INTRODUCTION

[Ivermectin (IVM)]

Ivermectin, a macrocyclic lactone derived from Streptomyces avermitilis, is FDA-approved antiparasitic agent (Crump A, 2017; Bai SH et al., 2016). It has been utilized by millions of people around the world exhibiting a wide margin of clinical safety. In parasites and helminths, ivermectin increases the activity of γ-aminobutyric acid (GABA) receptors or glutamate-gated chloride ion channels (Glu-Cl), which blocks the signal between neuron and muscle (Bai SH et al., 2016). In mammals, GABA-sensitive neurons are secured by the blood-brain barrier (BBB) within the central nervous system (CNS), protecting vertebrates against potentially harmful effects of IVM (Bai SH et al., 2016; Juarez M et al., 2018). IVM was reported that can inhibit the SERCA, sarcoplasmic/endoplasmic-reticulum Ca²⁺-ATPase. Recently, ivermectin was reported to have an anticancer effect, inducing autophagy or apoptosis in various cancer cell lines, including breast cancer, glioma, colon cancer and leukemia (Markowska A et al., 2019; Juarez M et al., 2018; Dou Q et al., 2016;

Liu Y et al., 2016; Melotti A et al., 2014; Sharmeen S et al., 2010; Draganov D et al., 2015). In addition, IVM induced mitochondrial dysfunction, which is caused by the generation of ROS or inhibition of respiratory complex I (Juarez M et al., 2018; Liu Y et al., 2016; Draganov D et al., 2015). Furthermore, IVM was induced immunogenic cell death by activation of P2X4/P2X7/Pannexin- 1 pathway (Draganov D et al., 2015), and it also inhibited the WNT-TCF pathway to suppress cancer proliferation (Melotti A et al., 2014).

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[Breast cancer]

Breast cancer is the most common malignancy in women, and one of the three most common malignancies worldwide, along with lung and colon cancer (Ferlay J et al., 2015; Torre LA et al., 2012). In 2015 there were 2.4 million estimated new cases and 523,000 estimated deaths worldwide in women, which correspond to about 29% of the total incident cancer cases and 14% of all cancer deaths (Fitzmaurice C et al., 2015). Breast cancer cells may overexpress specific receptors (estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2)) which, when activated can initiate downstream signaling resulting in the expression of genes for cancer cell proliferation, growth, survival, migration, angiogenesis and other vital cell cycle pathways (Pal SK et al., 2011; Yarden Y et al., 2001).

Triple-negative breast cancer (sometimes abbreviated TNBC) refers to any breast cancer that is defined by the lack of expression of ER, PR, and HER2 (Gluz O et al., 2009). Thus, TNBC is characterized by its unique molecular profile, aggressive nature, distinct metastatic patterns, and lack targeted therapies (Perou CM, 2011). Population-based studies have also demonstrated similar results with reduced breast cancer specific survival among those with TNBC, as compared with the luminal subtype (Carey LA et al., 2006).

Although triple negative breast cancers are associated with a generally poor breast cancer specific outcome. The conventional route to treat TNBC patients by taxol derivatives and anthracycline chemotherapy is still widely used until more “druggable” targets are identified (Gluz O et al., 2009). TNBC are resistant to anthracycline and taxane drugs, thus, it is urgently needed to develop new drugs with better efficiency and fewer side effects.

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[ER stress]

The endoplasmic reticulum (ER) is a major organelle for protein translocation, protein folding, and protein post-translational modifications that allow further transport of proteins to the Golgi apparatus and ultimately to vesicles for secretion or display on the plasma surface (Sano R et al., 2013).

The folding capacity of the ER is limited by tightly regulated expression of protein chaperones such as glucose related protein 78 (GRP78), GRP94, and calreticulin (CRT). The ER lumen has an oxidative environment, which is crucial for the formation of disulfide bonds via protein disulfide isomerase (PDI). It is thus understandable that many physiological and/or environmental perturbations including alterations in Ca²⁺ homeostasis, redox changes (Delic et al., 2012), accumulation of misfolded and aggregating proteins (Ellgaard and Helenius, 2001), elevated secretory protein synthesis, glucose deprivation (Ikesugi et al., 2006; Auf et al., 2010; Badiola et al., 2011), altered N -glycosylation (Olivari and Molinari, 2007), cholesterol overload (Kedi et al., 2009; Li et al., 2009), ischaemia (Bilecova-Rabajdova et al., 2010) and viral infection (Zhang and Wang, 2012), might cause an ER stress (Vannucal K et al., 2013). Physiological or pathological insults which overwhelm the folding capacity of the ER, a process named “ER-stress”, activate an evolutionary conserved cascade of signaling events known as the ER stress response, or unfolded protein response (UPR), a tightly orchestrated collection of intracellular signal transduction reactions designed to restore protein homeostasis (Ojha R et al., 2017). The UPR is mediated by three molecular sensors present on the membrane of the endoplasmic reticulum; PKR-like ER kinase (PERK), activated transcription factor 6 (ATF6) and inositol-requiring

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enzyme 1 alpha (IRE1) (Wang M et al., 2016). The ER resident BiP (Hsp70) binds the luminal tails of PERK, ATF6, and IRE1 to suppress their activity.

