Acetylation of α-tubulin by a Histone Deacetylase Inhibitor , Resminostat , Leads Synergistic Antitumor Effect with Docetaxel in Non-Small Cell Lung Cancer Models

Background: There is a growing body of clinical evidence to demonstrate that inhibition of histone deacetylase is effective in the treatment of various types of cancer. We examined whether acetylation of a non-histone protein α-tubulin was induced by resminostat and this acetylation exerts combination effects with docetaxel since α-tubulin was a target of docetaxel. Methods: The cytotoxicity of resminostat was evaluated in 11 human non-small cell lung cancer cell lines and the inhibitory activity of resminostat against histone deacetylase 6 was also determined. The stability of α-tubulin (the amount of polymerized and acetylated α-tubulin) was analyzed in NCI-H460 cells treated with resminostat and/or docetaxel. Caspase-3/7 activity was analyzed in NCI-H460, A549, NCI-H1975, and MOR/CPR cell lines treated with resminostat alone or in combination with docetaxel. The antitumor efficacies of resminostat and docetaxel were evaluated in xenograft mouse models. Results: Resminostat exhibited anti-proliferative activity in all 11 cell lines tested (IC50, 0.36-8.7 μmol/L) and also inhibited histone deacetylase 6 activities. The amounts of polymerized and acetylated α-tubulin and caspase-3/7 activity were increased by resminostat alone or the combination with docetaxel. This combination synergistically increased antitumor efficacy in all the tumor-bearing mice tested. Conclusions: These results demonstrate that the combination of resminostat and docetaxel offers a promising regimen in the treatment of non-small cell lung cancer.

(CDDP), docetaxel (DTX), and erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) [2].Among them, DTX inhibits the depolymerization of microtubules.This stabilizing effect is then followed by an increase in the polymerization of α-tubulin [3].Monotherapy with DTX, however, has proven insufficient to produce a satisfactory response; hence clinical study is focusing on its effect in combination with other agents.
One such agent is a histone deacetylase (HDAC) inhibitor.Inhibition of HDAC activity is followed by the accumulation of acetylated histones and acetylated non-histone proteins.HDAC regulates chromatin remodeling and is crucial in the reactivation of epigenetically silenced tumor suppressor genes [4,5].Inhibitors of HDAC have been reported to mediate transcriptional activity by modulating the acetylation status of nucleosomal histones proximal to gene promoters [6].In addition to chromatin remodeling, HDAC activity (mainly HDAC6) has been linked to the acetylation of non-histone proteins such as tumor suppressor p53 and α-tubulin [7][8][9].Excessive acetylation of α-tubulin leads to the stabilization of microtubules, resulting in cytotoxicity [10].

Introduction
Lung cancer is the leading cause of cancer-related death worldwide [1].A number of pharmacological agents are frequently used in the treatment of non-small cell lung cancer (NSCLC), including cisplatin effect of DTX is myelosuppression [17], whereas with resminostat, gastrointestinal disturbances are more common [12].This suggests that combination therapy with resminostat and DTX might offer a potent therapy against NSCLC, while avoiding synergistic adverse effects.In addition, DTX-based therapy enhances the stabilization of microtubules through a different biochemical mechanism than resminostat, which acetylates α-tubulin, suggesting a potential improvement in therapeutic efficacy.
The purpose of this study was to investigate the efficacy of combination therapy with resminostat and DTX against chemotherapyresistant NSCLC cells.Our hypothesis was that this combination might provide one of novel regimens for the clinical treatment of NSCLC.

Inhibition of HDAC activity in NCI-H460 cells
The NCI-H460 cells were treated with resminostat or SAHA for 60 min to determine the level of inhibitory activity of each of them against HDAC I/II.The HDAC I/II activity in these cells were subsequently determined using the HDAC-Glo TM I/II Assay and Screening System (Promega, Madison, WI, USA).The inhibitory activity of SAHA or resminostat against HDAC6 was also determined with the HDAC6 Fluorimetric Drug Discovery Kit (Enzo Life Sciences Inc., Farmingdale, NY, USA).

