Simplified Pretubulysin Derivatives and Their Biological Effects on

Article pubs.acs.org/jnp

Simplified Pretubulysin Derivatives and Their Biological Effects on Cancer Cells Rebekka Kubisch,†,‡ Matthias von Gamm,†,‡ Simone Braig,† Angelika Ullrich,§ Jens L. Burkhart,§ Laura Colling,† Jennifer Hermann,⊥ Olga Scherer,∥ Rolf Müller,⊥ Oliver Werz,∥ Uli Kazmaier,§ and Angelika M. Vollmar*,† †

Pharmaceutical Biology, Department of Pharmacy, Center for Drug Research, Ludwig-Maximilians-University, Butenandtstraße 5-13, D-81377 Munich, Germany § Institute for Organic Chemistry, Saarland University, P.O. Box 151150, D-66123 Saarbrücken, Germany ⊥ Department of Pharmaceutical Biotechnology, Saarland University, P.O. Box 151150, D-66123 Saarbrücken, Germany ∥ Pharmaceutical/Medicinal Chemistry, Friedrich-Schiller-University Jena, Philosophenweg 14, D-07743 Jena, Germany S Supporting Information *

ABSTRACT: Tubulin binding agents are a potent group of cancer chemotherapeutics. Most of these substances are naturally derived compounds. A novel substance class of destabilizing agents is the group of tubulysins. The tubulysins and their derivative pretubulysin have shown high efficacy in vitro and in vivo. Due to their complex chemical structures, one major bottleneck of the tubulysins is their accessibility. Biotechnological as well as chemical production is challenging, especially on larger scales. Thus, the synthesis of chemically simplified structures is needed with retained or improved biological activity. Herein is presented the biological evaluation of two pretubulysin derivatives [2-desmethylpretubulysin AU816 (1) and phenylpretubulysin JB337 (2)] in comparison to pretubulysin. Both 1 and 2 display a simplification in chemical synthesis. It was shown that both compounds exhibited potent biological activity against cancer cells. These simplified compounds inhibited tubulin polymerization in the nanomolar range. The cytotoxic effects of 1 and 2 were in a similar range, when compared with pretubulysin [IC50 (nM): pretubulysin: 0.6; 1: 10; 2: 100]. Furthermore, it was shown that cell cycle arrest is induced and migration is hampered in MDA-MB-231 breast cancer cells. In conclusion, 1 was shown to be about 10-fold more active than 2 and as potent as pretubulysin.

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Prominent TBAs in clinical use are the Vinca alkaloids, vinblastine and vincristine, as well as paclitaxel (Taxol).5 TBAs have shown great success in cancer treatment, but their chemical synthesis is often complicated due to their complex structures, so new compounds need to be developed and tested for their applicability to cancer treatment. Simplified chemical structures of these compounds can then be synthesized and compared to their original structures. One group of such compounds is the tubulysin class, a family of microtubule-depolymerizing natural products. The tubulysins are of myxobacterial origin and have shown to exert cytotoxic activity against numerous tumor cell lines.6,7 The tubulysins are a group with highly complex structures. A tubulysin precursor (pretubulysin) was described as exhibiting high cytotoxic potency against cancer cells and has been reported to be a potential antiangiogenic agent.8,9 Pretubulysin

atural compounds are a rich source for the development of anticancer therapeutics. Numerous broadly used therapeutics, such as doxorubicin or paclitaxel, are of natural origin. In fact, over 60% of anticancer drugs originate from natural products.1 Nevertheless, a major bottleneck of many naturally derived therapeutics is their restricted accessibility, as they often cannot be isolated from their source organism in sufficiently large quantities. Total chemical synthesis may circumvent this problem, but in many cases cannot be facilitated on an industrial scale, due to chemical structural complexity. Simplified and chemically accessible derivatives or precursors with an equivalent biological activity may be used to solve this issue. The microtubule system is one of the main targets in cancer therapy. In eukaryotic cells, microtubules are composed of continuously assembling and disassembling α,β-tubulin heterodimers that are essential for proliferation, trafficking, and migration.2−4 Tubulin binding agents (TBAs) interfere with these dynamics, ultimately resulting in mitotic arrest. Thus, TBAs hamper cell division of proliferating cells, e.g., cancer cells.5 © 2014 American Chemical Society and American Society of Pharmacognosy

