Bin Mei,Gao-lin Liang

a.CAS Key Lab oratory of Soft Matter Chemistry,Department of Chemistry,University of Science and Technology of China,Hefei 230026,China

b.Department of Biology,College of Life Science,Anhui Medical University,Hefei 230032,China

Paclitaxel Hydrogelator Delays Microtubule Aggregation

Bin Meia,b,Gao-lin Lianga?

a.CAS Key Lab oratory of Soft Matter Chemistry,Department of Chemistry,University of Science and Technology of China,Hefei 230026,China

b.Department of Biology,College of Life Science,Anhui Medical University,Hefei 230032,China

(Dated:Received on September 11,2016;Accepted on October 23,2016)

Paclitaxel(PTX)is one of the most efficient anticancer drugs for the treatment of cancers through β-tubulin-binding.Our previous work indicated that a PTX-derivative hydrogelator Fmoc-Phe-Phe-Lys(paclitaxel)-Tyr(H2PO3)-OH(1)could promote neuron branching but the underlying mechanism remains unclear.Using tubulin assembly-disassembly assay, in this work,we found that compound 1 obviously delayed more microtubule aggregation than PTX did.Under the catalysis of alkaline phosphatase,Fmoc-Phe-Phe-Lys(paclitaxel)-Tyr(H2PO3)-OH could self-assemble into nanofiber Fmoc-Phe-Phe-Lys(paclitaxel)-Tyr-OH with width comparable to the size of αβ-tubulin dimer.Therefore,we proposed in this work that nanofiber Fmoc-Phe-Phe-Lys(paclitaxel)-Tyr-OH not only inhibits the αβ-tubulin dimer binding to each other but also interferes with the plus end aggregation of microtubule. This work provides a new mechanism of the inhibition of microtubule formation by a PTX-derivative hydrogelator.

Paclitaxel,Hydrogelator,Microtubule,Aggregation

I.INTRODUCTION

Paclitaxel(PTX)has emerged as a standard chemotherapeutic drug for the treatment of various types of solid tumours such as breast,ovarian and prostate cancers[1-4].To overcome its hydrophobicity which is adverse for delivery,numerous delivery strategies have been explored experimentally and clinically [5-8].Among them,chemical modification of the C2′position of PTX to yield PTX hydrogelator has been proven to be an efficient method for its delivery[9-13]. Besides the similar preclinical and clinical effects to those of PTX,these PTX derivatives might have their own distinct advantages[9].Cline et al.found that PTX-derivate G5PAMAM dendrimers could affect microtubule structure by stabilizing microtubule polymerization or bundling the preformed microtubules[14].

Notably,PTX-hydrogelators have shown similar cytotoxicity on cancer cells to PTX itself[15,16].However,their cytotoxicity mechanism was ambiguous. That is,it is unknown whether the observed cytotoxicity was caused by PTX-induced stabilization of the microtubules,or by any other mechanism(s).Recent researches showed that even the peptide hydrogelators could induce cytotoxicity by the formation of nanofibers inside cells[17].Consequently,much attention had been paid to examining the amount of an anticancer drug (e.g.,PTX)delivered to the targeted site[9,15,16], but very tiny attention was directed toward examining the drug effect on microtubule aggregation,which is probably responsible for pharmacological effect[18].

Our previous work showed that a PTX-derivative Fmoc-Phe-Phe-Lys(taxol)-Tyr(H2PO3)-OH(1)(Fig.1) promoted neuron branching which was not observed in PTX group[9].At a low concentration of 10 nmol/L, compound 1 not only promoted neurite elongation as PTX did but also promoted axonal branching which was not achieved by PTX.We proposed that the promoted axonal branching was probably induced by the self-assembly of compound 1 along the microtubules which interfered with the aggregation of microtubules.

Following our previous study,in this work,we used compound 1 to study its effect on microtubule aggregation and used PTX as a control compound for parallel study.This may help to uncover the mechanism of compound 1 on promoting neuron branching.

II.EXPERIMENTS

A.Materials

All the starting materials were obtained from Adamas or Sangon Biotech.Commercially available reagents were used without further purification,unless noted otherwise.All other chemicals were reagent grade or better.

FIG.1 Chemical structures of compounds 1 and 2.