Anti-proliferative activity
The cells (A549, NCI-H460, NCI-H1975, and MOR/CPR) were seeded into a 96-well culture plate (2500 cells/well; 50 μL/well) and resminostat and/or DTX added the next day.The number of viable aprotinin; and a phosphatase inhibitor cocktail) for detection of polymerized α-tubulin.Cells lysates were clarified by centrifugation at 20,000 g for 10 min at 4 °C.The precipitate was lysed in RIPA buffer.For detection of other proteins (listed in Figure 3), the cells were lysed in RIPA buffer.

Statistical analysis
Group differences were analyzed by using a one-way analysis of variance (Dunnett).

Resminostat suppresses proliferation of NSCLC cells
Resminostat and SAHA showed similar inhibitory activity against HDAC I/II (cell-based assay) and HDAC6 (enzyme assay) (Table 1).The cytotoxic activity of resminostat was determined in NSCLC cells, including those with a KRAS mutation and chemotherapy-resistance cells was determined after 48 h incubation using the Cell Counting Kit-8 (Dojindo Molecular Technologies, Inc., Kumamoto, Japan).

Cell cycle in NCI-H460 cells
The NCI-H460 cells were exposed to resminostat (2-50 μmol/L) for 24 h.The cell cycle in these cells (5000 cells) was analyzed using the Cell Cycle Phase Determination Kit (Cayman Chemical Company, Ann Arbor, MI, USA) and a Guava easy Cyte TM flow cytometer (Merck Millipore, Darmstadt, Germany).
Additionally, no cross-resistance to resminostat was observed in EGFR-TKI-or CDDP-resistant cells.Resminostat-resistant NSCLC cells were not detected in 11 cell lines.The influence of resminostat on the cell cycle and related proteins was also determined to investigate the relationship between re-expressed proteins and cell cycle arrest.Resminostat induced expression of p21 accompanied by acetylation of histone H3.Subsequent degradation of cyclin D1 and dephosphorylation of RB ensued (Figure 2A).The cell cycle analysis revealed that resminostat induced G1 phase arrest at lower  and BIM, and caspase-3/7 activity were determined as indicators of induction of apoptosis.Resminostat decreased expression of XIAP and BCL-2, and induced expression of BIM (Figure 4A).Treatment with resminostat or DTX alone induced caspase-3/7 activity in all the tested NSCLC cells; in A549 cells, however, this effect was observed at higher concentrations (data not shown).In addition, combination treatment induced higher caspase-3/7 activity in NCI-H460, A549, and NCI-H1975 cells, than single-agent therapy, and this difference was significant (Figure 4B).

Combination of resminostat and DTX inhibits tumor growth in chemotherapy-resistant NSCLC cells in nude mice
The in vivo efficacy of resminostat in combination with DTX was subsequently determined.The antitumor efficacy was determined in two different schedules, intermittent administration schedule and daily administration schedule.In the former schedule, the mice were treated with resminostat on days 1-5, 8-12, 22-25, 29-33 (Figure 5A).
(2-5 μmol/L) and G2/M phase arrest at higher concentrations (20-50 μmol/L) (Figure 2B and Figure 2C).The status of the cell cycle and related proteins changed depending on the concentration used and duration of treatment.

Combination of resminostat and DTX synergistically promoted stabilization of microtubules and apoptosis
Excessive stabilization of microtubules leads to the suppression of cell division.The stabilization of α-tubulin in resminostat and/or DTX-treated cells was determined with respect to the polymerization and acetylation status of α-tubulin.Resminostat induced acetylation of α-tubulin and promoted its polymerization to the same extent as tubacin, a selective HDAC6 inhibitor.DTX induced polymerization of α-tubulin but not its acetylation.Combination treatment promoted polymerization to a greater extent than resminostat or DTX alone (Figure 3).Next, the effects of stabilization on apoptosis were examined.Expression of anti/pro-apoptotic proteins XIAP, BCL-2, due to reduced interaction between the transcription factor and HDAC2 [32].Change in apoptosis-related proteins influences the cytotoxicity of many antitumor agents.This indicates that resminostat might offer potent synergistic efficacy when used in combination with other antitumor agents.In the present in vivo study, resminostat combination therapy synergistically enhanced antitumor efficacy independent of the dosing schedule and cell line used.The cell lines used here represent several chemotherapy-resistant properties in clinical practice [14][15][16].The in vivo studies validated the effectiveness of the in vitro combination studies with respect to cytotoxicity and tubulin stabilization assays.No severe toxicity was observed in the mice with any of the agents used in the present study.Therefore, resminostat combination therapy is regarded as well tolerated.These results demonstrated that resminostat combination therapy offers a feasible option for NSCLC patients.

