AS-IV enhances the antitumor effects of propofol in NSCLC cells by inhibiting autophagy

Abstract Non-small cell lung cancer (NSCLC) is one of the most lethal malignant tumors. It has been shown that the general anesthetic agents, propofol and astragaloside IV (AS-IV) both exert antitumor effects in NSCLC. However, the effects of the combination of propofol with AS-IV in NSCLC remain unclear. Cell counting kit-8, and EdU and Transwell assays were performed to evaluate NSCLC cell viability, proliferation, and migration. Cell apoptosis and autophagy were observed by flow cytometric analysis and TUNEL and LC3 staining, respectively. AS-IV notably enhanced the anti-proliferative, pro-apoptotic, and anti-migratory properties of propofol in NSCLC cells. Moreover, AS-IV remarkably facilitated the anti-autophagy effect of propofol in NSCLC cells by downregulating LC3, Beclin 1, and ATG5. Significantly, the pro-apoptotic ability of the AS-IV/propofol combination in NSCLC cells was further enhanced by the autophagy inhibitor 3-MA, suggesting that autophagy plays a tumor-promoting role in NSCLC cells. Collectively, AS-IV could facilitate the antitumor abilities of propofol in NSCLC cells by inhibiting autophagy. These findings may be beneficial for future studies on the use of AS-IV and propofol for the treatment of NSCLC.


Introduction
Non-small cell lung cancer (NSCLC) is one of the most common malignancies, representing 80% of total lung cancer cases [1].The overall survival rate of NSCLC is poor due to delays in diagnosis and metastasis potential of this disease [2,3].Recently, even with aggressive treatment protocols, including complete surgical resection, radiotherapy, immunotherapy, and chemotherapy, relapse was developed in the majority of NSCLC patients (25-70%) within 5 years [4][5][6].Therefore, discovering novel efficient therapeutic approaches for NSCLC is urgently needed.
Local anesthetics are extensively applied in clinical cancer surgeries [7,8].Recently, evidence has shown that anesthetics also exhibit antitumor properties in multiple cancers, including NSCLC [9][10][11].Propofol, a general anesthetic agent administered during surgery, has been found to suppress the progression of NSCLC [12].Zhang et al. found that propofol could decrease NSCLC cell proliferation, migration, and invasion [13].Meanwhile, propofol was able to enhance the sensitivity of lung cancer cells to cisplatin in NSCLC [14].
It has been shown that traditional Chinese medicine combined with antitumor drugs could achieve favorable effects for cancer treatment [15,16].Astragalus membranaceus, a type of traditional Chinese medicine, possesses antiinflammatory, antioxidative, and antitumor effects [17][18][19].Astragaloside IV (AS-IV) is a main active component isolated from Astragalus membranaceus [20].Jia et al. found that AS-IV could reduce NSCLC cell proliferation and migration by inhibiting Akt/GSK-3β signaling [21].In addition, AS-IV could improve the response of NSCLC cells to cisplatin by inhibiting autophagy [22].However, the role of the combination of AS-IV and propofol in NSCLC remains unclear.Therefore, we aimed to explore the antitumor activities of AS-IV combined with propofol on NSCLC cells.In this study, we found that AS-IV could enhance the pro-apoptotic and anti-migratory activities of propofol in NSCLC cells.These findings may be beneficial for future studies on the use of AS-IV and propofol for the treatment of NSCLC.

Cell culture
Normal lung epithelial cells (BEAS2B) and NSCLC cell lines A549 and NCI-H1299 were obtained from Procell Life Science & Technology Co., Ltd.A549 and NCI-H1299 cells were maintained in complete medium consisting of DMEM (Thermo Fisher Scientific), 0.1% penicillin-streptomycin, and 10% fetal bovine serum (FBS, Thermo Fisher Scientific, category number: 26010-074) at 37°C in a humidified atmosphere of 5% CO 2 .Cells were maintained in a 75 cm 2 culture flask for culture.Cells were subcultured every 3 days with a maximum of passages not exceeding 25.When the cells grew to 70% confluence, they were collected and used for subsequent experiments.

EdU staining assay
The proliferative capacity of A549 and NCI-H1299 cells was assessed by EdU staining assay.The EdU assay was conducted using the Cell-Light EdU DNA cell proliferation kit (RiboBio).Briefly, NSCLC cells were treated with EdU solution (50 µM) for 2 h.After fixation with 4% paraformaldehyde (PFA), cells were stained with Apollo reagent for 30 min in darkness.The nuclear DNA was then stained with Hoechst 33342.Finally, EdU-positive cells were observed under a fluorescence microscope (OLYMPUS).

