**3. Results and discussion**

### **3.1 Effects of ivermectin on biological behaviors of ovarian cancers**

First, CCK8 experiments were used to measure cell proliferation changes between ovarian cancer cells (SKOV3; TOV-21G) and control cells (IOSE80), treated with and without ivermectin (**Figure 1**). Each type of cells was significantly inhibited by ivermectin with a dose-dependent relationship. The IC50 (half maximal inhibitory concentration) was 29.46 μM for IOSE80 cells, 20.85 μM for SKOV3, *The Anti-Cancer Effects of Anti-Parasite Drug Ivermectin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.95556*

### **Figure 1.**

lymphatic invasion (yes/no), primary therapy outcome success (complete remission/response, partial remission/response, progressive disease, and stable disease), additional radiation therapy (yes/no), survival time (days), tumor residual disease (no macroscopic disease, 1–10 mm, 11–20 mm, and > 20 mm), survival status

(http://ibl.mdanderson.org/tanric/design/basic/index.html) was used for survival analysis of lncRNAs in ovarian cancer. The large-scale CLIP-Seq data with starBasev 2.0 (http://starbase.sysu.edu.cn/mirCircRNA.php) was used to predict the EIF4A3 binding mRNAs. The Kaplan–Meier method relative to the log-rank test was used for survival analysis of mRNAs in ovarian cancers. Statistical significance was set as p value <0.05. GenCLiP 3 (http://ci.smu.edu.cn/genclip3/analysis.php) was used for pathway enrichment analysis of the association of EIF4A3-binding mRNAs and patient survival rates. The detailed procedure was described previously [4].

TRizol® Reagent (Invitrogen, CA, USA) was used to extract total RNAs of cells

TOV21G and A2780 treated with different concentration of ivermectin (0 μM, 10 μM, 20 μM, and 30 μM). The extracted total RNAs was reversely transcribed into cDNAs for qRT-PCR analysis of each lncRNA expression, including KIF9-AS1, HCG15, PDCD4-AS1, ZNRF3-AS1, ZNF674-AS1, LINC00565, SOS1-IT1, WWTR1- AS1, PLCH1-AS1, LINC00517, SNHG3, STARD13-IT1, AL109767.1, HOXC-AS3, LEMD1-AS1, and LBX2-AS1. Beta-actin was set as internal control for qRT-PCR

**2.7 LncRNA-based prognostic signature optimized with lasso regression for**

Lasso regression means least absolute shrinkage and selection operator regression, which was used to optimize and construct lncRNA-based prognostic signature, and the glmnet R package was used to measure the association between survival risk and lncRNA signature in ovarian cancers. Moreover, univariate and multivariate Cox regression, and Kaplan–Meier method were used to identify overall survivalrelated clinical characteristics described above in ovarian cancers to confirm the established lncRNA-based prognostic model. The detailed procedure was described

Benjamini–Hochberg (FDR) for multiple testing was used to correct the p values of IPA, GO, and KEGG analyses. Student's t test was used for qRT-PCR and western

(0 = alive, and 1 = dead), and PANCAN (Pan-Cancer Atlas). TANRIC

*Ovarian Cancer - Updates in Tumour Biology and Therapeutics*

**2.6 Ivermectin-related lncRNAs verified with qRT-PCR**

analysis. The detailed procedure was described previously [4].

blot data (p < 0.05) with data expression of mean SD (n = 3).

**3.1 Effects of ivermectin on biological behaviors of ovarian cancers**

First, CCK8 experiments were used to measure cell proliferation changes between ovarian cancer cells (SKOV3; TOV-21G) and control cells (IOSE80), treated with and without ivermectin (**Figure 1**). Each type of cells was significantly inhibited by ivermectin with a dose-dependent relationship. The IC50 (half maximal inhibitory concentration) was 29.46 μM for IOSE80 cells, 20.85 μM for SKOV3,

**ovarian cancers**

previously [4].

**206**

**2.8 Statistical significance**

**3. Results and discussion**

*Ivermectin suppressed ovarian cancer cell proliferation* in vitro*, measured with CCK8 (A-C), EdU (D-F), and clonogenic experiments (G, H). Reproduced from Li et al. [21], with copyright permission from nature springer publisher, copyright 2020.*

and 22.54 μM for TOV-21G (**Figure 1A**). The IC50 of ovarian cancers were significantly lower than the normal controls. Further, 20 μM ivermectin - slightly lower than IC50 – can effectively inhibit ovarian cancer proliferation (**Figure 1B** and **C**) [21]. For *in vivo* human trial, the highest FDA-approved ivermectin dose was 200 μg/kg for human use in anti-parasite; however, a study on 68 human subjects found that the dose up to 2,000 μg/kg still worked well without CNS toxicity. The mean area under the curve ratios for the 30 and 60 mg doses were 1.24 and 1.40, indicating a minimal accumulation of ivermectin [5, 22]. These data demonstrate that ivermectin was a well-tolerated safe drug. Second, EdU cell proliferation experiments also confirmed that ivermectin significantly suppressed cell proliferation of ovarian cancers with a time-dependent relationship (**Figure 1D**-**F**) [21]. Third, Clonogenic survival experiments confirmed that ivermectin effectively inhibited the formation of cell clones with a time-dependent relationship (**Figure 1G**-**H**) [21]. Moreover, 10 μM ivermectin cannot effectively inhibit cell proliferation of ovarian cancers, 30 μM ivermectin caused cell death of ovarian cancers, and 20 μM ivermectin was a suitable dose to significantly suppress growth and proliferation of ovarian cancer cells.

