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. 2023 Jul 14;24(14):11468.
doi: 10.3390/ijms241411468.

Synthesis, In Vitro, and In Vivo Investigations of Pterostilbene-Tethered Analogues as Anti-Breast Cancer Candidates

Guoxun Li  1 , Jian Li  1   2 , Wenqian Wang  1 , Xiaoqing Feng  1 , Xingkang Yu  1 , Shuo Yuan  1 , Wei Zhang  1 , Jialing Chen  1 , Caijuan Hu  1
Affiliations

Synthesis, In Vitro, and In Vivo Investigations of Pterostilbene-Tethered Analogues as Anti-Breast Cancer Candidates

Guoxun Li et al. Int J Mol Sci. .

Abstract

Pterostilbene has been found to be an active scaffold with anti-breast cancer (BC) action. In this study, fourteen pterostilbene-tethered analogues (2A-2N) were prepared and screened in vitro against MDA-MB-231 and MCF-7 cells. Meanwhile, their structures were characterized using 1H-NMR, 13C-NMR, and HRMS (ESI) spectroscopy techniques. Among them, analogue 2L displayed the most potent anti-proliferation effect on MDA-MB-231 (IC50 = 10.39 μM) and MCF-7 cells (IC50 = 11.73 μM). Furthermore, the meaningful structure-activity relationships suggested that the introduction of a saturated six-membered nitrogen heterocyclic ring into the side chain favored anti-BC capacity. Biological observations indicated that 2L could cause the typical morphological changes in apoptosis, namely an increase in reactive oxygen species level and a loss of mitochondrial membrane potential in BC cells. Importantly, 2L could induce mitochondrial-mediated apoptosis by regulating the expression of caspase-related proteins. Consistent with the results of our in vitro study, 2L apparently inhibited tumor growth in MDA-MB-231 xenograft mice without obvious toxicity. These findings revealed that 2L is expected to be a promising anti-BC lead compound that merits further investigations.

Keywords: anti-BC activity; mitochondrial apoptosis; pterostilbene; synthesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Chemical structures of pterostilbene-related derivatives (a–h) with anti-BC action.
Scheme 1
Scheme 1
The synthesis of PTE-based derivatives (2A–2N).
Figure 2
Figure 2
2L inhibited the proliferation of MDA-MB-231 (A) and MCF-7 (B) cells. The growth curve of BC cells treated with 2L (1.1, 3.3, 10 μM) for 0, 1, 2, and 3 days. The control group was treated with vehicle.
Figure 3
Figure 3
2L triggered apoptosis of BC cells. (A,D) Morphological alterations in 2L-treated MDA-MB-231 and MCF-7 cells captured using fluorescence microscopy. Representative photographs were obtained (scale bar: 50 μm). The white arrows represent the nucleus of apoptotic BC cells. (B,E) Flow cytometric analysis of apoptosis of BC cells treated with 2L. (C,F) The percentages of annexin V-positive BC cells. *** p < 0.001 vs. control.
Figure 4
Figure 4
2L triggered cell cycle arrest of BC cells. (A,C) Cell cycle distribution of BC cells was determined using the flow cytometry. (B,D) Statistical analysis for the cell cycle distribution of BC cells. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.
Figure 5
Figure 5
2L decreased MMP level in BC cells. (A,B) Fluorescence observation in MDA-MB-231 and MCF-7 cells under a fluorescence microscope (scale bar: 50 μm). (C,E) The variation in MMP level was measured using JC-1 assay kit. (D,F) Quantitative analysis of MMP in MDA-MB-231 and MCF-7 cells. *** p < 0.001 vs. control.
Figure 6
Figure 6
2L stimulated ROS production in MDA-MB-231 and MCF-7 cells and the role of ROS generation in 2L-induced apoptosis. (A,F) The fluorescence distribution of 2L-treated BC cells was observed and recorded (scale bar: 100 μm). (B,G) The variation in ROS level in BC cells was detected by flow cytometer. (C,H) Quantitative analysis of ROS in BC cells. (D,I) Apoptosis of cells was assessed by flow cytometry analyses after treatment with 2L and 2L + NAC. (E,J) Quantitative analysis of apoptosis rates by flow cytometry. *** p < 0.001 vs. control.
Figure 7
Figure 7
Effect of 2L on apoptosis and apoptosis-associated proteins. (A,E) The expression levels of Bcl-2, Bax, cleaved caspase-3, cleaved caspase-9, PAPR, and cleaved PARP proteins in BC cells were inspected using Western blot. (B,F) The relative quantification of protein levels was analyzed using ImageJ software (V1.8.0). (C,G) The apoptosis rates were assessed by flow cytometry. (D,H) Quantitative analysis of apoptosis rates using flow cytometry. ** p < 0.01 and *** p < 0.001 vs. control.
Figure 8
Figure 8
Anti-tumor efficacy of 2L in the MDA-MB-231 xenograft tumor model. (A) Digital images of tumor tissues from different experimental groups. (B) The change curve of tumor volume. (C) The average tumor volume was measured after the mice were sacrificed. (D) The measurement of tumor weight and TGI. (E) The variation of body weight in each group. (F) Western blot analysis of Bcl-2, Bax, PARP, and cleaved PARP proteins in tumor tissues. ** p < 0.01, *** p < 0.001 vs. control group.
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