Imbalance in intracellular chloride may be involved in

Previously, IVM was shown to target the Cl^- channels, such as γ-aminobutyric acid (GABA) receptors or glutamate-gated Cl^- ion channels (Glu-Cl) (Bai SH et al., 2016), which are expressed only in the nerve system. In addition, recent studies reported that IVM induces Cl^--dependent membrane hyperpolarization and cell death in leukemia cells, maybe via unknown Cl^--channel irrelevant to GABA receptor or Glu-Cl (Bai SH et al., 2016). Furthermore, the increase of the intracellular Cl^- by activation of chloride intracellular channel-1 (CLIC1) was shown to play a critical role in the paraptosis induced by a purified resin glycoside fraction (Zhu D et al., 2019). Thus, we tested whether imbalance intracellular Cl^- ion is also involved in IVM-induced paraptosis. First, we measured the changes in Cl^- levels using MQAE, an indicator of intracellular Cl^-. We found that while Cl^- was weakly detected only in organelles with similar morphology of mitochondria, a remarkable increase in Cl^- was observed in the cytosol, suggesting the release of Cl^- from these organelles.

To test whether IVM-induced vacuolation and cell death is linked to the activity of Cl^- channels, we examined the effects of various inhibitors of Cl -channels. We found that only DIDS (anion exchange inhibitor) and niflumic acid (Ca²⁺-activated Cl^- channel blocker) were inhibited cell death (Figure 23A) and ER vacuolation (Figure 23B). In particular, pretreatment with DIDS or niflumic acid inhibited IVM-induced ER vacuolation and permeabilization, but nor ER reorganization (Figure 24). When we further investigate the correlation among Cl^- imbalance and other signals associated with IVM-induced cell death, we found that either DIDS or niflumic acid did not block IVM-induced ER stress (Figure 25A) and IVM-induced increase in Ca²⁺ levels

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(Figure 25B). These results suggest that Cl^- channel may be activated downstream of ER stress and Ca²⁺, contributing to ER vacuolation, permeabilization and subsequent cell death.

In summary, IVM induces cancer cell death via catastrophic changes in the structure and function of ER. In this process, ER stress and imbalance of Ca²⁺

and Cl^- critically contribute to the anticancer effect of IVM.

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Figure 22. IVM induces the imbalance in Cl^- ion. (A) MDA-MB 435S cells were treated with 25 μM IVM for indicated time points. Treated cells were stained with 3 mM MQAE and observed under the fluorescent and phase contrast microscope. Scale bars, 20 μm.

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Figure 23. Cl^- channel blockers inhibit IVM-induced ER vacuolation and cell death. (A) MDA-MB 435S cells were pretreated with the indicated concentrations of Cl^- channel inhibitors for 30 min and further treated with 25 μM IVM for 24 h, and cellular viability was assessed using calcein-AM and EthD-1. Data represent the mean ± SD. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.005 vs. untreated cells; #P < 0.005 vs. cells treated with IVM. (B) MDA-MB 435S cells were pretreated with the indicated concentrations of Cl^- channel inhibitors for 30 min, and further treated with 25 μM IVM for 12 h, and observed by phase-contrast microscopy. Scale Bars, 20 μm.

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Figure 24. Cl^- channel blocker inhibits IVM-induced ER vacuolation and permeabilization. (A) MDA-MB 435S cells were pretreated with the 200 μM DIDS or 200 μM niflumic acid (NA) for 30 min, and further treated with 25 μM IVM for 12 h, and observed by confocal microscopy. Scale Bars, 10 μm.

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Figure 25. Inhibition of Cl^- channels may act downstream of IVM-induced ER stress and Ca²⁺ imbalance. (A) MDA-MB 435S cells pretreated with 200 μM DIDS, 200 μM Niflumic acid (NA) for 30 min and further treated with 25 μM IVM for indicated time points. Cell extracts were subjected to Western blotting of the indicated proteins, with β-actin used as a loading control. (B) MDA-MB 435S cells pretreated with 200 μM DIDS and NA for 30 min and further treated with 25 μM IVM for 12 h. The image was observed under the fluorescent and phase contrast microscope. Scale Bars, 20 μm.