### **3.2 Effects of ivermectin on cell cycle and apoptosis in ovarian cancers**

Flow cytometry was used to measure cell cycle and apoptosis of ovarian cancer cells treated with and without ivermectin (**Figure 2**) [21]. First, the cell proportion was significantly increased in G0/G phase, decreased in S phase, and no change in G2/M phase in the high concentration (20- and 30-μM) compared to the low concentration (0- and 10-μM) of ivermectin groups (**Figure 2A**-**C**). Second, compared to control group, the proportion of apoptosis cells was significantly increased

associated with reactive oxygen species (ROS) and energy metabolism pathways,

*Ivermectin inhibited cell migration of ovarian cancer cells TOV-21G relative to control cells A2780, analyzed with wound healing experiments. Reproduced from Li et al. [4], with copyright permission from nature springer*

mitochondrially encoded NADH dehydrogenase 5 (ND5), CytB, and ubiquinolcytochrome c reductase hinge protein (UQCRH) (**Figure 4**) [21]. Moreover, ivermectin directly regulated Rbp, CYP3A4, P2RX7, ABCB1, GLRB, ABCG2, P2RX4, P glycoprotein, Abcb1b, strychnine, cytokine, and insulin; and indirectly regulated TNF, APP, MAPK1, ERK1/2, MAPK3, MAPK13, ROS, NFKBIA, testosterone, and

**3.5 SILAC quantitative proteomics revealed the effects of ivermectin on key proteins in energy metabolism pathways in ovarian cancer cells**

SILAC quantitative proteomics was used to detect, identify, and quantify the key protein alterations in energy metabolic pathways in ovarian cancer cells treated with (SILAC: H) and without (SILAC: L) 20 μM ivermectin for 24 h (**Table 1**) [21]. This study found that ivermectin significantly reduced (i) the expression levels of glycolysis-related enzymes, including ADH5, ENO1, GPI, GAPDH, LDHA, LDHB, PFKP, and PKM; (ii) the Kreb's cycle-related enzymes, including ACON, PCK2, PDHB, MDH2, CS, IDH2, IDH3A, IDH3B, SUCLG2, and OGDHL; (iii) the OXPHOS-related enzymes, including CYTB, UQCRH, COX17, COX1, COX6C, COX4I1, COX2, COX7A2L, COX7A2, ATP6V0C, and ATP6; and (iv) the lactate

**3.6 RT-qPCR and Western blot confirmed the effects of ivermectin on the key molecules in energy metabolism pathways at the mRNA and protein levels**

in energy metabolism pathways in ovarian cancer cells treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) (**Figure 5**), and further western blot analysis confirmed the protein expression alterations of those corresponding key molecules (**Figure 6**) [21]. These key molecules included PFKP, and PKM in glycolysis pathway, PDHB, CS, IDH2, IDH3A, IDH3B, and OGDHL in Kreb's cycle pathway, ND2, ND5, CYTB, and UQCRH in oxidative phosphorylation pathway, MCT1, and MCT4 in lactate shuttle. These results clearly showed that ivermectin regulated energy

RT-qPCR analysis confirmed the mRNA expression alterations of key molecules

shuttle proteins MCT1 and MCT4, in ovarian cancer cells.

metabolism pathways in ovarian cancer cells.

including pyruvate kinase muscle (PKM), oxoglutarate dehydrogenase L (OGDHL), mitochondrially encoded NADH dehydrogenase 2 (ND2),

*The Anti-Cancer Effects of Anti-Parasite Drug Ivermectin in Ovarian Cancer*

*DOI: http://dx.doi.org/10.5772/intechopen.95556*

STAT3 [21].

**209**

**Figure 3.**

*publisher, copyright 2020.*

### **Figure 2.**

*Ivermectin blocked cell cycle progression (A, B, C) and promoted cell apoptosis (D, E) of ovarian cancer cells. Reproduced from Li et al. [21], with copyright permission from nature springer publisher, copyright 2020.*

in different concentration of ivermectin groups, with a dose-dependent relationship (**Figure 2D** and **E**).

### **3.3 Effect of ivermectin on cell migration in ovarian cancers**

Wound healing experiment was used to test the effect of ivermectin on cell migration of ovarian cancer cells. The results showed that cell migration was significantly inhibited in cells A2780 and TOV-21G after treatment of 20 μM and 30 μM ivermectin (**Figure 3**) [4].

### **3.4 Pharmaceutic molecular network predicted the association of ivermectin with ROS and energy metabolism**

Ingenuity Pathway Analysis (IPA) was used for pharmaceutic molecular network analysis of ivermectin. The results showed that ivermectin was significantly *The Anti-Cancer Effects of Anti-Parasite Drug Ivermectin in Ovarian Cancer DOI: http://dx.doi.org/10.5772/intechopen.95556*

**Figure 3.**

*Ivermectin inhibited cell migration of ovarian cancer cells TOV-21G relative to control cells A2780, analyzed with wound healing experiments. Reproduced from Li et al. [4], with copyright permission from nature springer publisher, copyright 2020.*

associated with reactive oxygen species (ROS) and energy metabolism pathways, including pyruvate kinase muscle (PKM), oxoglutarate dehydrogenase L (OGDHL), mitochondrially encoded NADH dehydrogenase 2 (ND2), mitochondrially encoded NADH dehydrogenase 5 (ND5), CytB, and ubiquinolcytochrome c reductase hinge protein (UQCRH) (**Figure 4**) [21]. Moreover, ivermectin directly regulated Rbp, CYP3A4, P2RX7, ABCB1, GLRB, ABCG2, P2RX4, P glycoprotein, Abcb1b, strychnine, cytokine, and insulin; and indirectly regulated TNF, APP, MAPK1, ERK1/2, MAPK3, MAPK13, ROS, NFKBIA, testosterone, and STAT3 [21].
