SKLB-23bb,AHDAC6-SelectiveInhibitor,Exhibits Superior and ... · Tubulin polymerization assay Turbidimetric assays of the microtubules were generally per-formed as described, with

Small Molecule Therapeutics

SKLB-23bb,AHDAC6-Selective Inhibitor, ExhibitsSuperior and Broad-Spectrum Antitumor Activityvia Additionally Targeting MicrotubulesFang Wang1, Li Zheng1, Yuyao Yi1,2, Zhuang Yang1, Qiang Qiu1, Xiaoyan Wang1,Wei Yan1, Peng Bai1, Jianhong Yang1, Dan Li1, Heying Pei1, Ting Niu1,2, Haoyu Ye1,Chunlai Nie1, Yiguo Hu1, Shengyong Yang1, Yuquan Wei1, and Lijuan Chen1,3

Abstract

Our previous study reported that SKLB-23bb, an orally bio-available HDAC6-selective inhibitor, exhibited superior antitu-mor efficiency both in vitro and in vivo in comparison withACY1215, a HDAC6-selective inhibitor recently in phase II clin-ical trial. This study focused on the mechanism related to theactivity of SKLB-23bb.Wediscovered that despite havingHDAC6-selective inhibition equal to ACY1215, SKLB-23bb showed cyto-toxic effects against a panel of solid and hematologic tumor celllines at the low submicromolar level. Interestingly, in contrast tothe reported HDAC6-selective inhibitors, SKLB-23bb was moreefficient against solid tumor cells. Utilizing HDAC6 stably knock-out cell lines constructed by CRISPR–Cas9 gene editing, weillustrated that SKLB-23bb could remain cytotoxic independent

of HDAC6 status. Investigation of the mechanism confirmed thatSKLB-23bb exerted its cytotoxic activity by additionally targetingmicrotubules. SKLB-23bb could bind to the colchicine site inb-tubulin and act as a microtubule polymerization inhibitor.Consistent with its microtubule-disrupting ability, SKLB-23bbalso blocked tumor cell cycle at G2–Mphase and triggered cellularapoptosis. In solid tumor xenografts, oral administration of SKLB-23bb efficiently inhibited tumor growth. These results suggestedthat SKLB-23bb was an orally bioavailable HDAC6 and micro-tubule dual targeting agent. The microtubule targeting profileenhanced the antitumor activity and expanded the antitumorspectrum of SKLB-23bb, thus breaking through the limitation ofHDAC6 inhibitors. Mol Cancer Ther; 17(4); 763–75. �2018 AACR.

IntroductionConsisting of 11 members, the classical histone deacetylase

(HDAC) enzyme family has emerged as an attractive target incancer therapy (1). In recent years, 5 HDAC pan-inhibitors (vor-inostat, romidepsin, belinostat, panobinostat, and chidamide)were approved for clinical application (2–4). The reported sideeffects of these drugs include fatigue, nausea, thrombocytopenia,and cardiotoxicity (5). To avoid these undesired responses, thedevelopment of isoform-selective inhibitors would be appre-ciated (6).

HDAC6 has long been referred to as a mysterious member ofthe HDAC family, for it harbors two catalytic domains and oneunique C-terminal zinc finger domain that bind ubiquitin (7).Located in the cytoplasm, HDAC6 mainly interacts with nonhis-tone proteins (8). Functionally, the inhibition of HDAC6 couldpotentially disrupt tumor protein homeostasis, block tumormigration, and suppress prosurvival signaling (9). Thus, mostlyapplied in combination with other chemotherapeutic agents inclinical trials, HDAC6 inhibitors were considered to be antitumorcandidates (10, 11). Especially in multiple myeloma, whichdepends highly on abnormal protein clearance, it was wellreported that the inhibition of HDAC6 could synergize withproteasome inhibitors to achieve significant clinical benefits(12–14).

Recently, the structure of the two catalytic domains of HDAC6was solved,which led tomoreunderstanding of themechanismofthis enzyme and elevated directions toward the design of selectiveinhibitors (15, 16). Tubacin and tubastatin A (Fig. 1)were the firstreported HDAC6-selective inhibitors (17, 18). Regretfully, theseagents did not have the therapeutic window for cancer treatmentdue to unfavorable pharmacokinetic profiles. In this decade,several new agents have been announced, and some individualcompounds have displayed antitumor potential, alone or syner-gistically with other drugs (19–22). The most clinically successfulexamples were ACY1215 (rocilinostat) and its analogue ACY241(refs. 23, 24; Fig. 1). ACY1215 has entered phase II for multiplemyeloma treatment. ACY1215 exhibited potential when admin-istrated in combination with bortezomib (23). Nevertheless,published data implied that the efficiency of ACY1215 as a single

1StateKey Laboratory ofBiotherapy, Collaborative InnovationCenter of Biother-apy and Cancer Center, West China Hospital of Sichuan University, Chengdu,China. 2Department of Hematology and Research Laboratory of Hematology,West China Hospital of Sichuan University, Chengdu, China. 3GuangdongZhongsheng Pharmaceutical Co., Ltd., Dongguan, Guangdong, China.

Note: Supplementary data for this article are available at Molecular CancerTherapeutics Online (http://mct.aacrjournals.org/).

F. Wang, L. Zheng, and Y. Yi contributed equally to this article.

Corresponding Author: Lijuan Chen, State Key Laboratory of Biotherapy,Collaborative Innovation Center of Biotherapy and Cancer Center, WestChina Hospital of Sichuan University, Chengdu 610041, China & GuangdongZhongsheng Pharmaceutical Co., Ltd., Dongguan, Guangdong 523325, China.Phone: 86-28-85164063; Fax: 86-28-85164060; E-mail: [email protected]

doi: 10.1158/1535-7163.MCT-17-0332

�2018 American Association for Cancer Research.

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agent was limited and its application was restricted to the tumortypes susceptible to protein homeostasis disruption (23, 25–27).

Our group previously reported an orally bioavailable HDAC6-selective inhibitor, SKLB-23bb, with antitumor capability(ref. 28; Fig. 1). SKLB-23bb showed therapeutic potential againsta panel of tumor types representing solid or hematologic malig-nancies both in vitro and in vivo. Interestingly, the efficiency of thisagent appeared to be superior against solid tumor models. In thisstudy, we found that although SKLB-23bb selectively inhibitedHDAC6, its antitumor activity was independent of this enzyme.Dramatically, it was revealed that SKLB-23bb could impact thecellular microtubule system by additionally targeting tubulin.These discoveries explained the superior and broad-spectrumantitumor efficiency of SKLB-23bb. This work also suggested thatHDAC6 inhibitors would optimally be administrated in thetumor setting in combination with other chemotherapeuticagents. Exploitation of compounds with additional targets apartof HDAC6 would also be encouraged (29).

