A Synthetic Dolastatin 10 Analogue Suppresses ... - ACS Publications

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A Synthetic Dolastatin 10 Analogue Suppresses Microtubule Dynamics, Inhibits Cell Proliferation, and Induces Apoptotic Cell Death Praveen Kumar Gajula,†,§ Jayant Asthana,‡,§ Dulal Panda,*,‡ and Tushar Kanti Chakraborty*,† †

CSIR-Central Drug Research Institute, Lucknow 226 001 India Department of Biosciences and Bioengineering, Indian Institute of Technology Bombay, Mumbai 400076, India

‡

S Supporting Information *

ABSTRACT: We have synthesized eight analogues (D1−D8) of dolastatin 10 containing several unique amino acid subunits. Of these agents, D5 was found to be most effective in inhibiting both HeLa cell proliferation and microtubule assembly in vitro. At low nanomolar concentrations, D5 inhibited the proliferation of several types of cancer cells in culture. D5 bound to tubulin with a dissociation constant of 29.4 ± 6 μM. D5 depolymerized microtubules in cultured cells and produced mulitpolar spindles. At its half-maximal inhibitory concentration (15 nM), D5 strongly suppressed the dynamics of individual microtubules in live MCF-7 cells. D5 increased the accumulation of checkpoint proteins BubR1 and Mad2 at the kinetochoric region and caused G2/M block in these cells. The blocked cells underwent apoptosis with the activation of Jun N-terminal kinase. The results suggested that D5 exerts its antiproliferative action by dampening microtubule dynamics.

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INTRODUCTION Dolastatin analogues are known to inhibit the proliferation of different kinds of cancer cells in culture.1−4 In addition, dolastatin 10 and its analogues have been shown to inhibit the growth of xenograft in tumor models.5,6 For example, dolastatin 10 was found to be highly effective in human ovarian carcinoma xenograft in nude mice.1 Further, an analogue of dolastatin 10 named TZT-1027 (1) has been shown to display potent antitumor effects in human tumors xenografted in nude mice from gastric, breast, colon, lung, liver, renal, and ovarian cancer lines.5 Another synthetic analogue auristatin PYE, of dolastatin 10, is effective in human colon tumor models.6 Dolastatin 10 was in phase II clinical trial for the treatment for various types of cancers including metastatic pancreatic cancer (ClinicalTrials.gov Identifier: NCT00003677), recurrent liver, bile duct, or gallbladder cancer (ClinicalTrials.gov Identifier: NCT00003557), advanced kidney cancer (ClinicalTrials.gov Identifier: NCT00003914), metastatic soft tissue sarcoma (ClinicalTrials.gov Identifier: NCT00003778), chronic myelo genous leukemia (ClinicalTrials.gov Identifier: NCT00003693), and chronic lymphocytic leukemia (ClinicalTrials.gov Identifier: NCT00005579). Some of the dolastatin 10 analogues like soblidotin or 1 are also in clinical trials for treatment of metastatic soft tissue sarcoma (ClinicalTrials.gov Identifier: NCT00064220) and metastatic nonsmall cell lung cancer (ClinicalTrials.gov Identifier: NCT00061854). 1 is active against many solid tumors like colon 26 adenocarcinoma, © 2013 American Chemical Society

B16 melanoma, and M5076 sarcomas as well as p388 leukemia.7 Dolastatins and their analogues are known to cause cell cycle arrest at mitosis by targeting microtubules.2,6,8,9 Dolastatins are shown to inhibit the assembly of microtubules both in vitro and in cells8,10 and are known to noncompetitively inhibit the binding of vinblastine to tubulin.11,12 It has been suggested that dolastatin 10 binds adjacent to the exchangeable GTP site on the β-tubulin.11,13,14 Although dolastatin 10 has been studied widely, its effects on the dynamic instability of individual microtubules in live cells are not known. Dolastatin analogues are active against different types of tumors; however, these agents are associated with various harmful side effects.15 For example, 1 has been found to cause myelosuppression (bone marrow toxicity) and peripheral neuropathy and local irritation at the injection site.15 Treatment with 1 is coupled with neutropenia and infusion arm pain.15 In addition, researchers are now interested in making potent synthetic derivatives of dolastatins because of low yields of these compounds from the source organisms.16 Further, cancer chemotherapy is associated with development of resistance against the existing drugs. The new synthetic derivatives are also being synthesized with the aim to overcome the resistance developed in cancer cells. Received: July 12, 2012 Published: February 27, 2013 2235

dx.doi.org/10.1021/jm3009629 | J. Med. Chem. 2013, 56, 2235−2245

Journal of Medicinal Chemistry

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Figure 1. Structures of dolastatin 10 and dolastatin 10 analogues.

estramustine are known to inhibit cell proliferation by suppressing microtubule dynamics.29−32 Here, we are reporting the effect of a dolastatin 10 analogue on microtubule dynamics in live MCF-7 cells. D5 dampened the dynamics of individual microtubules in live MCF-7 cells. The results suggested that the suppression of microtubule dynamics led to an increase in the mitotic index of the cells with the accumulation of check point proteins at the kinetochores and activated Jun N-terminal kinase (JNK) dependent apoptotic cell death.

