Abstract [44]graphic file with name ao4c08388_0009.jpg Benzyl isothiocyanate (BITC), a natural compound abundant in cruciferous vegetables, plays an important role in the chemoprevention of various human malignancies. However, the mechanism by which BITC inhibits tumor cell growth is not fully understood. This study combined network pharmacology, molecular docking, cellular experiments, and mouse tumor models to predict and validate the targets and mechanisms of BITC in the treatment of anaplastic thyroid carcinoma (ATC). A total of 10 key targets of BITC and ATC were selected for molecular docking. The key target genes of KEGG were mainly concentrated in the nuclear factor κB signaling pathway and apoptosis signaling pathway. The inhibitory effects of BITC on two ATC cell lines, 8505C and CAL-62, were dose-dependent and time-dependent, with IC[50] values of 27.56 and 28.30 μmol/L, respectively. BITC induced apoptosis in ATC cells. Pretreatment with autophagy inhibitor 3MA (2 mmol/L) significantly enhanced growth inhibition caused by BITC in ATC cells. Another autophagy inhibitor, HCQ (20 μmol/L), did not enhance the inhibitory effect of BITC. In CAL-62 xenografted nude mice, BITC (100 mg·kg^–1·d^–2, ip) significantly inhibited tumor growth. Our results indicate that BITC can inhibit the growth of ATC cells both in vitro and in vivo. Additionally, BITC disrupts autophagic degradation in ATC cells, inhibits the NF-κB pathway, and promotes apoptosis. 1. Introduction Based on the GLOBOCAN 2020 database of cancer incidence and mortality by the WHO International Agency for Research on Cancer, thyroid cancer (TC) has the ninth highest cancer incidence worldwide.^[45]1 TC is mainly divided into differentiated thyroid cancer (DTC), anaplastic thyroid cancer (ATC), and medullary thyroid cancer (MTC). ATC, an aggressive solid tumor, has a disease-specific mortality approaching 100%.^[46]2 ATC does not have the biological and functional characteristics of normal thyroid follicular cells. Most undifferentiated thyroid cancers are already advanced at the time of diagnosis, and they respond poorly to radioactive iodine therapy and endocrine therapy. Surgery and radiochemotherapy are also not very effective. Molecular targeted therapy, such as sorafenib, has serious side effects and requires exploration of new clinical treatment methods. Isothiocyanates (ITCs) are a class of R–N=S structure of the general formula of the compound, are the enzymatic hydrolysis of glucosinolates, with a high medicinal value of natural compounds, and found in cruciferous vegetables such as broccoli, watercress, Brussels sprouts, cabbage, and Japanese radish. The consumption of cruciferous vegetables was associated with a reduced risk of all-cause mortality, cancers, and depression. Dose–response analyses revealed that a 100 g/d increment was associated with a 10% decrease in the risk of all-cause mortality.^[47]3 ITCs present in crucifer’s vegetables have higher anticancerous property and can inhibit cell proliferation.^[48]4 Benzyl isothiocyanate (BITC) is a chemical that is beneficial to human health and is widely found in shallots, cress, mustard greens, and papaya seeds.^[49]5 BITC can effectively exert anticancer effects at concentrations (in vitro) or doses (in vivo) that are nontoxic to normal tissues.^[50]6 In addition, BITC can make tumors sensitive to chemotherapy, and have significant anticancer effects to a variety of human malignant tumors such as leukemia,^[51]7 breast cancer,^[52]8 prostatic cancer,^[53]9 lung cancer,^[54]10 pancreatic cancer,^[55]11 colon cancer,^[56]12 and hepatic carcinoma^[57]13. BITC has not been reported in the treatment of thyroid cancer. Network pharmacology, established by Hopkinsar in 2007, refers to a method that combines pharmacology, electronics, bioinformatics, and molecular biology to construct network relationships between chemical components, traditional Chinese medicine (TCM)-related targets, and disease pathways.^[58]14 Through network pharmacological analysis, we can predict more effective drug active targets, better explore the mechanism of action of drugs, and better understand the relationship between drugs and diseases. Although the anticancer activity of BITC has been demonstrated in many studies on human cancers, there are no reports on the application of BITC in ATC treatment. Our previous studies have shown that BITC inhibits the growth of human lung cancer cells and induces autophagy in these cells, which has a cytoprotective effect.