Special Issue: Special Issue in Honor of Otto Sticher Received: September 30, 2013 Published: January 17, 2014 536

dx.doi.org/10.1021/np4008014 | J. Nat. Prod. 2014, 77, 536−542

Journal of Natural Products

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is a tetrapeptide: N-methylated D-pipecolic acid is coupled to Lisoleucine, which is connected to C-desacetoxytubuvaline and tubuphenylalanine.10 When compared to tubulysin D, the chemical structure of pretubulysin is simplified in the tubuvaline moiety.10 Since the introduction of a labile N,Oacetal moiety is the crucial step in the chemical synthesis of tubulysin D, the production of pretubulysin is easier.10,11 Despite simplification of the original structure, pretubulysin is still highly complex and requires a challenging production. Thus, more simplified analogues were synthesized.12,13 Among these, the analogues AU816 (1) and JB337 (2) were revealed to be potent microtubule inhibitors. Both 1 and 2 inhibit the proliferation of proliferating endothelial cells (HMEC-1) in the nanomolar range (EC50 4.4 and 13.2 nM).8 Compound 1 lacks the α-methyl group of tubuphenylalanine and therefore a stereogenic center, while 2 was modified in the middle of the molecule by replacing the 1,3-thiazole structure with a simple benzene moiety.8,12,13 In this study, the activity of these two simplified derivatives in comparison to pretubulysin has been evaluated. Compounds 1 and 2 were demonstrated to exhibit high biological activity against breast cancer cells, by inhibiting tubulin polymerization, exhibiting cytotoxic effects, inducing cell cycle arrest, and hampering migration. Moreover, it was demonstrated that the cytotoxic potency of the simplified derivative 1 on cancer cells is equivalent to that of pretubulysin and vincristine, a compound that has long been used in the clinic.

analyze the inhibitory potency of the simplified pretubulysin derivatives 1 and 2 on the microtubule network of cancer cells, immunological detection of tubulin after treatment by these compounds was compared to pretubulysin-treated cells. Therefore, MDA-MB-231 cells were treated with the indicated compound for 24 h at concentrations between 1 and 1000 nM. Immunohistochemistry showed that starting at a concentration of 10 nM, pretubulysin and 1 had slight effects on disruption of the tubulin cytoskeleton (Figure 1). For 2, similar effects could be detected at 100 nM. Both 1 and 2 broke down the microtubule network within 24 h. When compared with pretubulysin, 1 was equally active, whereas approximately 10fold higher doses of 2 were needed to disrupt the tubulin network significantly. Similar effects were seen using bladder carcinoma cells (Figure S1, Supporting Information). In comparison, pretubulysin has been shown to alter tubulin structures in proliferating endothelial cells (HMEC-1), starting at 10 nM.8 AU816 (1) and JB337 (2) Exhibit Cytotoxic Effects on Breast Cancer Cells at the Nanomolar Range. In vitro, Vinca alkaloids exhibit cytotoxic activities at the nanomolar range in small-cell lung cancer cells.16 Pretubulysin has been shown to induce apoptosis in pancreatic cancer cells (L3.6pl) at 1 nM.9 In contrast, the cytotoxic effect of pretubulysin on nonmalignant cells is less profound. Peripheral blood mononuclear cells treated with pretubulysin showed initial cytotoxic effects starting at about 1 μM pretubulysin (Figure S2, Supporting Information). To compare the cytotoxicity of the derivatives, the cell viability (NADH status) of treated MDA-MB-231 breast cancer cells was determined with an incubation time of 72 h. Serial dilutions from 0.1 to 1000 nM were tested. As shown in Figure 2, the IC50 value of pretubulysin was 0.6 nM. The IC50 value of compound 1 was increased 10-fold (10.1 nM) vs pretubulysin. Compound 2 was less active in contrast to pretubulysin and 1, but still in a nanomolar range (IC50 100.2 nM). On comparing pretubulysin and its simplified derivatives to vincristine (IC50 1.6 nM), a tubulin-destabilizing agent in clinical use, pretubulysin and 1 showed efficacy in the same range in inducing cytotoxic effects. In contrast, 2 was about 100-fold less effective under the conditions used (Figure 2). The Cell Cycle of MDA-MB-231 Cells Is Arrested by AU816 (1) and JB337 (2). Microtubules are obligatory during mitosis to separate the chromosome pairs prior to cell division into two daughter cells. Thus, the inhibition of microtubule dynamics by TBAs blocks cell division and causes G2/M arrest. Pretubulysin previously has been shown to induce G2/M arrest at the nanomolar range in human hepatocellular carcinoma cells (HepG2).9 To compare the potency of inducing G2/M arrest of the cell cycle by pretubulysin, breast cancer cells treated with 1 and 2 were examined by flow cytometry. Compared to pretubulysin, 1 was slightly less active. At 10 nM, pretubulysin affected the cell cycle and significantly increased the G2/M population from 22% in control cells to more than 30%. In turn, while 1 did not increase the number of cells in the G2/M phase significantly at 10 nM, treatment of the cells with a 10-fold higher concentration resulted in an increased G2/M population (60%) and a reduction of the number of cells at the G1 phase (Figure 3A and B). Compound 2 had similar effects to 1 (Figure 3C). No effects on cell cycle were detected with 10 nM 2. Treatment with 100 nM compound 2 increased the G2/ M population from about 23% to 33%. In summary, it could be