In this study, we have synthesized and characterized eight analogues (D1−D8) of dolastain 10 (Figure 1 and Supporting Information). The salient feature in our approach to design these analogues was the use of versatile turn-inducing peptide building blocks called sugar amino acids (SAA), which have been extensively used by us and many others to mimic bioactive conformations in small peptides.17−26 The only conformational study of dolastatin 10 in solution in DMSO revealed a compact structure that folded around the central dolaisoleucine− dolaproline bond in its cis form, whereas the trans conformation exhibited a less ordered structure.27 On the basis of this study, we envisaged that substitution of the dolaproline residue of dolastatin 10 with a dipeptide isosteric sugar amino acid would induct the desired fold in the backbone of the molecule. Mannose and glucose-derived sugar amino acids were thus used as replacements for dolaproline residue. Besides, changes were also made in the dolaisoleucine and dolaphenylalanine components. The analogues were designed and synthesized as described in Supporting Information. The synthesized compounds were characterized by proton and C-13 NMR, IR, and mass spectral analysis (Supporting Information). On the basis of cell proliferation data and the effects on reconstituted microtubule assembly in vitro, D5 was selected from the compounds tested for further analysis. D5 has considerable structural differences from dolastatin 10 (Figure 1). For example, a dolavaline unit of dolastatin 10 has been replaced by its corresponding TFA salt in D5 and a dolaproline unit of dolastatin 10 has been replaced with the 6-amino-2,5anhydro-6-deoxy-D-mannonic acid Maa sugar amino acid in D5. In addition, thiazole moiety in dolastatin 10 has been removed in D5 keeping phenyl ring intact, while valine and dolaisoleucine units are same in both dolastatin 10 and D5 (Figure 1). In an equilibrium assay, D5 was found to bind to soluble tubulin with modest affinity. Although dolastatin 10 is known to depolymerize microtubules,12,28 its effect on the dynamics of individual microtubules in live cells is not known. Several of the successful anticancer drugs such as paclitaxel, vinblastine, and

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RESULTS Effects of Dolastatin 10 Analogues on Proliferation of HeLa Cells. All eight compounds (D1−D8) inhibited HeLa cell proliferation; among these agents, D1, D2, D3, and D5 were found to potently inhibit the proliferation of HeLa cells (Supporting Information Figure S1A). The half maximal proliferation inhibitory concentration (IC50) of D1, D2, D3, and D5 in HeLa cells was found to be 8.7 ± 0.3, 8.8 ± 0.3, 9.9 ± 0.4, and 6.8 ± 0.2 nM, respectively. These compounds were selected for further studies. Other dolastatin 10 analogues, D4, D6, D7, and D8, also inhibited the proliferation of HeLa cells within nanomolar range with an IC50 value of 16, 15, 16, and 12 nM, respectively. Although these analogues are having different substitutions, the antiproliferative activities of these compounds were not widely different. D1, D2, D3, and D5 Inhibited Assembly of Purified Tubulin. The effect of 5 μM D1, D2, D3, and D5 on the assembly of purified tubulin was examined by light scattering (Supporting Information Figure S1B). D1, D2, D3, and D5 inhibited tubulin polymerization by 19%, 21%, 10%, and 37% (data are average of two experiments), respectively, indicating that D5 inhibited tubulin polymerization more potently than the other compounds. Because D5 also inhibited HeLa cell proliferation more potently than the other compounds tested in this study, D5 was selected for further studies. Effects of D5 on the Assembly and GTPase Activity of Microtubules in Vitro. D5 inhibited the rate and extent of tubulin assembly in a concentration dependent manner and the 2236



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Figure 2. D5 inhibited the assembly and GTPase activity of reconstituted microtubules in vitro. (A) D5 inhibited the assembly of purified tubulin in vitro. Purified tubulin (10 μM) was polymerized in the absence (solid squares) or presence of 1 (solid circles), 5 (solid upward-pointing triangles), 7 (solid downward pointing triangles), and 10 μM D5 (left pointing solid triangles) as described in the Experimental Section. The experiment was performed two times. (B) D5 inhibited the assembly of MAP-rich tubulin. The assembly kinetics of MAP-rich tubulin (1 mg/mL) in the absence (solid squares) or presence of 1 (solid circles), 3 (solid upward-pointing triangles), 5 (left pointing solid triangles), and 10 μM (open triangles) of D5 was monitored by light scattering. The experiment was performed twice. (C) Transmission electron microscopic analysis of the effects of D5 (5 and 10 μM) on the assembly of purified tubulin. Scale bar equals 500 nm. (D) D5 decreased the GTPase activity of reconstituted microtubules in vitro. (E) Vinblastine (VB) decreased the GTPase activity of reconstituted microtubules in vitro..

10, and 20 μM of D5, respectively as compared to control. Under similar assembly conditions, the GTPase activity of microtubules was reduced by 16 ± 9, 37 ± 21, 45 ± 17, and 53 ± 13% in the presence 0.5, 1, 2, and 5 μM of vinblastine, respectively (Figure 2E). D5 Binds to Purified Tubulin in Vitro. D5 reduced the intrinsic fluorescence intensity of tubulin in concentration dependent manner, indicating that it binds to tubulin (Figure 3A). A dissociation constant for the binding of D5 to tubulin was determined to be 29.4 ± 6 μM from the binding isotherm (Figure 3B). D5 Induced Mitotic Block in HeLa Cells. D5 inhibited the proliferation of HeLa, MDA-MB-231, and MCF-7 cells in culture with IC50s of 6.8 ± 0.2, 13 ± 5, and 15 ± 7 nM, respectively (Figure 4A). Under similar conditions, nocodazole inhibited the proliferation of HeLa cells with an IC50 of 178 ±

half-maximal inhibition of the assembly of purified tubulin occurred in the presence of 6 μM D5 (Figure 2A). Under similar conditions, the assembly of purified tubulin was inhibited by 50% in the presence of 3 μM vinblastine (Supporting Information Figure S1C). D5 also decreased the rate and extent of the assembly of microtubule proteins (MAPrich tubulin) in vitro (Figure 2B). For example, 1 and 5 μM D5 inhibited microtubule polymerization by 13% and 48%, respectively. Electron microscopic analysis of the polymers showed that 5 μM D5 caused a significant inhibition of microtubule assembly and 10 μM of D5 induced aggregation of tubulin dimers (Figure 2C). Further, D5 inhibited the GTPase activity of microtubules in a concentration dependent manner (Figure 2D). For example, the amount of inorganic phosphate released was reduced by 17 ± 4, 42 ± 15, 50 ± 16, and 54 ± 15% in the presence of 2, 5, 2237