Materials and MethodsAntibodies and reagents

The antibodies against HDAC6, Ac-a-tubulin, Ac-H3, a-tubu-lin, p-H3, and PARP were purchased from Santa Cruz Biotech-nology. The antibodies against caspase-9, p-AKT, and Bax werepurchased fromCell SignalingTechnology. The antibodies againstcaspase-3 and AKT were purchased from Abcam. The antibodiesagainst Bcl-2 andBcl-XLwere purchased fromSigmaChemicalCo.The antibody against b-tubulin was purchased fromGenetex. Theantibodies against GAPDH and b-actin were purchased fromOrigene. The reagents of ACY1215, SAHA, colchicine, paclitaxel,and vinblastine were purchased fromMeilunbio. SKLB-23bb wassynthesized in laboratory as described previously (28).

Cell lines and cell cultureThe following cell lines were obtained from the ATCC: human

colon cancer cell lines hct116 and HT29; human ovarian cancer

cell lines A2780s and SKOV3; human lung cancer cell lines H460and A549; human breast cancer cell lines MCF-7 and MDA-MB-231; human melanoma cell line A375; human liver cancer cellline HepG2; human multiple myeloma cell lines ARD, U266,and RPMI-8226; human lymphoma cell lines Ramos, HBL-1,and Jeko-1; human leukemia cell lines MV4-11, K562 andLAMA84s. The multiple myeloma cell line MM1S was kindlyprovided by Dr. Li Zhang in West China Hospital (Chengdu,China). Cells were cultured in DMEM or RPMI1640 mediumaccording to the instructions from ATCC, with the mediumcontaining 10% FBS, 100 U/mL penicillin, and 100 mg/mLstreptomycin at 37 �C in an atmosphere of 5% CO2. All thecells were tested and authenticated by an AmpFlSTR IdentifilerPCR Amplification Kit purchased from Thermo Fisher Scientificin the year of 2016 in our laboratory. All the cell lines wereconfirmed to be mycoplasma negative via a PCR method. Thecell lines were used for experiments or implanted into immu-nodeficient mice between passages 6 and 14.

MTT assayAn MTT assay was performed to evaluate the drug cytotoxicity

against the tumor cell lines. MTT (3- (4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) was purchased from Sig-ma Chemical Co. Cells were treated with various concentrationsof SKLB-23bborACY1215 in96-well culture plates for 72hours infinal volumes of 200mL (5,000 cells perwell for adherent cells and20,000 cells per well for suspension cells). Then, 20 mL of MTT (5mg/mL in PBS) was added to each well and incubated for anadditional 1–4 hours. The plate was finally centrifuged at 1,000rpm for 5 minutes, and then the medium was removed. MTTformazan precipitate was dissolved in 150 mL of DMSO andshakenmechanically for 5minutes; then, the absorbance readingsat a wavelength of 570 nm were taken on a spectrophotometer(Molecular Devices). Cell viabilities of the tested groups werecalculated via comparison with the DMSO (vehicle)-treatedgroup. The IC50 values were calculated by curve fitting withGraphPad Prism 6.0 software.

Figure 1.

Chemical structure of SKLB-23bb andseveral reported HDAC6-selectiveinhibitors.

Wang et al.

Mol Cancer Ther; 17(4) April 2018 Molecular Cancer Therapeutics764



Western blottingCells were collected and washed twice with ice-cold PBS. Snap-

frozen tumor samples were pulverized in liquid nitrogen andwashed twicewith ice-cold PBS. The sampleswere lysedwith RIPAlysis buffer containing 1 mmol/L phenylmethylsulfonyl fluoride(PMSF). RIPA lysis buffer and PMSF were purchased from Beyo-time Biotechnology. The protein concentration was determinedby the Bradford protein assay. The samples were denatured insample buffer, and equal amounts of protein were separatedaccording to the molecular weight on an 8%–12% SDS-PAGEgel and transferred onto a polyvinylidene difluoride membrane.Membranes were blocked for 1 hour in 5% dried milk in TBST atroom temperature andprobedovernight at 4�Cwith the indicatedprimary antibodies diluted in "blocking buffer." Blots werewashed thrice for approximately 15 minutes with TBST andincubated with a horseradish peroxidase–conjugated species-spe-cific antibody diluted in blocking buffer for 1 hour at roomtemperature with rotation. After three additional washes, theenhanced chemiluminescent substrate (Abbkine) was added andthe images were captured via a ChemiScope 5300 chemilumi-nescent detection system (Clinx).

HDAC6 enzymatic activity assayThe cellular HDAC6 activity of the cell lines were measured by

an HDAC6 Activity Assay Kit (Fluorometric), purchased fromBiovision. The harvested cells (1–2 � 106) were washed twice inice-cold PBS and then lysed with 100 mL HDAC6 lysis buffersupplied in the kit. The protein amount in the lysed samples wasquantified via a Bradford protein assay and adjusted to 1 mg/mL.The rest of the experiment was conducted following the protocolof the kit. The fluorescence intensity was measured by a spectro-photometer (Molecular Devices).

CRISPR-Cas9 genome editingThe SgRNA sequences designed for HDAC6 knockout were as

follows: Sg1 forward: 50-CACCGTCCCTTGCAGTCCCACGATT-30;Sg2 reverse: 50-AAACAATCGTGGGACTGCAAGGGAC-30; Sg2 for-ward: 50-CACCGCCAGTGCTACAGTCTCGCAC-30; Sg2 reverse: 50-AAACGTGCGAGACTGTAGCACTGGC-30. The CRISPR-Cas9–basedHDAC6 gene knockout vectors were constructed via cloningthe annealed oligonucleotide pair into the plasmid pHKO23.The vectors were further transfected together with plasmidencoding packaging proteins into 293T cells. The viral super-natant was collected 48 hours after transfection.

Hct116 and A2780s cells were infected in the presence of2 mg/mL polybrene. The next day, the medium was replaced.After 3–5 days for gene editing progress, cells were selected bythe addition of 1 mg/mL puromycin. Knockout of HDAC6 wasverified by Western blotting.

