*2.5. T3, TOS, and T3E Also Enhanced the Sensitivity to Bortezomib through Protein Levels of NFE2L1 and NRF3 in Other Solid Cancer Cell Lines*

Based on our results, T3, TOS, and T3E may enhance the sensitivity to bortezomib in the H2452 cell line by modulating protein levels of NFE2L1. However, it is unclear whether the same effect can be achieved in other solid cancer cell lines. Therefore, we performed an evaluation of the effects of T3, TOS, and T3E on NFE2L1 and sensitivity to bortezomib in the lung adenocarcinoma cell line, A549, and the pancreatic cancer cell line, PANC1. As a result, the bortezomib alone group showed an increase in protein levels of unprocessed and processed NFE2L1 and mRNA expression levels of proteasome-related proteins compared to the control group, while the combination groups with bortezomib and T3, TOS, and T3E did not show a similar increasing tendency as bortezomib alone group in PANC1 and A549 (Figure 5a–d). In addition, the combination groups with bortezomib and T3, TOS, and T3E demonstrated a significant decrease in cell viability compared to the control group, bortezomib alone group, and the respective alone groups in PANC1 and A549 (Figure 5e,f).

These results suggest that T3, TOS, and T3E may enhance the sensitivity to bortezomib in different types of solid cancers by modulating protein levels of NFE2L1.

**Figure 4.** Effects of the combination of bortezomib and vitamin E on the viabilities. H2452 cells were treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 μM, T3 20 μM, TOS 20 μM, T3E 20 μM for 24 h, and cell viability was evaluated by the WST-8 assay. Data are means ± SD, n = 5. \* *p* < 0.05, \*\* *p* < 0.01 vs. as indicated. BTZ; bortezomib, TP; α-tocopherol, T3; α-tocotrienol, TOS; α-tocopheryl succinate, T3E; 6-O-Carboxypropyl-alpha-tocotrienol.

**Figure 5.** *Cont*.

**Figure 5.** Effects of vitamin E on NFE2L1 in another solid cancer cells. PANC1 (**a**) and A549 (**b**) cells were treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 μM, T3 20 μM, TOS 20 μM, T3E 20 μM for 12 h, and NFE2L1 protein levels were assessed by immunoblotting. Results are representative of three independent experiments. After PANC1 (**c**) and A549 cells (**d**) were treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 μM, T3 20 μM, TOS 20 μM, T3E 20 μM for 12 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. PANC1 (**e**) and A549 cells (**f**) were treated with bortezomib 50 nM or bortezomib 50 nM +TP 20 μM, T3 20 μM, TOS 20 μM, T3E 20 μM for 24 h. After the treatment, cell viability was evaluated by the WST-8 assy. Data are means ± SD, n = 5. \* *p* < 0.05, \*\* *p* < 0.01 vs. as indicated. TP; α-tocopherol, T3; α-tocotrienol, TOS; α-tocopheryl succinate, T3E; 6-O-Carboxypropyl-alpha-tocotrienol.
