Open access peer-reviewed chapter - ONLINE FIRST

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

By Xianquan Zhan and Na Li

Submitted: September 23rd 2020Reviewed: December 19th 2020Published: January 13th 2021

DOI: 10.5772/intechopen.95556

Downloaded: 18

Abstract

Ivermectin is an old, common, and classic anti-parasite drug, which has been found to have a broad-spectrum anti-cancer effect on multiple human cancers. This chapter will focus on the anti-cancer effects of ivermectin on ovarian cancer. First, ivermectin was found to suppress cell proliferation and growth, block cell cycle progression, and promote cell apoptosis in ovarian cancer. Second, drug pathway network, qRT-PCR, and immunoaffinity blot analyses found that ivermectin acts through molecular networks to target the key molecules in energy metabolism pathways, including PFKP in glycolysis, IDH2 and IDH3B in Kreb’s cycle, ND2, ND5, CYTB, and UQCRH in oxidative phosphorylation, and MCT1 and MCT4 in lactate shuttle, to inhibit ovarian cancer growth. Third, the integrative analysis of TCGA transcriptomics and mitochondrial proteomics in ovarian cancer revealed that 16 survival-related lncRNAs were mediated by ivermectin, SILAC quantitative proteomics analysis revealed that ivermectin extensively inhibited the expressions of RNA-binding protein EIF4A3 and 116 EIF4A3-interacted genes including those key molecules in energy metabolism pathways, and also those lncRNAs regulated EIF4A3-mRNA axes. Thus, ivermectin mediated lncRNA-EIF4A3-mRNA axes in ovarian cancer to exert its anticancer capability. Further, lasso regression identified the prognostic model of ivermectin-related three-lncRNA signature (ZNRF3-AS1, SOS1-IT1, and LINC00565), which is significantly associated with overall survival and clinicopathologic characteristics in ovarian cancer patients. These ivermectin-related molecular pattern alterations benefit for prognostic assessment and personalized drug therapy toward 3P medicine practice in ovarian cancer.

Keywords

  • ovarian cancer
  • ivermectin
  • anti-cancer effect
  • therapeutic targets
  • prognostic assessment
  • biomarker
  • predictive preventive personalized medicine

1. Introduction

Ivermectin is chemically derived from avermectin that was discovered and isolated from soil in Jan by Omura in 1973 [1]. It was approved by Federal Drug Administration (FDA) to use for anti-parasite drug in 1987, which has significantly improved global public health as an antiparasite medicine [2]. In 2015, its discovers Drs. Omura and Campbell earned the Nobel Prize in physiology or medicine [2]. Recent years, many studies have demonstrated that ivermectin has extensive roles in anti-bacteria, anti-virus, and anticancer, except for its anti-parasite effects [3, 4, 5]. Its anticancer effect has been shown by many in vitro and in vivo experiments in multiple cancers, including ovarian cancer, breast cancer, triple-negative breast cancer, cervical cancer, lung cancer, gastric cancer, colon cancer, glioblastoma, melanoma, and leukemia [4, 6], with a wide safe and clinically reachable drug concentration of anticancer according to its pharmacokinetic range in treatment of a parasite-infected patient [7]. It offers a promising opportunity to develop a new anticancer drug via drug repositioning of this existing compound with confirmed clinical safety [8].

Ovarian cancer, a very common cancer with high mortality and poor survival in women [9, 10], are involved in multiple signaling pathway network changes [11, 12]. Many intracellular molecules and signaling pathways would be the targets of ivermectin [13]. Ivermectin have shown a potential addition role for ovarian cancer treatment. For example, ivermectin can improve the chemosensitivity of overran cancer via targeting Akt/mTOR signaling pathway [14], and can inhibit PAK1-dependent growth of ovarian cancer cells via blocking the oncogenic kinase PAK1 [15]. Ivermectin also acts as a PAK1 inhibitor to induce autophagy in breast cancer [16]. Ivermectin can enhance p53 expression and cytochrome C release, and reduce the expression levels of CDK2, CDK4, CDK6, Bcl-2, cyclin E, and cyclin D1 in glioblastoma, those promoted the cancer cell apoptosis [17]. Ivermectin can inhibit cancer cell proliferation via decreasing YAP1 protein expression in the Hippo pathway [18]. Ivermectin represses WNT-TCF pathway in WNT-TCF-dependent disease [19]. Ivermectin can promote TFE3 (Ser321) dephosphorylation to block the binding between TFE3 and 14-3-3, and induce TFE3 accumulation in the nucleus of human melanoma cells [20]. Moreover, ivermectin also affects other signaling pathway network in human cancers, including oxidative stress, mitochondrial dysfunction, angiogenesis, epithelial-mesenchymal transition, drug resistance, and stemness in tumor [6]. Thereby, ivermectin demonstrates the potential therapeutic efficiency in multiple malignant tumors.

This book chapter discussed the anti-cancer effects of ivermectin on ovarian cancer in the following aspects: (i) ivermectin inhibited cell proliferation and growth, blocked cell cycle progression, and promoted cell apoptosis in ovarian cancer [4, 21]; (ii) ivermectin inhibited ovarian cancer growth through molecular networks to target the key molecules in energy metabolism pathways, including glycolysis, Kreb’s cycle, oxidative phosphorylation, and lactate shuttle pathways [21]; (iii) Integrated omics revealed that ivermectin mediated lncRNA-EIF4A3-mRNA axes in ovarian cancer to exert its anticancer capability [4, 13]; and (iv) lasso regression identified the prognostic model of ivermectin-related three-lncRNA signature (ZNRF3-AS1, SOS1-IT1, and LINC00565) that is significantly related to overall survival and clinicopathologic characteristics of ovarian cancers [4].

