DISCUSSION

Although surgical resection, radiotherapy, and chemotherapy have been used to treat tumors, cancer recurrence and acquirement of cancer resistance to further treatments lead to the failure in cancer therapies. Therefore, it is necessary to identify novel therapeutics to overcome to improve the effects of cancer therapy. Drug repositioning, sometimes referred to as ‘drug repurposing or drug recycling’, is a promising approach to overcome barrier to discovering and developing new drugs by identifying new therapeutic applications of known drugs (Turanli B et al., 2018).

Herein, we show that combination of various dihydropyridine calcium channel blockers (DHP-CCB), anti-hypertensive drugs, and bortezomib (Btz) synergistically killed MDA-MB 435S cells. Interestingly, while amlodipine, nicardipine, niguldipine, or felodipine plus bortezomib demonstrated a slight cytotoxicity in normal breast (MCF-10A) cells, combination of lercanidipine (Ler) and Btz did not induce cell death in these cells. Ler belongs to the third-generation of DHP-CCB, it shows greater vascular selectivity than other DHPs, including Amlo, Nigul, Nicar, and Felo (Meredith PA, 1999; Angelico P et al., 1999). Beneficial efficacy and safety of Ler has made it a flexible choice for the treatment of hypertension in a wide range of patients (Angelico P et al., 1999; Burnier M and Gasser UE, 2007). Here we have found that Ler effectively

enhances the anticancer effects of various PIs, including Btz, Cfz, and Ixz, and conserves normal breast cells in breast cancer cells. The synergistic effects of Ler/Btz were also observed in other types of cancer cells, including gastric, lung, pancreatic, and

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liver cancer cells. Our observation of cellular morphology dramatically triggered cellular vacuolation and subsequent cell death by combination of Ler and Btz, although the sub-lethal dose of Ler or Btz did not induce notable morphological changes. These results suggest that combination therapy with Ler and PIs can provide a safe and effective treatment strategy to improve the effectiveness of PI-based cancer treatment.

The vacuoles found prior to paraptosis were derived from mitochondria and the ER by Ler/Btz-induced cell death in cancer cells. In response to Ler/Btz, mitochondrial swelling was first observed at 8 h and peaked at 16 h, thereafter that mitochondria became contracted. In contrast, dilation of the ER first appeared after 12 h in Ler/Btz treated cells and remained until death-related cell detachment. The vacuolation of mitochondria and the ER, and finally cell death were effectively suppressed by CHX treatment, suggesting that new protein synthesis is required for this cell death. When we investigated that underlying mechanism by which Ler sensizes cancer cells to Btz-mediated cell death, we found that co-treatment with Ler and Btz significantly elevated accumulation of poly-ubiquitinated protein, phosphorylation of PERK and eIF2α, and increases of ATF and CHOP protein levels by Btz treatment. When we compared the expression of ER stress marker proteins in breast cancer cells and normal breast cells, the expression levels of ER stress proteins in normal cells were lower than those in cancer cells. These results suggest that Ler enhances the low-dose Btz-mediated ER stress selectively in cancer cells. We have previously shown that variety natural products, including curcumin (Yoon MJ et al., 2012; Yoon MJ et al., 2014a) and celastrol (Yoon MJ et al., 2014b), effectively kill cancer cells by inducing paraptosis. In

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that study, we showed that proteasome inhibition is necessary but not sufficient for paraptosis, and perturbation of Ca²⁺ homeostasis, particularly mitochondrial Ca²⁺ influx, critically contributes to the efficacious inducing paraptosis. In this study, Ler/Btz induced accumulation of Ca²⁺ in mitochondria and resultant increase cytoplasmic Ca²⁺

levels. Inhibition of MCU-mediated mitochondrial Ca²⁺ influx blocked Ler/Btz-induced mitochondrial swelling, the ER dilation, and subsequent cell death. Interestingly, pretreatment with MCU inhibitors blocked the Ler/Btz-induced MMP loss and increase of cytosolic Ca²⁺ levels. Scavenging of intracellular Ca²⁺ levels using BAPTA-AM effectively inhibited Ler/Btz-induced the ER dilation but not mitochondrial dilation.

These results indicate that MCU-mediated mitochondrial Ca²⁺ overload has a critical role in Ler/Btz-induced mitochondria dilation, subsequent ER dilation, and cell death, whereas increased cytoplasmic Ca²⁺ may play an important role in Ler/Btz-induced ER dilation at the late phase of Ler/Btz treatment.