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RESULTS AND DISCUSSION Pretubulysin Derivatives AU816 (1) and JB337 (2) Disrupt Tubulin Structures of Breast Cancer Cells. Tubulin-destabilizing agents, such as the Vinca alkaloids or the tubulysins, hamper tubulin polymerization.7,14 Tubulysins bind to the vinca domain of β-tubulin, hindering tubulin polymerization.15 Thus, these compounds induce mitotic arrest, leading to initiation of apoptosis in proliferating cells. To 537



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Figure 1. Microtubule staining of MDA-MB-231 cells treated with pretubulysin (PT), AU816 (1), or JB337 (2). Cells were seeded in eight-well microscopic slides and stimulated for 24 h with increasing (1, 10, 100, and 1000 nM) compound concentrations or DMSO (0.1%). α-Tubulin (green) and DNA (blue) were visualized by immunocytochemistry. Representative images are shown.

Figure 2. Antiproliferative effects of vincristine (A), pretubulysin (B), AU816 (1) (C), and JB337 (2) (D) against MDA-MB-231 cells. Cells were seeded in 96-well plates and treated with increasing concentrations (0.1, 1, 10, 100, 1000 nM). After 72 h, the CellTiterBlue assay was performed. VCR = vincristine, PT = pretubulysin. Results are displayed as percent of the mock-treated control. Data points represent the means ± SEM of three independent experiments performed in quadruplicate.

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Figure 3. Cell cycle analysis of MDA-MB-231 cells treated with pretubulysin and its analogues. MDA-MB-231 cells were treated with increasing concentrations (1−100 nM) of pretubulysin (PT) (A), AU816 (1) (B), JB337 (2) (C), or DMSO (0.01%) for 48 h. Permeabilized and propidium iodide stained cells were analyzed by flow cytometry. Bars represent the means ± SEM of three independent experiments performed in triplicate. Representative histograms for cell cycle analysis are displayed in the right panel, respectively.

shown that 1 and 2 are about 10-fold less effective when compared to pretubulysin. AU816 (1) and JB337 (2) Have Antimigratory Properties Similar to Pretubulysin. Growing evidence suggests that microtubules not only are crucial in mitosis and membrane trafficking but also contribute to metastasis and migration.17,18 Thus, TBAs not only induce mitotic arrest but also interfere with migratory processes. For instance, vincristine has been shown to cause a significant decrease in directional migration in GaMG and D-37MG cells,19 and epothilone has been shown to reduce lymph node metastasis of prostate cancer cells in vivo.20 The antimigratory potentials of tubulysin analogues were evaluated. In particular, it was tested whether on treatment with pretubulysin, 1 and 2 inhibit the migration of breast cancer cells into a wounded cell monolayer (wound healing or scratch assay). Scratches were inflicted into a dense monolayer of MDA-MB-231 cells that were treated subsequently with increasing compound concentrations and incubated for 18 h. Notably, during the time of the experiment (18 h), proliferation effects may be minor but cannot be totally excluded as taking part in wound closure. Both pretubulysin and 1 hampered wound closure in MDAMB-231 cells unnder similar concentrations. At 100 nM, 1 reduced the wound closure by about 35%, and 1 μM abrogated almost any cell migration (Figure 4A and B). In contrast, 2 was

