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Elevated level of neuroserpin is an indication for the resistance to gambogic acid-induced apoptosis and oxidative stress in triple-negative breast cancer cells

Ertan Kucuksayan
Ertan Kucuksayan
, 
Hakan Kucuksayan
Hakan Kucuksayan
, 
Mehmet Enes Sozen
Mehmet Enes Sozen
 and 
Aslinur Sircan-Kucuksayan
Aslinur Sircan-Kucuksayan
   | Apr 30, 2024
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Article Category: Original article
Published Online: Apr 30, 2024
Page range: 69 - 80
DOI: https://doi.org/10.2478/abm-2024-0010
Keywords
© 2024 Ertan Kucuksayan et al., published by Sciendo
This work is licensed under the Creative Commons Attribution 4.0 International License.

Figure 1.

Cytotoxic effect of GA on the BT-20 and MDA-MB-231 cells groups incubated for 24 h (n = 6 in each experiment). Values were mean ± SD. *Values significantly different from control, 50 nM, 100 nM, 200 nM, and 300 nM GA groups in the BT-20 cells (P < 0.01). **Values significantly different from control, 50 nM, 100 nM, and 200 nM GA groups in the MDA-MB-231 cells (P < 0.001). #Values significantly different from control, 50 nM, 100 nM, 200 nM, and 300 nM GA groups in the MDA-MB-231 and BT-20 cells (P < 0.001). +Values significantly different from control, 400 nM, 500 nM, 750 nM, 1,000 nM, 1,500 nM, 2,000 nM, and 2,500 nM GA groups in the BT-20 cells (P < 0.001). GA, gambogic acid,
Cytotoxic effect of GA on the BT-20 and MDA-MB-231 cells groups incubated for 24 h (n = 6 in each experiment). Values were mean ± SD. *Values significantly different from control, 50 nM, 100 nM, 200 nM, and 300 nM GA groups in the BT-20 cells (P < 0.01). **Values significantly different from control, 50 nM, 100 nM, and 200 nM GA groups in the MDA-MB-231 cells (P < 0.001). #Values significantly different from control, 50 nM, 100 nM, 200 nM, and 300 nM GA groups in the MDA-MB-231 and BT-20 cells (P < 0.001). +Values significantly different from control, 400 nM, 500 nM, 750 nM, 1,000 nM, 1,500 nM, 2,000 nM, and 2,500 nM GA groups in the BT-20 cells (P < 0.001). GA, gambogic acid,

Figure 2.

Cell pictures show the cell morphology and viability of MDA-MB-231 and BT-20 control and GA-treated cells (10× magnification). GA, gambogic acid.
Cell pictures show the cell morphology and viability of MDA-MB-231 and BT-20 control and GA-treated cells (10× magnification). GA, gambogic acid.

Figure 3.

mRNA levels of E-cadherin, N-cadherin, Bax, and neuroserpin in a dose-dependent manner following treatment by qRT-PCR. Data were presented as the mean ± standard deviation (n = 6). (A) E-cadherin relative mRNA expression level. *Values significantly different from control and 800 nM GA groups in the MDA-MB-231 cells (P < 0.01). #Values significantly different from control, 200 nM, and 400 nM GA groups in the BT-20 cells (P < 0.001). +Values significantly different from control group in the MDA-MB-231 and from control and 200 nM GA groups BT-20 cells (P < 0.01). (B) N-cadherin relative mRNA expression level. *Values significantly different from all groups in the BT-20 and MDA-MB-231 cells (P < 0.001). #Values significantly different from control group in the BT-20 and MDA-MB-231 cells (P < 0.01). (C) Bax relative mRNA expression level. *Values significantly different from control and 200 nM GA groups in the BT-20, and different from all groups in MDA-MB-231 cells (P < 0.01). (D) Neuroserpin relative mRNA expression level. *Values significantly different from control group in the BT-20, and different from all groups in MDA-MB-231 cells (P < 0.001). #Values significantly different from 800 nM GA group in the BT-20 cells (P < 0.001). GA, gambogic acid.
mRNA levels of E-cadherin, N-cadherin, Bax, and neuroserpin in a dose-dependent manner following treatment by qRT-PCR. Data were presented as the mean ± standard deviation (n = 6). (A) E-cadherin relative mRNA expression level. *Values significantly different from control and 800 nM GA groups in the MDA-MB-231 cells (P < 0.01). #Values significantly different from control, 200 nM, and 400 nM GA groups in the BT-20 cells (P < 0.001). +Values significantly different from control group in the MDA-MB-231 and from control and 200 nM GA groups BT-20 cells (P < 0.01). (B) N-cadherin relative mRNA expression level. *Values significantly different from all groups in the BT-20 and MDA-MB-231 cells (P < 0.001). #Values significantly different from control group in the BT-20 and MDA-MB-231 cells (P < 0.01). (C) Bax relative mRNA expression level. *Values significantly different from control and 200 nM GA groups in the BT-20, and different from all groups in MDA-MB-231 cells (P < 0.01). (D) Neuroserpin relative mRNA expression level. *Values significantly different from control group in the BT-20, and different from all groups in MDA-MB-231 cells (P < 0.001). #Values significantly different from 800 nM GA group in the BT-20 cells (P < 0.001). GA, gambogic acid.