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Figure 3. D5 binds to purified tubulin in vitro. (A) Effects of D5 on the intrinsic tryptophan fluorescence intensity of tubulin. Tubulin (2 μM) was incubated without (solid squares) and with different concentrations 5 (open circles), 10 (solid upward-pointing triangle), 25 (tilted squares), 40 (solid circles), 50 (open squares), 75 (solid tilted squares), and 100 μM (open triangles) of D5 in Pipes buffer pH 6.8 at 25 °C for 20 min. The emission spectra were recorded using 295 nm as the excitation wavelength. (B) Changes in fluorescence intensity of tubulin (ΔF) were plotted against concentration of D5 (R2 = 0.9903). The data were fitted in a binding equation as described in the Experimental Section. The reported dissociation constant is an average of five independent experiments.

20 nM. HeLa cells were incubated without or with different concentrations of D5 for 24 h. The flow cytometric analysis of the DNA content of the cells showed that D5 treatment induced G2/M block in HeLa cells. After incubating the HeLa cells with 30 nM of D5−9% and at 45 nM of D5−17%, cells were found to be in G2/M phase of cell cycle as compared to ∼4% G2/M cells in control (Figure 4B). D5 Caused a Modest Increase in Mitotic Index. HeLa cells were treated with different concentrations of D5 for either 24 or 48 h, then, the mitotic cells were counted by Hoechst 33258 staining of the DNA. The mitotic index was found to be 3.2, 6, 9, and 12 and 3, 7.5, 11, and 16 in the absence and presence of 7, 15, and 30 nM of D5 after 24 and 48 h of incubation, respectively. The antimitotic effect of D5 on HeLa cells was further analyzed by phosphohistone H3 (Ser10) staining. D5 treatment caused an increase in the number of phosphohistone H3 (Ser10) positive cell population in a concentration dependent manner. For example, 3 ± 0.3, 6.6 ± 0.6, 11 ± 0.5 and 14 ± 0.6% cells were found to be in mitotic phase in the absence and presence of 7, 15, and 30 nM of D5 after 24 h of incubation, respectively (Figure 4C). The effect of nocodazole on the mitotic progression of HeLa cells was compared with that of D5. As expected, nocodazole treatment induced mitotic block in HeLa cells in a dose dependent manner. The mitotic index was determined to be 4 ± 0.3%, 22 ± 1.5%, 28 ± 1%, 36 ± 2.6%, and 4 ± 0.2%, 14 ± 1.7%, 13 ± 1%, and 11 ± 0.57% after 24 and 48 h of incubation with the vehicle (0.1% DMSO) or 100, 200, and 300 nM nocodazole, respectively. D5 Depolymerized Interphase and Spindle Microtubules in HeLa and MCF-7 Cells in Culture. D5 perturbed microtubule organization in both interphase and mitotic HeLa cells (Figure 5A,B). Treatment of HeLa cells with 7 nM D5 did not cause a significant depolymerization of either interphase or mitotic microtubules, and the microtubule network were found to be similar to the vehicle treated (0.1% DMSO) control cells (Figure 5A,B). However, 15 nM D5 caused a significant depolymerization of spindle microtubules and chromosomes

Figure 4. (A) D5 inhibited the proliferation of HeLa, MDA-MB-231, and MCF-7 cells. HeLa (solid squares), MDA-MB-231 (solid circles), and MCF-7 (solid upward-pointing triangles) cells were treated without or with different concentrations of D5 (1−80 nM) for one cell cycle. The inhibition of cell proliferation was determined by counting the cells. Data are the average of three independent experiments. (B) D5 induced the G2/M cell cycle block in HeLa cells. Cells were treated with different concentrations (15, 30, and 45 nM) of D5 for 24 h and stained with propidium iodide (400 μg/mL). The DNA content of the cells was analyzed by a flow cytometer. Nocodazole (1 μM) treated cells were taken as a positive control. The experiment was done three times. The histogram with red color shows the percentage of cells stained with propidium iodide in G1 (first peak) and G2/M (second peak) phase. The hump with blue stripes shows the distribution of cells in S-phase. The data were fitted using Modfit LT software. The raw data (data line) is shown by a green arrow and fitted data line is shown by a blue arrow. (C) D5 increased the mitotic cell population. Cells were treated with different concentrations (7, 15, and 30 nM) of D5 for 24 h and stained with antibody against phosphohistone H3 (serine 10) and Hoechst 33258 was used for DNA staining.

were not organized properly at the metaphase plate. D5 (30 nM) strongly depolymerized microtubules in both interphase and mitotic cells. Further, D5-treatment produced multipolar spindles in HeLa cells undergoing mitosis. In addition, a brief treatment (3 h) with D5 (35 nM) caused strong depolymerization of interphase microtubules in HeLa cells (Supporting Information Figure S2). Similar to its action on microtubules in HeLa cells, D5 also depolymerized mitotic microtubules in MCF-7 cells (Supporting Information Figure S3). The depolymerization of micro2238