^[59]15 This study will investigate the therapeutic mechanism of BITC in ATC by means of network pharmacology, molecular docking, and experimental verification. 2. Results 2.1. Pharmacological Analysis of BTIC and ATC Network BITC 2D molecular structure is shown in [60]Figure [61]1 A. A total of 52 targets related to BITC and 1662 targets related to ATC were obtained. After the intersection of Venny2.1, a total of 22 common targets of BITC and ATC were obtained, as shown in [62]Figure [63]1B. Figure 1. [64]Figure 1 [65]Open in a new tab Pharmacological analysis of the BITC and ATC network. (A) Two-dimensional structure of BITC molecule; (B) Wayne diagram of intersection of BITC target and ATC target; (C) protein interaction (PPI) network; (D) according to the research design, the researchers combined default parameters applicable to most use cases or self-defined relevant parameters, applied the “degree” algorithm and statistical methods implemented in Cytoscape software to process and analyze the target data, and observed 10 key targets; (E–G) microshengxin GO analysis: (E) molecular function; (F) cell component; (G) biological processes; and (H) pathway enrichment analysis of potential targets of BITC treatment for ATC using the KEGG database. We obtained a PPI network with 22 nodes and 48 edges ([66]Figure [67]1 C). Nodes represent proteins, edges represent protein–protein interactions, and the larger the circle, the closer the connections made by its nodes. The cytoHubba plugin tool of Cytoscape software was used for topological analysis, and 10 key targets were selected through the “Degree” algorithm ([68]Figure [69]1D). GO and KEGG functional enrichment analyses and visualization of key target genes were performed. Key target genes are involved in these pathways ([70]Figure [71]1 E,F). KEGG functional enrichment results showed that key target genes were mainly enriched in NF-κB signaling pathway, apoptosis, and other pathways ([72]Figure [73]1H). These pathways and functions may play a key role in the pathogenesis of ATC. 2.2. Validation 2.2.1. Molecular Docking Results The structure diagram of BITC and the three-dimensional (3D) structure of the target protein were imported into AutoDockTools-1.5.7 software for molecular docking. The visualization results of the docking structure are shown in [74]Figure [75]2 , the molecular affinity was referred to the binding energy in [76]Table [77]1. In general, the binding affinity lower than −4.0 kal/mol indicates that the bindings are good interactions with lower numbers indicating stronger binding.^[78]14 BITC had a good docking activity with the target. Figure 2. [79]Figure 2 [80]Open in a new tab Molecular docking model of BITC for the ATC central target. (A) PLAU in NF-κB signaling pathway and (B) PARP1 in apoptosis. Table 1. Binding Energy of Molecular Docking. target PDB ID binding energy (kcal/mol) STAT3 [81]6NJS –4.6 AR [82]1E3G –5.6 APP [83]1AAP –4.6 PGR [84]3G8O –4.9 NR3C1 [85]1NHZ –5.8 MAOB [86]2XCG –6.1 PARP1 [87]1UK0 –4.2 PLAU [88]1C5W –4.9 CDK9 [89]3BLH –4.8 CYP1A2 [90]2HI4 –6.7 [91]Open in a new tab 2.2.2. BITC Can Induce Apoptosis of ATC Cells To verify the role of BITC, BITC was cultured with CAL-62 and 8505C cell lines. The results showed that BITC inhibited ATC cells in a concentration-dependent manner. BITC gradients (0, 10, 20, 30, and 40 μmol/L) were applied to CAL-62 and 8505C cells for 24 h. Compared with the blank group, BITC treatment effectively inhibited ATC cells in a concentration-dependent manner after 24 h ([92]Figure [93]3 A,B). The 24 h half-inhibition concentration of BITC in 8505C was 27.56 μmol/L (95% confidence interval CI: 26.66–28.50) and that of CAL-62 was 28.30 μmol/L (95% CI: 26.48–30.44). Figure 3. [94]Figure 3 [95]Open in a new tab BITC induced the apoptosis of two ATC cells. (A, B) 8505C (A) and CAL-62 (B) (comparison between groups, ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05); The 24 h semi-inhibitory concentration of BITC in 8505C was 27.56 μmol/L and that of CAL-62 was 28.30 μmol/L. After 24 h, cells were collected and counted by CCK-8. (C, D) BITC was added to CAL-62 and 8505C cell lines and cultured at 30 μmol/L for 24 h, flow cytometry was used to analyze the apoptosis of cells; compared with the control group, the apoptosis rate of 8505C and CAL-62 cells increased (**P < 0.01). The values represent the mean ± standard deviation of three independent experiments. BITC was added to CAL-62 and 8505C cell lines and cultured at a concentration of 30 μmol/L for 24 h. Compared with the control group, the apoptosis rates of 8505C and CAL-62 cells increased (P < 0.01), indicating that BITC can induce apoptosis of ATC cells ([96]Figure [97]3 C,D). 