2. Methods

2.1 Ovarian cancer cell biological behaviors affected by ivermectin

The normal ovarian cells IOSE80 and ovarian cancer cells TOV-21 and SKOV3 were treated with ivermectin to measure ivermectin-mediated ovarian cancer cell biological behavior changes. (i) IOSE80, TOV-21G, and SKOV3 were treated with ivermectin (0–60 μM) for 24 h, followed by the use of CCK8 to measure the IC50 of ivermectin in each cell. (ii) TOV-21G and SKOV3 were treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) for 24 h, followed by the use of EdU assay to measure DNA synthesis in each cell. (iii) TOV-21G and SKOV3 were treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) for 48 h, followed by clonogenic assay to measure the in vitro effects of ivermectin in each cell. (iv)TOV-21G and SKOV3 were treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) for 24 h, followed by flow cytometry to measure cell cycle and cell apoptosis changes in each cell. (v) When A2780 and TOV-21G seeded in 6-well plates were grown to approximately 90% confluency, followed by the use of 10-μl pipette tip to make an artificial wound, and then treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) for 24 h, and measure the wound healing. The relative percentage of wound healing = (the width of wound at 0 h − the width of wound at 24 h)/the width of wound at 0 h. The detailed procedure was described previously [4, 21].

2.2 Ivermectin-mediated pathway network predicted by ingenuity pathway analysis

The classical pathway network analysis software, Ingenuity Pathway Analysis (IPA) (http://www.ingenuity.com) [5] was used to predict ivermectin-related potential target molecules in three energy metabolism pathways. For this analysis, ivermectin and target genes in three energy metabolism pathways are all input into the IPA tool. The detailed procedure was described previously [21]. The predicted ivermectin-mediated targets in energy metabolism pathways were the basis for further experiment verification.

2.3 Ivermectin-mediated target molecule changes in energy metabolism pathways verified at the mRNA and protein levels

TOV-21G and SKOV3 were treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) for 24 h, and 48 h. At the 24 h time point, the RNAs were extracted for quantitative real-time PCR (qRT-PCR) analysis to measure the mRNA expression of target molecules (CS, PDHB, IDH2, IDH3A, IDH3B, PFKP, PKM, MCT1, MCT4, OGDHL, ND2, ND5, CYTB, and UQCRH) in energy metabolism pathways. At the 48 h time point, the proteins were extracted for Western blot analysis to measure the protein expression of target molecules (CS, PDHB, IDH2, IDH3A, IDH3B, PFKP, PKM, MCT1, MCT4, OGDHL, ND2, ND5, CYTB, and UQCRH) in energy metabolism pathways. The detailed procedure was described previously [21].

2.4 Ivermectin-mediated proteome changes in ovarian cancer identified by SILAC-based quantitative proteomics

SILAC (stable isotope labeling with amino acids in cell culture)-based quantitative proteomics was used to identified differentially expressed proteins in ovarian cancer TOV-21G treated with and without 20 μM ivermectin [13]. The identified differentially expressed proteins were used for molecular network and signaling pathway analyses to obtain ivermectin-related signaling pathway networks [13]. The detailed procedure was described previously [13].

2.5 Transcriptomics and clinical data of ovarian cancer patients extracted from TCGA database

Level 3 RNA-seq V2 transcriptomics data of 411 OC patients were extracted from The Cancer Genome Atlas (TCGA) data portal (http://cancergenome.nih.gov/) with the corresponding clinical data, including cancer status (with tumor or tumor-free), clinical stage (stages IIA, IIB, IIC, IIIA, IIIB, IIIC, and IV), neoplasm histologic grade (G1, G2, G3, G4, and GX), anatomic neoplasm subdivision (right, left, and bilateral), age at initial pathologic diagnosis (aged from 30 to 87), 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 (0 = alive, and 1 = dead), and PANCAN (Pan-Cancer Atlas). TANRIC (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].

2.6 Ivermectin-related lncRNAs verified with qRT-PCR

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

2.7 LncRNA-based prognostic signature optimized with lasso regression for ovarian cancers

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 survival-related clinical characteristics described above in ovarian cancers to confirm the established lncRNA-based prognostic model. The detailed procedure was described previously [4].

2.8 Statistical significance

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

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, 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.

Figure 1.

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.

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

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.

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].

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.

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 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].

Figure 4.

Pharmaceutic molecular network predicted the associations of ivermectin with reactive oxygen species (ROS) and energy metabolism pathways (A) Disease and functional analysis of ivermectin based on IPA database (B-G). The association of ivermectin with PKM (B), OGDHL (C), ND2 (D), UQCRH (E), ND5 (F), and CYTB (G). Reproduced from Li et al. [21], with copyright permission from nature springer publisher, copyright 2020.

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 shuttle proteins MCT1 and MCT4, in ovarian cancer cells.