Flow cytometric analysis
Flow cytometry analysis was used to evaluate the apoptosis of A549 and NCI-H1299 cells.The cells were collected, and the apoptotic cells were identified with an annexin V-FITC cell apoptosis detection kit (product No. C1063; Biotech Research Institute) according to the manufacturer's protocol.
In short, 1 × 10 6 cells were resuspended in 1 mL binding buffer, incubated with 5 µL of Annexin V-FITC for 15 min, and then incubated at 4°C with 5 µL of PI for 5 min in the dark.Fluorescence signals were collected by FACScan flow cytometry (Beckman Coulter, Inc. and then analyzed by FlowJo 8.7.1 software (FlowJo LLC).

TUNEL staining assay
The TUNEL assay was conducted using the In Situ Cell Death Detection Kit (Roche).Briefly, NSCLC cells were incubated with the TUNEL working solution for 1 h in darkness.The nuclear DNA was then stained with DAPI for 30 min.Finally, the apoptotic cells were captured under a fluorescence microscope.

Transwell migration assay
NSCLC cells (2 × 10 4 cells) suspended in serum-free DMEM were added to the upper compartment of 24-well migration chambers (Corning).Meanwhile, the lower compartment was filled with 500 µL of DMEM containing 10% FBS as the attracting agent.Cells in the upper chamber migrating through the polycarbonate membrane into the lower chamber with high nutritional content were considered to have high migration ability.Next 0.1% crystal violet was used to stain the migrated cells on the undersurface of the lower chamber at 24 h.Finally, the migrated cells were captured with a microscope.

Immunofluorescence assay
About 2 × 10 4 NSCLC cells were inoculated into a 24-well plate and fixed at room temperature with 4% PFA for 20 min.PBS containing 1% Triton X-100 was then added to permeate for 20 min.The cells were then incubated with anti-LC3 (ab192890, 1:1,000, Abcam) specific rabbit monoclonal antibody at 4°C overnight.The goat anti-rabbit IgG H&L (Alexa Fluor ® 647) secondary antibody (ab150079, 1:1,000, Abcam) was then incubated at room temperature in darkness for 2 h.The nuclei were stained with DAPI.Subsequently, the LC3-positive cells were observed using a fluorescence microscope.

Statistical analyses
All data were repeated at least three times independently.Data are shown as the mean value ± standard deviation and analyzed with Graphpad Prism 7.0.Statistical analysis was performed by one-way analysis of variance (ANOVA) followed by Tukey's test.p values <0.05 were considered statistically significant.

AS-IV enhanced the cytotoxic effect of propofol in NSCLC cells
To investigate the role of AS-IV and propofol in NSCLC cells, CCK-8 assay was conducted to determine the effects of AS-IV and propofol on the viability of BEAS2B, A549, and NCI-H1299 cells.As shown in Figure 1a, no significant change in BEAS2B cell viability was observed when the concentration of AS-IV was less than 20 ng/mL.As shown in Figure 1b and c, AS-IV (10, 20, or 40 ng/mL) notably inhibited the viability of NSCLC cells.The viability of both A549 and NCI-H1299 cells at 40 ng/mL AS-IV was reduced to below 60%. Figure 1d showed that compared with the control group, there was no statistical significance in cell viability of BEAS-2B cells treated with propofol when the concentration was 2.5, 5, 10 μg/mL.Compared with the control group, the cell viability decreased when the concentration of propofol was 20 μg/mL, which had statistical significance.Propofol (2.5, 5, 10, or 20 μg/mL) markedly reduced the viability of NSCLC cells (Figure 1e and f).Furthermore, 2.5 μg/mL propofol significantly decreased NSCLC cell viability and exhibited approximately 20% growth inhibition.The viability of both A549 and NCI-H1299 cells at 20 μg/mL propofol was reduced to below 50%, but it had no significant effect on the cell survival rate of BEAS2B.In particular, the combination of propofol (2.5 μg/mL) and AS-IV (5 ng/mL) markedly reduced NSCLC cell viability and exhibited approximately 50% growth inhibition (Figure 1g-i).Therefore, 2.5 μg/mL propofol and 5 ng/mL AS-IV were utilized in the following experiments.Collectively, AS-IV could enhance the cytotoxic effect of propofol in NSCLC cells.