2.2.3. Effect of BITC on ATC Cells Since many compounds may cause autophagy in different cell types, we investigated the effect of BITC on the autophagy status of ATC cells. LC3B II and P62 are widely used in autophagy detection. Western blot results showed that BITC induced the accumulation of LC3B II and P62 not only in concentration-based conditions but also in time-based conditions, as shown in [98]Figure [99]4 . Under the action of different concentrations of BITC, a high concentration promoted the conversion of ATC LC3B I to LC3B II, as shown in [100]Figure [101]4A,B. Moreover, the accumulation of LC3B II increased significantly when the BITC concentration was 10 μmol/L, so 10 μmol/L BITC was used for follow-up experiments. Figure 4. [102]Figure 4 [103]Open in a new tab BITC increased the expression levels of LC3B II and P62 protein ATC cells. (A, B) ATC cells were treated with 5, 10, 15, and 20 μmol/L BITC for 24 h. Whole cell lysate was prepared, and LC3B II and P62 levels were determined by Western blotting. GAPDH was used as a loading control. (C, D) ATC cells were treated with 10 μmol/L BITC for 4, 6, 12, and 24 h. Whole cell lysate was prepared, and LC3B II and P62 levels were determined by Western blotting. GAPDH was used as a loading control. Data were calculated from three independent experiments and expressed as the mean ± SEM. Compared with the control group, ****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05. 2.2.4. BITC Inhibits the ATC Mechanism On the basis of KEGG enrichment analysis and molecular docking results, we studied the effect of BITC on the NF-κB signaling pathway. ATC cells were treated with 10 μmol/L BITC for 24 h, and the protein expression was analyzed by Western blotting. As shown in [104]Figure [105]5 A,B, BITC inhibited the expression of NF-κB, Beclin-1, and Bcl-2 in 8505C and CAL-62 cells, while the expression of P62 increased, compared with the control group (P < 0.05). Figure 5. [106]Figure 5 [107]Open in a new tab BITC inhibits the ATC mechanism. (A, B) Effects of BITC on NF-κB, P62, Beclin-1, Bcl-2 in 8505C, and CAL-62 (*P < 0.05); (C) after ATC cells were treated with 10 μmol/L BITC for 24 h, LC3B mRNA level was detected by qPCR (****P < 0.0001); and (D–F) Western blot detection of LC3B protein expression (**P < 0.01, *P < 0.05). The expression of the LC3B gene and protein in ATC cells induced by BITC was detected by RT-PCR and Western blotting. After treating ATC cells with 10 μmol/L BITC for 24 h, LC3B gene and protein expression levels were detected, as shown in [108]Figure [109]5 . LC3B gene expression levels were significantly increased (P < 0.0001, [110]Figure [111]5C). The determination of the intracellular protein level showed that the relative expression level of LC3B was significantly higher than that of the control group, as shown in [112]Figure [113]5D,E (P < 0.05). 2.2.5. Autophagy Inhibitor 3MA Regulates the Effect of BITC on ATC Cells In order to prove that the inhibition effect of BITC on ATC cells is related to autophagy, we used the autophagy inhibitor 3MA to pretreat ATC cells for 2 h, then exposed to BITC, 3MA, BITC, and 3MA combined groups. The cell viability measured by CCK-8 is shown in [114]Figure [115]6 A. BITC could inhibit ATC cells, compared with the control group (P < 0.001). At the same time, 3MA could enhance the inhibitory effect of BITC (P < 0.0001). Figure 6. [116]Figure 6 [117]Open in a new tab BITC inhibited the activity of ATC cells. (A) 3MA enhanced the inhibitory effect of BITC on ATC cells. (B, C) 3MA increased the expression of LC3B II protein in BITC-induced ATC cells (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05). 3MA increased the expression of LC3B II in BITC-induced ATC cells. In order to further verify that the inhibition effect of BITC on ATC cells is related to autophagy, after 3MA pretreatment for 2 h, ATC cells were exposed to BITC and 3MA respectively, and the expression of LC3B II was detected by Western blot, as shown in [118]Figure [119]6 B,C. BITC promoted the conversion of LC3B I to LC3B II, compared with the control group (P < 0.05). 