Figure 4.

Percentage of ROS index in the MDA-MB-231 and BT-20 cell groups incubated for 12 h (n = 6 in each experiment). Values are mean ± SD. *Values significantly different from control groups in the BT-20 and MDA-MB-231 cells (P < 0.01). **Values significantly different from the 200 nM GA group in the BT-20 cells (P < 0.01). #Values significantly different from the 400 nM and 800 nM GA groups in the BT-20 cells (P < 0.001). +Values significantly different from 200 nM and 800 nM GA groups in the BT-20 and MDA-MB-231 cells (P < 0.001). GA, gambogic acid; ROS, reactive oxygen species.
Percentage of ROS index in the MDA-MB-231 and BT-20 cell groups incubated for 12 h (n = 6 in each experiment). Values are mean ± SD. *Values significantly different from control groups in the BT-20 and MDA-MB-231 cells (P < 0.01). **Values significantly different from the 200 nM GA group in the BT-20 cells (P < 0.01). #Values significantly different from the 400 nM and 800 nM GA groups in the BT-20 cells (P < 0.001). +Values significantly different from 200 nM and 800 nM GA groups in the BT-20 and MDA-MB-231 cells (P < 0.001). GA, gambogic acid; ROS, reactive oxygen species.

Figure 5.

DAPI staining of cells incubated for 12 hours in a dose-dependent manner of GA. DAPI fluorescence images of apoptotic cells with arrows indicating chromatin condensation and high blue fluorescence in the cell nucleus; Magnification ×20; h, hours. GA, gambogic acid.
DAPI staining of cells incubated for 12 hours in a dose-dependent manner of GA. DAPI fluorescence images of apoptotic cells with arrows indicating chromatin condensation and high blue fluorescence in the cell nucleus; Magnification ×20; h, hours. GA, gambogic acid.

Figure 6.

Percentage of apoptotic index in the cell groups incubated for 12 h (n = 6 in each experiment). Shiny DAPI-positive cells were quantified in the various experimental groups. Cells present in 6 high-powered fields (HPF; Magnification ×20) were counted in each experiment and the ratio of apoptotic cells was calculated as the percentage of the whole cell number. Values were mean ± SD. Statistical analysis was performed by one-way analysis of variance with all pairwise multiple comparison procedures done by Dunn’s test. *Values significantly different from control groups in the BT-20 and MDA-MB-231 cells (P < 0.01). **Values significantly different from the 200 nM GA group in the BT-20 cells (P < 0.01). #Values significantly different from the 400 nM and 800 nM GA groups in the BT-20 cells (P < 0.001). +Values significantly different from the 200 nM and 800 nM GA groups in the BT-20 and MDA-MB-231 cells (P < 0.001). GA, gambogic acid.
Percentage of apoptotic index in the cell groups incubated for 12 h (n = 6 in each experiment). Shiny DAPI-positive cells were quantified in the various experimental groups. Cells present in 6 high-powered fields (HPF; Magnification ×20) were counted in each experiment and the ratio of apoptotic cells was calculated as the percentage of the whole cell number. Values were mean ± SD. Statistical analysis was performed by one-way analysis of variance with all pairwise multiple comparison procedures done by Dunn’s test. *Values significantly different from control groups in the BT-20 and MDA-MB-231 cells (P < 0.01). **Values significantly different from the 200 nM GA group in the BT-20 cells (P < 0.01). #Values significantly different from the 400 nM and 800 nM GA groups in the BT-20 cells (P < 0.001). +Values significantly different from the 200 nM and 800 nM GA groups in the BT-20 and MDA-MB-231 cells (P < 0.001). GA, gambogic acid.

Figure 7.

Schematic summary of the changes in the expression profiles of GA-induced apoptotic pathway in the TNBC. GA, gambogic acid; TNBC, triple-negative breast cancer.
Schematic summary of the changes in the expression profiles of GA-induced apoptotic pathway in the TNBC. GA, gambogic acid; TNBC, triple-negative breast cancer.
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