When the levels of unfolded protein are elevated, BiP is titrated away, enhancing unfolded protein response (Bertolotti A et al., 2000; Shen J et al., 2002). BiP dissociates from UPR sensors, ATF6, IRE1, and PERK, in the presence of unfolded proteins. ATF6 translocates to the Golgi apparatus where it is subsequently cleaved by S1P and S2P. The cytosolic domain translocates to the nucleus where it promotes transcription of BiP, XBP1, proteins involved in lipid biosynthesis, and other chaperones. IRE1 and PERK both are oligomerized and auto-phosphorylated after dissociating from BiP. IRE1 splices incoming mRNAs, including Xbp1. Spliced Xbp1 is translated (XBP1s) and promotes transcription of BiP, EDEM1, proteins involved in lipid biosynthesis, and other chaperones. The product of unspliced Xbp1 translation (XBP1u) inhibits other components of the UPR, specifically XBP1s and ATF6.

Phosphorylated PERK, in turn, phosphorylates eIF2α, inhibiting general translation. Nevertheless, some proteins, such as ATF4, are preferentially translated. ATF4 promotes transcription of CHOP, GADD34, and proteins involved with amino acid regulation, redox homeostasis, and apoptosis (Lewy TG et al., 2017).

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[ER reorganization]

The ER forms a contiguous structure of interconnected sheets and tubules that spreads from the nuclear envelope to the cell cortex (Chen S et al., 2013).

ER reorganization is a structure characterized by aggregated or stacked ER membrane (Erik L. Snapp et al., 2003). ER reorganization is a reversible structure (Erik L. Snapp et al., 2003), and it can be removed by autophagy (Li X et al., 2016). ER reorganization is one of the physiologically redundant response and is known to be associated with diseases such as Emery-Dreifuss case, torsion dystonia, and Hodgkin's lymphoma (Fidziańska A et al., 2004;

Gonzalez-Alegre P et al., 2004; Parmley RT et al., 1976). ER reorganization is previously reported that it was induced by overexpression of ER membrane protein, including Cytochrome b(5)-GFP, Calnexin-GFP, STIM, ORAI3, and VAPB mutant, or by knockdown of Mcl-1, Syntaxin 18, a-SNAP (Erik L.

Snapp et al., 2003, Li X et al., 2016, Varadarajan S et al., 2012; Varadarajan S et al., 2013; Korkhov VM et al., 2009). And some chemicals such as Apogossypol (pan-Bcl-2 antagonist), Calmidazolium and A-7 (calmodulin antagonist), Thapsigargin, Cyclopiazonic acid, and 2,5-di-t-butyl-1,4-benzohydroquinone (SERCA inhibitor), PDMP (glucosylceramide synthase and sphingomyelin synthase inhibitor) were also reported that induced ER reorganization (Erik L. Snapp et al., 2003; Varadarajan S et al., 2012).

Imbalance of ion homeostasis (Ca²⁺, Na⁺) and disruption of vesicle trafficking are also one of the causes of ER reorganization. (Varadarajan S et al., 2012;

Varadarajan S et al., 2013; Korkhov VM et al., 2009). In addition, as a result of connectivity mapping analysis conducted using Apogossypol, it was confirmed that ER reorganization was also caused by various compounds such as ivermectin (anti-parasitic drug), thapsigargin (SERCA inhibitor),

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nordihydroguaiaretic acid (SERCA inhibitor, antioxidant), astemizole (antihistamine drug), troglitazone (antidiabetic and anti-inflammatory drug), mefloquine (anti-malaria drug), suloctidil (withdrawn peripheral vasodilators), terfenadine (anti-histamine drug) (Varadarajan S et al., 2012). Furthermore, ER reorganization is known to attenuate the ER to Golgi membrane trafficking and induce ER stress and UPR (Varadarajan S et al., 2012; Varadarajan S et al., 2013), but underlying mechanisms bout ER reorganization still remain unclear.

