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. 2021 Oct 19;22(20):11273.
doi: 10.3390/ijms222011273.

Oleanolic Acid's Semisynthetic Derivatives HIMOXOL and Br-HIMOLID Show Proautophagic Potential and Inhibit Migration of HER2-Positive Breast Cancer Cells In Vitro

Natalia Magdalena Lisiak  1 , Izabela Lewicka  1 , Mariusz Kaczmarek  2   3 , Jacek Kujawski  4 , Barbara Bednarczyk-Cwynar  4 , Lucjusz Zaprutko  4 , Blazej Rubis  1
Affiliations

Oleanolic Acid's Semisynthetic Derivatives HIMOXOL and Br-HIMOLID Show Proautophagic Potential and Inhibit Migration of HER2-Positive Breast Cancer Cells In Vitro

Natalia Magdalena Lisiak et al. Int J Mol Sci. .

Abstract

Approximately 20-30% of the diagnosed breast cancers overexpress the human epidermal growth factor receptor 2 (HER2). This type of cancer is associated with a more aggressive phenotype; thus, there is a need for the discovery of new compounds that would improve the survival in HER2-positive breast cancer patients. It seems that one of the most promising therapeutic cancer strategies could be based on the biological activity of pentacyclic triterpenes' derivatives and the best-known representative of this group, oleanolic acid (OA). The biological activity of oleanolic acid and its two semisynthetic derivatives, methyl 3-hydroxyimino-11-oxoolean-12-en-28-oate (HIMOXOL) and 12α-bromo-3-hydroxyimonoolean-28→13-olide (Br-HIMOLID), was assessed in SK-BR-3 breast cancer cells (HER2-positive). Viability tests, cell cycle assessment, evaluation of apoptosis, autophagy, and adhesion/migration processes were performed using MTT, clonogenic, cytofluorometry, Western blot, and qPCR. Both derivatives revealed higher cytotoxicity in studied breast cancer cells than the maternal compound, OA. They also decreased cell viability, induced autophagy, and (when applied in sub-cytotoxic concentrations) decreased the migration of SK-BR-3 cells.This study is the first to report the cytostatic, proautophagic (mTOR/LC3/SQSTM/BECN1 pathway), and anti-migratory (integrin β1/FAK/paxillin pathway) activities of HIMOXOL and Br-HIMOLID in HER2-positive breast cancer cells.

Keywords: Br-HIMOLID; HER2; HIMOXOL; autophagy; breast cancer; migration; oleanolic acid; oleanolic acid derivatives.

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Conflict of interest statement

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.