PathwayProtein IDGene nameProtein nameQ-valueIntensity HIntensity LRatio H/L
Glycolysis pathwayPFKAPPFKPATP-dependent 6-phosphofructokinase, platelet type0.00E+0014226000000255870000000.54
H3BQ34PKMPyruvate kinase7.46E-03107270000+
ODPBPDHBPyruvate dehydrogenase E1 component subunit beta, mitochondrial0.00E+0040728000016495000000.46
K4EN11GAPDHGAPDH (Fragment)0.00E+0000/
ENOAENO1Alpha-enolase0.00E+00546870000001256600000000.44
F5GXY2LDHAL-lactate dehydrogenase A chain (Fragment)1.00E+0010379000294700000.34
Q5U077LDHBL-lactate dehydrogenase0.00E+0027852000000669900000000.42
A0A0A0MTS2GPIGlucose-6-phosphate isomerase (Fragment)1.00E+00566850001385200000.44
Q6IRT1ADH5S-(hydroxymethyl)glutathione dehydrogenase0.00E+00130810000035137000000.45
B3KUV2ACSS2cDNA FLJ40707 fis, clone THYMU2026835, highly similar to Acetyl-coenzyme A synthetase, cytoplasmic9.53E-039455200257580000.73
H3BRS6ADPGKADP-dependent glucokinase (Fragment)5.31E-0411465000184130000.69
AL1B1ALDH1B1Aldehyde dehydrogenase X, mitochondrial0.00E+00698210001967500000.45
ALDH2ALDH2Aldehyde dehydrogenase, mitochondrial0.00E+0081224000018226000000.44
AL3A2ALDH3A2Aldehyde dehydrogenase family 3 member A20.00E+002250000003943600000.55
AL9A1ALDH9A14-trimethylaminobutyraldehyde dehydrogenase0.00E+0052902000013224000000.48
A0A024QZ64ALDOCFructose-bisphosphate aldolase0.00E+00110480000026507000000.43
H0YDD4DLATAcetyltransferase component of pyruvate dehydrogenase complex (Fragment)0.00E+0053072000012511000000.46
A0A024R713DLDDihydrolipoyl dehydrogenase0.00E+0063217000018438000000.52
Q6FHV6ENO2ENO2 protein0.00E+0061819000028871000000.26
ENOBENO3Beta-enolase0.00E+002158100004823400000.59
B4DG62HK1cDNA FLJ56506, highly similar to Hexokinase-10.00E+00161700000040758000000.53
HKDC1HKDC1Hexokinase HKDC10.00E+001328500005684300000.30
PCKGCPCK1Phosphoenolpyruvate carboxykinase, cytosolic [GTP]0.00E+0012677001603700000.07
A0A384MTT2PCK2Epididymis secretory sperm binding protein0.00E+0040319000010325000000.56
A0A024RBX9PDHA1Pyruvate dehydrogenase E1 component subunit alpha0.00E+0045749000013530000000.49
PFKALPFKLATP-dependent 6-phosphofructokinase, liver type0.00E+00124250000025673000000.52
A0A024R0Y5PFKMATP-dependent 6-phosphofructokinase0.00E+00167760000037688000000.47
Q6P6D7PGAM1Phosphoglycerate mutase0.00E+0011906000000304090000000.36
A0A3B3ITK7PGM1Phosphoglucomutase-10.00E+0072145000016419000000.43
PGM2PGM2Phosphoglucomutase-20.00E+001441800004235800000.40
A0A024R5Z9PKM2Pyruvate kinase1.00E+00355410001254300000.54
Kreb’s cycleODPBPDHBPyruvate dehydrogenase E1 component subunit beta, mitochondrial0.00E+0040728000016495000000.46
B4DJV2CSCitrate synthase0.00E+00242850000053387000000.45
IDHPIDH2Isocitrate dehydrogenase [NADP], mitochondrial0.00E+00128120000029943000000.46
IDH3AIDH3AIsocitrate dehydrogenase [NAD] subunit alpha, mitochondrial0.00E+0026860000011193000000.40
A0A087WZN1IDH3BIsocitrate dehydrogenase [NAD] subunit, mitochondrial0.00E+001426300004771800000.41
OGDHLOGDHL2-oxoglutarate dehydrogenase-like, mitochondrial0.00E+00177070001199700000.56
O75944ACONAconitase (Fragment)0.00E+00048304000
A0A384MTT2PCK2Epididymis secretory sperm binding protein0.00E+0040319000010325000000.56
Q0QF37MDH2Malate dehydrogenase (Fragment)0.00E+005856200000144060000000.42
A0A024R325SUCLG2Succinate--CoA ligase [GDP-forming] subunit beta, mitochondrial0.00E+002322100007798000000.41
Q71UF1ACO2Aconitate hydratase, mitochondrial1.00E+00012950000
A0A024R1Y2ACLYATP-citrate synthase0.00E+00203390000044907000000.46
H0YDD4DLATAcetyltransferase component of pyruvate dehydrogenase complex (Fragment)0.00E+0053072000012511000000.46
A0A024R713DLDDihydrolipoyl dehydrogenase0.00E+0063217000018438000000.52
Q6IBS5DLSTDLST protein0.00E+0060154000013387000000.53
A0A0S2Z4C3FHEpididymis secretory sperm binding protein (Fragment)0.00E+00149870000038495000000.43
IDH3GIDH3GIsocitrate dehydrogenase [NAD] subunit gamma, mitochondrial0.00E+00554460002303800000.54
ODO1OGDH2-oxoglutarate dehydrogenase, mitochondrial0.00E+003250900009496100000.43
A0A494C101PCPyruvate carboxylase, mitochondrial (Fragment)7.83E-043454600196850000.28
PCKGCPCK1Phosphoenolpyruvate carboxykinase, cytosolic [GTP]0.00E+0012677001603700000.07
A0A024RBX9PDHA1Pyruvate dehydrogenase E1 component subunit alpha0.00E+0045749000013530000000.49
A0A024QZ30SDHASuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial0.00E+00109650000029508000000.44
SDHBSDHBSuccinate dehydrogenase [ubiquinone] iron–sulfur subunit, mitochondrial0.00E+001430300005163600000.37
D3DVH1SDHCSuccinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa, isoform CRAa0.00E+00680600001301500000.46
B7ZAF6SUCLA2Succinate--CoA ligase [ADP-forming] subunit beta, mitochondrial0.00E+001149000007262000000.33
Q6IAL5SUCLG1Succinate--CoA ligase [ADP/GDP-forming] subunit alpha, mitochondrial0.00E+0026158000011057000000.34
Oxidative phosphorylationD2Y6X2ND5NADH dehydrogenase subunit 5 (Fragment)5.33E-044125200245910000.41
A0A1B0TCA9CYTBCytochrome b (Fragment)3.59E-0353396000597650000.55
Q567R0UQCRHUQCRH protein0.00E+002520500005469000000.51
C9J8T6COX17Cytochrome c oxidase copper chaperone0.00E+007643200530200000.36
Q6FGA0COX7A2LCOX7A2L protein5.34E-0422802000128040017.81
U3L4G0ATP6ATP synthase subunit a0.00E+001936000003559000000.73
X2C5C9COX1Cytochrome c oxidase subunit 17.89E-0421347000575430000.38
A0A346M047COX2Cytochrome c oxidase subunit II (Fragment)0.00E+00104660000024060000000.38
H3BNI4ATP6V0CV-type proton ATPase proteolipid subunit1.00E+0011819000337880000.47
Q496I0COX7A2COX7A2 protein0.00E+002235500006876600000.32
COX6CCOX6CCytochrome c oxidase subunit 6C0.00E+0024314000578990000.34
COX41COX4I1Cytochrome c oxidase subunit 4 isoform 1, mitochondrial0.00E+00105700000025246000000.40
AT12AATP12APotassium-transporting ATPase alpha chain 21.00E+0025608000481530000.50