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[ER vacuolation]

ER vacuolation is the structure that have a balloon-like shape due to ER dilation. The vacuolization of ER components seems to be due to the osmotic pressure increase resulting from high quantities of misfolded proteins in the ER (Andrey V et al., 2016). The compensation of osmotic pressure by water diffusion into the ER leads to the formation of vacuoles. Furthermore, dysfunction of big conductance calcium-activated potassium channels (BKCa) induced ER vacuolation (Hoa N et al., 2009). BKC inhibition was induced increase of K⁺ in ER and mitochondria, and the K⁺ mediated by osmotic effects leads to the formation of vacuoles (Bury M et al., 2013). Collectively, increase of osmotic pressure in ER via accumulation of misfolded protein or ion imbalance can lead to the ER vacuolation (Andrey V et al., 2016). And some chemicals such as curcumin, celastrol, ophiobolin A that can induce ER stress when it was treated with the cancer cells (Lee D et al., 2016). In Zika virus infection, heme depletion, Also, these are reported that ER vacuolation was appeared (Monel B et al., 2017; Petrillo S et al., 2018). In addition, dysfunctional ERAD, such as treatment of proteasome inhibitor, HSP90 inhibition, overexpression of dominant negative VCP, can affect the intracellular proteostasis and induced ER vacuolation (Mimnaugh EG, 2006).

Moreover, ER vacuolation is phenotype of paraptosis that is non-apoptotic cell death mode accompanied the ER and mitochondria dilation (Lee D et al., 2016). Therefore, induction of ER vacuolation can lead the cytotoxicity.

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[ER permeabilization]

ER permeabilization is a phenomenon that induced leakage of ER luminal protein from ER to cytosol caused by loss of ER membrane integrity.

According to previous studies, ER membrane permeabilization depend on the proapoptotic Bcl-2 members Bax and Bak (Wang X et al., 2011). Under ER stress condition by palmitate, suppression of IRE1 signaling led to the accumulation of the BH3 domain-containing protein Bnip3, which in turn triggered the oligomerization of Bax and Bak in the ER membrane and ER membrane permeabilization (Kanekura K et al., 2015; Kanekura K et al., 2015). ER permeabilization can be assessed using stable cell line expressed ER luminal fluorescent protein S the ER-localized fluorescent protein is released into the cytosol and occupies all the cellular space in cells undergoing ER stress (Wang X et al., 2011; Kanekura K et al., 2015; Kanekura K et al., 2015). Homeostatic alterations in the ER play roles in the pathogenesis of chronic human disorders, such as type 1 and type 2 diabetes, myocardial infarction, stroke, and neurodegeneration, as well as inherited disorders including Wolfram syndrome, which is characterized by b cell death and neurodegeneration (Wang S et al., 2012; Fonseca SG et al., 2011). The ER membrane permeabilization occur in acquired pathological states associated with ER dysfunction, namely, ischemia/reperfusion injury and stroke, as well as Wolfram syndrome, an inherited disease state (Kanekura K et al., 2015)

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[Paraptosis]

The most current anticancer therapies, including chemo-, radio‐ , and immuno-therapy, primarily activate the apoptosis in cancer cells (Fulda S, 2009). But inherent or acquired resistance of cancer cells to various proapoptotic treatments leads to therapeutic failure (Moulder S, 2010). Thus, a better understanding of alternative, non-apoptotic cell death pathways may facilitate the design of novel therapeutics against malignant cancer cells.

Paraptosis is one of programmed cell death mode accompanied by dilation of the ER and mitochondria. It lacks the apoptotic features, including activation of caspase, DNA fragmentation, chromatin condensation, and the formation of apoptotic body. Several reports have shown that paraptosis requires de novo protein synthesis, activation of mitogen-activated protein kinases (MAPKs), and AIP-1/Alix decrease (Sperandio S et al., 2004). Also, recent reports demonstrated that ionic imbalance of Ca²⁺ (Yoon MJ et al., 2012; Yoon MJ et al., 2014) and K⁺ (Bury M et al., 2013), generation of ROS (Yoon MJ et al., 2010; Ghosh K et al., 2016), and perturbation of cellular proteostasis due to proteasomal inhibition (Yoon MJ et al., 2010; Yoon MJ et al., 2014), and disruption of sulfhydryl homeostasis (Kar R et al., 2009; Kim IY et al., 2017;

Seo MJ et al., 2019) are involved in the paraptosis. However, the underlying mechanism of paraptosis for triggering dilation of ER and mitochondria is not fully understood.

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Here, we show that IVM kills various cancer cells via paraptosis. Interestingly, the ER is structurally altered, including the ER reorganization, vacuolation, and permeabilization. IVM increases lipid droplet, relevant to IVM-induced ER reorganization, but not ER vacuolation, ER permeabilization, and cell death. IVM upregulates ER stress, and reduction of ER stress by CHX pretreatment effectively inhibits IVM-induced ER vacuolation, ER permeabilization, and cell death. In addition, IVM increases the intracellular Ca²⁺ and Cl^-, and these related to IVM-induced ER vacuolation, ER permeabilization, and cell death. Collectively, our results show that IVM treatment may provide a therapeutic strategy against breast cancer cell via induction of paraptosis mediated by ER stress and imbalance of Ca²⁺ and Cl -homeostasis.

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