ANTICANCER RESEARCH 27: 729-736 (2007)

Antiproliferating Activity of the Pironetin against - and -resistant Human Small Cell Lung H69 Cells

MITSUZI YOSHIDA1, YUKI MATSUI1, YOSHINORI IKARASHI1, TAKEO USUI2, HIROYUKI OSADA2 and HIRO WAKASUGI1

1Pharmacology Division, National Cancer Center Research Institute, 5-1-1 Tsukiji Chuo-ku, Tokyo 104-0045; 2Antibiotics Laboratory, RIKEN Discovery Research Institute, 2-1 Hirosawa Wako-shi, Saitama 351-0198, Japan

Abstract. Pironetin, isolated from sp., is a for anticancer . are composed potent inhibitor of assembly and the first of ·- and ‚-tubulin heterodimers. Microtubule- compound identified that covalently binds to ·-tubulin at targeted drugs previously reported have a characteristic Lys352. We examined whether pironetin is an effective agent ability to bind directly to different sites of ‚-tubulin in vitro. against human small cell H69 cells, including Therefore, they are classified into vinca , and two cell lines resistant to the microtubule-targeted drugs types depending upon their binding site on vindesine (H69/VDS) and paclitaxel (H69/Txl) that interact microtubules. with ‚-tubulin. Pironetin was found to be effective against Pironetin (Figure 1), isolated from Streptomyces sp., is a these resistant cells as well as their parental cells. In addition, potent inhibitor of microtubule assembly (1) and the first pironetin inhibited the growth of human leukemic K562 compound identified that covalently binds to ·-tubulin at multidrug-resistant cells (K562/ADM), which have mdr1 gene Lys352 (2). In our study, we examined whether pironetin expression, as well as the parental K562 cells. In these cell shows antiproliferating activity against clinically employed lines, including the parental and resistant cells, pironetin microtubule-targeted drug-resistant H69 human small cell caused complete mitotic arrest; in addition, apoptosis lung cancer cells and the multidrug-resistant K562 human inductions by 30 and 100 nM pironetin were observed. In this leukemic cells. study, the new mitotic inhibitor, pironetin, was found to be effective not only against human tumor cell lines resistant to Materials and Methods microtubule-targeted drugs, but also multidrug-resistant cells with mdr1 gene expression. These results suggest that Chemicals. Pironetin was kindly supplied from Nippon Kayaku Co., pironetin is a useful agent for overcoming drug resistance in Ltd (Tokyo, Japan). Vindesine sulfate was purchased from Shionogi (Osaka, Japan). Paclitaxel (Taxol®), (Adriamycin®), cancer chemotherapy. propidium iodide, Nonidet® p 40, sperminetetrahydrochloride, trypsin, trypsin inhibitor, ribonuclease A, monoclonal anti-P- Microtubules are a cytoskeletal element essential in the glycoprotein antibody (Clone F4) and peroxidase-conjugated anti- segregation of during cell division, as well as mouse IgG were purchased from Sigma (St. Louis, MO, USA). in axon transport, secretory processes and the maintenance Polyclonal anti-caspase-3 antibody was purchased from Cell of specific cell morphology. Thus, they are essential in all Signaling Technology (Danvers, MA, USA). Pironetin and paclitaxel eukaryotic cells and are among the most successful targets were dissolved in dimethyl sulfoxide (DMSO) and used at final concentrations of DMSO below 0.001% and 0.06%, respectively.

Cell lines. The human small-cell lung cancer H69 cells and leukemic K562 and HL-60 cells were grown in RPMI-1640 medium Correspondence to: Dr. Mitsuzi Yoshida, Pharmacology Division, supplemented with 10% fetal bovine serum. Vindesine-resistant National Cancer Center Research Institute, 5-1-1 Tsukiji Chuo-ku, (H69/VDS) and paclitaxel-resistant (H69/Txl) H69 cells were Tokyo 104-0045, Japan. Tel: +81 3 3542-2511, Fax: +81 3 3542- established in our laboratory as reported elsewhere (3, 4). H69/VDS 1886, e-mail: [email protected] and H69/Txl resistant cell lines were maintained in the presence of vindesine (10 nM) and paclitaxel (20 nM), respectively, in the Key Words: Pironetin, mitotic inhibitor, ·-tubulin, ‚-tubulin, culture medium. Doxorubicin-resistant K562 cells (K562/ADM) resistant cells, vindesine, paclitaxel, mdr1, apoptosis, caspase-3, were used as positive control for typical multidrug-resistant cells sub-G1. expressing the mdr1 gene (5).