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Table 1. Effects of D5 on the Parameters of the Dynamic Instability of Microtubules in MCF-7 Cellsa parameters growth rate (μm/min) shortening rate (μm/min) growth length (μm) shortening length (μm) growth time (min) shortening time (min) pause time (min) % shortening time % growth time % pause time dynamicity (μm/min) rescue frequency (events/min) catastrophe frequency (events/min) rescue frequency (events/μm) catastrophe frequency (events/μm)

control 15 16.4 1.6 1.6 0.9 0.6 1 27 37 35 10 10 4 0.68 0.5

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

3 5 0.4 0.5 0.1 0.2 0.2 8 6 8 3 2 1.2 0.22 0.1

D5 (15 nM) 11 12 1.1 0.9 0.5 0.4 1.5 19 21 59 5 12 3 0.9 0.7

± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2b 2b 0.4b 0.3b 0.2b 0.2 0.3b 9b 8b 15b 2b 3e 1c 0.4e 0.3d

Data are the mean ± SD. n = 20 microtubules in each case. bP < 0.001. cP < 0.05. dP < 0.01. eStatistically not significant. (Significance test was done by One-way ANOVA). a

Figure 5. D5 perturbed microtubule network in HeLa cells. Effects of D5 on the (A) interphase and (B) mitotic microtubules of HeLa cells are shown. HeLa cells were incubated in the absence or presence of different concentrations (7, 15, 30 nM) of D5 for 24 h. Microtubules (red) and DNA (blue) are shown. Scale bar equals 10 μm. (C) D5 suppressed the dynamics of individual microtubules in live MCF-7 cells. Life history traces of microtubules in absence and presence of 15 nM D5 are shown. Each trace represents a single microtubule. Twenty microtubules were measured for each experimental condition.

got accumulated at the kinetochoric region indicated that D5 treatment disrupted the kinetochore microtubule attachment and tension (Figure 6A,B).

tubules was apparent from the Western blot wherein the polymeric fraction of tubulin was found to decrease with increasing concentration of D5 with a concomitant increase in the soluble pool of tubulin in the D5-treated MCF-7 cells (Supporting Information Figure S4). The ratio of polymeric to soluble tubulin was determined to be 1.2, 0.8, 0.6, and 0.5 in control, 30 nM D5, 60 nM D5, and 2 nM vinblastine treated cells, respectively. D5 Treatment Suppressed Microtubule Dynamics in GFP-Tubulin Transfected MCF-7 Cells. Microtubules of the control cells (vehicle-treated) were found to be highly dynamic as reported earlier (Figure 5C).32−35 D5 (15 nM) visibly suppressed the dynamic instability of microtubules in MCF-7 cells (Figure 5C). For example, it reduced the growth rate by ∼27%. In the presence of 15 nM D5, the average growth and shortening length of the microtubules in MCF-7 cells were found to be decreased by 31 and 44%, respectively (Table 1). The pause time (the phase of the microtubule where it is neither growing nor shortening) was increased by 50%. In the presence of 15 nM D5, the length based catastrophe (a transition from a growth or a pause phase to a shortening phase) and rescue frequency (a transition from a shortening phase to a growth or a pause phase) were found to be increased by 40% and 32%, respectively (Table 1). The dynamicity of the microtubules (dimer exchange at the end of the microtubules per unit time) was decreased by 50%. D5 Perturbs Microtubule−Kinetochore Attachment and the Tension Across the Kinetochores. BubR1 and Mad2 are involved in recognizing the spindle attachment and tension.36,37 Mad2 and BubR1 proteins are localized to the kinetochoric region and lead to the metaphase arrest until the defect is corrected. In the presence of D5, Mad2 and BubR1

Figure 6. D5 caused accumulation of Mad2 and BubR1 at the kinetochores of HeLa cells. HeLa cells were treated with either the vehicle (0.1% DMSO) or different concentrations (15, 30, and 45 nM) of D5 for 24 h. The cells were stained with (A) BubR1, and (B) Mad2 antibodies. DNA was visualized using Hoechst 33258 dye, and Hec1 (red) staining was performed to locate kinetochores. Scale bars equal to 10 μm.

D5 Induced Apoptosis in HeLa Cells. HeLa cells were incubated with vehicle (0.1% DMSO) or with different concentrations of D5 for 24 h. Vehicle treated HeLa cells remained viable after 24 h, while D5 treated cells were found at various stages of apoptosis. After 24 h, D5 (7 and 15 nM) treated cells were stained positive for annexin V and weakly stained for propidium iodide, indicating that the cells were in the early or in the mid apoptotic phase. However, cells treated with 30 nM D5 were stained positive for both annexin V and propidium iodide, indicating them to be in the late apoptotic phase (Figure 7A). In a separate experiment, cells were incubated with either D5 (35 nM, ∼5 IC50) or nocodazole (700 nM, ∼5 IC50) for 10 and 24 h, respectively, and the number of apoptotic cells was quantified by FACS analysis using cells stained with annexin V 2239