ATPGATP5F1CATP synthase subunit gamma, mitochondrial0.00E+0061073000017528000000.61
ATPDATP5F1DATP synthase subunit delta, mitochondrial0.00E+001732900003786600000.59
ATP5IATP5MEATP synthase subunit e, mitochondrial0.00E+001398600004165800000.28
ATPKATP5MFATP synthase subunit f, mitochondrial0.00E+001466000003696600000.57
E9PN17ATP5MGATP synthase subunit g, mitochondrial0.00E+0050181000010398000000.45
Q5QNZ2ATP5PBATP synthase F(0) complex subunit B1, mitochondrial0.00E+00107490000024863000000.46
ATP5HATP5PDATP synthase subunit d, mitochondrial0.00E+005250700009655100000.39
ATPOATP5POATP synthase subunit O, mitochondrial0.00E+00149560000030243000000.56
VPP1ATP6V0A1V-type proton ATPase 116 kDa subunit a isoform 10.00E+00980380005573000000.35
R4GN72ATP6V0D1V-type proton ATPase subunit d 10.00E+002585300008067200000.31
VATAATP6V1AV-type proton ATPase catalytic subunit A0.00E+00119180000032188000000.37
VATB2ATP6V1B2V-type proton ATPase subunit B, brain isoform0.00E+0058339000023106000000.35
A0A024R9I0ATP6V1C1V-type proton ATPase subunit C0.00E+001057900005243000000.36
Q53Y06ATP6V1E1ATPase, H+ transporting, lysosomal 31 kDa, V1 subunit E isoform 10.00E+002261900004575400000.46
A4D1K0ATP6V1FV-type proton ATPase subunit F0.00E+00727470002870100000.62
A0A024R883ATP6V1G1V-type proton ATPase subunit G0.00E+001216600001508900000.58
A0A024R7X3ATP6V1HV-type proton ATPase subunit H0.00E+00337830001663300000.37
COX15COX15Cytochrome c oxidase assembly protein COX15 homolog0.00E+00525070001603500000.47
A0A343FH12COX3Cytochrome c oxidase subunit 30.00E+002518000006206700000.40
H3BNX8COX5ACytochrome c oxidase subunit 5A, mitochondrial0.00E+0048964000014517000000.67
COX5BCOX5BCytochrome c oxidase subunit 5B, mitochondrial0.00E+002373700007549900000.33
CX6B1COX6B1Cytochrome c oxidase subunit 6B10.00E+0027819000010286000000.28
CY1CYC1Cytochrome c1, heme protein, mitochondrial0.00E+004261900008767700000.51
Q5T1Z0LHPPPhospholysine phosphohistidine inorganic pyrophosphate phosphatase9.74E-03020832000
D8VCQ0ND4NADH–ubiquinone oxidoreductase chain 4 (Fragment)3.36E-03360480074939000.44
Q7Z518NDUFA10NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 10, mitochondrial0.00E+00468790001943200000.31
NDUADNDUFA13NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 130.00E+00434610002617800000.34
NDUA2NDUFA2NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 20.00E+00356770001650300000.24
NDUA4NDUFA4Cytochrome c oxidase subunit NDUFA40.00E+009954200010414000000.28
NDUA5NDUFA5NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 50.00E+001263700004405400000.46
NDUA8NDUFA8NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 80.00E+00757710002265600000.33
NDUA9NDUFA9NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 9, mitochondrial0.00E+00381340002122500000.38
H3BNK3NDUFAB1Acyl carrier protein (Fragment)0.00E+00913830002201400000.41
NDUB1NDUFB1NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 10.00E+00525720001047800000.46
H3BPJ9NDUFB10NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 100.00E+00684000003535500000.41
NDUBBNDUFB11NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 11, mitochondrial0.00E+00404080001921100000.31
C9JKQ2NDUFB3NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 3 (Fragment)7.84E-0419660000912170000.33
NDUB4NDUFB4NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 40.00E+00157640001296600000.40
NDUB8NDUFB8NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 8, mitochondrial0.00E+00388970001341100000.34
A0A3B3IT57NDUFB9NADH dehydrogenase [ubiquinone] 1 beta subcomplex subunit 90.00E+00479090001782500000.26
E5KRK5NDUFS1Mitochondrial NADH–ubiquinone oxidoreductase 75 kDa subunit0.00E+008763500014240000000.27
NDUS2NDUFS2NADH dehydrogenase [ubiquinone] iron–sulfur protein 2, mitochondrial0.00E+002552100005558000000.40
NDUS3NDUFS3NADH dehydrogenase [ubiquinone] iron–sulfur protein 3, mitochondrial0.00E+0030355000010071000000.38
H0Y9M8NDUFS4NADH dehydrogenase [ubiquinone] iron–sulfur protein 4, mitochondrial (Fragment)0.00E+00227760001246200000.20
Q6IBA0NDUFS5NADH dehydrogenase (Ubiquinone) Fe-S protein 5, 15 kDa (NADH-coenzyme Q reductase)0.00E+0013539000836310000.36
B7Z4P1NDUFS7cDNA FLJ58024, highly similar to NADH–ubiquinone oxidoreductase 20 kDa subunit, mitochondrial3.38E-03896660001465300001.06
E9PKH6NDUFS8NADH dehydrogenase [ubiquinone] iron–sulfur protein 8, mitochondrial (Fragment)0.00E+0025384000709880000.38
G3V0I5NDUFV1NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrial0.00E+0025550000984240000.34
Q9UEH5NDUFV224-kDa subunit of complex I (Fragment)0.00E+001208100004071300000.33
IPYR2PPA2Inorganic pyrophosphatase 2, mitochondrial0.00E+0081573000017438000000.42
A0A024QZ30SDHASuccinate dehydrogenase [ubiquinone] flavoprotein subunit, mitochondrial0.00E+00109650000029508000000.44
SDHBSDHBSuccinate dehydrogenase [ubiquinone] iron–sulfur subunit, mitochondrial0.00E+001430300005163600000.37
D3DVH1SDHCSuccinate dehydrogenase complex, subunit C, integral membrane protein, 15 kDa, isoform CRAa0.00E+00680600001301500000.46
A0A024R5E5TCIRG1V-type proton ATPase subunit a0.00E+001394500002273000000.73
QCR9UQCR10Cytochrome b-c1 complex subunit 90.00E+002603300004918900000.52
QCR7UQCRBCytochrome b-c1 complex subunit 70.00E+002083300005233000000.37
QCR1UQCRC1Cytochrome b-c1 complex subunit 1, mitochondrial0.00E+00132640000037721000000.43
QCR2UQCRC2Cytochrome b-c1 complex subunit 2, mitochondrial0.00E+00171840000036361000000.43
A0A384NPX8UQCRFS1Cytochrome b-c1 complex subunit Rieske, mitochondrial0.00E+00799040002115400000.34
QCR8UQCRQCytochrome b-c1 complex subunit 80.00E+00885360002378900000.57
Lactate shuttleB4E106MCT1cDNA FLJ53399, highly similar to Monocarboxylate transporter 10.00E+00237990001154200000.53
MOT4MCT4Monocarboxylate transporter 40.00E+0081832000021037000000.38