Figures

Figure 1
Figure 1
The first poses of the docked OA (A), HIMOXOL (B), and Br-HIMOLID (C) ligands with surroundings amino acids within the cavity (LigPlot+ v.2.2 software [22,23]).
Figure 2
Figure 2
The RMSD plot for the ligand within ligand–protein complex during the MD productive phase was calculated for the complex of OA (red), HIMOXOL (green), or Br-HIMOLID (blue) and the 3pp0.pdb cavity.
Figure 3
Figure 3
Contribution of oleanolic acid and its derivatives to breast cancer cells viability. SK-BR-3 cells were treated for 24 or 72 h with a high range of concentrations of studied compounds, i.e., 1–100 µM. The mean of three experiments ±SD is shown. Statistically significant difference versus control samples is shown: * p < 0.05; ** p < 0.005; *** p < 0.001.
Figure 4
Figure 4
Influence of OA and its derivatives on the colonies formation of SK-BR-3 cells. The cells were plated in each well of a 6-well plate at 100 cells/mL and were allowed to adhere for 24 h. They were then treated with studied compounds in three different concentrations, corresponding to 0.5× IC50, 1× IC50, or 1.5× IC50 for 24 h. After the specified time interval, media was replaced and the cells were grown for ten days with one media change on the fourth day. The colonies formed were fixed with formaldehyde and stained with crystal violet. The wells were then washed, air-dried, and the colonies were enumerated. The experiment was repeated at least two times; × ±SD, p < 0.05. Statistically significant difference versus control samples is shown: * p < 0.05; ** p < 0.005; *** p < 0.001.
Figure 5
Figure 5
Evaluation of cell cycle modulation and proapoptotic potential of OA and its derivatives. SK-BR-3 cells were treated with studied compounds and 0.5×, 1×, or 1.5× IC50 values were applied followed by immunodetection. (A) Immunoidentification of target proteins was subjected to densitometry analysis. (B), normalized to GAPDH. Gefitinib (Gef, 20 µM) was used as a positive control. The mean value of three experiments ±SD is shown; p < 0.05. (C). Statistically significant difference versus control samples is shown: * p < 0.05; ** p < 0.005.
Figure 6
Figure 6
Evaluation of autophagy induction by OA and its derivatives. SK-BR-3 cells were treated with studied compounds, i.e., 0.5× 1× or 1.5× IC50 respective values were applied followed by monodansylcadaverine (MDC) staining (mean fluorescence, MFI, detection) and immunodetection. (A) Bars represent relative MFI of MDC staining in the indicated samples versus corresponding controls; B, immunoidentification of target proteins was subjected to densitometry analysis normalized to GAPDH. Gefitinib (Gef, 20 µM) was used as a positive control. The mean of three independent experiments is shown with p < 0.05 calculated from paired data. × ±SD, p < 0.05 (B). Statistically significant difference versus control samples is shown: * p < 0.05; ** p < 0.005; *** p < 0.001. (C) Evaluation of autophagy induction by OA and its derivatives in breast cancer cells. Cells were treated with studied compounds, i.e., 0.5×, 1×, or 1.5× IC50 respective values for 24 h. Gefitinib (Gef, 20 µM) was used as a positive control. Expression of target genes (MAPLC3, ECN1, SQSTM (p62), RICTOR (mTOR) at the mRNA level was performed using qPCR (C). GAPDH was used as a reference gene. The mean of three independent experiments ±SD is shown with p values calculated from paired data. Statistically significant difference versus control samples is shown: * p < 0.05; ** p < 0.005; *** p < 0.001.
Figure 7
Figure 7
Influence of OA and its derivatives on migration of SK-BR-3 cells. (A) Effect of OA and its derivatives, HIMOXOL and Br-HIMOLID, on wound healing in SK-BR-3 breast cancer cells. Cells were scraped with a pipette tip and treated with OA or its derivatives in a concentration corresponding to 0.5× IC50 for 24 h. The photos represent cell migration under the microscope at 100 × magnification before (Control, 0 h) and after scratch. The experiments were repeated three times. *** p < 0.001 compared to the control group. (B) Contribution of OA and its derivatives to adhesion and migration of breast cancer cells. Evaluation of integrin β1, FAK, and paxillin protein accumulation in the SK-BR-3 cells (8 × 104) was performed using immunodetection followed by densitometry analysis. Cells were treated with OA and its derivatives (Br-HIMOLID and HIMOXOL) in a concentration corresponding to 0.5× IC50 for 24 h. The mean of three independent experiments ±SD is shown with p values calculated from paired data. Statistically significant difference versus control samples is shown: * p < 0.05; ** p < 0.005; *** p < 0.001. (C) Contribution of OA and its derivatives to the expression of genes contributing to a migration-associated pathways in breast cancer cells. SK-BR-3 cells (8 × 104) were treated with OA, Br-HIMOLID, or HIMOXOL for 24 h. Expression of target genes (integrin β1, FAK, and paxillin) was evaluated using specific primers and qPCR, relative to housekeeping gene GAPDH. Data from three independent experiments are shown as mean ±SD. The mean of three independent experiments ±SD is shown with p-values calculated from paired data. Statistically significant difference versus control samples is shown: * p < 0.05; ** p < 0.005; *** p < 0.001.
Figure 8
Figure 8
Structure of studied compounds.
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