less potent in comparison to pretubulysin, as the wound closure in MDA-MB-231 cells was not affected at a concentration of 100 nM. However, the highest concentration tested drastically decreased wound closure to values seen for pretubulysin and 1 (Figure 4C). Importantly, effective concentrations in this assay were 10- to 100-fold increased compared to other assays shown. This may be a result of the relatively high cell number and thus the increased amount of polymerized tubulin when compared to other in vitro assays. It was shown earlier that the growth inhibitory potency of microtubule-depolymerizing agents is dependent on the amount of polymerized tubulin.21,22 Indeed, cytotoxicity was also decreased in the present study if the total cell number was increased (Figure S3, Supporting Information). Taken together, it was observed that wound closure of MDA-MB-231 cells was affected by treatment with both analogues. Compound 1 was as potent as pretubulysin in hampering wound closure, and 2 showed a 10-fold lesser efficacy. Vincristine-Resistant Cancer Cells Are Sensitive to Pretubulysin, AU816 (1), and JB337 (2). Resistance to chemotherapy is a major hurdle in cancer treatment.23 Thus, the development of new compounds is a key issue in contemporary drug development. Vincristine-resistant leukemia cells were used to determine whether pretubulysin and its derivatives are able to exhibit cytotoxic effects in these cells. 539



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In conclusion, 1 and 2 exhibit biological activity against breast cancer cells. Pretubulysin, 1, and 2 inhibit tubulin polymerization, exhibit cytotoxic effects, induce cell cycle arrest, and hamper migration. Even the viability of chemoresistant lymphoma cells was reduced by pretubulysin and its derivatives. The potency of 2 was around 10-fold less when compared to pretubulysin. Moreover, 1 showed an equivalent biological potency to pretubulysin and thus represents a potential, chemically simplified alternative for further in vivo studies.

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EXPERIMENTAL SECTION

General Experimental Procedures. The test compounds pretubulysin, 1, and 2 were synthesized and provided in pure form by the group of U. Kazmaier.10,12,13 Vincristine was purchased from Sigma-Aldrich, St. Louis, MO, USA. Cell Culture. All cell lines were cultured at 37 °C under a humidified atmosphere with 5% CO2. MDA-MB-231 cells were obtained from ATCC and cultivated in DMEM high glucose [PAA Laboratories (Cö lbe, Germany) or PAN Biotech (Aidenbach, Germany)], and CCRF-CEM-VCR-R were obtained from Maria Kavallaris (Children’s Cancer Institute Australia for Medical Research, Randwick, New South Wales, Australia) and cultivated in RPMI-1640 medium (PAA Laboratories, Cölbe, Germany). DMEM and RPMI1640 media were supplemented with 10% FCS (Biochrom AG, Berlin, Germany). Immunostaining and Laser Scanning Microscopy. Cells were seeded in eight-well μ-slides (Ibidi, Martinsried, Germany) and treated with the compounds pretubulysin, 1, and 2 at concentrations between 1 and 1000 nM for 24 h. After treatment, the medium was discarded and cells were incubated for 2 min with cell extraction buffer [80 mM PIPES pH 6.8, 1 mM MgCl2, 5 mM EGTA-K, and 0.5% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA)] to remove monomeric and dimeric tubulin subunits in order to prevent intensive background staining. Next, cells were fixed by addition of glutaraldehyde to the extraction buffer to a final concentration of 0.5% for 10 min. The supernatant was discarded, and residual glutaraldehyde was reduced by addition of freshly prepared 0.1% NaBH4 solution and incubation for 7 min. Cells were washed with PBS (three times, 5 min) followed by incubation with BSA (0.2% in PBS) for 10 min to block unspecific binding sites. Subsequently, cells were incubated with the primary antiα-tubulin antibody [Abcam, Cambridge, UK (ab18251)] in 0.2% BSA in PBS for 1 h and washed thereafter with PBS (four times, 5 min). Then, a solution of the secondary fluorescent antibody (Abcam AlexaFluor 488 secondary antibody) and Hoechst 33342 (SigmaAldrich, St. Louis, MO, USA) was added and incubated for 45 min (protected from light, gentle shaking). Thereafter, cells were washed again with PBS (four times, 5 min, protected from light), embedded in FluorSave Reagent mounting medium, and covered with glass coverslips. Stained microtubule structures were analyzed using a laser scanning microscope (LSM 510 META, Zeiss, Jena, Germany). Cytotoxicity Assay (CellTiterBlue). Cytotoxicity was determined using the CellTiterBlue assay (Promega, Madison, WI, USA) according to the manufacturer’s instructions. In brief, cells were seeded in 96-well plates and treated for 72 h. Thereafter, the CellTiterBlue reagent was added to each well. After incubation at 37 °C for 2 h, fluorescence was measured using a microplate reader (SpectraFluor Plus, Tecan, Männedorf, Switzerland). Results are shown as the percent of the mock-treated control. Cell Cycle and Apoptosis. For cell cycle analysis, MDA-MB-231 cells were seeded in 12-well plates and grown to 60% confluence. Thereafter, cells were treated for 48 h with the compounds or with a corresponding amount of DMSO as control. Next, cells were harvested by analogy to the protocol of Riccardi and Nicoletti and analyzed by flow cytometry.24 Detached cells were permeabilized in hypotonic fluorochrome solution [0.1% (v/v) Triton X-100, 0.1% (w/v) sodium citrate, in PBS], supplemented with 50 μg/mL propidium iodide. Subsequently, cells were incubated for at least 30 min at 4 °C. For cell cycle analysis, flow cytometric measurements were made on a