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Table I. Sensitivities of H69, H69/VDS and H69/Txl cells to vindesine, paclitaxel and pironetin in the MTT assay.

IC50 (nM)

Cell line Vindesine Paclitaxel Pironetin

H69 5.5±0.3 57±1.0 17.5±0.9 Figure 1. Chemical structure of pironetin. H69/VDS 55±4.0 530±40 20.3±2.3 (10.0)* (9.3) (1.2)

H69/Txl 53±5.0 2350±260 11.5±0.4 Antitumor activity in vitro (MTT assay). Sensitivities of tumor and (9.6) (41.2) (0.7) resistant cell lines to antitumor drugs were examined with a 3-(4,5- dimethyl-2-thiazoyl)-2,5-diphenyltetrazolium bromide (MTT) *Values in parentheses show relative resistance compared to H69 cells. assay, as outlined elsewhere (6). Briefly, H69/VDS and H69/Txl cells were cultured in vindesine- and paclitaxel-free medium for 1 week before the assay; H69 (104 cells/ml), H69/VDS (104 cells/ml), H69/Txl (2x104 cells/ml), K562 (2x103 cells/ml), and K562/ADM (2x103 cells/ml) were then seeded into a 96-well microplate in a The DNA contents were analyzed with the FACSCalibur volume of 180 Ìl. A 20-Ìl aliquot of various concentrations of automated system (BD Biosciences, San Jose, CA, USA) using antitumor drugs was added to each well. Because H69 and its CellQuest software (BD Biosciences). resistant cell lines grow so slowly that their doubling times are over 50 h, differing from the circa 12-h doubling time of K562 cell lines, Immunoblot analysis. Apoptosis caused by pironetin was also H69 and the resistant cell lines were cultured for 7 days and K562 detected by immunoblot analysis using polyclonal anti-caspase-3 and its adriamycin-resistant cells were cultured for 4 days at 37ÆC antibody. Cells were cultured with pironetin and lysed in M-PER mammalian protein extraction reagent (Pierce, Rockford, IL, in an atmosphere of 5% CO2 in air. A 50-Ìl aliquot of MTT (2 mg/ml) dissolved in Ca2+, Mg2+-free Dulbecco's phosphate- USA) containing 1 mM dithiothreitol (DTT) and protease buffered saline was added to each well and the plates were inhibitors. Equal amounts of total protein were separated by 4% incubated for 4 h. The MTT-formazan crystals were dissolved in to 20% (W/V) SDS-PAGE and transferred electrophoretically to DMSO and the optical density of each solution was measured at polyvinylidene difluoride (PVDF) membranes. Expression of P- 570 nm and 630 nm using a Microplate Manager III (BIO-RAD glycoprotein in tumor cell lines was also examined by immunoblot Laboratories, Hercules, CA, USA) analysis program on a analysis using monoclonal anti-P-glycoprotein antibody. Macintosh computer interfaced with a Benchmark Microplate Immunoblot stainings were performed as described elsewhere (8). Reader (BIO-RAD Laboratories). The IC50 was defined as the drug concentration required to Results inhibit cell growth to 50% that of the control. The relative resistance was defined as the IC50 of the resistant subline/IC50 of Antiproliferating activity of pironetin against vindesine- and the parental cell line. paclitaxel-resistant H69 cells. Human small cell lung cancer H69 cells were sensitive to vindesine and paclitaxel; the Apoptosis assay (flow cytometry). Apoptosis induction by pironetin was determined with flow cytometry by the detergent-trypsin concentration-response curves of H69 to vindesine and method of Vindelov (7). The cells (5x105) were suspended in 200 paclitaxel obtained are shown in Figure 2. From these Ìl of citrate buffer including 5% dimethylsulfoxide. Samples were figures, the IC50s of H69 for vindesine and paclitaxel were frozen rapidly in polypropylene tubes immersed in a mixture of estimated as 5.5±0.3 nM and 57±1.0 nM, respectively dry ice and acetone and then stored in a freezer at –80ÆC. Stock (Table I). H69/VDS cells resistant to vindesine showed ® solution: Trisodium citrate (3.4 mM), 0.1% Nonidet p 40, 1.5 about tenfold resistance to vindesine compared to parental mM sperminetetrahydro-chloride and Tris(hydroxymethyl)- H69 cells. H69/Txl cells showed 41.2-fold resistance to aminomethane were dissolved in distilled water and the pH was adjusted to 7.6. The stock solution was used as the basis for paclitaxel compared to H69 cells. Moreover, at preparing solution A, B and C. Before analysis, the samples were concentrations up to 1 ÌM, paclitaxel promoted the growth thawed rapidly in a water bath at 37ÆC and then 1800 Ìl of of H69/Txl cells as shown in Figure 2B. H69/Txl and solution A (0.003% trypsin in the stock solution) was added to H69/VDS cells were also resistant to vindesine and the tube and mixed gently. After 10 min at room temperature, paclitaxel, with their relative resistances of 9.6 and 9.3, 1500 Ìl of solution B (0.05% trypsin inhibitor and 0.01% respectively. However, H69/Txl and H69/VDS cells as well ribonuclease A in the stock solution) were added. The solutions as the parental cells were sensitive to pironetin (Figure 3). were again mixed by inversion of the tube and after 10 min at room temperature, 1500 Ìl ice-cold solution C (0.042% Pironetin caused growth inhibition of the resistant H69/Txl propidium iodide and 0.12% spermine tetrahydrocloride in the cells to a higher degree than the parental H69 cells, with a stock solution) were added. The solutions were mixed for 15 min. relative resistance of 0.7 (Table I).