Table 1.

SILAC quantitative proteomics revealed the protein expression changes of key molecules in energy metabolic pathways in ovarian cancer cells TOV-21G treated with (SILAC: H) and without (SILAC: L) 20 μM ivermectin for 24 h. - means the protein expressed in L group but not in H group. + means the protein expressed in H group but not in L group. /means the protein with expressed value 0 in both H and L groups. Ratio H/L means the ratio of the ivermectin-treated group (SILAC: H) to the no ivermectin-treated group (SILAC: L). Reproduced from Li et al. [21], with copyright permission from nature springer publisher, copyright 2020.

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

RT-qPCR analysis confirmed the mRNA expression alterations of key molecules 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 metabolism pathways in ovarian cancer cells.

Figure 5.

RT-qPCR confirmed the effects of ivermectin on the mRNA expressions of key molecules in the energy metabolism pathways in ovarian cancer cells (a-f). The effects of different concentration of ivermectin (0, 10, 20, and 30 μM) on mRNA expressions of PFKP, PKM, CS, PDHB, IDH2, IDH3A, IDH3B, OGDHL, ND5, ND2, CYTB, UQCRH, MCT1, and MCT4. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Reproduced from Li et al. [21], with copyright permission from nature springer publisher, copyright 2020.

Figure 6.

Western blot confirmed the effects of ivermectin on the protein expressions of key molecules in the energy metabolism pathways in ovarian cancer cells. n = 3. *p < 0.05, **p < 0.01, ***p < 0.001. Reproduced from Li et al. [21], with copyright permission from nature springer publisher, copyright 2020.

3.7 Ivermectin regulated lncRNA-EIF4A3-mRNA axis in ovarian cancer cells

Our quantitative mitochondrial proteomics data identified 1198 differentially mitochondrial proteins (mtDEPs) in human ovarian cancer tissues relative to control ovary tissues [11, 23]. Six RNA-binding proteins among those 1198 mtDEPs were identified, including EIF4A3, SFRS1, IGF2BP2, UPF1, C22ORF28, and EWSR1. Of them, only EIF4A3 was predicted to bind to the mRNA of key molecules in energy metabolism pathways. Further, Starbase predicted 3636 EIF4A3-biding mRNAs in various cancer; and of them, 306 EIF4A3-binding mRNAs was associated with ovarian cancer survival rate. Among 306 EIF4A3-binding mRNAs, the protein expressions of 116 EIF4A3-binding mRNAs and EIF4A3 were found to be inhibited by ivermectin, identified by SILAC quantitative proteomics in ovarian cancer cells treated with and without ivermectin (Table 2) [4].