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Discussion

Previously, IVM was reported that have anticancer effect in various cancer cells (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), but the underlying mechanism was still uncleared. In this study, we observed that IVM induced cell death accompanied by sequential changes of the ER structure, including reorganization, vacuolation, and permeabilization of the ER. When we observed YFP-ER cells and Sec61-GFP cells stained with ER-Tracker Blue-White DPX by confocal microscopy, both YFP-ER and Sec61-GFP showed reticular ER structure in untreated cells without the fluorescence of ER-Tracker White DPX. ER-Tracker Blue-White DPX, a cell permeable dye that selectively labels the ER possibly due to its hydrophobic nature to make them compatible for partitioning into the ER membrane environment (Wijesooriya CS et al., 2019; Cloe L et al., 2000), and ER-Tracker Blue-White DPX is preferentially attached to the thiol groups

(Nandi S et al., 2018). And, in our data. ER-Tracker Blue-White DPX was colocalized Sec61-GFP fluorescence. These results suggested that ER-Tracker Blue-White DPX indicated the ER membrane. In both YFP-ER and Sec61-GFP cells treated with IVM for 4 h, green or yellow fluorescence of round shape was exactly colocalized with ER-Tracker Blue-White DPX. In addition, EM showed the stacked membrane structure of round shape around the nuclei at this time. These ER structures were different from those with the fluorescence of YFP-ER-(+) inside of the ER-derived vacuoles and Sec61-(+) around the derived vacuoles at 8 h of IVM treatment. In addition, ER-Tracker Blue-White DPX was detected only outside of the ER vacuoles. These

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results suggest that IVM-induced ER membrane stacking or aggregation precedes the ER-derived vacuolation. Very interestingly, from 16 h of IVM treatment, phase-contrast microscopy showed that ER-derived vacuoles, which were evidently detected at 8 h of IVM treatment, disappeared. The fluorescence of YFP-ER cells was diffusively scattered in the cellular space including the cytosol and nuclei, except the cellular regions, which were presumed to be megamitochondria. Structurally, the ER is a network of membranes found throughout the cells and connected to the nucleus. Since rough ER is attached to the nuclear envelope that surrounds the nucleus (Dultz E et al., 2007; Gia K. Voeltz et al., 2002), nuclear membrane integrity also may be also disrupted, when the ER membrane lose its integrity. As a result, ER luminal protein might be diffused also into the nuclei at late phase of IVM treatment. The fluorescence of Sec61-GFP reappeared with less stacked patterns in the cytosol and was exactly co-localized with that of ER-Tracker Blue-White DPX. These results suggest that the boundaries of the ER-derived vacuoles and the nuclei are disrupted and the squeezed and stacked ER membrane structures lost the tension due the permeabilization of the ER. In ER permeabilization, Sec61-GFP and ER-tracker were co-localized in re-formed ER reorganization structure. But Its size was larger and less compacted than the ER reorganization.

ER reorganization is a structure characterized by aggregated or stacked ER membrane (Erik L. Snapp et al., 2003). Previous studies reported that ER reorganization was induced by imbalance of ion homeostasis (Ca²⁺, Na⁺), overexpression of ER membrane protein, disruption of vesicle trafficking (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). Among these stimuli

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of ER reorganization, imbalance of ion homeostasis is also reported as the stimulus of ER vacuolation (Yoon MJ et al., 2012; Yoon MJ et al., 2014).

Previously, tunicamycin, an ER stress inducer, was reported to induce ER reorganization, and after induction of ER reorganization, UPR signals were activated (Varadarajan S et al., 2012). ER stress is one of the causes to induce ER vacuolation (Lee D et al., 2016). Therefore, there may exist some extent of correlation between ER reorganization and ER vacuolation, although further detailed study is required. In this study, we observed vesicle-like lipid droplet in electron microscopy image. Therefore, we examined whether the formation of lipid droplets is associated with IVM-induced changes in the ER structure. We found that addition of oleic acid, an inducer of lipid droplet (Lucía C. Lagrutta et al., 2017), promoted IVM-induced ER reorganization and pretreatment with triacsin C, an inhibitor of acyl-CoA synthetase which is known to inhibit the formation of lipid droplets (Dechandt CRP et al., 2017), effectively reduced it. However, either oleic acid or triacsin C, did not affect IVM-induced vacuolation and permeabilization of the ER and subsequent cell death. These results suggest the possibility that IVM-induced ER reorganization is closely linked to lipid homeostasis, but not to other changes in the ER structure and cell death.