Immunofluorescence stainingWild-type A2780s or HDAC6 knock-out A2780s cells were

seeded into 6-well plates, and then treated with compounds atthe indicated concentrations for 16 hours. The cells were fixedwith 4% paraformaldehyde and then penetrated with PBS con-taining 0.5% Triton X-100. After blocking for 30 minutes in 5%goat serum albumin at room temperature, the cells were incu-bated with anti-a-tubulin antibody, or in the colabeling proce-dure, the cells were incubated with anti-b-tubulin and anti-Ac-a-tubulin antibodies at room temperature for 1 hour. Then, thecells were washed three times with PBS following staining with

fluorescent secondary antibodies and labeling of the nuclei with4,6-diamidino-2-phenylindole (DAPI). The cells were finallywashed thrice and visualized using a fluorescence microscope(Olympus).

EBI competition assayThe EBI competition assay was generally conducted using the

samemethod reported previously (30). The recommendedMDA-MB-231 cell linewasused. Cellswere seeded in6-well plates at 5�105 cells/well. After cell adherence, compounds were added to thecells for 4 hours. Then, EBI (TRC Biomedical Research Chemicals,Canada) was added at 100 mmol/L and incubated for 1.5 hours.Thereafter, the cells were harvested, and cell extracts were pre-pared. The b-tubulin and EBI: b-tubulin adduct bandwas detectedvia Western blotting. GAPDH was also examined to approve theequal protein loading.

Tubulin polymerization assayTurbidimetric assays of the microtubules were generally per-

formed as described, with some modification (31). Microtubuleprotein was purchased from Cytoskeleton. Tubulin (3 mg/mL)was incubated in the polymerization buffer (0.1 mol/L PIPES, 2mmol/LMgCl2, 0.5mmol/L EGTA, pH 6.9) at 4�C for 30minuteswhile the 96-well plate was prewarmed to 37�C. Before the start ofthe measurement, various compounds at the indicated concen-trations were added to the wells. Then, guanosine triphosphate(GTP)was added to the tubulin to achieve a final concentration of1mmol/L andmixed. The tubulin-containing sample was rapidlydiluted into the 96-well plate (100 mL per well). Immediately, thechange in absorbance at 340 nmwas kineticallymeasured at 37�Cby a thermostatically controlled spectrophotometer (MolecularDevices).

Cell-cycle analysisCells were plated on 6-well culture plates. After cell adher-

ence, the cells were treated with the indicated concentrations ofSKLB-23bb or ACY1215 for another 24 hours. Cells werewashed with PBS two times and then fixed with 75% ethanolovernight. Then, the cells were washed with PBS three times,and stained with PI (50 mg/mL) for 20 minutes. The cells werethen subjected to flow cytometry (BD FACSCalibur) for cell-cycle analysis.

Clonogenic assaysA2780s and hct116 cells treated with the indicated concentra-

tion of SKLB-23bb were washed with PBS, trypsinized, andreseeded into 6-well plates at 300 cells per well. The HDAC6knockout cells were seeded into 6-well plates at 150 cells per well.The colonies were allowed to form for 10–14 days. At the end ofthe culture, cells were washed with PBS, fixed with methanol for30 minutes, and stained with 0.5% crystal violet for 20 minutes.After careful washing, the images were taken, and colonies werecounted manually.

Annexin V-FITC/PI apoptosis assayCells were plated on 6-well culture plates. After cell adherence,

cells were treated with the indicated concentrations of SKLB-23bbfor 48 hours. Then, the cells were collected and subjected to anAnnexinV/PI Apoptosis Detection kit (Miltenyi Biotec) for stain-ing according to the manufacturer's instructions, and finallyanalyzed by flow cytometry (BD FACSCalibur).

A HDAC6 and Microtubule Dual Targeting Antitumor Agent

www.aacrjournals.org Mol Cancer Ther; 17(4) April 2018 765



Mitochondrial membrane potential detectionHct116 and A2780s cells were seeded into 6-well plates. After

cell adherence, the cells were treated with 1000 nmol/L of SKLB-23bb for the indicated time points. Then, the cells were collected,washed, and stained according to the instructions in the JC-1 kit(Keygen Biotech), and finally analyzed by flow cytometry (BDFACSCalibur).

Antitumor activity in vivoAll animal studies were approved by the Animal Care and Use

Committee of Sichuan University (Chengdu, Sichuan, China).Five- to 6-week-old female Balb/C athymic nude mice or 6- to 8-week-old female NOD/SCID mice were purchased from theBeijing HFK Bioscience Company. Mice were implanted with theindicated number of cells suspended in 100-mL PBS into the rightflank. When the xenografts developed, and the average tumorvolume reached 100 mm3, the mice were randomly divided intothe indicated groups and treatment began. The length and widthof the tumors were measured, and the tumor volume (mm3) wascalculated by the formula: p/6� length�width2. Tumor volumesand body weights were measured three times a week. The anti-tumor activity of the compound was evaluated by tumor inhibi-tion ¼ (1�tumor weight of treat group/tumor weight of controlgroup) � 100%.

Statistical analysisData obtained in the cellular studies were assessed by unpaired

Student t test. The tumor weight or tumor volume data wereanalyzed by ANOVA following a Dunnett multiple comparisonstest. A P < 0.05 indicated a significant difference.

ResultsSKLB-23bb showed antitumor activity against a panel of tumorcell lines in low submicromolar grade

The effect of SKLB-23bb on tumor cell viability was testedagainst a panel of tumor cell lines from different origins. SKLB-23bb exhibited activity with IC50 values under 100 nmol/L,against most of the cell lines checked (Table 1). In comparison,the IC50s of ACY1215 were all above 1000 nmol/L, except for theJeko-1 cell line (983 nmol/L). It could be implied that some otherreported HDAC6-selective inhibitors were inferior compared toSKLB-23bb. According to the literature, the C1A agent inhibitedproliferation of a panel of human tumor cell lines at the lowmicromolar range (19). Another compound, HPOB, had no effecton tumor cell viability alone (20). Recently, a new candidatetermed WT161 was reported (21). WT161 significantly enhancedthe activity of bortezomib on multiple myeloma, but failed toexhibit an obvious effect on cell viability in single-compoundexperiments.Hence, the outcome suggested that SKLB-23bbhad asuperior cytotoxic effect on tumor cell lines compared withACY1215, or the other known HDAC6-selective inhibitors.