3MA did not block BITC-induced LC3B coupling; in fact, it unexpectedly enhanced LC3B II expression, compared with BITC alone (P < 0.05), also with the control group (P < 0.0001). 2.2.6. LC3B Expression and CCK-8 Cell Viability Were Detected in ATC Cells in the Presence of HCQ To determine whether the increased expression of LC3B II was due to the production and accumulation of autophagosomes, we used another autophagy inhibitor, chloroquine (HCQ), which blocks the transition of autophagosomes to autophagolysosomes. After HCQ pretreatment for 2 h, ATC cells were exposed to BITC and HCQ, respectively. Western blot detected the expression of LC3B II, as shown in [120]Figure [121]7 A,B. Combined treatment of BITC and HCQ, had a significant additive effect on the accumulation of LC3B II, compared with BITC treatment alone (P < 0.05), also with the control group (P < 0.001). As shown in [122]Figure [123]7, CCK-8 assay showed that BITC could inhibit ATC cell viability (P < 0.0001). However, HCQ did not enhance the inhibitory effect of BITC, compared with BITC alone (P > 0.05), as shown in [124]Figure [125]7C. Figure 7. [126]Figure 7 [127]Open in a new tab Effects of BITC regulated by HCQ on ATC. (A, B) HCQ increased the expression of LC3B II protein in BITC-induced ATC cells and (C) HCQ did not increase the inhibitory effect of BITC on ATC cells (****P < 0.0001, ***P < 0.001, **P < 0.01, and *P < 0.05). 2.2.7. In Vivo Effect of BITC CAL-62 cells were subcutaneously injected into the mice. BITC was administered every other day for 21 days, and the weight of the nude mice was measured. As shown in [128]Figure [129]8 D, compared with the control group, the treated mice did not lose weight, and there was no significant difference, indicating that BITC had no toxic effect at this dose. Compared with the control group ([130]Figure [131]8C), the tumor volume and weight of BITC treatment group were significantly reduced, and the tumor growth rate was significantly slower, indicating that BITC could significantly delay tumor growth (P < 0.05). In vitro, the comparison of tumor with the control group is shown in [132]Figure [133]8B. Tumor tissue protein was extracted, and Western blots were used to detect LC3B II, Bcl-2, Beclin-1, P62, and NF-κB, as shown in [134]Figure [135]8E,F. LC3B II and P62 expressions were increased, compared with the control group (P < 0.05), while the expressions of Bcl-2, Beclin-1, and NF-κB decreased (P < 0.05). Figure 8. [136]Figure 8 [137]Open in a new tab Effects of BITC on the proliferation of transplanted tumors in nude mice. (A) Tumor volume in the control group and experimental group; (B) tumor weight in vitro; (C) tumor growth in the experimental group after treatment (*P < 0.05); (D) weight change of nude mice; (E) Western blot of LC3B II, Bcl-2, Beclin-1, P62, and NF-κB in transplanted tumors in nude mice; (F) LC3B II, Bcl-2, Beclin-1, P62, and NF-κB protein expression compared with the control group (*P < 0.05). 3. Discussion In this study, combined with network pharmacology and molecular docking, the target and mechanism of BITC in the treatment of ATC were predicted. It was found that the KEGG functional enrichment results showed that key target genes are mainly concentrated in chemical carcinogens-receptor activation, tryptophan metabolism, drug metabolization-cytochrome P450, prostate cancer, NF-κB signaling pathway, 5-hydroxytryptaminergic synapses, oocyte meiosis, apoptosis, and other signaling pathways. To further investigate the mechanism of BITC in the treatment of ATC, we selected two cell lines 8505C and CAL-62 to verify the network pharmacology and molecular docking results. The NF-κB signaling pathway consists of receptor and receptor proximal signal adaptor protein, IκB kinase complex, IκB protein, and NF-κB dimer. In apoptosis, NF-κB has an antiapoptotic effect in many cases. For example, NF-κB can inhibit the expression of proapoptotic proteins such as Bax and Bak while promoting the expression of antiapoptotic proteins such as Bcl-2 and Bcl-xL, thereby inhibiting apoptosis. Moreover, molecules involved in the signaling pathway of NF-κB activation are usually involved in the regulation of apoptosis. Upon stimulation, NF-κB is activated and breaks down the IκB protein. This activation opens the NF-κB signaling pathway. The role of the NF-κB signaling pathway in apoptosis is to control apoptosis through the regulation of cytokines in the tissue microenvironment, including proliferation factors, inflammatory factors, cell survival factors, and various hormones. In addition to its antiapoptotic effect in apoptosis, NF-κB can also be involved in the growth of malignant tumors, promoting the expression of tumor-related genes, cell cycle regulation, and cancer cell survival. Our previous research suggests that dehydroxym ethylepoxyquinomycin (DHMEQ) and taxane enhanced the induction of apoptosis. In ATC cells, dual effects of taxanes exist: they bind to microtubules, impair mitosis, and induce apoptosis. However, at the same time, they can also induce NF-κB activation, leading to cell survival. DHMEQ can block the nuclear translocation of NF-κB and promote apoptosis.