Figure 4. Migration (wound closure) of MDA-MB-231 cells during treatment with pretubulysin (A) or its analogues AU816 (1) (B) and JB337 (2) (C). MDA-MB-231 cells were grown to 100% confluence. After scratch infliction, cells were stimulated with increasing concentrations of pretubulysin (PT) analogues or DMSO (0.1%) as indicated and incubated for 18 h. Scratched areas were analyzed with an ImageJ plug-in and expressed as wound closure relative to control. Representative images are displayed in the right panel. Bars represent the means ± SEM of three independent experiments performed in quintuplicate.

The resistance of CCRF-CEM-VCR-R cells in comparison to control cells (CCRF-CEM) again was confirmed. Vincristine induced apoptosis within 48 h at the nanomolar range in control cells, whereas, in vincristine-resistant cells, apoptosis was induced only slightly at 1 μM (20%) (Figure S4, Supporting Information). To analyze if vincristine-resistant lymphoblastic leukemia cells are sensitive to analogues 1 and 2, CCRF-CEM-VCR-R cells were treated for 72 h. For comparability of the applied concentrations to cytotoxic effects, a concentration of 2.5 μM vincristine was chosen. At 2.5 μM, vincristine displayed a threshold concentration at which the vincristine-resistant cells start to respond to the treatment. At 2.5 μM, the viability of the vincristine-resistant cells was about 80%. As shown in Figure 5A, pretubulysin showed cytotoxic effects at 1 μM (70%), which were even greater at 2.5 μM (30%). Similar effects were detected using 1 (Figure 5B). Both compounds were more effective than vincristine itself. In contrast, 2 showed cytotoxic effects at 2.5 μM and thus an equal potency to vincristine (Figure 5C). As described previously, CCRF-CEM-VCR-R cells have an increased amount of polymerized tubulin, and thus higher quantities of vincristine are needed to induce apoptosis.22 The present work shows that due to the high potency of the tubulysin derivatives, they are able to exhibit cytotoxic effects even in resistant cells. 540



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Figure 5. Cytotoxic effects of tubulin destabilizer against vincristine-resistant leukemia cells. Pretubulysin (A), AU816 (1) (B), and JB337 (2) (C) against vincristine-resistant lymphoma cells (CCRF-CEM-VCR-R); cells were seeded in 96-well plates and treated for 72 h with pretubulysin (PT), AU816 (1), JB337 (2) (2.5, 1, 0.1, 0.01 μM), or vincristine (2.5 μM). To determine cytotoxic effects, a CellTiterBlue assay was performed. Results are shown as percentages of the mock-treated control. Bars represent the means ± SEM of three independent experiments performed in quadruplicate [*p < 0.05 (t test)].