Interestingly, SKLB-23bb seemed to be more efficient againstsolid tumor cell lines, with IC50s ranging from 31.32 nmol/L to82.81nmol/L,while higher data towardhematologic tumorswereobserved (IC50s ranging from49.80nmol/L to121.28nmol/L). Incontrast, ACY1215 showed better efficiency against hematologictumor cell lines with IC50s between 983 nmol/L to 4,497 nmol/L,which is consistent with the fact that most publications aboutthis drug were related to hematologic models (23, 25–27). TheIC50s of ACY1215 against the solid tumor cell lines were all above

4,000 nmol/L, and theH460, A549, and A2780s cell lines appear-ed to be robust against ACY1215 treatment. In our earlier report(28), the inhibitory effect of SKLB-23bb against the colon cancerhct116 xenograft model was also superior to that toward theMV4-11 and Ramos xenografts, which represent hematologicmalignances. Integrating the preexisting information, it could beinferred that SKLB-23bb possessed a broad antitumor spectrum,especially toward solid tumor models. Thus, it would be impor-tant to investigate the underlying mechanism.

SKLB-23bb selectively inhibited cellular HDAC6, but theantitumor activity of SKLB-23bb was independent of HDAC6

The extraordinary advantage of SKLB-23bb compared withACY1215 triggered our curiosity to further investigate the rela-tionship between the antitumor activity of SKLB-23bb and itscellular target HDAC6. We first screened the basic expressionlevel of HDAC6 in several cell lines. As presented in Fig. 2A, inthe panel of the solid tumor cell lines, hct116, A2780s, SKOV3,A549, and MDA-MB-231 highly expressed HDAC6, while theH460 and MCF-7 cell lines exhibited a relatively low HDAC6expression level. In the six hematologic tumor cell lines inves-tigated, the HDAC6 expression level was high in MM1S, U266,HBL-1, and Jeko-1, and low in the RPMI-8226 and Ramos. TheHDAC6 enzymatic activity of the cell lines was also measured.Interestingly, the cellular HDAC6 activity did not strictly cor-relate with the protein level among the cell lines (Supplemen-tary Fig. S1A).

For further investigation, the hct116, A2780s, and U266 celllines were selected, as they displayed high HDAC6 expressionlevels or enzymatic activity among solid and hematologic celllines. The effect of the compounds on the acetylation levels ofa-tubulin and H3 were evaluated. As shown in Fig. 2B andSupplementary Fig. S1B, after SKLB-23bb or ACY1215 treatment,the level of Ac-a-tubulin increased significantly at low concentra-tions of 10 or 100 nmol/L, while that of Ac-H3 did not obviouslyincrease until higher doses were used, suggesting that both agents

Table 1. Cytotoxic effects of SKLB-23bb andACY1215 against various tumor celllines

IC50 � SD (nmol/L)Tumor type Cell line SKLB-23bb ACY1215

Colon Hct116 38.56 � 13.05 6,246 � 562HT29 71.94 � 37.41 4,145 � 274

Lung H460 78.25 � 32.01 >10,000A549 75.45 � 29.95 >10,000

Ovarian A2780s 36.68 � 13.65 >10,000SKOV3 40.17 � 8.70 4,783 � 397

Breast MCF-7 82.81 � 12.29 4,541 � 74MDA-MB-231 66.2 � 14.83 4,237 � 622

Melanoma A375 48.30 � 14.90 5,472 � 374Liver HepG2 31.32 � 8.00 5,658 � 267Multiple myeloma MM1S 67.40 � 7.61 1,334 � 453

RPMI-8226 49.80 � 23.62 2,521 � 799ARD 93.44 � 27.56 4,497 � 513U266 86.94 � 16.32 1,354 � 67

B-Cell lymphoma Ramos 73.86 � 8.01 2,624 � 821HBL-1 121.28 � 49.9 1,070 � 67Jeko-1 115.8 � 26.74 983 � 45

Leukemia LAMA-84s 83.55 � 5.08 2,431 � 274K562 116.56 � 18.08 1,461 � 128MV4-11 67.64 � 20.35 2,531 � 358

NOTE: The time point for analysis was 72 hours. IC50 values were calculated anddata were expressed as means � SDs. At least two independent experimentsagainst each cell line were performed

Wang et al.




selectively inhibited the deacetylase activity of HDAC6, whichtakes a-tubulin as substrate, not class I HDACs that catalyze thedeacetylation of histones. As a reference, the pan-inhibitor, SAHA,triggered the increase of both H3 and a-tubulin acetylation levels,with no obvious difference (Fig. 2B; Supplementary Fig. S1B).

The superior antitumor activity of SKLB-23bb implied addi-tional mechanisms or targets for several reasons. It could beinferred that, at the concentration of 100 nmol/L, both SKLB-23bb and ACY1215 remarkably elevated the Ac-a-tubulin level,but these two agents showed great difference in the cytotoxicprofiles. In the observation of the A2780s cell line, it was evidentthat ACY1215 induced acetylation of a-tubulin at the concentra-tion of 100 nmol/L, while the IC50 for this agent to affect A2780s

cell viability was above 10,000 nmol/L, indicating that inhibitionofHDAC6was not sufficient to achieve antitumor response in thismodel. Therefore, if HDAC6 was the unique target of SKLB-23bb,this compound should also be inactive towards A2780s. Contra-dictorily, the cytotoxic IC50 of SKLB-23bbonA2780swas as lowas36.68 nmol/L (Table 1). Furthermore, it could be observed thatamong the cell lines investigated, the HDAC6 expression level orenzyme activitywas poorly correlatedwith the sensitivity to SKLB-23bb (Fig. 1A; Supplementary Fig. S1A; Table 1).

To clarify whether the cytotoxicity of SKLB-23bb and ACY1215was dependent on HDAC6, we established HDAC6 stablyknocked out cell lines and assessedwhether the antitumor activityof SKLB-23bb or ACY1215 differed between the HDAC6-null cell

Figure 2.