B.Methods

1.Synthesis and purification of compound 1

The syntheses of compound 1 are facile and straightforward as described previously[13].Briefly, tetrapeptide Fmoc-Phe-Phe-Lys(Boc)-Tyr(H2PO3)-OH was prepared with solid phase peptide synthesis (SPPS)according to the protocol[19].The Boc protecting group was cleaved with 95%TFA in DCM for 3 h at room temperature to yield Fmoc-Phe-Phe-Lys-Tyr(H2PO3)-OH after HPLC purification.The C2′hydroxyl group of taxol was coupled with succinic anhydride,activated by N-hydroxysuccinimide,and coupled with Fmoc-Phe-Phe-Lys-Tyr(H2PO3)-OH through the reaction of amino and carboxyl groups to yield compound 1.

HPLC purification and analyses were performed on a Shimadzu UFLC system equipped with two LC-20AP pumps and an SPD-20A UV-Vis detector using a Shimadzu PRC-ODS column,and on an Agilent 1200 HPLC system equipped with a G1322A pump and an in-line diode array UV detector using an Agilent Zorbax 300SB-C18 RP column,with CH3CN(0.1%of TFA) and ultrapure water(0.1%of TFA)as the eluent,respectively.Electrospray ionization-mass spectrometry (ESI-MS)spectra of compound 1 were recorded on a LCQ Advantage MAX ion trap mass spectrometer (Thermo Fisher).

2.Cryo-transmission electron microscopy(cryo-TEM)

The compound 1 solutions of in 0.01 mol/L phosphate-bufferred saline(1wt%,pH=7.4)was incubation with(or without)300 U/mL of alkaline phosphatase(ALP)for 12 h.Then the solutions were applied to a carbon-coated 400-mesh Cu EM specimen grids freshly glow dis-charged.The samples were then preserved by staining with 0.75%(w/w)uranyl formate solution.Images were recorded at a magnification of 62,000 on a 4096×4096 CCD detector(FEI Eagle)with a Tecnai F20 electron microscope(FEI)operating at an acceleration voltage of 200 kV.The sizes of the nanostructures in cryo-TEM images were calculated with Image J.

3.Microtubule imaging assay

To obtain the action of compound 1 on microtubule, we transfected cherry-tubulin plasmids into HeLa cells [20],and established stable cell lines expressing cherrytubulin.Then the cells were treated with compound 1 or PTX at 10μmol/L for 2 h,fixed in 4%paraformaldehyde for 30 min at room temperature prior to imaging.

4.Tubulin purification and assembly-disassembly assay

Microtubule proteins were purified from fresh rat brain through 3 cycles of assembly-disassembly according to Miller and Wilson[21,22],and stored at-70°C as a pellet in PEM buffer(pH=6.8,100 mmol/L PIPES, 1 mmol/L EGTA,1mmol/L MgSO4).

Purified tubulin at 0.2 mg/mL was polymerized in PEM buffer added with 1 mmol/L GTP at pH 6.8 in the absence or presence of PTX(or compound 1)at 4°C.The microtubule polymerization was monitored by the turbidity at 350 nm and 35°C using a Beckman DU 640 temperature controlled spectrophotometer for 60 min.The time of median turbidity absorption (Tmedian)about tubulin aggregation was calculated by the following equation:Tmedian=(Tmin+Tmax)/2.

C.Statistical analysis

All the data are analysed using Origin 8.0.All the bars in Fig.2 represent standard error of the mean (SEM).The data were compared and analysed using the one-way analysis of variance test(ANOVA).

III.RESULTS AND DISCUSSION

A.Hydrogelation of compound 1

We used cryo-TEM to characterize the morphology of the nanostructures formed in the solutions.As shown in Fig.3(a),cryo-TEM image of compound 1 in the absence of ALP showed regular nanoparticles with an average diameter of 14.2 nm.In the presence of ALP, the phosphate group on compound 1 was cleaved to yield the hydrogelator Fmoc-Phe-Phe-Lys(taxol)-Tyr-OH(2)which self-assembled into nanofibers with an average width of 7.2 nm(Fig.3(b)).

