**1. Introduction**

Stable isotope labeling with amino acids in cell culture (SILAC) is a polypeptidelabeling technology developed by the Thermo Fisher company of the United States in 2002 [1]. Heavy isotopes (13C or 15N) and light isotopes (12C or 14N) are used to label two essential amino acids (L-lysine and L-arginine) that are contained in a cell-cultured medium, respectively. After the cells were cultured with essential amino acids for 6–10 generations, all proteins were labeled with heavy isotopes or light isotopes. The cellular proteins stimulated by different treatment factors are analyzed by mass spectrometry (MS) to obtain the qualitative and quantitative proteome data [2]. SILAC generally allows heavy and light isotopes-labeled sample cells at the early stage of the experimental workflow, so the variability caused by the sample handling process was minimized [3]. SILAC was widely used in quantitative proteomics to study pathogenesis, drug target, protein modification and dynamics, protein-molecule interaction, and screen special functional proteins [4]. SILAC showed outstanding performance for quantification and dynamics of phosphosites in colorectal cancer with the treatment of the epidermal growth factor receptor (EGFR)-blocking antibody cetuximab, rendering it the effective method for cellular signaling study in cell culture models [5]. In terms of identification of protein-molecule interaction, SILAC combined with various affinity purifications of protein experimental setups could be used to distinguish specific complexes from nonspecific ones [6]. One study performed SILAC to overcome the most challenging problem in defining specific partners in protein complexes. The cells containing an affinity tagged protein were cultured in a light isotopic medium, while wild-type cells were grown in a heavy isotopic medium. The results of MS showed that specific partners appear as isotopically light [7]. SILAC also offers numerous opportunities to discover potential biomarkers and therapeutic targets for some drugs [8]. SILAC in combination with other developed approaches made SILAC more popular; for example, these SILAC labels in pulse or pulse-chase scenarios could be used to measure macromolecular dynamics on time scales of several hours [9]. An MS-based approach combining dynamic-SILAC labeling with isobaric mass tagging was well used to understand protein degradation and synthesis in cellular systems [10]. SILAC provided an effective scheme to comprehensively and systematically qualify and quantify complex mammalian cell proteome, which would promote progress in the medical field.

Ivermectin, marketed in 1981, was commonly used as a broad-spectrum antiparasitic compound. It was approved to treat onchocerciasis (150–200 μg kg−1 body weight), scabies (200 μg kg−1 body weight), lymphatic filariasis (150–200 μg kg−1 body weight), demodicosis (200 μg kg−1 body weight), strongyloidiasis (200 μg kg−1 body weight), pediculosis (400 μg kg−1 body weight), and filariasis (due to Mansonella ozzardi, 6 mg as a single dose) [11].

Because ivermectin mainly targets chloride-dependent channels (γ-aminobutyric acid and glutamate), its safety could be fine in higher animals. In humans, especially the blood-brain barrier can reduce ivermectin delivery to the central nervous system [12]. The safety of ivermectin has been proved with clinical studies on children, infants, and pregnant women. A study including 170 infants and children with the treatment of oral ivermectin (mean dose = 223 μg kg−1) showed good tolerance, and only seven subjects occurred mild adverse events [13]. A study including 893 pregnant women with the oral treatment of ivermectin also showed good tolerance, and no patients were reported to generate serious events (stillbirths, neonatal death, low birth weight, spontaneous abortions, preterm births, and congenital anomalies) [14].

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

Those studies proved that ivermectin was safe enough to be used in human diseases, but there was still insufficient evidence to prove no adverse side effects. The highest ivermectin dose was 200 μg/kg, which was approved by FDA. However, some patients without serious events have used 10 times more than the FDA-approved dose [15]. All those made ivermectin more likely to achieve success in clinical application. In recent days, studies found that ivermectin was effective in a completely new range of diseases, such as neurological disorders, antiviral (e.g., dengue, HIV, and encephalitis), antibacterial (e.g., Buruli ulcer and tuberculosis), anticancer (melanoma, lymphoid leukemia, lung cancer, glioblastoma, and breast cancer) [11]. The functions and mechanisms of ivermectin on anticancer generated interest and excitement in the scientific community. Ivermectin suppresses breast cancer by disrupting cellular signaling in the process and activating cytostatic autophagy through mediating PAK1 expression [16]. Ivermectin showed a synergistic effect with the chemotherapy agents by increasing cell death in leukemia cells. Some researchers, who aimed at overcoming cancer, claimed that ivermectin could be rapidly advanced into clinical trials [17]. Further study on molecular network, signaling pathway, and key biological processes of ivermectin would provide more useful information about this multifaceted "wonder" drug.

This chapter describes that SILAC identifies differentially expressed proteins (DEGs) in ivermectin-treated ovarian cancer cells in the following aspects: (i) ovarian cancer cell culture—TOV21G and labeled with heavy and light SILAC reagents; (ii) ivermectin treatment of SILAC-labeled TOV21G cells and protein preparation; (iii) the quality of SILAC-labeled protein samples with 1D SDS-PAGE; (iv) trypsin-digestion of SILAC-labeled proteins; (v) each fraction was subjected to LC-MS/MS analysis; and (vi) bioinformatics analysis (signaling pathway and biological process). SILAC can be a useful and effective method to detect protein alterations and dynamic changes in living cells, and the results would provide scientific data to further clarify molecular mechanisms of ivermectin in ovarian cancer.