^[138]15 Po et al.^[139]16 found that BITC could reduce the expression of NF-κB in the study of the mechanism of BITC treatment of gastric adenocarcinoma. Batra^[140]17 et al. found BITC can significantly reduce the transcriptional activity of NF-κB. Similar to our results, BITC can induce NF-κB of 8505C and CAL-62. After treatment of ATC cells 8505C and CAL-62 with 10 μmol/L BITC for 24 h, Western blot assay showed decreased expression of NF-κB and Bcl-2 proteins, promoting apoptosis of cancer cells. At the same time, we also found that after BITC treatment, autophagy-related proteins LC3B and Beclin-1 expression showed different changes in which LC3B expression increased, while Beclin-1 expression decreased. Beclin-1, a mammalian homologue of yeast Atg6/Vps30, was identified as a Bcl-2 interacting protein by yeast two-hybrid screening. Beclin-1 acts as an interface for three different cellular cascades: autophagy, differentiation, and apoptosis. Although basal Beclin-1 levels do not cause autophagy or differentiation, consumption of Beclin-1 disrupts the process of autophagy and differentiation, leading to the activation of apoptosis. Importantly, the interaction between Beclin-1 and the antiapoptotic proteins Bcl-2 and Bcl-XL determines the direction of Beclin-1-driven apoptosis and autophagy processes. Depending on the nature of the stimulus, Beclin-1 is involved in different cellular processes; for example, the Beclin-1-Bcl-XL complex becomes apoptotic or antiautophagous under nonautophagy stimulation and becomes antiapoptotic and autophagous under autophagy stimulation.^[141]18 Prerna^[142]19 mentioned that when the stress level exceeds the threshold, the highly upregulated autophagy process begins to decline and transition from autophagy to apoptosis. It is difficult to describe the transition from autophagy to apoptosis. At this stage, the Bcl-2 phosphorylation mediated by c-Jun N-terminal protein kinase 1(JNK1) reaches a maximum, resulting in the destruction of the Beclin-1-Bcl-2 complex and Bcl-2-Bax complex and in the dissociation of Bcl-2. This is conducive to the initiation of apoptosis by proapoptotic proteins. In addition, the increase in Beclin-1 levels also decreases due to Becpase-mediated Beclin-1 cleavage. Collectively, an “on–off switch” is turned on in the cell, promoting the transition from autophagy to apoptosis. At present, BITC is known to have anticancer effects, and the mechanisms of its anticancer effects are induced apoptosis^[143]20 and autophagy.^[144]16 The effect of autophagy on cancer depends on many factors, including tumor microenvironment, cancer type and stage, and genetic background.^[145]21 Our previous study also found that cancer-associated fibroblasts (CAFs) had higher autophagy levels than normal fibroblasts (NFs). Inhibition of autophagy by inhibitors or gene regulation could reduce the effect of CAFs, but it did not completely block this effect. Autophagy promoted the effect of CAFs on the migration and invasion of lung cancer cells to a certain extent.^[146]22 In other previous studies, BITC was shown to have different effects on different cell types. BITC induced protective autophagy in lung cancer cells,^[147]10 prostate cancer cells,^[148]9 and colon cancer cells,^[149]23 but BITC induced autophagy in breast cancer cells resulting in cell death.^[150]24 It has been reported that autophagy can maintain the survival of gastric cancer cells and affect the resistance of anticancer drugs.