FIG.2 Hydrogelator compound 1 delayed the tubulin aggregation in tubulin self-assembly assay in v itr o.(a)The turbidity changes of tubulin assembly at 350 nm.Polymerization of purified tubulin at 0.2 mg/mL was carried out at 35°C initiated by 1%GTP(control),with 10 nmol/L PTX or 10 nmol/L compound 1 for 60 min.(b)Aggregation times of the median turbidity(Tmedian)in(a).(p＜0.01).

FIG.3 Cryo-EM images of 1wt%compound 1 in PBS (pH=7.4,0.01 mol/L)in the(a)absence or(b)presence of 300 U/mL ALP at 37°C for 12 h.

FIG.4 Proposed mechanism of compound 1 inhibiting the aggregation of microtubule.The αβ-tubulin dimer has 8 nm in length and 4.6 nm in diameter.

B.Tubulin purification and assembly-disassembly assay

Tubulin assembly-disassembly assay was carried out to investigate the effect of compound 1 on microtubule aggregation.Purified tubulin solutions at 0.2 mg/mL in PEM buffer at pH=6.8 were added with 10 nmol/L PTX or compound 1 at 35°C,respectively.As shown in Fig.2,both PTX and compound 1 induced the aggregation of the tubulins under this condition.However, compared with the control tubulins that aggregated at 19.0 min,tubulins incubated with PTX or compound 1 aggregated at 24.1 or 36.1 min,respectively.

As we know,the soluble αβ-tubulin subunits are firstly converted into a growing microtubule from microtubule nucleation[23].Microtubule nucleation in cells occurs from templates,either the central organizer[24, 25]or the severed end of a pre-existing micro-tubule [26-28].In neuron,the serving parts of microtubule was one of the nucleation inducing the neuron branching[29].We propose herein that,under the catalysis of ALP,compound 1 was converted to compound 2 and thereafter self-assembled into the nanofibers alongside the tubulin subunits which delayed their attachment to the serving parts of microtubule.This destabilization of the nascent plus ends effectively affected the persistent elongation of microtubule.Meanwhile,the self-assembling process of compound 2 itself could also delay the aggregation of microtubule[17].This also explained why compound 1 could promote neuron branch observed in our previous work[9].

We suppose the mechanism through the basic parameters of tubulin and microtubule[30].As shown in Fig.4,the αβ-tubulin dimer has 8 nm in length and 4.6 nm diameter.Our result showed that,under the catalysis of ALP,compound 1 could form nanofiber compound 2 with 7.2 nm diameter.When the tubulin dimer was attacked by nanofiber compound 2,it was hard for the dimers to attach with each other.Another reason is that,since the inner diameter of microtubule is about 15 nm and the width of nanofiber compound 2 is about 7.2 nm,it is hard for more than two of nanofiber compound 2 to fill in the inner side of a microtubule.Therefore,nanofiber compound 2 is supposed to attach to the interface of the microtubule,as shown in Fig.4.In the meantime,the nanofibers also mulched on the severing plus end,affecting the attachment of αβ-tubulin to the nucleation and thus delaying microtubule aggregation.

Therefore,besides the two above-mentioned mechanisms proposed by Cline et al.,we also propose that our PTX-derivative compound 1 could delay microtubule aggregation by interfering with the plus end aggregation of microtubule.

IV.CONCLUSION

In summary,a PTX-derivative Fmoc-Phe-Phe-Lys(Taxol)-Tyr(H2PO3)-OH(1)was synthesized which had similar effect on microtubule aggregation to that of PTX.However,compound 1 heavily delayed microtubule aggregation than PTX did.Under the catalysisof ALP,compound 1 could self-assemble into nanofiber compound 2 with width comparable to the size of αβtubulin dimer.Therefore,we proposed in this work that nanofiber compound 2 not only inhibits the αβ-tubulin dimer binding to each other but also interferes with the plus end aggregation of microtubule.This work provides a new mechanism of the inhibition of microtubule formation by a PTX-derivative hydrogelator and collection of the evidences to verify the hypothesis is undergoing.

V.ACKNOWLEDGMENTS

This work was supported by the Ministry of Science and Technology of China(No.2016YFA0400904) and the National Natural Science Foundation of China (No.U1532144 and No.21675145).

[1]E.K.Rowinsky and R.C.Donehower,N.Eng.J.Med. 332,1004(1995).