There are two basic types of ER, such as rough ER and smooth ER (Schwarz DS et al., 2016). Rough ER is called rough because it has ribosomes attached to its surface and it look like sheets or disks of bumpy membranes. In contrast, smooth ER looks like tubes and important in the creation and storage of lipids and steroids (Gia K. Voeltz et al., 2002). Since ER reorganization is known to be derived from smooth ER and IVM increases lipid droplets at early phase and then induces ER reorganization, suggesting that disruption of lipid

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homeostasis related to smooth ER may be critically involved in IVM-induced ER reorganization.

The ER is the central organelle for protein translocation, protein folding, and protein post-translational modifications (Sano R et al., 2013). When the ER undergoes excessive workload over the capacity of protein folding, ER stress is induced, and to cope with this stress, UPR signaling pathway is activated.

In the present study, IVM increased the expression of ER stress-related proteins.

ER stress is one of the causes of paraptosis, because many paraptosis inducers accumulates misfolded proteins and blocking of protein synthesis inhibits the induction of paraptosis (Sperandio S et al., 2004; Lee D et al., 2016). In addition, ER vacuolation is a key morphological feature of paraptosis. Since we found that IVM induces ER vacuolation prior to cell death, we investigated whether IVM-induced cell death is associated paraptosis. When we pretreated MDA-MB 435S cells with CHX, a protein synthesis inhibitor, IVM-induced ER stress, ER vacuolation, ER permeabilization, and subsequent cell death. These results suggest that the increase of ER stress play a critical role in IVM induced paraptosis.

Interestingly, IVM-induced ER reorganization was rather increased by pretreatment of CHX. Since CHX was previously shown to increase the levels of cholesterol esters (Suzuki M et al., 2012), a source of lipid droplet, CHX-mediated further increase in ER reorganization in IVM-treated cancer cells may be due to the increase in cholesterol levels. These results suggest that disruption of protein homeostasis more critically contribute to IVM-induced cell death than the disruption of lipid homeostasis.

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In addition, paraptosis was shown to be mediated by the mitogen-activated protein kinase (MAPK), such as MEK-2 or JNK (Sperandio S et al., 2004). In our data, IVM activated MAPKs, such as JNK, ERK, and p38. Pretreatment with U0126, a specific inhibitor of MEK, and SB203580, a specific inhibitor of p38, delayed IVM-induced vacuolation and permeabilization of the ER and subsequent cell death. Collectively, these results suggest that ER stress and MAPK pathway are positively involved in IVM-induced cell death and IVM may induce paraptosis in MDA-MB 435S cells.

ER stress can be induced also by imbalance of Ca²⁺ homeostasis, disruption of thiol homeostasis, and increased intracellular ROS (Delic et al., 2012;

Ellgaard et al., 2001; Yoon MJ et al., 2010; Ghosh K et al., 2016). When we tested the possible involvement of Ca²⁺ in IVM-induced paraptosis, IVM increased intracellular Ca²⁺ homeostasis, and IVM-induced vacuolation and permeabilization of the ER, and cell death was inhibited by pretreatment of BAPTA-AM, an intracellular Ca²⁺ chelator. These results suggest that disruption of Ca²⁺ homeostasis play a crucial role in IVM-induced paraptosis.

Interestingly, pretreatment with BAPTA-AM did not affect IVM-induced ER stress responses, except CHOP induction, whereas CHX effectively blocked IVM-induced increase of intracellular Ca²⁺ levels. Therefore, these results suggest that Ca²⁺ imbalance may not be a main cause of IVM-induced ER stress or Ca²⁺ levels may be increased downstream of IVM-induced ER stress.

IVM is known to kill the parasites via induction of membrane hyperpolarization due to irreversible opening of Cl^- channels such as γ-aminobutyric acid (GABA) receptors or glutamate-gated chloride channels (Glu-Cl) (Crump A, 2017). In addition, IVM was reported to induce

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membrane hyperpolarization via the increase of intracellular Cl^- in leukemia cells (Sharmeen S et al., 2010). Furthermore, a purified resin glycoside fraction induced paraptosis via activated Cl^- intracellular channel 1 (CLIC1)-mediated increase in intracellular Cl^- levels (Zhu D et al., 2019). Therefore, we tested whether imbalance of Cl^- was involved in IVM-induced paraptosis.