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Figure 7. D5 induced apoptosis in HeLa cells. (A) HeLa cells were treated with either vehicle (0.1% DMSO) or different concentrations (7, 15, 30 nM) of D5 for 24 h. The panels display Annexin V and PI staining of the cells. (B) D5 caused genomic DNA fragmentation in HeLa cells. Cells were incubated with different concentrations (7, 15, 30 nM) of D5 for 40 h. (C) D5 treatment activated JNK and caspase-3. HeLa cells were treated with 30 nM D5 for 24 h. Western blot analysis showed the activation of Jun N-terminal kinase (phosphorylated JNK), caspase-3 and further the caspase-3 dependent cleavage of PARP.

fragmentation of procaspase-3 into two smaller functional fragments (Figure 7C). The activated caspase-3 resulted in the cleavage of PARP (poly ADP-ribose phosphate polymerase) (Figure 7C). Effects of D5 on HeLa Cell Proliferation in Combination with Colchicine or Vinblastine. D5, colchicine, and vinblastine inhibited the proliferation of HeLa cells with a median dose (Dm) of 8.9, 17.8, and 2.3 nM, respectively (Figure 8A−C). Colchicine (10 nM) in combination with 3 and 5 nM D5 inhibited the proliferation of HeLa cells by 55 ± 2.5 and 66 ± 1.5%, respectively, while colchicine (15 nM) in combination with 3 and 5 nM D5 inhibited the proliferation of HeLa cells by 66 ± 2 and 76 ± 2%, respectively. The combination index (CI) values for the combination of colchicine (10 nM) with 3 and 5 nM D5 was found to be 0.75 ± 0.06 and 0.64 ± 0.03, respectively (Figure 8D), whereas CI value for colchicine (15 nM) with 3 and 5 nM D5 was found to be 0.67 ± 0.05 and 0.541 ± 0.04, respectively (Figure 8D). Vinblastine (1 nM) in

and propidium iodide (Supporting Information Figures S5A and S5B). After 10 h of D5 and nocodazole treatment, 22.4 and 14.7% of the cells were found to be undergoing apoptosis while 65.3 and 50.5% cells were undergoing apoptosis after 24 h of D5 and nocodazole treatment, respectively (Supporting Information Figures S5A and S5B). Further, D5 treatment was found to induce DNA fragmentation in HeLa cells, as evident from the laddering pattern of genomic DNA. This supported the finding that D5 treated HeLa cells were undergoing apoptosis (Figure 7B). Phosphorylated JNK1 is the active form of the JNK1. Activation of JNK1 is an early event of the onset of apoptosis. D5 treatment activated JNK1 (Jun N-terminal kinase 1) and caspase-3 in HeLa cells. The level of phosphorylated JNK was found to increase in the D5treated HeLa cells as compared to the vehicle treated cells, indicating the involvement and activation of JNK in D5induced apoptosis. Western blotting of D5-treated HeLa cells also showed the activation of procaspase-3, leading to the 2240



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Figure 8. D5 inhibited the proliferation of HeLa cells synergistically with colchicine and antagonistically with vinblastine. The median effect plots for the inhibition of cell proliferation in the presence of D5 (A), colchicine (B), and vinblastine (C) are shown. Combination indices for the combination of D5 with either colchicine (Col) (D) or vinblastine (VB) (E) are shown. Data are the average of three experiments.

containing natural dolastatin 10.27 The variation in the chirality of sugar amino acid may alter the bioactivity and to address this issue, both “2,5-cis” and “2,5-trans” tetrahydrofuran ring containing analogues were prepared, the latter having an enhanced propensity to induce turn in the backbone.20 Besides, the chirality at position C18 has been reported to be particularly important for the tubulin inhibitory activity of dolastatin 10.39 The reversal of chirality at positions C18 alone or together with change in chirality at position C19 renders dolastatin ineffective in inhibiting tubulin polymerization.39 Two of the analogues described here, D5 with “2,5-trans” and D6 having “2,5-cis” ring junctions, maintain the chirality at C18 position same as that of dolastain 10. Detail conformational analysis of the analogues is currently in progress, which will provide a better understanding of the structure activity relationships. In this work, D5, one of the synthesized analogues, was found to inhibit the proliferation of several types of cancer cells in culture at low nanomolar concentration range. Using fluorescence spectroscopy, we are able to determine the binding constant of D5 to tubulin in equilibrium. D5 bound to purified tubulin with a dissociation constant of 29.4 ± 6 μM, indicating that it binds to tubulin with low affinity. D5 increased the lag phase of tubulin assembly, indicating that it inhibited the nucleation step of the assembly. D5 inhibited the assembly of purified tubulin with an IC50 of 6 μM. Dolastatin 15, dolastatin 10, vinblastine, and rhizoxin inhibited the polymerization of tubulin with an IC50 of 23, 1.2, 1.5, and 6.8 μM, respectively.10

combination with 3 and 5 nM D5 inhibited the proliferation of HeLa cells by 26 ± 1.5 and 34 ± 1.7%, respectively, whereas vinblastine (2 nM) in combination with 3 and 5 nM D5 inhibited the proliferation of HeLa cells by 38 ± 2 and 44 ± 1.5%, respectively. Vinblastine (1 nM) in combination with 3 and 5 nM D5 produced the CI values of 2 ± 0.14 and 1.83 ± 0.13, respectively (Figure 8E), while the combination of vinblastine (2 nM) with 3 and 5 nM D5 resulted in the CI values of 1.94 ± 0.15 and 1.82 ± 0.1, respectively (Figure 8E). The results indicated that D5 exerts synergistic effects on the inhibition of proliferation of HeLa cells when combined with colchicine while it displays antagonistic effects when combined with vinblastine.

