Microwave ablation combined with Lawsone significantly inhibit the proliferation and promote apoptosis of breast cancer cells in vitro compared with the single treatment groups
In vitro, we simulated residual breast cancer cells after microwave ablation by heating (45℃, 10 min) breast cancer cells in a water bath. In the preliminary experiment, we found that Lawsone (Fig. 1A) had a significant inhibitory effect on the activity of all types of breast cancer cells, and then we selected triple-negative breast cancer cells (MDA-MB-231 and SUM-1315) with strong proliferation and migration abilities as subjects in the subsequent experiment. Lawsone repressed the growth of breast cancer cells in a dose-dependent manner, and the half inhibitory concentration (24 h) of Lawsone on MDA-MB-231 and SUM-1315 breast cancer cells was 118.07 µM and 145.19 µM, respectively (Fig. 1B). The selected cell treatment concentration in subsequent in vitro experiments was 100µM. CCK8 assay, clone formation assay and EdU assay all manifested that compared with the control group, the proliferation ability of breast cancer cells in the heat treatment group did not change prominently, but significantly diminished in the Lawsone treatment group (Fig. 1C-F). By comparison with the control group, heat treatment group or Lawsone treatment group, the growth ability and proliferation ability of MDA-MB-231 and SUM-1315 breast cancer cells in the combined treatment group were significantly reduced. This indicated that the susceptibility of heat-treated breast cancer cells to Lawsone treatment was significantly enhanced.
Cell cycle arrest and increased apoptosis are two important considerations that inhibit the growth of breast cancer cells. Firstly, the results of flow cytometry declared that in comparison with the control group, the cell cycle progression of breast cancer cells in the heat treatment group had no significant change, and the quantity of breast cancer cells in the G0/G1 phase in the Lawsone treatment group was conspicuously increased. By comparison with the single treatment group, the breast cancer cells treated with heat treatment combined with Lawsone were prominently arrested in G0/G1 phase, and the quantity of cells in S phase was markedly decreased (Fig. 1H). In addition, flow cytometry analysis after Annexin V-FITC/PI staining showed that the total apoptosis rate of breast cancer cells treated with Lawsone after heat treatment was conspicuously higher than that of control group and single treatment group (Fig. 1J). The above results were also confirmed by Western blot (Fig. 1L). In contrast with the control group and the single treatment group, the expression level of G1/S phase related checkpoint proteins (such as CyclinD1 and CDK4) and anti-apoptotic protein (Bcl2) were obviously reduced, and the expression of pro-apoptotic protein (Bax) was markedly increased in breast cancer cells treated with heat treatment combined with Lawsone. In comparison with the control group, the protein expression level of the heat treatment group had no significant change, while the Lawsone treatment group had a certain degree of decrease or increase.
Microwave ablation combined with Lawsone significantly inhibit the migration and invasion of breast cancer cells in vitro compared with single treatment groups
The wound healing experiment and transwell migration experiment showed that the migration ability of MDA-MB-231 and SUM-1315 breast cancer cells treated with Lawsone after heat treatment was more significantly reduced by comparison with the control group, heat treatment group and Lawsone treatment group (Fig. 2A,C). The migratory ability of breast cancer cells was enhanced after heat treatment, but decreased after Lawsone treatment in comparison with the control group. Lawsone has a stronger inhibitory effect on the migration ability of breast cancer cells after heat treatment. The results of the transwell invasion assay were also conformed with the above results (Fig. 2D). Epithelial-mesenchymal transition (EMT) is a recognized key event in the process of tumor metastasis (19). The protein expression level of EMT-related molecules was detected, and it was found that the epithelial cell marker E-cadherin was more significantly up-regulated in MDA-MB-231 and SUM-1315 breast cancer cells treated with Lawsone after heat treatment compared with the single treatment group, and mesenchymal cell markers and transcription factors (N-cadherin, Vimentin, Snail, Slug) were significantly down-regulated (Fig. 2G). Compared with the control group, epithelial cell markers were down-regulated and mesenchymal cell markers and transcription factors were up-regulated in breast cancer cells treated with heat treatment, while the opposite was true after Lawsone treatment. In addition, the relative expression of mesenchymal cell markers (Vimentin) and epithelial cell markers (E-cadherin) in MDA-MB-231 and SUM-1315 cells in four different treatment groups was evaluated by immunofluorescence assay, and the consequences were also consistent with those described above (Fig. 2H).