HDAC6 basic expression in tumor celllines and HDAC inhibition of SKLB-23bb, ACY1215 and SAHA. A, Analysisof basal HDAC6 expression levelsamong a panel of tumor cell lines byWestern blotting. b-Actin wasdetected as protein loading control.The relative expression level waspresented according to densitometryanalysis and normalized based onb-actin level. B, Western blot analysisof acetylated a-tubulin, acetylatedhistoneH3 in hct116, A2780s, andU266cell lines after 6 hours of treatmentwith SKLB-23bb, ACY-1215, or SAHAatthe indicated concentrations. GAPDHwas used as a loading control.





lines and their respective parent cell lines. Via the application ofCRISPR-Cas9 system, HDAC6 knock-out hct116 and A2780s(hct116�HDAC6, A2780s�HDAC6) cell lines were stably con-structed. Losses of cellular HDAC6 and the correlated elevationof Ac-a-tubulin were confirmed (Fig. 3A and B). The effects ofSKLB-23bb and ACY1215 on cell viability were subsequentlyevaluated on these cell lines and their parent wild-type cell lines.SKLB-23bb was still active against the HDAC6 knockout celllines compared with the parent cell lines, with IC50 values ranging

from 41.50 to 53.55 nmol/L in the HDAC6 knockout cells and39.79 to 45.98 nmol/L in the parent cell lines, respectively. Whentreating the parent hct116 cell line, the IC50 value of ACY1215was5.21 mmol/L. However, the IC50s of ACY1215 in the knockoutcell lines were 10.14 and 11.92 mmol/L, respectively, indicatinga 1.95- to 2.29-fold increase (Fig. 3A). Similar results werealso observed in A2780s cell lines, the IC50 of ACY1215 towardthe wild-type cell line was 10.14 mmol/L, while the IC50s in theknockout cell lines were 20.09 and 28.94 mmol/L, respectively,

Figure 3.

Analysis of the cytotoxic effects of SKLB-23bb and ACY1215 against HDAC6 knockout cell lines compared with the parent ones. A, hct116 background.B, A2780s background.

Wang et al.




with a 1.98- to 2.85-fold increase (Fig. 3B). These findings indi-cated that SKLB-23bb could remain efficient in the absenceof HDAC6.

The proliferation trends of the HDAC6 knockout cell lineswere also compared with their parent cell lines. The results of aclonogenic assay indicated that in the hct116 and A2780s celllines, the loss of HDAC6 had minimal effect on the rate of cellgrowth (Supplementary Fig. S2). These findings might explainthe reason underlying the lack of efficiency of ACY1215 on solidtumor models, as our observation suggested that in such models,depletion or inhibition of HDAC6 was not sufficient to achievesatisfying therapeutic outcome.

SKLB-23bb additionally functioned as a microtubulepolymerization inhibitor independent of cellular HDAC6status

The finding that SKLB-23bb was active toward cell linesregardless of their HDAC6 status inferred that this agent hasan additional target. As the similar quinazoline structure wasapplied in the microtubule-targeting agent MPC6827 (32),we speculated that SKLB-23bb might affect the microtubulesystem and experiments were conducted for validation. Weassessed whether SKLB-23bb could impact the cellular micro-tubule system in situ via immunofluorescence staining in theA2780s cell line. As illustrated in the left of Fig. 4A, whentreated with 100 nmol/L of SKLB-23bb, the normal arrange-ment of the microtubules was disrupted, and a higher concen-tration of SKLB-23bb (500 nmol/L) caused significant disrup-tion of microtubule assembly. Moreover, from the properties ofthe observed microtubule morphologic changes triggered bySKLB-23bb, we could predict that SKLB-23bb acted as a micro-tubule polymerization inhibitor, as both SKLB-23bb and thereference compound colchicine resulted in relatively diffusedcellular microtubules (33). Cellular microtubules appearedin a more aggregated fashion after treatment with paclitaxel,a known tubulin polymerization enhancer (34). Furthermore,the modality of the microtubule arrangement in the ACY1215-treated group tended to be similar to that of the vehicle controlgroup. We subsequently investigated the effects of these com-pounds on microtubules in the HDAC6 knockout A2780s cellsand similar results were observed (Fig. 4A, right). These obser-vations indicated that SKLB-23bb additionally targeted micro-tubules, and this effect was independent of HDAC6.

In distinct approaches, we discovered that SKLB-23bb couldinduce acetylation of a-tubulin and inhibit tubulin polymeriza-tion. It would be important to clarify the relationship between thetwo effects of this compound, as HDAC6 is a microtubule-asso-ciated deacetylase (35, 36). Thus, a microtubule and acetylatedmicrotubule costaining immunofluorescence experiment wasconducted. It could be observed that SKLB-23bb simultaneouslyinhibited microtubule polymerization and induced a-tubulinacetylation at 100 nmol/L and 500 nmol/L (Supplementary Fig.S3). For comparison, the HDAC6 inhibitor ACY1215 enhancedAc-a-tubulin but had no observed effect on microtubule mor-phology. Colchicine inhibited microtubule polymerization andpaclitaxel enhanced microtubule polymerization, but the twotubulin targeting agents had no obvious effect on themicrotubuleacetylation status. The phenotype of the HDAC6 knock-out cellswas similar to that of the ACY1215 group with enhanced Ac-a-tubulin, which is consistent with the previous finding that theloss of HDAC6 was correlated with the elevation of a-tubulin

acetylation level (Supplementary Fig. S3). Thus, the outcomesuggested that the effects of SKLB-23bb on microtubule acetyla-tion and microtubule polymerization were both independent.

An N,N0-ethylene-bis(iodoacetamide; EBI) competition assaywas conducted to investigate the binding site of SKLB-23bb ontubulin. In this method, the reference agent colchicine couldengage the colchicine site in b-tubulin, preventing the EBI probefrom forming a secondary b-tubulin band (EBI-tubulinadduct), which could be detected in Western blotting (30). Asshown in Fig. 4B, SKLB-23bb dose-dependently reduced thequantity of the EBI-tubulin adduct, with a maximum effectachieved at 50 mmol/L, confirming that SKLB-23bb binds tothe colchicine site. Vinblastine was used as a negative controlto illustrate that no false results were obtained. Vinblastinebinds to the vinblastine site, that is nonoverlapping with thecolchicine site (37, 38).

An in vitro tubulin polymerization assay was subsequentlyconducted to further evaluate the direct interaction betweenSKLB-23bb and tubulin. As displayed in Fig. 4C, it was confirmedthat SKLB-23bb inhibited tubulin polymerization. AlthoughSKLB-23bb impacted tubulin assembly to a weaker extent com-pared with colchicine, a lag at the starting period of tubulinpolymerization was obvious at the concentration of 10 mmol/L.These findings illustrated that SKLB-23bb was a microtubulepolymerization inhibitor.