[2]T.M.Mekhail and M.Markman,Expert Opin.Pharmacother.3,755(2002)

[3]A.R.Hanauske,Anticancer Drugs 2,29(1996).

[4]Y.Wei,Z.Xue,Y.Ye,Y.Huang,and L.Zhao,Arch. Pharm.Res.37,728(2014).

[5]N.I.Marupudi,J.E.Han,K.W.Li,V.M.Renard,B. M.Tyler,and H.Brem,Expert.Opin.Drug Saf.6,609 (2007).

[6]P.B.Schif f,J.Fant,and S.B.Horwitz,Nature 277, 665(1979).

[7]P.B.Schiffand S.B.Horwitz,Biochemistry 20,3247 (1981).

[8]O.N.Zefirova,E.V.Nurieva,H.Lemcke,A.A.Ivanov, D.V.Shishov,D.G.Weiss,S.A.Kuznetsov,and N.S. Zefirov,Bioorg.Med.Chem.Lett.18,5091(2008).

[9]J.Wei,H.Wang,M.Zhu,D.Ding,D.Li,Z.Yin,L. Wang and Z.Yang,Nanoscale 20,9902(2013).

[10]H.Wang and Z.Yang,Nanoscale 4,5259(2012).

[11]J.Liu,L.Zhang,Z.Yang,and X.Zhao,Int.J. Nanomedicine 6,2143(2011).

[12]Y.Yuan,L.Wang,W.Du,Z.Ding,J.Zhang,T.Han, L.An,H.Zhang,and G.Liang,Angew.Chem.Int.Ed. 54,9700(2015).

[13]B.Mei,Q.Miao,A.Tang,and G.Liang,Nanoscale 7, 15605(2015).

[14]E.N.Cline,M.H.Li,S.K.Choi,J.F.Herbstman, N.Kaul,E.Meyhofer,G.Skiniotis,J.R.Baker,R.G. Larson,and N.G.Walter,Biomacromolecules 14,654 (2013).

[15]H.M.Wang,J.Wei,C.B.Yang,H.Y.Zhao,D.X. Li,Z.N.Yin,and Z.M.Yang,Biomaterials 33,5848 (2012).

[16]Y.Gao,Y.Kuang,Z.F.Guo,Z.Guo,I.J.Krauss,and B.Xu,J.Am.Chem.Soc.131,13576(2009).

[17]Y.Kuang and B.Xu,Angew Chem.Int.Ed.52,6944 (2013).

[18]K.Park,J.Control.Release 158,355(2012).

[19]B.Bacsa and C.O.Kappe,Nat.Protoc.2,2222(2007). [20]A.Matov,K.Applegate,P.Kumar,C.Thoma,W. Krek,G.Danuser,and T.Wittmann,Nat.Methods 7,761(2010).

[21]H.P.Miller and L.Wilson,Methods Cell Biol.95,3 (2010).

[22]M.Castoldi and A.V.Popov,Protein Expr.Purif.32, 83(2003).

[23]M.Wieczorek,S.Bechstedt,S.Chaaban,and G.J. Brouhard,Nat.Cell Biol.17,907(2015).

[24]M.Moritz,M.B.Braunfeld,J.W.Sedat,B.Alberts, and D.A.Agard,Nature 378,638(1995).

[25]Y.Zheng,M.L.Wong,B.Alberts,and T.Mitchison, Nature 378,578(1995).

[26]K.McNally,A.Audhya,K.Oegema,and F.J.McNally, J.Cell Biol.175,881(2006).

[27]M.Srayko,A.Kaya,J.Stamford,and A.A.Hyman, Dev.Cell 9,223(2005).

[28]J.J.Lindeboom,M.Nakamura,A.Hibbel,K. Shundyak,R.Gutierrez,T.Ketelaar,A.M.Emons, B.M.Mulder,V.Kirik,and D.W.Ehrhardt,Science 342,1245533(2013).

[29]P.W.Baas,Neuron 78,3(2013).

[30]S.Sahu,S.Ghosh,B.Ghosh,K.Aswani,K.Hirata,D. Fujita,and A.Bandyopadhyay,Biosens.Bioelectron. 47,141(2013).

?Author to whom correspondence should be addressed.E-mail:gliang@ustc.edu.cn,Tel:+86-551-63607935,FAX:+86-551-63600730