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DISCUSSION AND CONCLUSIONS

Dolastatin 10 analogues are in various stages of clinical trials for the treatment for several types of cancers. It has been suggested that the C-terminal portion of dolastatin 10 is important for cytotoxicity, as the tripeptides lacking dolaproline and dolaphenine units lack inhibitory activity against L1210 cells.38 Here, we have used sugar amino acid to mimic the DAP (dolaproline) residue of dolastatin 10 and synthesized eight analogues of dolastatin 10. The rationale of selecting sugar amino acid was based on the conformational studies on various sugar amino acid oligomers and sugar amino acid based peptides carried out previously.17−26 Preliminary conformational analysis of these analogues suggested that the dihedral angles of sugar amino acids in these molecules render the backbone structures closely resembling that of the DAP 2241



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D5 Suppresses Microtubule Dynamics and Induces Mitotic Block. At its half-maximal proliferation inhibitory concentration, D5 strongly suppressed the dynamic instability of interphase microtubules in live MCF-7 cells without having any visible effect on the interphase microtubule network. D5 treatment decreased the rates and extents of both the growth and shortening excursions of microtubules and caused a strong increase in the duration of the pause state of microtubules. Considering its low binding affinity toward tubulin, it can be argued that the binding of a few molecules of D5 at the end of microtubules could inhibit the dynamic instability of microtubules. D5 perturbed microtubules of both interphase and mitotic cells; however, the depolymerizing effect of D5 on microtubules was more pronounced in the mitotic cells as compared to that of the interphase cells. Bipolar spindle assembly was also lost in the cells treated with ≤2 IC50 D5. D5 also produced multipolar mitotic cells, indicating that D5 inhibited centrosome maturation and also inhibited the centrosome coalescence like griseofulvin.40 In D5 treated cells, microtubules failed to properly attach to kinetochores, leading to reduced tension across the sister kinetochores. It was found in earlier studies that the main components involved in wait anaphase signal are Mad and Bub family proteins.41,42 The mitotic block may be due to the accumulation of check point proteins like BubR1 and Mad2, which check the kinetochore microtubule attachment and tension across the kinetochores.36,37,43 Mad2 localizes to the unattached kinetochores and moves off when there is proper microtubule attachment to the kinetochores. BubR1 move off the kinetochores when there is adequate tension across the kinetochores.36,43 Both BubR1 and Mad2 were found to be accumulated at the kinetochores of the D5 treated cells, suggesting that the suppression of the dynamic instability of microtubules by D5 activated mitotic check point leading to the mitotic block. D5 Treated Cells Undergo Caspase Dependent Apoptosis. D5 treatment induced apoptotic cell death in HeLa cells. During apoptosis, several proteins are activated and cleaved to become functional. For example, JNKs are known to become activated through phosphorylation in the cells undergoing apoptosis.44,45 We found that the phosphorylation level of Jun-N-terminal kinases (JNK) increased in D5-treated HeLa cells, suggesting that D5 treatment activated Jun Nterminal kinase in these cells, representing an early event prior to the onset of apoptosis. Further, D5 treatment generated an active caspase-3 by the cleavage of procaspase-3, which was further confirmed by the cleavage of PARP, a downstream target of caspase-3. The results together suggested that D5 induces apoptosis in HeLa cells by activating the caspase-3 pathway. Nocodazole treatment induced a stronger mitotic block than that of D5, which could be due to the different mode of action of the two compounds. It may be possible that nocodazole mainly activates cell death pathway after prolonged accumulation of the cells in the mitosis while mitotically blocked cells immediately die in the presence of D5. Alternatively, D5 may activate the apoptotic pathways without blocking the cells at mitosis. Previously, it was indicated that microtubule-targeting drugs can cause apoptotic cell death without inducing mitotic block.46,47 For example, at low concentrations, paclitaxel caused apoptotic cell death without inducing mitotic arrest in human breast cancer BCap37 and the human epidermoid carcinoma KB cells.46 Another tubulin binding agent vinflunine caused

50−70% cell death in SK-N-SH neuroblastoma cells under certain concentrations without inducing G2/M arrest in these cells.47 Low concentrations of paclitaxel and vinflunine are known to suppress microtubule dynamics without grossly affecting the polymerized amount of tubulin.30,48 It may be possible that the suppression of microtubule dynamics by D5 also induced apoptotic cell death prior to G2/M entry, resulting in a weak mitotic block. Dolastatin 10 has also been reported to increase the mitotic index in L1210 murine leukemia12 and prostate cancer cells;49 however, relatively higher concentrations of dolastatin 10 were required to induce mitotic block than to inhibit proliferation in these cells.12,49 In conclusion, the evidence obtained in this study suggested that D5 inhibits the proliferation of cancer cells by suppressing microtubule dynamics, and the results indicated that D5 may have antitumor potential. Dolastatin series of compounds are known to exhibit strong side effects; therefore, the toxicity of D5 needs to be evaluated first for its possible use in cancer chemotherapy.