SKLB-23bb blocked the cell cycle progress at G2–M phaseThe effect of SKLB-23bb on the cell-cycle distribution was

assessed in the hct116 and A2780s cell lines. The results indi-cated that SKLB-23bb dose-dependently blocked the cell cycle atG2–M phase (Fig. 5A). Further examination of p-H3, a well-reported mitotic marker, by Western blotting confirmed the cell-cycle arrest caused by SKLB-23bb treatment (Fig. 5B). Moreover,no obvious effect on cell-cycle distribution was shown wheninvestigation was made on ACY1215 (Supplementary Fig. S4),which is consistent with its report on lymphoma models (25).These data inferred that the G2–Mphase cell-cycle arrest inducedby SKLB-23bb was due to the tubulin binding effect, as trigger-ing of G2–M phase cell-cycle arrest is a critical feature ofmicrotubule-binding agents (39).

SKLB-23bb inhibited the clonogenic potential of tumor cellsand triggered apoptosis

The clonogenic assay is awell approbatedmethod of testing theantitumor efficiency of chemotherapeutic agents (40). As shownin Fig. 6A, SKLB-23bb could inhibit the clonogenic potential ofboth hct116 and A2780s cells in a concentration-dependentmanner. To evaluate whether SKLB-23bb could trigger tumor celldeath, hct116 and A2780s cells were treated with the indicatedconcentrations of SKLB-23bb and dual-stained by Annexin V andPI. The apoptotic cell populations were analyzed by flow cyto-metry. The outcomes displayed that after incubation of increasingdoses of SKLB-23bb, the Annexin V–positive populationincreased, inferring that SKLB-23bb induced tumor cell apoptosis(Fig. 6B). For further demonstration, the effects of SKLB-23bb oncaspase-9, caspase-3, and the PARP axis were investigated. TheWestern blotting results indicated that the intrinsic apoptosisactivator caspase-9, the effector caspase-3 and the cell deathbiomarker PARP were activated by SKLB-23bb in both time andconcentration-dependent manners in hct116 and A2780s cells(Fig. 6C). The activationof caspase-9 couldbe related to the loss of





mitochondrial membrane potential (MMP). To verify, a JC-1probe detection was conducted. The outcome of the flow cyto-metry analysis indicated that the lowering of the red fluorescenceintensity was accompanied by the increase of the green fluores-cence intensity, which labeled MMP loss (Supplementary Fig.S5A). The status of some key proteins related to the intrinsicapoptosis pathway was also investigated via Western blotting.SKLB-23bb reduced the level of oncoprotein AKT as well as itsphosphorylated form in a time-dependent manner (Supplemen-

tary Fig. S5B). The protein levels of Bax, Bcl-2, andBcl-XLwere alsoanalyzed. The results indicated that SKLB-23bb treatment upre-gulated the proapoptotic protein Bax and suppressed the anti-apoptotic member Bcl-XL. Another antiapoptotic protein, Bcl-2,was not affected by SKLB-23bb treatment (Supplementary Fig.S5B). Theupregulationof Bax and suppression of Bcl-XL suggestedthe breaking of the balance between the two groups of MMP-regulating factors, the elevation of the Bax/Bcl-XL ratio and theactivation of the intrinsic apoptosis pathway.

Figure 4.

SKLB-23bb acted as a microtubule polymerization inhibitor via targeting the colchicine site in b-tubulin subunit. A, Effect of SKLB-23bb and other referencecompounds on the organization of cellular microtubule network. Tubulin are shown in green and the Nuclei in blue. Col, colchicine; PTX, paclitaxel. B, EBIassay to confirm Colchicine site binding by SKLB-23bb. MDA-MB-231 cells were treated by the indicated concentrations of compounds for 4 hours. Next, EBI (100mmol/L) was added, and after 1.5 hours, the cells were harvested and cell extracts were prepared for Western blot analysis using anti-b-tubulin antibody.EBI could result in the formation of a b-tubulin adduct (a second immunoreactive band) that migrates faster. Compounds that could preoccupy the colchicine-site inb-tubulin prevent the formation of the EBI: b-tubulin adduct. 1, b-tubulin band; 2, EBI:b-tubulin adduct band. GAPDH were also probed to confirm equalsample loading. Vin, vinblastine; Col, colchicine. C, Inhibition of tubulin polymerization. Tubulin was at 3 mg/mL and preincubated at 4�C for 30 minutes, then thecompounds at the indicated concentrations and guanosine triphosphate (GTP) were added to start the tubulin polymerization reactions. The reaction wasmonitored at OD340 nm at 37�C. Col, colchicine.

Wang et al.




SKLB-23bb inhibited tumor growth in various xenograftsrepresenting different tumor types, via dual targetingofHDAC6and microtubules

SKLB-23bb had good pharmacokinetic profiles and was orallybioavailable (28). HBL-1 xenografts, representing a B-cell lym-phoma model, were established utilizing NOD/SCID mice. Asreports suggested that ACY1215 could exhibit therapeutic poten-tial against lymphomas, the activity of SKLB-23bb and ACY1215were compared at the same dose and routine in this model. Bothadministrated three times a week orally at the dose of 40 mg/kg,SKLB-23bb resulted in significant tumor growth inhibition with a58.22% tumor-inhibitory rate, while ACY1215 showed only a26.75% tumor-inhibitory rate (Supplementary Fig. S6; Supple-mentary Table S1).

To thoroughly evaluate the therapeutic potential of SKLB-23bbagainst solid tumor models, hct116, A2780s, and MCF-7 xeno-grafts were established on Balb/c nude mice and SKLB-23bb wasscheduled for oral administration at various doses three times aweek. SKLB-23bb treatment significantly inhibited tumor growthin the three models (Fig. 7A–C). As presented in Table 2, thehct116 model representing colon cancer had tumor inhibitionrates of 51.28%, 60.37%, and 66.05% at the doses of 12.5mg/kg,25 mg/kg, and 50 mg/kg, respectively. In ovarian cancer A2780sxenografts, the tumor-inhibitory rate of the lowest dose, 3 mg/kg,was 30.65%, and the data of the 6mg/kg, 12.5mg/kg, and 25mg/kg groups were 62.19%, 75.50%, and 77.39%, respectively. Sim-ilar results were also observed in the breast cancer MCF-7 model,the inhibitory rates of the 12.5 mg/kg, 25 mg/kg, and 50 mg/kggroups were 30.57%, 50.51%, and 65.65%, respectively.