Microwave ablation combined with Lawsone treatment significantly inhibit the growth and metastasis of residual breast cancer in vivo compared with the single treatment groups
To evaluate the therapeutic effect of microwave ablation combined with Lawsone treatment on breast cancer in vivo, MDA-MB-231 cells were transplanted into BALB/c nude mice and treated according to group. At the end of the experiment, mice were sacrificed and xenograft tumors were removed (Fig. 3A). In contrast with the control group, there was no conspicuous change in the growth ability of residual tumors after microwave ablation, but it was significantly reduced after Lawsone treatment. By comparison with the control group, microwave ablation group and Lawsone treatment group, the growth rate of tumors in the microwave ablation combined with Lawsone treatment group was slower, and the volume and weight of tumors were smaller (Fig. 3B,C). This indicated that Lawsone inhibited the growth of residual tumors after microwave ablation more significantly than that without microwave ablation (surgical resection). Ki67 is a marker of proliferating cells. Immunohistochemical staining of tumor tissues manifested that the expression of Ki67 in tumor tissues of the combined treatment group was significantly lower than that of the other three groups, which confirmed the above results (Fig. 3D). In addition, to estimate the effect of different treatments on lung metastasis in mice, a lung metastasis model experiment was performed. Lung tissues of mice in the four treatment groups were removed (Fig. 3F) and stained with HE (Fig. 3G). The results indicated that the number and size of lung metastases in the heat treatment combined with Lawsone treatment group were significantly reduced in contrast with the control group and the single treatment groups. The number and size of pulmonary metastasis increased in the heat-treated group and decreased in the Lawsone-treated group in contrast with the control group. These results demonstrate that Lawsone has stronger inhibitory ability on lung metastasis of breast cancer cells after microwave ablation than without microwave ablation.
Lawsone treatment significantly decrease the expression of NCAPG and induce autophagy in residual breast cancer after microwave ablation
To further investigate the mechanism of Lawsone's inhibitory effect on residual breast cancer cells after microwave ablation, we performed transcriptome sequencing in the microwave ablation group and microwave ablation combined with Lawsone treated breast cancer tissues. GO and KEGG enrichment analysis manifested that down-regulated genes were frequently involved in DNA replication, cell cycle, cell proliferation, cell migration, autophagy and other biological processes, and involved in mTOR and PI3K-AKT signaling pathways (Fig. 4A-D). Combined with the sequencing analysis results and bioinformatics analysis, we selected NCAPG as the research object, that is, as a possible target of Lawsone (Figure S1A,B) (Table 1). UACLAN database analysis showed that NCAPG was significantly overexpressed in all types of tumors and paired/unpaired breast cancer tissues (especially triple-negative breast cancer) (Figure S1C-E), and had a good diagnostic significance for breast cancer (AUC = 0.954, Figure S1F). Western blot analysis showed that the protein expression level of NCAPG in breast cancer tissues was prominently increased in comparison with adjacent normal tissues (Figure S1G). In addition, high expression of NCAPG was associated with shortened OS (P = 0.037), PFI (P = 0.019) and DSS (P = 0.006) in breast cancer patients(Figure S1I). High expression of NCAPG was also associated with poor prognosis in patients with N2 stage (P = 0.02), pathological stage 2 (P = 0.003) and pathological stage 3 (P = 0.013) (Figure S1J). Western blot and qRT-PCR experiments manifested that the mRNA and protein expression of NCAPG in heat-treated MDA-MB-231 and SUM-1315 cells were significantly decreased after treated with Lawsone (Fig. 4E,F). Furthermore, in addition to cell proliferation and migration, the regulation of autophagy, a biological process, was significantly enriched. Western blot analysis of the expression of autophagy-related proteins manifested that, in comparison with the control group, heat treatment group and Lawsone treatment group, Beclin1, ATG5 and Atg7 were more significantly up-regulated, LC3-II/LC3-I ratio was more significantly increased, and P62 level was more significantly reduced in the combined treatment group (Fig. 4G). At the same time, in contrast with the control group, the levels of autophagy-related proteins were increased or decreased to a certain extent after Lawsone treatment, but there was no conspicuous change after heat treatment. The effect of Lawsone on autophagy induction in heat-treated breast cancer cells was stronger than that without heat treatment. The inversion of LC3-I to lipidated LC3-II and the distribution of endogenous LC3 puncta are two classic indicators of autophagy regulation (20). Immunofluorescence assay was employed to detect the distribution of endogenous LC3 puncta, and the results showed that the LC3 puncta in breast cancer cells were markedly increased in the combined treatment group compared with the other groups (Fig. 4H). In comparison with the control group, the LC3 puncta increased to a certain extent after Lawsone treatment, but did not change significantly after heat treatment. In addition, the formation of autophagosomes was observed more directly by TEM, and the results showed that breast cancer cells treated with heat treatment combined with Lawsone showed more accumulation of double or multimembrane structures (autophagosomes) than those treated with heat treatment.