In the present study, we found that the intracellular Cl^- was enriched in mitochondria-like organelles. When we measured Cl^- ion, using MQAE, a fluorescent Cl^- ion indicator (Koncz C et al., 1994), Cl^- was enriched in untreated YFP-Mito cells (Figure 30). IVM treatment induced the release of the increased Cl^- into the cytosol. Several Cl^- channels, including CLIC4 and IMAC, are known to localize in mitochondria (Brian O’Rourke, 2007) and activation of CLIC1 was shown to induce paraptosis by increasing intracellular Cl^- (Zhu D et al., 2019). When we tested whether chloride imbalance was involved in IVM-induced paraptosis, we found that calcium-activated chloride channel (CACC) inhibited IVM-induced cellular responses.

both DIDS and niflumic acid, which are known to inhibit the Ca²⁺-activated Cl^- channels, decreased IVM-induced increase of Cl^- and its release into the cytosol (Figure 30). In addition, both niflumic acid and DIDS inhibited IVM-induced loss of mitochondrial membrane potential (Figure 31). Furthermore, niflumic acid and DIDS inhibited the vacuolation and permeabilization of the ER and subsequent `cell death (Figure 24). Taken together, IVM-induced increase in Ca²⁺ and subsequent activation of Cl^- channel may critically contribute to the catastrophic changes in the ER structure and subsequent cell death.

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[ER permeabilization and IRE1]

ER permeabilization is accompanied by the loss of ER membrane integrity, which leads to the leakage of ER luminal proteins from the ER lumen to the cytosol. Previous studies showed that ER membrane permeabilization depends on the proapoptotic Bcl-2 members, including Bax and Bak (Wang X et al., 2011). In addition, IRE1 signaling was shown to prevent ER membrane permeabilization mediated by Bax and Bak and the death of cells experiencing ER stress (Kanekura K et al., 2015; Kanekura K et al., 2015). In the present study, we observed the leakage of ER luminal proteins into the cytosol and nuclei in YFP-ER stable cell line at very late phase of IVM treatment, suggesting that ER permeabilization may irreversibly contribute to IVM-induced cell death. IVM-induced ER permeabilization, in spite of the progressive increase in the present study, suggest that IRE1 activation does not have any role in IVM-induced permeabilization or the extent of IRE1 activation was not enough to block its cell death.

[Mitochondria and paraptosis]

In the present study, we focused on the massive change of ER and its underlying mechanism of IVM-induced paraptosis-associated cell death.

Since paraptosis is also accompanied by mitochondrial dilation (Lee D et al., 2016), we examined the effect of IVM on mitochondrial morphology and its function. We found that IVM induces a slight mitochondrial dilation (Figure 26). Increased intracellular ROS was reported to be associated with paraptosis (Yoon MJ et al., 2010; Ghosh K et al., 2016) and IVM was shown to inhibit

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the respiratory complex 1, resulting in the functional impairment of mitochondria (Wang J et al., 2018). Although ROS levels were only slightly increased IVM, pretreatment with GSH or NAC did not affect IVM-induced cellular responses. These results suggest that ROS may not be critically involved in the anticancer effects of IVM. However, basal oxygen consumption rate was decreased, and ATP synthesis was also reduced (Figure 29). Therefore, IVM-induced mitochondrial dysfunction may contribute to its cytotoxic effect in cancer cells.

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Figure 26. IVM induces mitochondria dilation. YFP-Mito/435S cells were treated with 25 μM IVM for indicated time points and observed under the confocal microscope.

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Figure 27. IVM induces the loss of mitochondrial membrane potential.

YFP-Mito/435S cells were treated with 25 μM IVM or 20 μM CCCP for indicated time points. Treated cells were stained with 200 nM TMRM and observed under the confocal microscope.

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Figure 28. induced ROS generation is not important for IVM-induced ER vacuolation and cell death. (A) MDA-MB 435S cells treated with 25 μM IVM for indicated time points were stained with 2.5 μM H2 DCF-DA and observed under the fluorescent and phase contrast microscope. Scale bars, 20 μm. (B) MDA-MB 435S cells were pretreated with the indicated concentrations of NAC and GSH for 30 min and further treated with 25 μM IVM for 24 h, and cellular viability was assessed using calcein-AM and EthD-1. Data represent the mean ± SD. One-way ANOVA and Bonferroni’s post hoc test. *P < 0.005 vs. untreated cells; #P < 0.005 vs. IVM-treated cells. (C) MDA-MB 435S cells were pretreated with the indicated concentrations of Cl -channel inhibitors for 30 min, and further treated with 25 μM IVM for 12 h, and observed by phase-contrast microscopy. Scale Bars, 20 μm.