Protein IDsProtein namesGene namesQ-valueScoreIntensity HIntensity LRatio H/L
A0A0S2Z4C6Serine/threonine-protein phosphatase 2B catalytic subunit alpha isoformPPP3CA01064500400002.321E+090.17
A0A024R7B0Ubiquitin-like protein 5UBL503334000001563600000.20
A0A024R9A9Ubiquitin-conjugating enzyme E2 TUBE2T07545800002161500000.22
A0A494C101Pyruvate carboxylase;Pyruvate carboxylase, mitochondrialPC033454600196850000.28
A0A1W2PNM1Hydroxyacyl-coenzyme A dehydrogenase, mitochondrialHADH011853890003322000000.30
Q496I0Cytochrome c oxidase subunit 7A2, mitochondrialCOX7A20102235500006876600000.32
Q149N6Dedicator of cytokinesis protein 4DOCK4042402200121530000.33
Q15036Sorting nexin-17SNX17092545600642950000.33
J3KN67Tropomyosin alpha-3 chainTPM303442080002254300000.34
Q8WZ82Ovarian cancer-associated gene 2 proteinOVCA20822175000922940000.34
G3V0I5NADH dehydrogenase [ubiquinone] flavoprotein 1, mitochondrialNDUFV10925550000984240000.34
J3QLR828S ribosomal protein S23, mitochondrialMRPS2308599570001275100000.35
J3KSI828S ribosomal protein S7, mitochondrialMRPS70956198000981080000.35
B4DP80NAD(P)H-hydrate epimeraseAPOA1BP047810140003479800000.37
C9JFE4COP9 signalosome complex subunit 1GPS10251899300004473200000.38
Q9Y3B739S ribosomal protein L11, mitochondrialMRPL11051727900052135000.38
Q9Y333U6 snRNA-associated Sm-like protein LSm2LSM20231885700004237800000.38
P63261ActinACTG102123.864E+091.674E+100.38
Q07954Prolow-density lipoprotein receptor-related protein 1LRP1053971000524280000.38
Q68E01Integrator complex subunit 3INTS3011328730001136100000.38
K7EKI439S ribosomal protein L4, mitochondrialMRPL409419470001219000000.39
V9GZ56U6 snRNA-associated Sm-like protein LSm4LSM404899880002173300000.39
A0A0S2Z4T1DNA replication licensing factor MCM3MCM301871.033E+093.186E+090.40
A0A0S2Z3L0Electron transfer flavoprotein subunit alpha, mitochondrialETFA0446232800001.382E+090.40
Q8NFH5Nucleoporin NUP53NUP350812066300003988700000.40
Q5T7C4High mobility group protein B1HMGB10982.916E+091.141E+100.40
A0A024R8M4Phosphoribosyl pyrophosphate synthase-associated protein 1PRPSAP1020882270002006700000.41
G3V2D5Zinc finger protein 36, C3H1 type-like 1ZFP36L10186238500150190000.41
A6NMQ3Alpha-endosulfineENSA05802310002063300000.41
A0A0J9YYL3Poly(U)-binding-splicing factor PUF60PUF6001354689100001.281E+090.42
A0A481SVJ4Matrix-remodeling-associated protein 7MXRA7024184200241670000.42
J3KTF8Rho GDP-dissociation inhibitor 1ARHGDIA0561.44E+093.59E+090.42
P49736DNA replication licensing factor MCM2MCM20909371400002.544E+090.43
K7EJH0Kinetochore protein Spc24SPC2407912780001960000000.43
Q9BRA2Thioredoxin domain-containing protein 17TXNDC170387452400001.838E+090.43
P15531Nucleoside diphosphate kinase ANME10203123800009660900000.43
D3DVA5Rho guanine nucleotide exchange factor 2ARHGEF201626979000802460000.44
B7ZM10Exportin-6XPO6077924200148220000.44
G8JLD3ELKS/Rab6-interacting/CAST family member 1ERC10441796300004284300000.44
B2R7W3Breast carcinoma amplified sequence 2BCAS2015381900001771900000.44
C9JJ1928S ribosomal protein S34, mitochondrialMRPS34010698620001584900000.44
A0A0S2Z4Q4Hepatocyte growth factor-regulated tyrosine kinase substrateHGS010521650001210600000.44
Q53Y51D-dopachrome decarboxylaseDDT0362597700006721600000.45
A0A024R8U9Pyrroline-5-carboxylate reductase 1, mitochondrialPYCR1020723600002651000000.45
Q6FHQ0Histone-binding protein RBBP7RBBP701602.134E+094.792E+090.45
P29144Tripeptidyl-peptidase 2TPP201735230600001.368E+090.45
Q9UP83Conserved oligomeric Golgi complex subunit 5COG50514333000323720000.46
E9PID8Cleavage stimulation factor subunit 2CSTF20391282400002394600000.46
A0A024R496Calcium-binding protein 39CAB390241984300004186500000.46
Q6IAP9U4/U6 small nuclear ribonucleoprotein Prp4PRPF40421681900006214800000.46
A0A024RB32Prostaglandin E synthase 3PTGES301102.02E+095.058E+090.46
E9PMG1RalBP1-associated Eps domain-containing protein 1REPS10813894000281020000.46
P28066Proteasome subunit alpha type-5PSMA501071.677E+094.011E+090.46
I3L2G3Ketosamine-3-kinaseFN3KRP0716246000544770000.46
A0A0S2Z4Z0RNA-binding protein 14RBM140836064700001.446E+090.46
A8K651Complement component 1 Q subcomponent-binding protein, mitochondrialC1QBP01093.184E+097.587E+090.47