Subsequently, the keymolecular events SKLB-23bb triggered inthe xenografts were evaluated. Mice bearing hct116 xenografts

were given a single oral 50 mg/kg dose of SKLB-23bb. Analysis ofthe protein levels of the tumor samples indicated that SKLB-23bbinduced selective acetylation ofa-tubulin, phosphorylation ofH3

and activation of PARP (Fig. 7D; Supplementary Fig. S7). Takentogether, our data inferred that the molecular modulations thatresulted from SKLB-23bb treatment could be retained in exper-imental animal systems, proposing that SKLB-23bb could beeffective in the treatment of various tumor types.

DiscussionIn previous study, we report an orally bioavailable, HDAC6-

selective inhibitor SKLB-23bb with a broad antitumor spectrum.In this work, interests focused onwhy SKLB-23bb, comparedwithACY1215, an agent in clinical trials, exhibited superior antitumorefficiency, especially toward tumor models that tended to berobust against HDAC6 inhibition. Through the research proce-dure, it was discovered that SKLB-23bb additionally targetstubulin.

Altered tubulin acetylation status could influence cell beha-viors, such as cytoskeleton organization, intracellular cargo trans-porting, and cell migration (41–44)). Whether HDAC6 couldimpact the basic tubulin assembly process via modulation ofacetylation of the a-tubulin subunit is still unclear (45, 46). Thus,it would be essential to clarify whether SKLB-23bb directly targetstubulin or if its impact on the microtubule system was causedindirectly by the agent's inhibition of HDAC6. In our experimen-tal procedures,we found that SKLB-23bb could affectmicrotubulemodality independently of the HDAC6 status, and the HDAC6inhibitor ACY1215 failed to impact the cellular microtubulearrangement and cell-cycle distribution (Fig. 4A; Supplementary

Figure 5.

SKLB-23bb blocked tumor cell cycle at G2–M phase. A, Effects of SKLB-23bb on cell cycle in hct116 and A2780s cells. Data shown are representative of twoindependent experiments. Right, Graph summarizing the cell-cycle distribution effected by SKLB-23bb. B, Analysis of p-H3 level. Concentration of SKLB-23bb, 200nmol/L; Concentration of ACY1215, 10 mmol/L. Right, relative expression level summarized on the basis of densitometry analysis.





Figs. S3 and S4). The opinion that another HDAC inhibitor, TSA,could not influence microtubule polymerization or depolymer-ization (47) also suggested that, despite their epigenetic modify-ing function, HDAC inhibitors could not disrupt the basic micro-tubule assembly disassembly progress. Furthermore, the evidencethat SKLB-23bb simultaneously disrupted cellular microtubulesand induced a-tubulin acetylation (Supplementary Fig. S3) dem-onstrated that SKLB-23bb was a dual targeting agent.

Considering the "dose–effect" relationship, it could be discov-ered that the cellular concentration required for SKLB-23bb toeffectively inhibit HDAC6, impact the microtubule system andsubsequently block the cell cycle at G2–M phase were all lowerthan 100 nmol/L. The cytotoxic IC50 values of SKLB-23bb againstmost tumor cell lines were also under 100 nmol/L (Table 1).Taken together, these clues suggested that for both the targets ofHDAC6 and tubulin, SKLB-23bb has good "on-target effect (48)".

Microtubule-targeting agents were well reported to trigger theinstinct apoptotic pathway (32, 49), with familiar progressincluding disruption of BCL2 family proteins, mitochondrialmembrane potential loss, caspase-9 activation, and caspase-3activation (50). The HDAC6 inhibitors ACY1215 and WT161were reported to trigger the unfolded protein response, leading tocaspase family activation (21, 26). Hence, the signaling pathwaysSKLB-23bb impacted and the effect of SKLB-23bb to induceapoptosis were in accordance with the two cellular targets.

The final evaluation of the antitumor activity of SKLB-23bbutilizing xenografts representing various tumor types indicatedthat SKLB-23bb was active in vivo. In HBL-1model, ACY1215 wasevaluated for comparison in the samedose, frequency and routineas SKLB-23bb. Rationally, in clinical trials, ACY1215 was admin-istrated orally (10). However, the activity of ACY1215 as a single

agent was limited and inferior to SKLB-23bb, which is consistentwith the data fromB-cell lymphomaRamos xenografts assessed inour previous work (28). The published data describing the effi-ciency of ACY1215 against solid tumor models suggested that itwas not activewhen administrated alone (51).On the basis of thisinformation, ACY1215 was no longer engaged in the studies ofsolid tumor xenograft models.

As seen in the 2 models of hct116 and A2780s, 12.5 mg/kgor 6 mg/kg TIW oral dosing of SKLB-23bb exhibited signi-ficant antitumor effect, respectively. Notably, in the MCF-7xenograft, the outcome of 12.5 mg/kg group was not satisfying.The probable explanation is that MCF-7 exhibited relatively lowHDAC6 expression and enzyme activity (Fig. 2A; Supplemen-tary Fig. S1A). Thus, the threshold required for SKLB-23bb toexhibit potential toward this model depended more on theagent's microtubule-targeting ability.

In this study, data suggested that ACY1215 was active onlytoward specific tumor types, typically, multiple myeloma andlymphoma models (Table 1). The probable explanation is thatthese tumor subtypes tend tobehighly dependent on cellularmis-folded protein clearance, in which HDAC6 participated as a keyfactor in the proteasome-independent proteolysis mechanism.Thus, these tumors could be more susceptible to HDAC6 inhi-bition. Moreover, based on the results shown in this study as wellas other ACY1215 reports, the possibility that the undesired class IHDAC inhibition contributed to the activity of this compoundcould not be excluded, which was supported by the fact thatACY1215 could elevate histone H3 acetylation level at concentra-tions above 1 mmol/L (Fig. 2B), and, in correlation, the cytotoxiceffects did not appear until such concentrations were reached. Thepharmacodynamic results in a recent clinical trial might indicate

Figure 6.