Table 1
Patients’ characteristics and their association with NCAPG expression
Characteristic
|
Low expression of NCAPG
|
High expression of NCAPG
|
p
|
n
|
532
|
533
|
|
T stage, n (%)
|
|
|
< 0.001
|
T1
|
171 (16.1%)
|
104 (9.8%)
|
|
T2
|
268 (25.2%)
|
347 (32.7%)
|
|
T3
|
78 (7.3%)
|
59 (5.6%)
|
|
T4
|
14 (1.3%)
|
21 (2%)
|
|
N stage, n (%)
|
|
|
0.346
|
N0
|
251 (24%)
|
256 (24.5%)
|
|
N1
|
177 (16.9%)
|
172 (16.4%)
|
|
N2
|
52 (5%)
|
64 (6.1%)
|
|
N3
|
43 (4.1%)
|
31 (3%)
|
|
Pathologic stage, n (%)
|
|
|
0.003
|
Stage I
|
110 (10.6%)
|
70 (6.7%)
|
|
Stage II
|
278 (26.7%)
|
328 (31.5%)
|
|
Stage III
|
127 (12.2%)
|
111 (10.7%)
|
|
Stage IV
|
8 (0.8%)
|
10 (1%)
|
|
Age, n (%)
|
|
|
0.001
|
<=60
|
267 (25.1%)
|
321 (30.1%)
|
|
> 60
|
265 (24.9%)
|
212 (19.9%)
|
|
Histological type, n (%)
|
|
|
< 0.001
|
Infiltrating Ductal Carcinoma
|
308 (32.1%)
|
449 (46.8%)
|
|
Infiltrating Lobular Carcinoma
|
159 (16.6%)
|
43 (4.5%)
|
|
PR status, n (%)
|
|
|
< 0.001
|
Negative
|
103 (10.1%)
|
235 (23.1%)
|
|
Indeterminate
|
1 (0.1%)
|
3 (0.3%)
|
|
Positive
|
404 (39.8%)
|
270 (26.6%)
|
|
ER status, n (%)
|
|
|
< 0.001
|
Negative
|
60 (5.9%)
|
177 (17.4%)
|
|
Indeterminate
|
0 (0%)
|
2 (0.2%)
|
|
Positive
|
448 (44.1%)
|
330 (32.4%)
|
|
HER2 status, n (%)
|
|
|
0.003
|
Negative
|
290 (40.4%)
|
258 (36%)
|
|
Indeterminate
|
6 (0.8%)
|
6 (0.8%)
|
|
Positive
|
59 (8.2%)
|
98 (13.7%)
|
|
PAM50, n (%)
|
|
|
< 0.001
|
Normal
|
35 (3.3%)
|
5 (0.5%)
|
|
LumA
|
420 (39.4%)
|
131 (12.3%)
|
|
LumB
|
30 (2.8%)
|
172 (16.2%)
|
|
Her2
|
20 (1.9%)
|
62 (5.8%)
|
|
Basal
|
27 (2.5%)
|
163 (15.3%)
|
|
Lawsone inhibit the proliferation of residual breast cancer cells after microwave ablation mainly by targeting NCAPG
In order to further explore the role of NCAPG in the inhibition of proliferation and migration of breast cancer cells by combined treatment, we transfected LV-NCAPG into heat-treated MDA-MB-231 and SUM-1315 breast cancer cells to achieve stable overexpression of NCAPG (Fig. 5A,B). CCK8 assay, clone formation assay and EdU assay showed that compared with the negative control transfection, the proliferation ability of heat-treated breast cancer cells treated with Lawsone after overexpressing NCAPG was significantly increased (Fig. 5C-F). At the same time, the growth ability of heat-treated breast cancer cells without Lawsone treatment after overexpression of NCAPG was also significantly increased. These results indicated that overexpression of NCAPG could significantly attenuate repression of Lawsone on the proliferation of heat-treated breast cancer cells. The effect of NCAPG overexpression on cell cycle progression and apoptosis was evaluated by flow cytometry. The results manifested that the number of breast cancer cells in G0/G1 phase was significantly reduced, the number of S-phase cells was significantly increased, and the total cell apoptosis rate was also significantly decreased in heat-treated breast cancer cells after overexpression of NCAPG and treatment with Lawsone in comparison with those without overexpression (Fig. 5H,J). These results demonstrated that overexpression of NCAPG could reverse the cell cycle