-231 (B) cancer cell lines. Fraction Affected (Fa) vs Combination Index (CI) plots derived from Chou-Talalay median-effect analysis for MCF-7 (C) and MDA-MB-231 (D) cell lines, respectively. These plots illustrate the relationship between the fraction of affected cells (Fa) and the combination index (CI), providing insights into the synergistic, additive, or antagonistic effects of the drug combination. Additionally, the terms ED30, ED50, and ED75 denote the doses at which 30, 50, and 75% of cells or organisms are effectively inhibited

Fig. 3

Changes in the expression of apoptosis-related proteins (Bax, Bcl-2, Casp-9) and heat shock proteins (Hsp27, HSP70, HSP90) in MCF-7 cells after a 48-h treatment with BIIB021, ABT-263, and BIIB021 + ABT-263. Gene expression (A) and protein expression (B) changes in apoptosis-related proteins (Bax, Bcl-2, pro-Casp-9) and HSPs (HSP27, HSP70, HSP90), the abundance of apoptosis-related proteins and HSPs in MCF-7 (C). (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 4

Changes in the expression of apoptosis-related proteins (Bax, Bcl-2, Casp-9) and heat shock proteins (Hsp27, HSP70, HSP90) in MDA-MB-231 cells after a 48 h treatment with BIIB021, ABT-263, and BIIB021 + ABT-263. Gene expression (A) and protein expression (B) apoptosis-related proteins (Bax, Bcl-2, pro-Casp-9) and HSPs (HSP27, HSP70, HSP90), the abundance of apoptosis-related proteins and HSPs in MCF-7 (C). (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001)

Fig. 5

A Protein–protein interaction network analysis of heat shock proteins (HSP90, HSP70, HSP27) and apoptosis-related proteins (Bax, Bcl-2, Casp-9) using the STRING database and visualized with Cytoscape v3.10.2. This network highlights the interactions and potential functional relationships between these proteins. B Pathway enrichment analysis using KEGG and Gene Ontology (GO) biological processes performed with ClueGo v2.5.10 and CluePedia v1.5.10 via Cytoscape v3.10.2