O60506Heterogeneous nuclear ribonucleoprotein QSYNCRIP01213.657E+098.092E+090.47
P42345Serine/threonine-protein kinase mTORMTOR014522060001447800000.47
A8K878Mesencephalic astrocyte-derived neurotrophic factorMANF0386044600001.613E+090.47
A0MNN4Shwachman-Bodian-Diamond syndrome isoform 1SMU10603213200006798400000.47
A0A024R8B1TBC1 domain family member 13TBC1D130420484000742490000.47
Q9UHR4Brain-specific angiogenesis inhibitor 1-associated protein 2-like protein 1BAIAP2L10621391000418550000.47
Q5SRT3Chloride intracellular channel protein 1CLIC102991.295E+102.924E+100.47
A0A0S2Z5I7Ribosome maturation protein SBDSSBDS0163445400008088600000.48
Q13505Metaxin-1MTX1010457370001097700000.49
J3KS15Peptidyl-tRNA hydrolase ICT1, mitochondrialICT10.01229017000456290000.50
Q53HN4DNA fragmentation factor subunit alphaDFFA0312723900005397100000.50
P38919Eukaryotic initiation factor 4A-IIIEIF4A30771.57E+093.534E+090.50
B4DY09Interleukin enhancer-binding factor 2ILF20962.642E+096.06E+090.50
E9PF19Transducin beta-like protein 2TBL20281094000002139700000.50
A0A087WXS7ATPase ASNA1ASNA10638501900001.755E+090.51
O43324Eukaryotic translation elongation factor 1 epsilon-1EEF1E10296624600001.236E+090.51
Q15717ELAV-like protein 1ELAVL10922.093E+094.32E+090.51
Q9UMS4Pre-mRNA-processing factor 19PRPF1901278971500002.364E+090.52
P14324Farnesyl pyrophosphate synthaseFDPS0261.03E+092.078E+090.52
P28070Proteasome subunit beta type-4PSMB40255120900001E+090.52
Q0VGA5SARS proteinSARS01491.216E+092.647E+090.53
A0A024RCX8Peptidyl-prolyl cis-trans isomerase-like 1PPIL10101228100003650700000.53
Q9H8H0Nucleolar protein 11NOL110522443000800630000.54
E7EX90Dynactin subunit 1DCTN101318231500001.803E+090.54
Q05D78Double-strand break repair protein MRE11AMRE11A0324064000563500000.54
H7C440DIS3-like exonuclease 2DIS3L20413696000211170000.54
Q9Y3U860S ribosomal protein L36RPL360345445400007702000000.55
Q9UJZ1Stomatin-like protein 2, mitochondrialSTOML201198662300001.683E+090.55
Q567R6Single-stranded DNA-binding protein, mitochondrialSSBP10605618200001.077E+090.55
Q15084Protein disulfide-isomerase A6PDIA603231.201E+102.023E+100.56
Q15645Pachytene checkpoint protein 2 homologTRIP130272766700004929300000.56
A0A024R6K8Epididymis secretory sperm binding proteinWARS0801.769E+092.803E+090.56
Q9NPD3Exosome complex component RRP41EXOSC40252399800004905700000.57
Q9UHB9Signal recognition particle subunit SRP68SRP680683439100006876400000.57
F5H0P4Porphobilinogen deaminaseHMBS09383220001294900000.57
Q96SB4SRSF protein kinase 1SRPK10491235100001990500000.57
Q9Y6W5Wiskott-Aldrich syndrome protein family member 2WASF2022690820001512500000.57
A0A024R8S5Protein disulfide-isomeraseP4HB03001.656E+102.799E+100.57
A0A2X0SF71Rho GTPase-activating protein 17ARHGAP17028594670001165900000.58
P09496Clathrin light chain ACLTA075086500007657000000.58
R4GMU1GDH/6PGL endoplasmic bifunctional proteinH6PD0420789000345250000.60
Q8NCN5Pyruvate dehydrogenase phosphatase regulatory subunit, mitochondrialPDPR0310312000281160000.60
Q8WY22BRI3-binding proteinBRI3BP0.012509970061467000.62
Q6IB29Probable rRNA-processing protein EBP2EBNA1BP20630468000618250000.62
O15031Plexin-B2PLXNB2018309860002212400000.62
C9JYQ960S ribosomal protein L22-like 1RPL22L1066168600001.011E+090.66
Q9UNS2COP9 signalosome complex subunit 3COPS30561579000002941800000.66
A0A0G2JNZ5GlucosylceramidaseGBA061185700001875100000.67
A0A140VK17EH domain-binding protein 1EHBP1046547700170180000.71
F5GWG3Retinoic acid-induced protein 3GPRC5A0111491900001857900000.76
S4R3V8Lipolysis-stimulated lipoprotein receptorLSR0426182000343320001.05
Q9NWT6Hypoxia-inducible factor 1-alpha inhibitorHIF1AN017046940000NaN
B7ZBQ1Mediator of RNA polymerase II transcription subunit 20MED200.012018220000NaN
H3BR38Target of rapamycin complex subunit LST8MLST803013473000NaN
P5281539S ribosomal protein L12, mitochondrialMRPL120.01200NaN
A0A024R1I3Pyridoxal phosphate phosphatasePDXP06040714000NaN
A0A2R8YDS2Ras/Rap GTPase-activating protein SynGAPSYNGAP102018400000NaN
J3KQA0Synaptotagmin-1SYT10260339450000NaN
Q5W0C6Torsin-3ATOR3A03016493000NaN
B4DSK7Mediator of RNA polymerase II transcription subunit 1MED102014356000NaN
Q99549M-phase phosphoprotein 8MPHOSPH8012015782000NaN