AS-IV enhanced the anti-proliferative, pro-apoptotic, and anti-migratory properties of propofol in NSCLC cells
Next we explored the effects of AS-IV and propofol on the proliferation, apoptosis, and migration of NSCLC cells.As indicated in Figure 2a and b, propofol (2.5 μg/mL) significantly suppressed NSCLC cell proliferation.As expected, the combination of AS-IV with propofol further inhibited the proliferation of NSCLC cells (the proportion of Edupositive cells was decreased by 2-fold) compared to that of cells in the propofol treatment alone group (Figure 2a  and b).Additionally, propofol treatment obviously resulted in increased NSCLC cell apoptosis (Figure 3a and b).Interestingly, AS-IV further strengthened propofol-induced NSCLC cell apoptosis (cell apoptosis above 35%) (Figure 3a and b).Furthermore, propofol or AS-IV treatment led to a remarkable decrease in NSCLC cell migration (Figure 4a and b).Meanwhile, the combination of AS-IV and propofol further reduced the migratory ability of NSCLC cells (the number of migrating cells decreased by more than 3-fold) compared to the propofol treatment alone group (Figure 4a and b).Collectively, AS-IV enhanced the anti-proliferative, pro-apoptotic, and anti-migratory properties of propofol in NSCLC cells.

Combination of AS-IV and propofol suppressed NSCLC cell autophagy and ERK1/2 signaling
Autophagy is a highly conserved cellular proteolysis process that plays an important role in cancer development [24].Thus, we next explored whether AS-IV and propofol could affect NSCLC cell autophagy.As shown in Figure 5a-f, AS-IV (5 ng/mL) group and propofol (2.5 μg/mL) group significantly reduced LC3, Beclin 1, ATG5, and p-ERK/ERK levels in NSCLC cells.Interestingly, the combination of AS-IV and propofol further down-regulated LC3, Beclin 1, ATG5, and p-ERK/ERK levels in NSCLC cells.In summary, the combination of AS-IV and propofol suppressed NSCLC cell autophagy.

AS-IV combined with propofol triggered NSCLC cell apoptosis by inhibiting autophagy
Since the AS-IV/propofol combination could induce NSCLC cell apoptosis and suppress cell autophagy, we then focused on the interaction between apoptosis and autophagy in NSCLC cells.The pro-apoptotic effect of the AS-IV/propofol combination on NSCLC cells was further enhanced by treatment with the autophagy inhibitor 3-MA, as shown by the decreased level of Bcl-2 and increased level of cleaved caspase 3 (Figure 6a-e).Collectively, the combination of AS-IV and propofol could trigger NSCLC cell apoptosis by inhibiting autophagy.