^[151]25 On the basis of the results of network pharmacology and molecular docking, we further verified the apoptosis and autophagy-related content of the BITC treatment mechanism of ATC by cell experiments and found that BITC effectively inhibited the proliferation activity of two kinds of ATC cells in time and concentration-dependent manners. High concentration of BITC promoted the transformation of ATC from LC3B I to LC3B II under different concentrations of BITC, increasing the expression of P62. To further verify the correlation with autophagy, qPCR showed changes in gene expression. LC3B gene expression level was significantly increased in CAL-62 and 8505C cell lines compared with the control group. Compared with the control group, the relative expression of LC3B protein in both types of cells in the BITC group increased. LC3B II and P62 are well-known and commonly applied autophagy markers.^[152]26 When autophagy degradation occurs, LC3 II detaches from the outer membrane, while both LC3 II from the inner membrane and P62 are degraded along with the cargo by lysosomal proteases.^[153]26 Some studies have reported that LC3B and P62 proteins increase with the inhibition of autophagy degradation. Po et al.^[154]16 found that BITC simultaneously induced the accumulation of LC3B II and P62 proteins in AGS cells, suggesting that BITC might impair autophagy degradation. There is evidence that Class III PI3K is essential for LC3 coupling (conversion of LC3B I to LC3B II) to initiate the autophagy process.^[155]27 In addition, since the BITC-induced LC3B II level elevation remains high in the presence of Class III PI3K inhibitors, it is considered that BITC-induced LC3B coupling is not dependent on Class III PI3K. Another study reported that LC3B coupling occurred even in MEF (mouse embryonic fibroblasts) cells with Class III PI3K-deficient cells when autophagy degradation was reduced.^[156]28 They speculate that BITC-induced LC3B II accumulation in AGS cells may not be an autophagy inducing signal but may be the result of reduced autophagy degradation. The results of this study showed that in the presence of IPI3K/AKT/mTOR pathway inhibitor 3MA, BITC significantly enhanced its inhibitory effect on ATC cell viability, and the difference was significant. The expression of LC3B II protein was significantly increased by Western blotting, the difference was significant compared with other groups, which was consistent with the results of Po et al.^[157]16 Therefore, we believe that BITC-induced LC3B coupling is not dependent on Class III PI3K but may be the result of reduced autophagy degradation. On the other hand, BITC increased the mRNA levels of LC3B in ATC cells, which suggests that the host ATC cells may generate more autophagy proteins as a response against the BITC-induced stress. HCQ blocks autophagy flux by inhibiting the hydrolytic ability of the autolysosome. They increase the pH of the autolysosome and block the activity of acid proteases and other enzymes.^[158]29 Thus, the fusion and degradation of intracellular autophagolysosomes were inhibited. HCQ treatment of cells resulted in the aggregation of LC3B II, and the observed changes in LC3B II represented only changes in the number of autophagosomes. In this study, LC3B II protein expression induced by BITC was significantly increased in the presence of HCQ. 3MA is an inhibitor targeting PIK3C3/Vps34 (phosphatidylinositol 3-kinase catalytic subunit type 3), one of the components of the PI3KC3 complex, which is recruited to autophagosome precursor structures during phagocytosis and is involved in promoting endocytosis as well as the formation and maturation of autophagosomes and autophagic vesicles.^[159]30 BITC and PI3K inhibitor 3MA induced synergistic cell death but with lysosomal degradation inhibitor HCQ did not increase the inhibitory effect of BITC on ATC cell viability, suggesting that 3MA and BITC may complement each other to inhibit autophagy by inhibiting initiation and degradation steps of autophagy. In addition, in the presence of autophagy inhibition, if the basic autophagy of ATC cells has a prosurvival effect, the combination of autophagy inhibitor and BITC will result in more dead cells. This study shows that for HCQ, the mechanism may overlap with BITC, and may compete with BITC. However, 3MA increased BITC-induced cell death, indicating a tumor-promoting effect of basal autophagy in ATC cells. This suggests that BITC-induced autophagy inhibition may be beneficial in the treatment of ATC. In this study, due to limited time and resources, the relative toxicity of BITC to normal cells was not examined. However, toxicity testing of BITC against normal cells has been carried out in some studies. Studies have shown that BITC inhibits cell growth, promotes G2/M phase arrest, and triggers apoptosis of oral cancer OC2 cells, with minimal toxicity to normal PBMCs.