Conclusions
Our study indicates that a rational strategy in combination with resminostat and DTX is thought to a promising regimen in the treatment of chemothrapy-resistant NSCLC.Based on the present results, we are proceeding with a phase I/II study in NSCLC patients to further investigate the safety and efficacy of this regimen.
In the latter schedule, the mice were treated with resminostat on days 1-5, 8-12, 15-19 (Figure 5B).There was no difference on antitumor efficacy.Monotherapy with resminostat or DTX yielded a moderate decrease in tumor growth, whereas combination therapy resulted in an appreciable improvement in antitumor efficacy in all the NSCLC tumors tested (Figure 5B-5E) ( p < 0.05 vs. Control).None of the mice treated with these agents showed any severe macroscopic adverse effects.

Discussion
The present results revealed that combination therapy with resminostat and DTX was effective against chemotherapy-resistant NSCLC cells.This combination therapy exerted a synergistic effect, resulting in an increase in antitumor efficacy against several NSCLC cells both in vitro and in vivo, especially with respect to the stabilization of microtubules.Resminostat exhibitsits cytotoxicity on chemotherapy-resistant NSCLC cells through various mechanisms.In this study, no cross-resistance to resminostat was observed in EGFR-TKI-or CDDP-resistant or KRAS mutation cells.The difference in the cytotoxic effect of resminostat and DTX is likely due to the mode of action of each reagent.In general, tubulin-targeting agents exert strong cytotoxicity, whereas HDAC inhibitors exert moderate cytotoxicity [18,19].HDAC inhibitors have several modes of action, including tubulin acetylation, HSP90 inhibition, and pro-apoptosis protein re-expression [20][21][22][23].An agent with several modes of action is less likely to induced resistance due to specific mechanisms.In fact, in the present study, no cross-resistance to resminostat was detected in other chemotherapy-resistant cell lines.This suggests that the combination of resminostat and DTX could be effective in the treatment of NSCLC.
Combination therapy with resminostat and DTX appears to be a suitable option with respect to its mode of action, i.e., the stabilization of microtubules.Increased levels of acetylated α-tubulin with resminostat might be due to inhibition of HDAC6 (Table 1).Acetylated α-tubulin contributes to the stabilization of microtubules, whereas DTX inhibits α-tubulin depolymerization.Thus, used in combination, these two agents exert a synergistic effect in increasing polymerization of α-tubulin, resulting in its stabilization.In addition, these results correlated with those of the apoptosis experiments, which indicated synergistic activation of caspase-3/7.
Resminostat suppressed the proliferation of tumor cells in the G1 phase, and DTX killed other cells in the G2/M phase through stabilization of microtubules.Resminostat induced p21 expression, which was followed by a decrease in cyclin D1 expression and phosphorylation of RB.This arrest of the G1 phase may have been due to this decrease in phospho-RB and cyclin D1 [24][25][26][27].The stabilization of α-tubulin had a significant influence on the cell cycle, resulting in arrest at the G2/M phase [28].In the present study, acetylation of α-tubulin led to stabilization of microtubules, resulting in G2/M phase arrest in cells treated with high concentrations of resminostat.In an earlier study, resminostat induced G1 phase arrest in 3 out of 4 myeloma cell lines associated with upregulation of p21, and G2/M phase arrest in 2 out of 4 cell lines at different concentrations.The cause of this G2/M phase arrest remains to be identified [11].However, resminostat exhibits multiple modes of action, including induction of p21 expression and acetylation of α-tubulin.Thus, both G1 and G2/M phase arrest induced by resminostat appears to be due to its exposure conditions.
The combination of resminostat and DTX exerted a synergistic antitumor effect against chemotherapy-resistant NSCLC cells in both the in vitro and in vivo studies.Resminostat alone increased expression of pro-apoptotic while decreasing expression of antiapoptotic proteins.Induction of BIM expression might be due to enhancement of FOXO3A and E2F1 promoter activity [29,30].It has been reported that low BIM expression can lead to resistance to TKI treatment [31].In the present study, induction of BIM expression may have led to synergistic cytotoxicity against chemotherapyresistant cell lines.Reduced expression of BCL-2 may be caused by decreased binding of its transcription factor to the BCL-2 promoter