1.Synergistic cell death induced by loperamide plus bortezomib is neither apoptosis not autophagic cell death
7. Combined treatment with loperamide and proteasome inhibitors does not induce cell death in normal colon cells
Next, we examined the effect of loperamide plus other proteasome inhibitors (Carfilzomib, MLN9708,Epoxomicin, MG-132) on DLD-1 cells. We found that co-treatment with loperamide enhanced cell death, when combined with other proteasome inhibitors, similar to bortezomib (Fig. 31). When we examined the effect of loperamide and/or any proteasome inhibitor on the viability of CCD-112 and CCD-841 normal colon cells, neither a single treatment nor any combined treatment did induce cell death (Fig. 32). These results suggest that the combined treatment with loperamide and any proteasome inhibitor may selectively kill colon cancer cells, sparing normal cells. When we further examined the expression of CHOP and Noxa in these normal colon cells, treatment with loperamide and/or bortezomib did not up-regulate CHOP and Noxa, differently from those in DLD-1 cells (Fig. 33). These results indicate that the combination treatment with loperamide and bortezomib may selectively kill colon cancer cells via up-regulation of CHOP and Noxa.
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Figure 31.Loperamide enhances the death of DLD-1 cells treated with other proteasome inhibitors.DLD-1 cells were treated with the indicated concentrations of combined treatment with 20 μM loperamide and other proteasome inhibitors (Carfilzomib, MLN9708, Epoxomicin, MG-132)for 24 h and then viability was assessed using calcein-AM and EthD-1.* p<0.05 vs.
Control.
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Figure 32.Loperamide and/or proteasome inhibitors do not induce cell death in normal colon cells.Normal colon cells were treated with the indicated concentrations of combined treatment with 20 μM loperamide and other proteasome inhibitors (Carfilzomib, MLN9708, Epoxomicin, MG-132)for 24 h and then viability was assessed using calcein-AM and EthD-1.
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Figure 33.Loperamide and/or bortezomib do not induce CHOP and Noxa expression in normal colon cells. Normal colon cells were treated with 20 μM loperamide and 40 nM bortezomib for the indicated time points were subjected to Western blotting of the indicated proteins. α-tubulin was examined to verify equal loading.
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IV. DISCUSSION
Bortezomib is a 26S proteasome inhibitor that was approved by the Food and Drug Administration for the treatment of relapsed/refractory multiple myeloma and mantle cell lymphoma (Richardson et al., 2003; Richardson et al., 2005). Previous studies proposed that apoptosis induced by bortezomib requires the up-regulation of pro-apoptotic members of the Bcl-2 protein family, including Noxa, Bax, and Bik (Fribley et al., 2006; Li et al., 2008; Perez-Galan et al., 2007; Qin et al., 2005; Voortman et al., 2007; Zhu et al., 2005). In contrast, accumulation of anti-apoptotic factors, including Mcl-1, was reported to confer cancer cells resistance against bortezomib. On the other hand, bortezomib-induced apoptosis was associated with Mcl-1 cleavage regardless of Mcl-1L accumulation (Gomez-Bougie et al., 2007). In our study, we demonstrated that combination of loperamide and bortezomib could induce the synergistic killing of various colon cancer cells. Loperamide, an FDA-approved antidiarrhea drug, acts on the μ-opioid receptors in the mesenteric plexus of large intestines (Loetchutinat et al., 2003). The cell death by the combination treatment was non-apoptotic and non-autophagic
in DLD-1 cells (Fig. 1-9). Since we observed dramatic ER-derived vacuolation prior to cell death by loperamide plus bortezomib, we hypothesized that this cell death might be related to ER stress. Indeed, loperamide plus bortezomib induced a marked induction of CHOP/GADD153, a marker of the ER stress signaling. In addition, co-treatment with loperamide further enhanced bortezomib-induced Noxa up-regulation. Knockdown of CHOP or Noxa could effectively rescue from the cell death by loperamide plus bortezomib, suggesting their critical involvement. In addition, we found that Noxa was down-regulated by CHOP
６１