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EXPERIMENTAL SECTION

Materials. Mouse monoclonal anti α-tubulin IgG, alkaline phosphatase conjugated antimouse IgG, FITC (fluorescein isothiocyanate) conjugated antimouse IgG, FITC-conjugated antirabbit IgG, bovine serum albumin, and Hoechst 33258 dye were purchased from Sigma (St. Louis, MO, USA). Annexin V/propidium iodide apoptosis detection kit and mouse monoclonal anti-BubR1 IgG was purchased from BD Pharmingen (San Diego, USA). Rabbit polyclonal anti-Mad2 IgG was purchased from Bethyl Laboratories (Montgomery, USA). Anti Mouse IgG-Alexa 568 conjugate was purchased from Invitrogen (Carlsbad, CA, USA). Rabbit polyclonal antiphosphosphistone−H3 IgG, rabbit polyclonal anti-JNK1 and pJNK IgG, mouse monoclonal anti-procaspase-3 IgG, and rabbit polyclonal anti-PARP IgG were purchased from Santa Cruz Biotechnology (CA, USA). Fetal bovine serum was purchased from Biowest (Nuvaille, France). All other reagents were of analytical grade and obtained from either Sigma (St. Louis, MO, USA) or Himedia (Mumbai, India). Cell Culture. Human cervical carcinoma (HeLa), human breast adenocarcinoma (MCF-7), and metastatic breast adenocarcinoma (MDA-MB-231) cells were obtained in September 2010 from cell repository of National Centre for Cell Science (NCCS), Pune, India. NCCS characterized the cells by mt-rDNA sequence to confirm the species. These cell lines were found to be free of mycoplasma. Further authentication has not been done. Human cervical carcinoma (HeLa) cells and breast adenocarcinoma (MCF-7) cells were cultured in Eagle’s Minimal Essential Medium containing 10% fetal bovine serum, 2.2 g/L sodium bicarbonate, and 1% antibacterial and antimycotic solution containing streptomycin, amphoterecin B, and penicillin. Cells were grown at 37 °C in a humidified atmosphere of 5% carbon dioxide and 95% air in incubator34 (Sanyo, Tokyo, Japan). The tested compounds were dissolved in dimethyl sulfoxide (DMSO). Determination of IC50 of D5 in HeLa, MCF-7, and MDA-MB231 Cells. HeLa, MCF-7, and MDA-MB-231 cells (1 × 105 cells/mL) were grown in 96-well tissue culture plates at 37 °C for 24 h.33 Then the medium was replaced with fresh medium containing vehicle (0.1% DMSO) or different concentrations of D5 (1 − 80 nM), and the cells were grown for an additional 24 or 48 h. Both attached and floating cells were harvested and counted after staining with trypan blue.50 Percentage inhibition of cell proliferation was plotted against D5 concentration using Origin Pro 7.5 software. Immunofluorescence Microscopy. Cells (0.6 × 105 cells/mL) were grown as monolayers on glass coverslips for 24 h.32−34 Then the medium was replaced with fresh medium containing vehicle (0.1% DMSO) or different concentrations of D5, and the incubation continued for further 24 h (HeLa cells) or 48 h (MCF-7 cells). The cells were then fixed with 3.7% formaldehyde at 37 °C for 30 min and immunostained using respective antibodies.32,34,51 The DNA was 2242



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incubation, 1 mM GTP was added to the assembly mixture and then polymerized at 37 °C for 10 min. The reaction was stopped by using 10% (v/v) of 7 M perchloric acid. The amount of inorganic phosphate released was determined by the ammonium molybdate malachite green assay.54,56 Determination of Dissociation Constant Using Tryptophan Fluorescence of Tubulin. Tubulin (2 μM) was incubated without and with different concentrations of D5 in 25 mM Pipes buffer pH 6.8 for 15 min at 25 °C. The effect of D5 on the fluorescence of tubulin was monitored using 295 and 336 nm as the excitation and emission wavelength, respectively. D5 does not have a significant absorption either at the excitation or emission wavelength; therefore, no inner filter effect correction was needed. In addition, experiments were performed using a 0.3 cm path length fluorescence cell. Fluorescence changes were fitted into the following equation,

