*2.6. Atorvastatin Did Not Affect NFE2L1 and NRF3 under Bortezomib Treatment*

Vitamin E and its derivatives are well known to have a cholesterol-lowering effect. It also has been reported that NFE2L1 is involved in the regulation of cholesterol [33]. Based on these findings, we speculated that the cholesterol-lowering effect of T3, TOS, and T3E could moderate the protein levels of NFE2L1. Therefore, finally, we evaluated

the effect of atorvastatin, a cholesterol-depleting agent, on NFE2L1 under bortezomib treatment. We observed no significant differences between the bortezomib alone group and the combination group with bortezomib and atorvastatin in protein levels of unprocessed and processed NFE2L1 (Figure 6a), mRNA expression levels of PSMB7 (Figure 6b) and the accumulation of ubiquitinated proteins (Figure 6c). These results suggest that the cholesterol-lowering effect does not affect NFE2L1 under proteasome inhibition.

**Figure 6.** Effects of atorvastatin on NFE2L1 in treatment bortezomib. H2452 cells were treated with bortezomib 50 nM or bortezomib 50 nM +atorvastatin 10 μM for 24 h, and NFE2L1 (**a**) and ubiquitinated protein (**c**) levels were assessed by immunoblotting. α-Tubulin protein levels served as the loading control. A densitometric analysis was performed as described in the Materials and Methods section. (**b**) After H2452 cells were treated with bortezomib 50 nM or bortezomib 50 nM +atorvastatin 10 μM for 24 h, and the mRNA levels of PSMB7 were assessed by real-time quantitative PCR as described in the Materials and Methods section. RPL32 mRNA levels served as the loading control. Data are means ± SD, n = 3. \* *p* < 0.05 \*\* *p* < 0.01 vs. the control. BTZ; bortezomib, statin; atorvastatin.

#### **3. Discussion**

In this study, we initially examined the effects of TP, T3, TOS, and T3E on NFE2L1 and proteasome homeostasis under bortezomib treatment. We observed that T3, TOS, and T3E (but not TP) suppressed the increase in protein levels of NFE2L1, as well as the expression levels of transcriptional target genes such as proteasome-component proteins (PSMA7, PSMB7, and PSMC4) and proteasome maturation proteins (POMP). Furthermore, it was observed that T3, TOS, and T3E inhibited the recovery of proteasome activity under and after bortezomib treatment and that the combination with bortezomib and T3, TOS, and T3E significantly reduced cell viability compared to them alone.

Under proteasome inhibition, NFE2L1 is known to synthesize new proteasomes by promoting the transcription of proteasome-related genes to maintain proteasome homeostasis. It has been reported that their inhibition prevented recovery of proteasome activity during proteasome inhibition and greatly enhanced the sensitivity to proteasome inhibitors in solid tumor cells such as breast cancer [14]. Since similar events to these reports were observed in the present study, T3, TOS, and T3E may also enhance sensitivity to bortezomib in solid cancer cells by targeting NFE2L1 under proteasome inhibition. This suggests that T3, TOS, and T3E may be candidate adjunctive agents for solid cancer treatment with bortezomib. On the other hand, NFE2L2 and NFE2L3, which is a transcription factor belonging to the leucine zipper family like NFE2L1, has also been reported to be involved in the recovery of proteasome activity under proteasome inhibition [5,14,34,35]. Since T3, TOS, and T3E strongly inhibited the recovery of proteasome activity, they may also affect NFE2L2 and NFE2L3. However, further studies are needed to clarify the effects of T3, TOS, and T3E on NFE2L2 and NFE2L3. We also observed the NFE2L1 inhibitory effects in T3, TOS, and T3E but not in TP. When comparing TP and T3, it is known that T3 is more readily taken up in cells than TP due to the presence of a double bond in the side chain [36,37]. Therefore, T3 may have a more immediate and full effect on NFE2L1 in comparison to TP. Additionally, TOS and T3E are derivatives that block the antioxidant group in vitamin E and are not consumed as antioxidants like TP and T3. Therefore, TOS and T3E may show stronger inhibitory effects on NFE2L1 than TP.

This study revealed that TOS and T3E, which are vitamin E derivatives with blocked antioxidant groups, also exerted inhibitory effects on NFE2L1, while atorvastatin, a cholesterollowering agent, did not exert inhibitory effects on NFE2L1. This suggests that T3, TOS, and T3E may exert inhibitory effects on NFE2L1 without implicating the antioxidant and cholesterol-lowering effects that have been identified in vitamin E. However, in this study, we did not confirm the effects of T3, TOS, and T3E on proteins such as NGLY, p97 and Hrd1, which are involved in the release of NFE2L1 from the endoplasmic reticulum into the cytoplasm, and additional investigations are needed to elucidate the mechanism of NFE2L1 inhibition by T3, TOS, and T3E.

DDI2 is a molecule which involve in the maturation of NFE2L1, and its inhibition suppress the function of NFE2L1 [15,16,38]. In the present study, we found that T3, TOS, and T3E suppressed the increase in protein levels of NFE2L1 under proteasome inhibition without affecting the protein level of DDI2 (Figure S1), suggesting that they are a new type of NFE2L1 inhibitor. Additionally, T3, TOS, and T3E may also affect molecules involved in NFE2L1 protein regulation, which suggests that T3, TOS, and T3E may be good tools for investigating NFE2L1 regulation.