^[160]31 Similarly, BITC has been found to induce apoptosis in breast cancer cells but has no effect on normal breast MCF-10A cells.^[161]32 These studies show that BITC is selectively toxic to tumor cells and safe for use in the treatment of ATC. 4. Conclusions In summary, this study combined network pharmacology, molecular docking, and in vitro experiments to predict and verify its anti-ATC targets and mechanisms. BITC could damage the autophagy degradation of ATC cells, inhibit the expression of NF-κB, and promote apoptosis. Therefore, the mechanism of ATC cell death induced by BITC is involved in promoting apoptosis in addition to damaging autophagy degradation. The results of this study provide the basis for BITC as a novel anticancer drug and support autophagy as a promising therapeutic target for ATC. 5. Materials and Methods 5.1. Target of BITC Was Obtained The isothiocyanate benzyl ester SMILES structural formula was obtained from the PubChem database ([162]https://pubchem.ncbi.nlm.nih.gov/). It was then imported into PharmMapper ([163]http://lilab-ecust.cn/pharmmapper/) database to find the target of BITC. Then, from the Genecards database ([164]https://www.genecards.org/), the species was defined as “Homo sapiens” and the target of ATC was searched with the keyword “ATC”. Intersection targets were with Venny2.1 ([165]https://bioinfogP.cnb.csic.es/tools/venny/). 5.2. Construction of Protein Interaction (PPI) Network, Cluster Analysis, and Screening of Key Targets Intersection targets were uploaded to the STRING ([166]https://cn.string-db.org/) database, the restricted species was “Homo sapiens”, exported, and saved as TSV format. Cytoscape software was imported to map the protein interaction network. Topological analysis was performed using the cytoHubba plugin tool of Cytoscape software to sort and screen key targets by the “Degree” algorithm. 5.3. Bioinformatics Bioinformatics ([167]http://www.bioinformatics.com.cn/) comes in the form of the bubble chart GO (gene ontology) and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. Enrichment of the GO analysis included molecular functions (MF), cell composition (CC), and biological process (BP). 5.4. Validation 5.4.1. Molecular Docking According to the Pubchem structure formula of BITC, the structure diagram of BITC was drawn with PyMOL software and stored in MOL2 format for use. The protein molecule corresponding to the required target was searched in Uniprot ([168]https://www.uniprot.org/) database, the protein structure that meets the requirements was selected, and the PDB ([169]https://www.rcsb.org/) database jumped to download the 3D structure of the target protein. AutoDockTools-1.5.7 software was used to optimize the protein structure, such as removing water, and molecular docking was started. Finally, PyMOL (TM) 2.5.2 was used to map and present the results. 5.4.2. Cell Lines and Cell Culture Two human ATC cell lines, CAL-62 and 8505C cells, were used in this study. CAL-62 cell was obtained from the cell bank of the Chinese Academy of Sciences; 8505C cells were purchased from the Institute of Shanghai Academy of Biological Sciences. CAL-62 and 8505C cells used in the experiment were cultured in high-sugar DMEM medium (Xiao peng, Shanghai, China) or RP MI1640 culture (Xiao peng, Shanghai, China) base containing 10% fetal bovine serum (FBS) and 1% double antibody (100 U/mL penicillin and streptomycin, Solarbio, Beijing, China), respectively. Incubation was carried out in a sterile incubator (Chengdu, China) at 37 °C, 5% CO[2], and constant temperature and humidity. The cells were inoculated as monolayers in a culture dish with a cell-to-total culture medium ratio of 1:10. Each time 90% confluence is reached, a subculture was performed. 5.4.3. Cell Viability Assay The effect of BITC on ATC cell viability was evaluated by a Cell Counting Kit-8 (CCK-8) assay (Yeasen, Shanghai, China). CAL-62 and 8505C cells (5000 cells/ml) were placed in 96-well plates with 100 μL per well, cultured for 24 h, and treated with different concentrations of BITC (252492-5G, Sigma-Aldrich, Germany) for 24 h. Then, the cell viability was measured by the CCK-8 method. 