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FACSCalibur instrument (BD) by recording fluorescence in the linear mode. The percentage of cells in the different cell cycle phases was determined using the flow cytometry analysis software FlowJo 7.6 (Tree Star, Ashland, OR, USA). For apoptosis (sub-G1) analysis flow cytometric measurements were done by recording fluorescence in the logarithmic mode. The percentage of cells in the subG1 phase was determined using the flow cytometry analysis software FlowJo 7.6. Analysis of Cell Migration (Wound-Healing Assay). The wound-healing assay, also known as a scratch assay, is a well-developed method to determine cell migration in vitro.25 The method was modified for MDA-MB-231 cells. In brief, cells were seeded in 96-well plates (2 × 105 per well) and grown to 100% confluence. Scratches of approximately 1 mm were inflicted into the monolayer using a scratching device with 96 spring-mounted PTFE tips. Detached cells were removed by washing twice with PBS+ (137 mM NaCl, 2.68 mM KCl, 8.10 mM Na2HPO4, 1.47 mM KH2PO4, 0.25 mM MgCl2 in H2O), and the remaining cells then were incubated for approximately 16 h until the scratch in control cells was almost closed. Cells were cultivated in either culture medium containing DMSO at a concentration being present at the highest compound dilutions (set as 100% migration) or culture medium containing increasing concentrations of pretubulysin or its derivatives 1 and 2. A control plate was left untreated and directly fixed after preparation of the scratch (set as 0% migration). After incubation for 16 h, cells were washed with PBS and fixed with 4% formaldehyde. Fixed cells were stained with a solution of crystal violet (0.5% crystal violet, 20% methanol, in water) for 15 min, washed with water, and air-dried. An image was taken of each well on an Eclipse Ti (Nikon) inverted light microscope system with 4× objective lens, LED light, and automatic sample table. Images were acquired with the software NISElements AR (Nikon, Tokyo, Japan). Areas on the images not covered by cells were determined with a modified ImageJ26 plug-in, originally developed by S. Gallagher, W. Ashby, F. Cordeliéres, and L. Larue and published under a GNU license agreement. The plug-in was modified in four aspects with the objective to increase the accuracy and simplify further analytical steps. Cell-covered areas were calculated from the scratch areas, subtracted from the 0%-migration control, and expressed as fold decrease of wound closure as compared to DMSO-treated cells.

ASSOCIATED CONTENT

S Supporting Information *

Microtubule staining of bladder carcinoma cells (T-24) treated with pretubulysin (PT), AU816 (1), or JB337 (2). α-Tubulin (green) and DNA (blue) were visualized by immunocytochemistry. Representative images are shown (Figure S1). MTT assay of pretubulysin-treated primary monocytes (Figure S2). Viability of MDA-MB-231 cells under conditions of the migration assay: 2 × 105 cells per well, 24 h compound incubation (Figure S3). Apoptosis induction in control cells vs vincristine-resistant cells. CCRF-CEM or CCRF-CEM-VCR-R cells were treated for 48 h with vincristine (A) or pretubulysin (B). Apoptosis induction (DNA fragmentation) was determined by FACS analysis (Figure S4). These materials are available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +49(0) 89-2180-77168. Fax: +49(0)89-2180-77170. Author Contributions ‡

R. Kubisch and M. von Gamm contributed equally.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

We kindly thank M. Kavallaris for providing the CCRF-CEM and the CCRF-CEM-VCR-R cell lines. This project was funded by the German Research Foundation (DFG, FOR 1406, Vo 376-14/15, and WA1648/3-1). 541



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DEDICATION Dedicated to Prof. Dr. Otto Sticher, of ETH-Zurich, Zurich, Switzerland, for his pioneering work in pharmacognosy and phytochemistry.

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REFERENCES

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Simplified Pretubulysin Derivatives and Their Biological Effects on

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