Table 2.

The proteins of 116 EIF4A3-biding mRNAs and EIF4A3 were inhibited by ivermectin, identified with SILAC quantitative proteomics in ovarian cancer cells treated with (H) and without (L) ivermectin. Reproduced from Li et al. [4], with copyright permission from nature springer publisher, copyright 2020.

Moreover, TCGA transcriptomics analysis found that 16 lncRNAs had binding sites with EIF4A3 and associated with ovarian cancer survival rate, including SNHG3, HCG15, PDCD4-AS1, KIF9-AS1, ZNRF3-AS1, ZNF674-AS1, LINC00565, SOS1-IT1, WWTR1-AS1, PLCH1-AS1, LINC00517, STARD13-IT1, LEMD1-AS1, AL109767.1, HOXC-AS3, and LBX2-AS1 [23]. Further, RT-qPCR analysis of these 16 lncRNA expressions in ovarian cancer cells treated with ivermectin (0 μM, 10 μM, 20 μM, and 30 μM) compared to control cells, which found 9 lncRNAs (PDCD4-AS1, ZNRF3-AS1, HCG15, KIF9-AS1, LINC00565, ZNF674-AS1, AL109767.1, SOS1-IT1, and LBX2-AS1) were significantly affected by ivermectin (Figure 7) [4].

Figure 7.

RT-qPCR analysis revealed the effects of ivermectin on lncRNAs in ovarian cancers relative to control cells. Reproduced from Li et al. [4], with copyright permission from nature springer publisher, copyright 2020.

These findings clearly demonstrated that ivermectin regulated lncRNA-EIF4A3-mRNA axis in ovarian cancer cells, and these mRNAs included the key molecules in energy metabolism pathways in ovarian cancer cells.

3.8 The prognostic model of ivermectin-related three-lncRNA signature for ovarian cancers identified and optimized by lasso regression

Based on those nine ivermectin-mediated lncRNAs in ovarian cancers, survival analysis and lasso regression were used to identify and optimize the prognostic model of ivermectin-related three-lncRNA signature (ZNRF3-AS1, SOS1-IT1, and LINC00565) (Figure 8) [4]. This prognostic model was significantly related to overall survival and clinicopathologic characteristics in ovarian cancer patients [4], which might benefit for prognostic assessment and personalized drug therapy toward 3P medicine practice in ovarian cancer.

Figure 8.

Lasso regression identified and optimized the prognostic model of ivermectin-related three-lncRNA signature in ovarian cancers. (A and B). Lasso regression complexity is controlled by lambda using the glmnet R package. (C). Overall survival analysis of three-lncRNA signature between high-risk and low-risk groups. Reproduced from Li et al. [4], with copyright permission from nature springer publisher, copyright 2020.

4. Conclusions

Ivermectin, as an old, common, and classic anti-parasite drug, has demonstrated its effective in vitro anti-cancer efficiency for ovarian cancer. Ivermectin significantly inhibited cell proliferation, growth and migration, blocked cell cycle progression, and promoted cell apoptosis of human ovarian cancer cells. Drug pathway network analysis of ivermectin revealed that it was significantly related to the key molecules of four energy metabolism pathways, and RT-qPCR and immunoaffinity blot analyses found that ivermectin significantly regulated these key molecules for those energy metabolism pathways, including PFKP in glycolysis, IDH2 and IDH3B in Kreb’s cycle, ND2, ND5, CYTB, and UQCRH in oxidative phosphorylation, and MCT1 and MCT4 in lactate shuttle. The integrative analysis of TCGA transcriptomics and mitochondrial proteomics in ovarian cancer revealed that 16 survival-related lncRNAs were mediated by ivermectin, which were further confirmed with RT-qPRC in human ovarian cancer cells. SILAC quantitative proteomics analysis revealed that the expressions of RNA-binding protein EIF4A3 and 116 EIF4A3-interacted genes were extensively inhibited by ivermectin. Those 116 EIF4A3-interacted proteins included those key molecules in four energy metabolism pathways, and those lncRNAs regulated EIF4A3-mRNA axes. Thus, ivermectin mediated lncRNA-EIF4A3-mRNA axes in ovarian cancer to exert its anticancer activities. Moreover, lasso regression identified the prognostic model of ivermectin-related three-lncRNA signature (ZNRF3-AS1, SOS1-IT1, and LINC00565), which was significantly associated with overall survival and clinicopathologic characteristics of ovarian cancer patients. These ivermectin-related molecular pattern alterations benefit for prognostic assessment and personalized drug therapy in the context of 3P medicine practice in ovarian cancer.

Moreover, one must realize that these achieved data about the anti-cancer activities of ivermectin in ovarian cancers are derived from the in vitro cell models. It is necessary to expand it into the in vivo animal experiments and pre-clinical and clinical experiments for its real application in ovarian cancers.

Acknowledgments

The authors acknowledge the financial supports from the Shandong First Medical University Talent Introduction Funds (to X.Z.), the Hunan Provincial Hundred Talent Plan (to X.Z.), and the grants from China “863” Plan Project (Grant No. 2014AA020610-1 to XZ).

Conflict of interest

We declare that we have no financial and personal relationships with other people or organizations.

Author’s contributions

X.Z. conceived the concept, designed the manuscript, wrote and critically revised the manuscript, coordinated and was responsible for the correspondence work and financial support. N.L. participated in preparing figures, and partial literature analysis.

Acronyms and abbreviations

FDAFederal Drug Administration
mtDEPsdifferentially mitochondrial proteins
RT-qPCRquantitative real-time PCR
SILACstable isotope labeling with amino acids in cell culture
TCGAThe Cancer Genome Atlas

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Xianquan Zhan and Na Li (January 13th 2021). The Anti-Cancer Effects of Anti-Parasite Drug Ivermectin in Ovarian Cancer [Online First], IntechOpen, DOI: 10.5772/intechopen.95556. Available from:

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