stained using Hoechst 33258 dye. The images were visualized by TE Eclipse 2000U fluorescence microscope (Nikon, Tokyo, Japan) and analyzed with Image-Pro Plus software. Microtubule dynamics was done in EGFP-tubulin transfected MCF-7 cells using FV500 confocal laser scanning microscope (Olympus, Tokyo, Japan). The kinetic parameters were calculated as described earlier.32 Western Blotting. MCF-7 cells were grown as a monolayer and then treated without or with either 30 and 60 nM D5. Soluble and polymer tubulin fractions of the treated cells were prepared, and Western blotting was performed as described earlier.32 Apoptosis Detection Assays. Annexin V/Propidium Iodide Staining. HeLa cells were grown in the absence and presence of different concentrations (7, 15, and 30 nM) of D5 for 24 h. Control and treated cells were stained using the annexin V/propidium iodide apoptosis kit. For FACS, analysis cells were treated with either D5 or nocodazole for 10 or 24 h and then stained with annexin V and propidium iodide using annexin V/propidium iodide apoptosis kit as described earlier.32,34,52 DNA Fragmentation Assay. HeLa cells were incubated without and with different concentrations (7, 15, and 30 nM) of D5. After 40 h of incubation, cells were trypsinized and collected by centrifugation. The cells were lysed in buffer (50 mM Tris pH 8.0, 10 mM EDTA, 0.5% sarcosine, and 0.5 mg/mL proteinase K) and incubated at 50 °C for 1 h in the heating block. RNase A (1 mg/mL) was added, and incubation was further extended for 1 h. The samples were allowed to come to 25 °C. Gel electrophoresis was done using 2% agarose.53 The fragmented genomic DNA was visualized after staining with ethidium bromide using UV gel documentation system. JNK, Caspase-3, and PARP Assay. HeLa cells were incubated without and with 30 nM of D5 for 24 h. The cells were washed twice in phosphate buffered saline. Cell lysates were prepared, and Western blotting was done using respective antibodies against JNK1, phosphorylated JNK, procaspase-3, and PARP. Determination of Effect of D5 on Cell Cycle of HeLa Cells. HeLa cells were grown for 24 h (one cell cycle) without or with different concentrations (15, 30, and 45 nM) of D5. Samples for flow cytometry were prepared as described recently.32 In brief, the cells were trypsinized, washed twice in phosphate buffered saline and fixed in 70% ethanol for 2 h. Then propidium iodide (400 μg/mL) and RNase A (1 μg/mL) were added and further incubated on ice for 2−3 h. DNA content of the cells was quantified in a flow cytometer (FACS Aria special order system, Becton Dickinson, USA), and the data were analyzed using the Modfit LT program (Verity Software, ME, USA). Microtubule Polymerization Assay. Goat brain tubulin (10 μM) was incubated without or with different compounds D1, D2, D3, and D5 (5 μM) in microtubule assembly buffer (1 M monosodium glutamate, 3 mM MgCl2, 1 mM EGTA, and 25 mM Pipes pH 6.8) on ice for 20 min. Then GTP (1 mM) was added to the reaction mixture. The assembly of tubulin was monitored by 90° light scattering at 400 nm for 30 min at 37 °C using a fluorescence spectrophotometer.54,55 A similar experiment was done with D5 (1, 5, 7, and 10 μM). MAP-rich tubulin (1 mg/mL) was incubated without or with different concentrations of D5 (1, 3, 5, and 10 μM) in PEM buffer (25 mM Pipes pH 6.8 containing 3 mM MgCl2 and 1 mM EGTA) for 15 min on ice, and then 1 mM GTP was added to the reaction mixture. The assembly kinetics was monitored as described above for the purified tubulin. Electron Microscopy. Tubulin (10 μM) was incubated without or with different concentrations of D5 (5 and 10 μM) for 20 min on ice. The polymerization reaction was performed as described above. Polymers were fixed using 0.5% of glutaraldehyde, transferred to Formvar carbon coated electron microscopy grids (Electron Microscopy Sciences, USA), and negatively stained using 2% uranyl acetate. Samples were dried in an infrared lamp for 10−30 min then visualized under electron microscope (Philips Tecnai FE 5000).55 Effect of D5 on GTPase Activity of Tubulin. Tubulin (10 μM) was incubated without or with different concentrations of either D5 (2, 5, 10, and 20 μM) or vinblastine (0.5, 1, 2, and 5 μM) in microtubule assembly buffer (1 M monosodium glutamate, 3 mM MgCl2, 1 mM EGTA, and 25 mM Pipes pH 6.8) on ice for 10 min. After the

(

ΔF = (ΔFmax )(([P0] + [L0] + Kd) −

)

([P0] + [L0] + Kd)2 − 4[P0][L0]) /2[P0]

ΔF was the change in the fluorescence intensity of tubulin in the presence of D5, ΔFmax was the maximum change in the fluorescence intensity of tubulin when it was saturated with D5, and P0 and L0 are the concentration of tubulin and D5, respectively.57,58 The dissociation constant (Kd) for D5 binding to tubulin was calculated using the Graph Pad Prism 5 software (Graph Pad Software, CA, USA). The experiments were performed using a fluorescence spectrophotometer (JASCO FP-6500, Japan). Determination of Combination Index (CI). HeLa cells were individually treated with 3 and 5 nM D5, 10, and 15 nM colchicine and 1 and 2 nM vinblastine for 24 h, and the cells were also treated with the combination of D5 along with either colchicine or vinblastine. The combination index was calculated from the equation.59,60 CI = (D)1/(Dx )1 + (D)2/(Dx )2 (D)1 and (D)2 are the concentrations of compound 1 and compound 2 that produce the given effect when used in combination, where (Dx)1 and (Dx)2 are the concentrations of compound 1 and compound 2 that also produce the same effect when used alone. The Dx and Dm values were calculated as described earlier from the median dose plot.59−61 CI < 1 suggests synergism, CI = 1 suggests additive effect, and CI > 1 indicates antagonism.

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ASSOCIATED CONTENT

S Supporting Information *

Synthesis and characterization of the compounds D1−D8;. Effect of compounds D1−D8 on the proliferation of HeLa cells, effects of D1, D2, D3, and D5 on the assembly of purified tubulin and S1C shows the effect of vinblastine on the assembly of tubulin, effect of short exposure of D5 on the interphase microtubules in HeLa cells, effects of D5 on the mitotic microtubules of MCF-7 cells, D5 reduced the amount of polymerized tubulin in MCF-7 cells, D5 induced apoptosis in HeLa cells. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*For D.P.: phone, +91-222-5767838; fax, +91-222-5723480; Email, [email protected]. For T.K.C.: phone, +91-522-2623286; fax, +91-522-2623405; E-mail, [email protected], [email protected]. Author Contributions §

P.K.G. and J.A. contributed equally to the work.

Notes

The authors declare no competing financial interest. 2243



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ACKNOWLEDGMENTS The work was supported by a DAE-SRC fellowship from Government of India to D.P., J. C. Bose Fellowship, DST, Government of India, to T.K.C., UGC, New Delhi, for fellowship to P.K.G. and J.A. We thank Bhavya Jindal, Sonia Kapoor, and Ankit Rai for critical reading of the manuscript.

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ABBREVIATIONS USED FITC, fluorescein isothiocyanate; GTP, guanosine-5′-triphosphate; PIPES, piperazine-N,N′-bis(2-ethanesulfonic acid); EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N′,N′-tetraacetic acid; DMSO, dimethyl sulfoxide; PBS, phosphate buffered saline

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