5.4.4. Flow Cytometry The cells were inoculated on a 12-well plate with a density of 2.5 × 10^5 cells/mL and a complete medium containing cell suspension of 1 mL per well. Cells reached 70–80%, treated with different drugs for 24 h, cell survival ratio was observed under a microscope, and apoptosis was detected according to the instructions of the kit (Annexin V-FITC/PI Apoptosis Kit, Yeasen, Shanghai, China). The cells were treated with BITC for 24 h (24 h IC[50], concentration reduces growth by 50% within 24 h). After 24 h, the cells were collected and washed with PBS (Servicebio, Wuhan, China), and then washed with 1× binding buffer. Cells were resuspended with 100 μL of binding buffer per tube, followed by 10 μL of Annexin V-FITC and 5 μL of PI. Apoptosis should be detected by flow cytometry (ACEA) within 1 h after 15 min of dark staining. 5.4.5. Western Blot Analysis CAL-62 and 8505C cells (2.5 × 10^6 cells/pores) were cultured in 6-well plates for 24 h. After pretreatment with 3-methyladenine (3MA, M486160, Aladdin, Shanghai, China) or hydroxychloroquine sulfate (HCQ, H141480, Aladdin, Shanghai, China) for 2 h, the cells were then exposed to 10 μmol/L BITC, 2 mmol/L 3MA, 10 μmol/L BITC combined with 2 mmol/L 3MA, 20 μmol/L HCQ, and 10 μmol/L BITC combined with HCQ for 24 h. The total protein of CAL-62 and 8505C cells was obtained by using RIPA buffer (Solarbio, Wuhan, China). An equal amount of protein (10 μg) was added to each 10% SDS-PAGE column (Solarbio, Wuhan, China) and separated. The transferred PVDF (Thermo) film was taken, put in skim milk powder, and nonspecific closure on the table for 2 h. Then, incubated with primary antibody at 4 °C for 14 h. The primary antibodies are as follows: NF-κB (10745-1-AP, 1:1000, Sanying, Wuhan, China), LC3B (JJ0906-6, 1:1000, Hua’an, Hangzhou, China), Beclin-1 (A7353, 1:1000, ABclonal, Wuhan, China), and Bcl-2 (SZ10-03, 1:1000, Hua’an, Hangzhou, China). The PVDF membrane was placed in the corresponding secondary antibody solution (Goat Anti-Rabbit or Goat Anti-Mouse IgG, 1:5000, anola, China), incubated at room temperature for 2 h, incubated with ECL kit (Solarbio, Wuhan, China) for 4 min, and imaged on the visual working system. ImageJ software was used to analyze the protein gray values in the bands; GAPDH (10494-1-AP, 1:5000, Sanying, Wuhan, China) was used as a reference to analyze the relative protein expression levels in each group. 5.4.6. Reverse Transcription-Polymerase Chain Reaction (RT-PCR) The cells were inoculated into 12-well plates with a density of 2.5 × 10^5 cells/mL, when the length reached 70–80%, they were treated with BITC (10 μmol/L) for 24 h, and RNA content was extracted and detected. The expression of LC3B was detected by qPCR. Each sample was set up with three multiple pores. GAPDH was used as the internal reference, and the relative expression of target RNA was measured by 2^–△△Ct. The expression of the target gene was detected by Western blot. The siRNA double strand sequence of GAPDH and LC3B Primer is as follows: GAPDHF: 5′-GGAGCGAGATCCCTCCAAAAT-3′; GAPDHR: 5′-GGCTGTTGTCATACTTCTCATGG-3′; LC3BF: 5′-GAT GTC CGA CTT ATT CGA GAG C-3′; LC3BR: 5′-TTG AGC TGT AAG CGC CTT CTA-3′. Real-time fluorescence quantitative PCR was conducted using the qPCR instrument (7500 real-time Fluorescence Quantitative PCR, ABI). The experiment was conducted according to the instructions of SYBR qPCR SuperMix Plus (Novoprotein Scientific, Shanghai, China) kit. All samples were analyzed repeatedly. GAPDH was used as an internal control. 5.4.7. Mouse Tumor Model and BITC Therapy Male BALB/c nude mice aged 4–5 weeks were provided by Beijing Vital River Laboratory Animal Technology Co. Ltd. (Beijing, China). The use and care of mice during the study were in line with the guidelines of the Tianjin Medical University Institutional Animal Care and Use Committee. The mice were maintained in a climate-controlled environment with a 12 h light/12 h dark cycle of food and water. CAL-62 cells were injected subcutaneously into the mice. The mice were randomly divided into two groups (5 in each group) and treated with intraperitoneal injection of BITC (100 mg/kg) and normal saline. The drug was administered every other day for 21 days, and the weight of the nude mice was measured. The tumor tissue was dissected, and Western blot was performed. 5.4.8. Statistical Analysis GraphPad Prism 9.0 software was used to draw graphs and perform statistical analysis. All values were expressed as mean ± SEM. Statistical differences were assessed by one-way ANOVA with Tukey’s posthoc analysis. P < 0.05 was considered as a threshold for the statistically significant differences. Acknowledgments