arrest and apoptosis induction induced by combined treatment in breast cancer cells. In addition, the expression of G1/S phase related checkpoint proteins (such as CyclinD1 and CDK4) and anti-apoptotic protein Bcl2 were significantly increased, and the expression of pro-apoptotic protein Bax was significantly decreased in the combined treatment group after overexpression of NCAPG (Fig. 5L). To further evaluate the effect of NCAPG overexpression on Lawsone treatment of residual breast cancer after microwave ablation in vivo, we designed and performed xenograft tumor model experiments. During the experiment, the changes in tumor volume and tumor mass were recorded, and the results manifested that after overexpression of NCAPG, the tumor growth rate was accelerated, and the tumor volume and weight increased significantly in the combined treatment group (Figure S2A-C). The results of immunohistochemistry manifested that the expression levels of Ki67 and NCAPG in tumor tissues were significantly increased and the expression level of autophagy-related protein Beclin1 was conspicuously decreased in the combined treatment group after overexpression of NCAPG (Figure S2D).
Lawsone inhibit the migration and invasion ability of residual breast cancer cells after microwave ablation mainly by targeting NCAPG
In the following, we investigated the role of NCAPG in the inhibition of migration and invasion of breast cancer cells by combined treatment. The results manifested that the migration and invasion ability of heat-treated MDA-MB-231 and SUM-1315 breast cancer cells treated with Lawsone after overexpression of NCAPG were significantly enhanced compared with transfection of negative control (Fig. 6A-D). In addition, the protein expression levels of EMT related molecules were detected, and the results manifested that mesenchymal cell markers and transcription factors (N-cadherin, Vimentin, Snail, Slug) were significantly up-regulated and epithelial cell markers (E-cadherin) were significantly down-regulated in the combined treatment group after overexpression of NCAPG (Fig. 6G). The results of immunofluorescence assay also demonstrated that the expression of Vimentin was conspicuously up-regulated relative to E-cadherin in MDA-MB-231 and SUM-1315 breast cancer cells after overexpression of NCAPG, which was consistent with the above results. These results indicated that overexpression of NCAPG could significantly alleviate the inhibition of EMT progression in breast cancer cells by combined treatment. To further evaluate the effect of NCAPG overexpression on the lung metastasis ability of breast cancer treated with microwave ablation combined with Lawsone in vivo, we designed and conducted lung metastasis model experiments. Take out the lung tissue for photography and HE staining. The results showed that the number and size of lung metastases in the combined treatment group were significantly increased after overexpression of NCAPG (Figure S2E). In short, overexpression of NCAPG can significantly attenuate the inhibition of heat treatment combined with Lawsone on the migration and invasion ability of breast cancer cells.