VDAC (voltage-dependent anion channel) is a Ca²⁺ transport channel which locates in the outer membrane of mitochondria (OMM) and highly permeable to Ca²⁺ (Liao Y et al., 2015). Thus, we tested whether treatment with DIDS, a VDAC inhibitor

(Benítez-Rangel E et al., 2015), affects the cell death induced by combined treatment with Ler and Btz. In contrast to the effect of Ru360, DIDS did not affect this cell death.

Ruthenium red (RR) or Ru360 was shown to induce VDAC1 channel closure in a time-dependent manner and stabilize the channel in a low conduction state (Shoshan-Barmatz V et al., 2017). Therefore, this result shows that DIDS may suppress only VDAC, but Ru360 may block both VDAC and MCU, resulting in the greater blocking effect of

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Ru360 than that of DIDS on Ler/Btz-induced cell death.

Considering the loss of MMP with mitochondrial swelling and subsequent deformation at late time in Ler/Btz treatment, we found that the gradual loading of mitochondrial Ca²⁺ to mitochondria may cause mitochondrial dilation and immediately make the mitochondrial permeability transition pore (mPTP) open (Bernardi P and von Stockum S, 2012; Baumgartner HK et al., 2009; Deniaud A et al., 2008). As a result, mitochondrial Ca²⁺ may be released into the cytosol (Gunter TE and Sheu SS, 2009;

Smaili SS and Russell JT, 1999). Supporting this idea, the menadione (vitamin K3 prodrug), or ER stress inducers, including A23187, thapsigargin, and tunicamycin, were shown to increase the mitochondrial Ca²⁺ influx. Long term elevation of the mitochondrial Ca²⁺ exceeding the threshold may result in mitochondrial membrane permeability (Baumgartner HK et al., 2009; Deniaud A et al., 2008).

While CHX treatment completely inhibited Ler/Btz-induced up-regulation of ER stress marker proteins, Ru360 partially blocked it. However, BAPTA-AM had no effect on it.

In addition, increased expression of ER stress marker proteins was followed by mitochondrial Ca²⁺ overload and subsequent increase in cytosolic Ca²⁺ levels. These results suggest that Ler initially enhances Btz-mediated ER stress and ER dilation by enhancing the activity of the PIs and later by disrupting Ca²⁺ homeostasis. However, the underlying mechanism by Ler/Btz increases the level of mitochondrial Ca²⁺ remains to be clarified. We previously showed that inhibition of MCU significantly inhibited curcumin- or celastrol-induced mitochondrial dilation and subsequent the ER dilation (Yoon MJ et al., 2012; Yoon MJ et al., 2014). The ER was found to be a major source of

６３ treatment. We found that pretreatment with dantrolene (RyR antagonist) or 2-APB (IP₃R antagonist) did not inhibit the Ler/Btz-induced cell death. In addition, high concentration of 2-APB, which is known to inhibit SOCE (DeHaven WI et al., 2008), also had no effect on Ler/Btz-induced cell death. Although the cause of increased Ca²⁺

following treatment with Ler/Btz is not clear, we cannot exclude the possibility that the unidenfitied Ca²⁺ release channel in the ER, other than RyR and IP3R, may contribute to acuumulation of mitochondrial Ca²⁺. Since how cytosolic Ca²⁺ levels are increased following mitochondrial Ca²⁺ overload is not also understood, Further study is necessary to decipher the detailed mechanism by which Ler/Btz disupts Ca²⁺

homeostasis leading to paraptosis. Previously the ER vacuolation was proposed to be associated with the massive formation of misfolded proteins in the ER lumen, an increase in the resulting osmotic pressure, and consequently the influx of water from the cytoplasm (Mimnaugh EG et al., 2006). In addition, additional work is needed to clarify whether there is a direct association between increases in the cytosolic Ca²⁺ level and accumulation in the ER of misfolded proteins, or whether these two mechanisms contribute independently to the ER dilation (Figure 29).

The combination therapy of Ler and PIs has many advantages, including strong

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therapeutic effects, better safety (e.g., the possibility of minimizing toxicity with low PIs administration), the possibility of expanding the applicability of PIs to solid tumors, and the opportunity to lower treatment costs using fewer expensive PIs with Ler, relatively inexpensive (approximately ~ 2 US dollars/20 mg). In addition, since safety profiles, drug dynamics, and metabolism of Btz or Ler have already been established, clinical trials using Ler and PIs can be initiated relatively easily (Tan CRC et al., 2019;

Epstein M, 2001). In the future, our current findings should be confirmed through in vivo experiments that could facilitate successful translations into clinic.

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Figure 29. Hypothetical model of Ler/Btz-induced paraptosis

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