knockdown, suggesting the possible mechanistic link between the CHOP and Noxa of DLD-1 cells. We also found that the synergistic cell death may be dependent on ROS generation. While ROS play an important role in physiological cellular functions by activating several enzymatic cascades and transcription factors (Droge, 2002), excessive ROS signals, however, are detrimental, causing Ca2+ overload, mitochondrial depolarization, lipid peroxidation, transcription factor activation and DNA damage, and lead to apoptotic and/or non-apoptotic cell death (Yanet al., 2006). In our study, loperamide plus bortezomib induced the generation of mitochondrial superoxide (MitoSOX-red). Scavenging of ROS by antioxidants (NAC and GSH) and MnSOD mimetic (MnTBAP) attenuated not only the vacuolation but also cell death induced by combined treatment. Aggresome has cytoprotective response that is activated in response to proteasome inhibition perhaps by shuttling ubiquitinated proteins to lysosomes for degradation (Garcia-Mata et al., 2002). We found that co-treatment with loperamide could disrupt aggresome formation induced by bortezomib, possibly contributing to the cell death by the combination. Since active protein synthesis appears to be required for induction of vacuolation and aggresome formation (Wasik et al., 2011; Steffan et al., 2008), we tested the effect of the translation inhibitor cycloheximide on vacuolation and cell death by loperamide plus bortezomib. We found that pretreatment with cycloheximide almost completely inhibited not only the dilation of the ER but also cell death(data not shown). We further tested whether cyclohemixide pretreatment has any effect on the key signals involved in the cell death by loperamide plus bortezomib. Interestingly, co-treatment with cycloheximide remarkably reduced the accumulation of poly-ubiquitinated proteins as well as induction of CHOP and Noxa. Taken together, these results indicate that protein synthesis is required for ER-derived dilation and
６２
subsequent cell death induced by the combined treatment. When we analyzed the cell cycle following treatment with loperamide and/or bortezomib, bortezomib-induced G2/M arrest was abrogated by co-treatment with loperamide without increase in subG1 cell population. Therefore, we speculate that loperamide-mediated cell cycle progression may interfere bortezomib-induced G2 phase, accelerating protein synthesis and aggravating proteotoxicity. Further study on the effect of loperamide and/or bortezomib on cell cycle is required to understand the underlying mechanism of proteotoxicity by the combined treatment. Furthermore, the combined treatment with loperamide and bortezomib could induce intracellular and mitochondrial Ca2+
accumulation. BAPTA-AM pretreatment alleviated the ER dilation but not cell death, suggesting that the increased intracellular Ca2+ levels may be related with ER dilation but not cell death. However, it remains to be clarified how the combined treatment with loperamide and bortezomib induces ROS generation and induction of CHOP/Noxa during the progression of cell death.
Because both bortezomib and loperamide are used in the clinic, safety of the respective drug is already approved. It has been reported that diarrhea is commonly seen with the use of bortezomib in the treatment of multiple myeloma and mantle cell lymphoma patients. In the pivotal studies with this agent, diarrhea occurred in 51% of patients, with 8% of the events being severe (Berenson et al., 2005). In addition to diarrhea, peripheral neuropathy is also a significant side effect of bortezomib. Peripheral neuropathy induced by bortezomib is occurred in 37-44% of clinical trial patients, requiring dose modification and potential changes in the treatment plan when it occurs (Cavaletti et al., 2010; Argyriou et al., 2010). In addition to the antidiarrheal effect, loperamide has been shown to demonstrate the analgesic effect (Kumar et
６３
al., 2012). Therefore, co-treatment with loperamide is expected to not only enhance the
anti-cancer effect of bortezomibbut also reduce the required dose of bortezomib, contributing to minimize its side effects. Furthermore, since combination of loperamide and bortezomib effectively induces cell death in various colon cancer cells, but not in normal cells, this combination regimen may provide a safe and effective cancer therapeutics against colon cancer that is resistant to anti-cancer effect of the proteasome inhibitor.
In conclusion, our results show that combined treatment with loperamide and bortezomib effectively kills colon cancer cells via up-regulation of CHOP and Noxa, ROS generation, and disruption ofaggresomes.
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