Inhibition of autophagy can significantly alleviate the anti-proliferation, anti-migration and anti-invasion effects of Lawsone on residual breast cancer cells after microwave ablation
Previous studies have shown that Lawsone significantly down-regulated NCAPG and significantly induced autophagy in residual breast cancer cells after microwave ablation, and the key role of NCAPG in anti-cancer effect has been verified. Therefore, we further explored the role of autophagy in this process. Indeed, both increased autophagosome formation and obstruction of the autophagosome-lysosome fusion process lead to the accumulation of LC3-II and LC3-positive puncta. Since chloroquine (CQ) can block autophagosome-lysosome fusion and lead to autophagosome accumulation, to distinguish between the above two possibilities, we detected LC3-II levels in breast cancer cells in the combined treatment group in the presence of CQ. The results indicated that the expression of LC3-II in heat-treated MDA-MB-231 and SUM-1315 breast cancer cells treated with CQ and Lawsone was conspicuously increased in comparison with that without CQ treatment (Fig. 7A). In addition, 3-methyladenine (3-MA) is an inhibitor of autophagy in the early stage, and the expression of LC3-II in heat-treated breast cancer cells was significantly reduced after 3-MA and Lawsone treatment. These results indicated that Lawsone significantly induced early stage autophagosome formation after treatment of heat-treated breast cancer cells. To explore the biological significance of the increased autophagy induction, we examined the changes in the proliferation, migration and invasion ability of breast cancer cells treated with autophagy inhibitors, and the results indicated that CQ and 3-MA conspicuously blocked the inhibitory effects of Lawsone on the proliferation, migration and invasion of heat-treated breast cancer cells (Fig. 7B-E). In addition, Beclin1, ATG5 and ATG7 are key regulators of autophagy and biomarkers in the early stage of autophagy formation. To further confirm whether the anti-cancer effect of Lawsone on heat-treated breast cancer cells could be achieved by inducing autophagy, we used siRNA to silence the expression of Beclin1, ATG5 and ATG7 in heat-treated breast cancer cells, and then treated with Lawsone (Fig. 7F). Clone formation assay, EdU assay, transwell migration and invasion assay indicated that compared with negative control siRNA, knockdown of Beclin1, ATG7 and ATG5 in breast cancer cells treated with combination treatment could significantly restore cell proliferation activity and migration and invasion ability (Fig. 7G-J). That is, the anti-cancer effect of Lawsone on residual breast cancer cells after microwave ablation may be mainly achieved by inducing inhibitory autophagy.
The anticancer effect of Lawsone on residual breast cancer cells after microwave ablation may be mainly achieved by down-regulating NCAPG/AURKB/AKT/mTOR axis to induce autophagy
Previous studies have indicated that both overexpression of NCAPG and inhibition of autophagy can significantly attenuate the anticancer effect of Lawsone on heat-treated breast cancer cells. Therefore, we would like to further explore whether there is an association between NCAPG and autophagy. Sequencing results showed that mTOR pathway and PI3K/AKT pathway were significantly enriched. PI3K/AKT/mTOR pathway, a key negative regulator of autophagy, is frequently activated in cancer cells, and is also involved in regulating cell cycle, growth, EMT, migration and invasion of cancer (21, 22). Therefore, we investigated the regulation of AKT/mTOR pathway and autophagy in heat-treated breast cancer cells treated with Lawsone before and after overexpression of NCAPG. The results demonstrated that p-S473-AKT and p-S2448-mTOR were conspicuously down-regulated and Beclin1 and LC3-II were markedly up-regulated after Lawsone treatment on heat-treated MDA-MB-231 and SUM-1315 breast cancer cells. After overexpression of NCAPG in heat-treated breast cancer cells combined with Lawsone treatment, p-S473-AKT and p-S2448-mTOR were conspicuously up-regulated, and Beclin1 and LC3-II were markedly down-regulated (Fig. 8A). The results of immunofluorescence assay also manifested that LC3 puncta in MDA-MB-231 and SUM-1315 breast cancer cells in the combined treatment group were significantly reduced after overexpression of NCAPG (Fig. 8B). These results indicated that overexpression of NCAPG could prominently inhibit the induction of autophagy by combination treatment through up-regulation of AKT-mTOR pathway. These data indicate that Lawsone may exert its anticancer effect on heat-treated breast cancer cells by down-regulating NCAPG, inhibiting AKT/mTOR pathway and inducing autophagy. To further elucidate the mechanism of NCAPG regulating downstream pathways, we used Co-Immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS) to precipitate and analyze NCAPG interacting proteins in MDA-MB-231 breast cancer cells, respectively. According to the correlation score and functional enrichment analysis, we selected AURKB as the possible interacting protein of NCAPG. AURKB (Aurora kinase B) is a serine/threonine-9 kinase involved in regulating various stages of cell division (23). The results of bioinformatics analysis indicated that AURKB was significantly highly expressed in various tumors and breast cancer (especially triple-negative breast cancer), and was obviously associated with poor prognosis and had good diagnostic value (Figure S3A-E). More noteworthy, AURKB was significantly positively correlated with NCAPG (P < 0.001), and negatively correlated with Beclin1 (P < 0.001) (Figure S3F,G). Immunoprecipitation and western blot confirmed that AURKB and NCAPG could co-precipitate in breast cancer cells (Fig. 8D). Western blot was used to detect the expression of related proteins in the combined treatment group after overexpression of NCAPG and/or knockdown of AURKB, and the results demonstrated that knockdown of AURKB significantly inhibited the regulation of overexpression of NCAPG on AKT/mTOR pathway and autophagy (Fig. 8E).