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Figure 29. IVM decreases the basal respiration and ATP synthesis. (A) Cellular OCR was measured using the XF analyzer as described in Materials and Methods in MDA-MB 435S cells treated with 25 μM IVM for 8 h. (B) Bar histograms showing ATP production in MDA-MB 435S cells treated with IVM for 24 h. ATP production was measured by the luminometer.

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Figure 30. Ivermectin induces the release of Cl^- enriched in mitochondria.

(A) YFP-Mito/435S cells were stained with 3 mM MQAE and observed by confocal microscopy. (B) YFP-Mito/435S cells were treated with 25 μM IVM or 20 μM CCCP for 12 h. Treated cells were stained with 3 mM MQAE and observed under the fluorescent and phase contrast microscope.

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Figure 31. DIDS and NA inhibit the imbalance of Cl^- and loss of mitochondrial membrane potential. MDA-MB 435S cells were pretreated with DIDS or NA and/or treated with 25 μM IVM for 12 h. Treated cells were stained with 3 mM MQAE and 200 nM TMRM and observed under the fluorescent and phase contrast microscope.

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REFERENCE

1. Andrey V. Shubin, Ilya V. Demidyuk, Alexey A. Komissarov, Lola M.

Rafieva, and Sergey V. Kostrov. Cytoplasmic vacuolization in cell death and survival. Oncotarget. 2016 Aug 23; 7(34): 55863–55889.

2. Andrey V. Shubin, Ilya V. Demidyuk, Alexey A. Komissarov, Lola M.

Rafieva, and Sergey V. Kostrov. Cytoplasmic vacuolization in cell death and survival. Oncotarget. 2016 Aug 23; 7(34): 55863–55889.

3. Bai SH, Ogbourne S. Eco-toxicological effects of the avermectin family with a focus on abamectin and ivermectin. Chemosphere. 2016 Jul;154:204-214.

4. Bertolotti A, Zhang Y, Hendershot LM, Harding HP, Ron D. Dynamic interaction of BiP and ER stress transducers in the unfolded-protein response. Nat Cell Biol. 2000 Jun;2(6):326-332.

5. Brian O’Rourke. Mitochondrial Ion Channels. Annu Rev Physiol. 2007;

69: 19–49.

6. Bury M, Girault A, Mégalizzi V, Spiegl-Kreinecker S, Mathieu V, Berger W, Evidente A, Kornienko A, Gailly P, Vandier C, Kiss R.

Ophiobolin A induces paraptosis-like cell death in human glioblastoma cells by decreasing BKCa channel activity. Cell Death Dis. 2013;4:e561.

7. Carey LA, Perou CM, Livasy CA, Dressler LG, Cowan D, Conway K, Karaca G, Troester MA, Tse CK, Edmiston S, Deming SL, Geradts J, Cheang MC, Nielsen TO, Moorman PG, Earp HS, Millikan RC. Race, breast cancer subtypes, and survival in the Carolina Breast Cancer Study.

JAMA. 2006 Jun 7;295(21):2492-502.

8. Chawla, A., Chakrabarti, S., Ghosh, G. & Niwa, M. Attenuation of

- 68 -

yeast UPR is essential for survival and is mediated by IRE1 kinase. The Journal of cell biology 193, 41–50, 2011

9. Chen S, Novick P, Ferro-Novick S. Chen S1, Novick P, Ferro-Novick S. Curr Opin Cell Biol. 2013 Aug;25(4):428-433.

10. Cole L, Davies D, Hyde GJ, Ashford AE. ER-Tracker dye and BODIPY-brefeldin A differentiate the endoplasmic reticulum and golgi bodies from the tubular-vacuole system in living hyphae of Pisolithus tinctorius. J Microsc. 2000 Mar;197(Pt 3):239-49.

11. Crump A. Ivermectin: enigmatic multifaceted 'wonder' drug continues to surprise and exceed expectations. J Antibiot (Tokyo). 2017 May;70(5):495-505.

12. Crump A. Ivermectin: enigmatic multifaceted ‘wonder’ drug continues to surprise and exceed expectations. J Antibiot (Tokyo). 2017 May;70(5):495-505.

13. Dou Q, Chen HN, Wang K, Yuan K, Lei Y, Li K, Lan J, Chen Y, Huang Z, Xie N, Zhang L, Xiang R, Nice EC, Wei Y, Huang C. Ivermectin

13. Dou Q, Chen HN, Wang K, Yuan K, Lei Y, Li K, Lan J, Chen Y, Huang Z, Xie N, Zhang L, Xiang R, Nice EC, Wei Y, Huang C. Ivermectin