SKLB-23bb triggered apoptosis in hct116 and A2780s cell lines. A, SKLB-23bb inhibited hct116 and A2780s cells' ability to form colonies. Cells were exposed tothe indicated concentrations of SKLB-23bb for 48 hours, the clonogenic assay was conducted as described in the Materials and Methods section. Datashown are representative of two independent experiments. Right, graph summarizing the result of the clonogenic assay. Colonies were manually counted.�� , P <0.01; �� , P <0.001.B, Flow cytometric histograms of apoptotic hct116 andA2780s cells after 48-hour treatment of SKLB-23bb at the indicated concentrations.Data shown are representative of two independent experiments. Right, graph summarizing the percentage of cells found in the different regions of thebiparametric flowcytometric histograms, in which the Aþ/PI� and the Aþ/PIþ region represent the apoptotic population. C, Effects of SKLB-23bb onPARP, caspase-3, and caspase-9 in hct116 and A2780s cells examined by Western blotting. Concentration of SKLB-23bb in the analysis of increasing timepoints: 1,000 nmol/L. Arrow, cleaved caspase-9; asterisk, nonspecific band.

Wang et al.




the same opinion, as high doses of ACY1215 also increasedhistone acetylation in the tested samples (10).

In the effort to establish HDAC6 stably knockout cell lines, itwas shown that the loss of HDAC6 was insufficient to impact cellgrowth in the hct116�HDAC6� and A2780s�HDAC6� cell lines(Supplementary Fig. S2), suggesting that HDAC6 is less criticalin the aspect of tumor survival in the two cell lines. This obser-vation may explain the reason why HDAC6-selective inhibitorsACY1215 and ACY241 are mainly evaluated in combinationapproaches.

In summary, ourwork determined the advantage of SKLB-23bbagainst ACY1215 in terms of the cancer therapeutic potential.According to our literature research, SKLB-23bbmight be the firstorally bioavailable HDAC6 and tubulin dual-targeting agent withtumor therapy potential. The certification of tubulin as an addi-tional target of SKLB-23bb gifted this compound with broadantitumor spectrum and superior activity. In the future, it maybe worthy to evaluate SKLB-23bb in various tumor types, espe-cially solid tumor subtypes that HDAC inhibitors are rarely testedin clinical, thus breaking through the bottleneck restricting

Figure 7.

SKLB-23bb inhibited tumor growth in various xenografts, via dual targeting of HDAC6 and microtubules. A–C, In vivo antitumor activity of SKLB-23bb in variousdoses against hct116, A2780s, and MCF-7 xenografts, respectively. Left, time curve of tumor growth. Points, mean; bars, SD; Right, bar chart of tumor weight.�� , P < 0.01; �� , P < 0.001, �� , P < 0.0001, compared with vehicle control (n � 6). D, Analysis of protein levels of hct116 xenograft tissue. Tumor bearingmice were orally administrated by SKLB-23bb at the dose of 50 mg/kg once. Then mice were sacrificed at the indicated time points and xenograft tissues werecollected for Western blotting.

Table 2. Summary of the antitumor activity of SKLB-23bb against solid tumor xenografts

Administration Toxicity

Model GroupDose(mg/kg) Schedule Route

Max bodyweight loss (%) Death

Tumor inhibitionrate (%)

hct116 Vehicle — TIW p.o. 4.9 0/7 —

SKLB-23bb 12.5 TIW p.o. 4.2 0/7 51.28SKLB-23bb 25 TIW p.o. 3.8 0/7 60.37SKLB-23bb 50 TIW p.o. 5.4 0/7 66.05

A2780s Vehicle — TIW p.o. 4.8 0/7 —

SKLB-23bb 3 TIW p.o. 2.7 0/7 30.65SKLB-23bb 6 TIW p.o. 2.9 0/7 62.19SKLB-23bb 12.5 TIW p.o. 2.7 0/7 75.50SKLB-23bb 25 TIW p.o. 6.9 0/7 77.39

MCF-7 Vehicle — TIW p.o. 4.5 0/6 —

SKLB-23bb 12.5 TIW p.o. 2.1 0/6 30.57SKLB-23bb 25 TIW p.o. 3.9 0/6 50.51SKLB-23bb 50 TIW p.o. 1.8 0/6 65.65

Abbreviation: BIW, twice a week; p.o., orally; TIW, thrice a week.





HDAC6-selective inhibitors and HDAC pan inhibitors. SKLB-23bb might also serve as an example and encourage the designand development of small molecules with multiple targets incancer therapy (52).

Disclosure of Potential Conflicts of InterestNo potential conflicts of interest were disclosed.

Authors' ContributionsConception and design: F. Wang, L. Zheng, C. Nie, Y. Wei, L. ChenDevelopment of methodology: L. Zheng, Y. Yi, Q. Qiu, Y. Hu, L. ChenAcquisition of data (provided animals, acquired and managed patients,provided facilities, etc.): F. Wang, L. Zheng, Y. Yi, X. Wang, W. Yan, P. Bai,J. Yang, D. Li, H. Pei, L. ChenAnalysis and interpretation of data (e.g., statistical analysis, biostatistics,computational analysis): F. Wang, L. Zheng, Y. Yi, H. YeWriting, review, and/or revision of the manuscript: F. Wang, L. Zheng, Y. Hu,L. Chen

Administrative, technical, or material support (i.e., reporting or organizingdata, constructing databases): F. Wang, Z. Yang, Q. Qiu, T. Niu, Y. Hu, S. YangStudy supervision: C. Nie, Y. Hu, S. Yang, L. Chen

AcknowledgmentsThis work was supported by the National Natural Science Foundation of

China (U1402222, to L. Chen; 81703344, to Z. Yang), and GuangdongInnovative Research Team Program (grant 2011Y073, to L. Chen). The authorsthank Ronghong Zhang, Xuefei He, Mengshi Hu, Linlin Xue, Xue Yuan forassistance in xenograft studies. The authors thankMinghai Tang for checking thepurity of the synthesized compounds. The authors thank Yuxi Wang, Chen-gyong Wu and Hua Jiang for providing their valuable advices.

The costs of publication of this articlewere defrayed inpart by the payment ofpage charges. This article must therefore be hereby marked advertisement inaccordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received April 16, 2017; revised September 7, 2017; accepted January 24,2018; published first April 2, 2018.

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2018;17:763-775. Mol Cancer Ther Fang Wang, Li Zheng, Yuyao Yi, et al. MicrotubulesBroad-Spectrum Antitumor Activity via Additionally Targeting SKLB-23bb, A HDAC6-Selective Inhibitor, Exhibits Superior and

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SKLB-23bb,AHDAC6-SelectiveInhibitor,Exhibits Superior and ... · Tubulin polymerization assay Turbidimetric assays of the microtubules were generally per-formed as described, with