Discussion
Recently, Chinese traditional medicine has been used in the treatment of NSCLC [25,26].AS-IV isolated from Astragalus membranaceus has been recognized to exert anticancer effects in NSCLC [21].In this research, we found that AS-IV could enhance the antitumor effects of propofol in NSCLC cells by suppressing autophagy.
Propofol, an anesthesia drug, has been proven to act as an antitumor agent in multiple cancers [27,28].For example, propofol inhibits the development of bladder cancer [29] and colon cancer [30] by regulating miR-145-5p or JAK2/ STAT3 signaling pathway.Propofol increases miR-486-5p levels in NSCLC cells and xenograft tumor tissues in a N6methyladenosine (m6A) dependent manner, thereby inactivating the Ras associated protein 1 (RAP1)-NF-kappaB (NF-κB) axis to increase cisplatin sensitivity in NSCLC [23].Propofol inhibits the development of NSCLC by inhibiting the circ-RHOT1/miR-326/Prognostic Significance of Forkhead Box M1 (FOXM1) [13] axis and miR-21/PTEN/AKT [12] axis.AS-IV inhibits the progression of NSCLC  5 In addition, propofol could trigger NSCLC cell apoptosis by inactivating ERK1/2 signaling and upregulating p53-upregulated modulator of apoptosis [31].These results demonstrate that propofol can function as a tumor suppressor in cancer through miRNA, JAK2/STAT3, RAP1/NF-κB, PTEN/AKT, and ERK1/2 signaling pathways.On the basis of these previous studies, our results showed that propofol treatment resulted in a significant decrease in NSCLC cell proliferation and migration and an increase in cell apoptosis.These results further confirmed the antitumor effects of propofol in NSCLC.
Recently, combination therapy has attracted increasing attention in cancer treatment due to the advantages of reduced toxicity, synergistic antitumor effects, and diminished acquired resistance [32][33][34].Propofol regulates Wnt/β-Catenin, HIF-1 signaling pathway, circ-ERBB2/miR-7-5p/FOXM1 axis, inhibits NSCLC cell proliferation, invasion, and glycolysis, and accelerates cell apoptosis [14,35,36].AS-IV inhibits AMPK signaling pathway, Akt/GSK-3β/β-Catenin, endoplasmic reticulum stress signaling pathway, and autophagy signaling pathway and evidence in previous studies also proved AS-IV inhibits the proliferation of NSCLC and promotes apoptosis [21,22,37].This study found that the combination of propofol and AS-IV could further inhibit ERK1/2 signaling in NSCLC cells.Therefore, the combined action of propofol and AS-IV can synergistically act on multiple signal pathways, thereby inducing apoptosis in lung cancer cells.The combination of propofol and sevoflurane remarkably suppressed the migration and invasion of lung adenocarcinoma cells compared to single drugs [38].The combination of propofol and paclitaxel obviously induced apoptosis in prostatic cancer cells compared to paclitaxel alone [39].In addition, AS-IV and curcumin synergistically inhibited tumor growth in hepatocellular carcinoma [40].These results predict that the therapeutic effect of the combination may be more significant than that of the single medication.Whether AS-IV would produce synergistic effects with propofol in NSCLC has not been reported.While the present study conducted a preliminary exploratory study of this blank, in this study, we found that AS-IV could strengthen the antitumor effects of propofol in NSCLC cells.Meanwhile, the combination of AS-IV and propofol exhibited broad antitumor activity compared to single drug treatment.
Autophagy is a self-degradative system that can exert tumor-promoting or antitumor effects in different contexts [41,42].Propofol could enhance tumor sensitivity to cisplatin   AS-IV inhibits the progression of NSCLC  9 in gastric cancer by inhibiting autophagy [43].In addition, AS-IV was able to induce vulvar squamous cell carcinoma cell apoptosis and autophagy by regulating TGF-β/Smad signaling [44].Moreover, AS-IV was found to sensitize NSCLC cells to cisplatin treatment by inhibiting autophagy [22].In this study, AS-IV significantly suppressed NSCLC cell autophagy, and propofol slightly suppressed NSCLC cell autophagy.As expected, the combination of AS-IV and propofol further inhibited NSCLC cell autophagy compared to single drug treatment by inhibiting LC3, Beclin1, and ATG5.In addition, inhibition of autophagy by 3-MA further increased the effect of combination-induced NSCLC cell apoptosis.These results showed that autophagy played a tumor-promoting effect in NSCLC cells.Collectively, combined AS-IV with propofol was able to trigger NSCLC cell apoptosis by inhibiting autophagy.
However, there are some limitations to this study.In this study, the effects of AS-IV and propofol via autophagy on the proliferation, migration, and apoptosis of NSCLC cells were investigated only at the cellular level in vitro and were not further verified at the in vivo level.In the future, it is necessary to construct an animal model of subcutaneous tumor of NSCLC to further study the antitumor effect of AS-IV combined with propofol.The lack of clinical trial data is also one of the limitations of this study.In addition, in this study, coadministration of AS-IV and propofol inhibited autophagy in lung cancer cells by further downregulating the level of the autophagy protein Beclin1, but the specific molecular mechanism still needs further investigation.In addition to that, we only explored the antitumor effects of propofol in NSCLC cells, future studies are suggested to explore the combined effect of other anesthetics, such as sevoflurane and dexmedetomidine, with AS-IV in NSCLC cells.These limitations need to be carefully addressed before this study can be translated into clinical practice.

Conclusion
In conclusion, AS-IV enhanced the antitumor effects of propofol in NSCLC cells by inhibiting autophagy.These findings might pave the way for the application of AS-IV and propofol in NSCLC in the future.

Figure 4 :
Figure 4: AS-IV enhanced the anti-migratory property of propofol in NSCLC cells.(a) A549 and (b) NCI-H1299 cells were treated with AS-IV (5 ng/mL) and/or propofol (2.5 μg/mL) for 48 h.Cell migration was assessed by Transwell assay (200×).The combination of AS-IV and propofol further reduced the migratory ability of NSCLC cells compared with propofol treatment alone.*p < 0.05, **p < 0.01.

Figure 6 :
Figure 6: The combination of AS-IV and propofol triggered NSCLC cell apoptosis by inhibiting autophagy.A549 cells were pretreated with 5 mM 3-MA prior to 12 h and then treated with propofol or propofol plus AS-IV for 48 h.Cell apoptosis was assessed by flow cytometric analysis (a) and TUNEL assay (b) (×200×).(c)-(e) Western blot assay was applied to determine Bcl-2 and cleaved caspase 3 levels in A549 cells.Combination of AS-IV and propofol could trigger NSCLC cell apoptosis by inhibiting autophagy.*p < 0.05, **p < 0.01.