**3.1 Proteomic profile based on SILAC in ivermectin-treated TOV-21G cells**

The flow chart of SILAC quantitative proteomics was shown to summarize the overall analysis process for the identification of ivermectin-related proteins (**Figure 1**).

In total, 4447 proteins were identified with SILAC quantitative proteomics in human ovarian cancer cells treated with ivermectin. The ratio of "heavy"/"light"

**Figure 1.**

*The flow chart of SILAC quantitative proteomics analysis of ovarian cancer cells treated with and without ivermectin.*

*The Use of Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Study… DOI: http://dx.doi.org/10.5772/intechopen.102092*

labeling samples was obtained, including 97.91% proteins with ratio < 1, and 2.09% proteins with ratio > 1. The MS/MS spectra of tryptic peptides EYQDLLNVK (**Figure 2A**) and VVQGSLDSLPQAVR (**Figure 2B**) are taken as examples. For peptide EYQDLLNVK (gene name = NEFM), the excellent b-ion and y-ion series were obtained with high signal-to-noise (S/N) (**Figure 2A**). For peptide VVQGSLDSLPQAVR (gene name = PKC1), the excellent b-ion and y-ion series were also obtained with high signal-to-noise (S/N) (**Figure 2B**).

The fold-changes of some identified proteins were very striking; for example, those upregulated proteins (ratio > 2), including histone H2A, progranulin, cathepsin Z, SWI/SNF-related matrix-associated actin-dependent regulator of chromatin subfamily A-like protein 1, beta-mannosidase, GRAM domain-containing 1C, BMP-2-inducible protein kinase, ribosomal protein L3, ubiquitin-conjugating enzyme E2, PIK3R1, LIM domain-containing protein 1, retinal guanylyl cyclase 1, telomerasebinding protein EST1A, and COX7A2L protein. Some of them have been reported in ovarian cancers. For example, recent studies demonstrated that PI3K/AKT/mTOR and ERK1/2 signaling pathways were involved in this chemoresistance. Progranulin was upregulated in epithelial ovarian cancer cell lines and associated with cisplatin resistance through regulating AKT/mTOR and ERK1/2 signaling pathways [21]. Progranulin is also involved in the process of cartilage development, progression, wound healing, and inflammation in ovarian cancer [22]. Additionally, one study showed that progranulin could directly activate cancer-associated fibroblasts to

**Figure 2.**

*The MS/MS spectra of tryptic peptides with SILAC labeling. (A) MS/MS spectrum of tryptic peptide EYQDLLNVK (gene name = NEFM). (B) MS/MS spectrum of tryptic peptide VVQGSLDSLPQAVR (gene name = PKC1).*

induce the epithelial-mesenchymal transition process of epithelial ovarian cancer cells [23]. Copy number loss of PIK3R1 most commonly occurs in ovarian cancer, which would activate AKT and p110-independent JAK2/STAT3 signaling and renders ovarian cancer cells vulnerable to AKT inhibitors [24]. CD97 can activate NF-κBdependent JAK2/STAT3 pathway, consequently playing an important role in migratory, invasive capacity, and drug-resistant in ovarian cancer cells [25].

Some downregulated proteins (ratio < 0.1) were also very striking; for example, anion exchange protein, Rho guanine nucleotide exchange factor 16, dynein assembly factor 1, glucoside xylosyltransferase 1, glutamine synthetase, insulin-like growth factor-binding protein 2, microtubule-associated protein RP/EB family member 3, myelin proteolipid protein, neurochondrin, neurofilament medium polypeptide, phosphoenolpyruvate carboxykinase, ANKUB1, and TASOR 2. Some of them play an important role in the pathogenesis of ovarian cancer. For example, the most prominent effects of insulin-like growth factor-binding protein 2 in ovarian cancer include promoting driving invasion, proliferation, and suppressing apoptosis. The area under the ROC curve of insulin-like growth factor-binding protein 2 in detecting ovarian cancer was 0.815 (95% CI: 0.721–0.910, P < 0.001), further studies are needed to confirm its diagnostic performance at an early stage of ovarian cancer [26].

The glutamine metabolism could be a novel therapeutic target against cisplatin resistance in various cancers. Glutamine synthetase can take part in the reprogramming of glutamine metabolism to induce cisplatin resistance in A2780 ovarian cancer cells [27]. Anion exchanger 2 is a sodium-independent chloride/bicarbonate transporter, which is implicated in the regulation of membrane potential and intracellular potential of hydrogen (pH value). Anion exchanger 2 was highly expressed in ovarian cancer tissues compared to adjacent non-tumor lesions with quantitative proteomics analysis [28]. Those identified proteins in ovarian cancer cells treated with and without ivermectin with SILAC quantitative proteomics discovered reliable and effective biomarkers and drug targets for the anticancer process of ivermectin [8].

#### **3.2 Ivermectin-mediated molecular pathway in human ovarian cancer cells**

In total, 89 statistically significant molecular pathways were enriched based on those 4447 ivermectin-related proteins with KEGG pathway analysis (**Table 1**).

These molecular pathways demonstrated that ivermectin was involved in multiple cancer-related molecular pathways, such as mismatch repair process, ErbB signaling pathway, HIF-1 signaling pathway, cell-cycle regulation, ubiquitin-mediated proteolysis, AMPK signaling pathway, apoptosis, ferroptosis, proteoglycans, and central carbon metabolism in cancer. These molecular pathways also indicated that ivermectin was involved in multiple cancer pathogenesis, such as energy metabolism pathways, immunity-related pathways, stromal element-related pathways, RNA regulation pathways, hormone signaling pathways, and biosynthesis of substances. Different pathways enriched different proteins, whereas some pathways shared the same proteins. These data showed that ivermectin has a complex influence on various signaling pathways. The results were consistent with many studies previously. Ivermectin induced PAK1-mediated cytostatic autophagy both *in vitro* and *in vivo*, which might be one of the PAK1 inhibitors and inhibits the growth of ovarian cancer, glioblastoma, breast cancer, and NF2 tumors [29]. Another investigation found that ivermectin induced apoptosis in HeLa cells by upregulating Bax and p53 expressions, enhancing cytochrome c release, decreasing the levels of CDK2, CDK6, cyclin E, and cyclin D1 [30]. The primary immunogenic features—immunogenic cell death (ICD)

*The Use of Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Study… DOI: http://dx.doi.org/10.5772/intechopen.102092*



*The Use of Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) to Study… DOI: http://dx.doi.org/10.5772/intechopen.102092*


#### **Table 1.**

*Statistically significant pathways identified with ivermectin-related proteins with KEGG pathway enrichment analysis.*

included the release of high-mobility-group protein B1, secreted ATP, and surfaceexposed calreticulin. Recent data supported that ivermectin could kill human triplenegative breast cancer cells through mechanisms of ICD, which induced pannexin-1 channel opening and cell death [31]. Ivermectin was also reported as an RNA helicase inhibitor, which reduced precursor and mature microRNAs potentially inhibiting cell invasion and proliferation [32]. Additionally, ivermectin effectively targets angiogenesis through decreasing membrane potential, mitochondrial respiration, ATP levels, and increasing mitochondrial superoxide, and the effects proliferation, capillary network formation, and survival in human brain microvascular endothelial cell [33]. According to the cross-talking between different signaling pathways, more favorable evidence indicated that ivermectin in combination with other drugs exhibited more powerful anticancer effects, including daunorubicin, anti-BRAF V600 inhibitors, cytarabine, paclitaxel, and tamoxifen [34]. Obviously, the detailed mechanisms of ivermectin remain unclear. However, the application of new drugs brought ones to better health.

### **3.3 Ivermectin-mediated biological processes in human ovarian cancer cells**

A total of 61 statistically significant biological processes were enriched based on those 4447 ivermectin-related proteins with GO analysis (**Figure 3**). These biological processes indicated that ivermectin was involved in multiple cancer-related biological processes, such as negative/positive regulation of canonical Wnt signaling pathway, cysteine-type endopeptidase activity involved in the apoptotic process, innate immune response-activating signal transduction, protein targeting to membrane, T-cell receptor signaling pathway, regulation of protein ubiquitination, activation of protein kinase activity, regulation of transcription by RNA polymerase II, and DNAbinding transcription factor activity. These results were consistent with many studies previously. For example, CTNNB1 (catenin beta 1, IMPβ1) in the biological process of protein polyubiquitination, was responsible for the nuclear entry of cargoes.

#### **Figure 3.**

*Statistically significant biological processes (BPs) identified with ivermectin-related proteins with GO enrichment analysis.*

Ivermectin can impact thermal stability and α-helicity of IMPα and IMPβ1 by binding to the IMPα armadillo repeat domain [35]. CASP3 in the biological process of protein kinase regulator activity is a member of the cysteine-aspartic acid protease (caspase) family. SK-MEL-28 cells were treated with different concentrations of ivermectin (2.5 μM, 5 μM, and 10 μM). Ivermectin enhanced the apoptosis effect by the upregulation of caspase-3 activity [36]. Also, PAK1 in the biological process of protein kinase regulator activity binds to and inhibits the activity of cyclin-cyclin-dependent kinase 2 or -cyclin-dependent kinase 4 complexes, and thus functions as a regulator of cell-cycle progression at G1. Ivermectin inhibited cancer stem cells formation by regulating the binding of PAK1/Stat3 complex and the IL-6 promoter [37]. YAP1 in the biological process of positive regulation of canonical Wnt signaling pathway was involved in the development, growth, repair, and homeostasis of multiple cancers. Ivermectin inhibited YAP1 nuclear expression and nuclear accumulation in gastric cancer cells. Moreover, in xenografts of gastric cancer cells, ivermectin suppressed tumor growth by regulating YAP1 nuclear expression [38]. Those identified proteins in ovarian cancer cells treated with and without ivermectin based on the SILAC method play important roles in multiple cellular signaling pathways and have broad

biological activities. Those findings provide basic data for further study of ivermectin in ovarian cancer.