730 Yoshida et al: Antiproliferating Activity of Pironetin

Figure 2. Concentration-response curves of H69, H69/VDS and H69/Txl cells to (A) vindesine and (B) paclitaxel. Values are average ±S.D. of three independent experiments performed in quintuplicate with H69 (●), H69/VDS (■), and H69/Txl (▲) cells. The cells were cultured for 7 days with various concentrations of vindesine or paclitaxel. Cell growth was measured using the MTT assay, as described in Materials and Methods.

Table II. Sensitivities of K562 and K562/ADM cells to doxorubicin and pironetin in the MTT assay.

IC50 (nM)

Drugs K562 K562/ADM Relative resistance

Doxorubicin 52±5.0 4500±500 86.5

Pironetin 17.3±0.8 16±1.3 0.9

Antiproliferating activity of pironetin against K562 and K562/ADM cells. Figure 4A shows that K562/ADM was strongly resistant to doxorubicin compared to the parental K562 cells. A relative resistance of 86.5 was calculated from this figure (Table II). However, K562/ADM cells showed the same sensitivity to pironetin as the parental K562 cells (Figure 4B). K562/ADM cells are known to be a typical multidrug-resistant cell line that expresses the mdr1 gene Figure 3. Concentration-response curves of H69, H69/VDS, and H69/Txl product, P-glycoprotein (Figure 5). Thus, this result cells to pironetin. Values are average ±S.D. of three independent experiments performed in quintuplicate with H69 (●), H69/VDS (■), and suggested that pironetin was not a substrate for P- H69/Txl (▲) cells. The cells were cultured for 7 days with various glycoprotein and that P-glycoprotein did not affect the concentrations of pironetin. Cell growth was measured using the MTT assay. efflux of pironetin.

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Figure 4. Concentration-response curves of K562 and K562/ADM cells to (A) doxorubicin and (B) pironetin. Values are average ±S.D. of three independent experiments performed in quintuplicate with K562 (●) and K562/ADM (■) cells. The cells were cultured for 4 days with various concentrations of doxorubicin and pironetin.

Apoptosis induction by pironetin. Pironetin exhibits antitumor activity by apoptosis induction via microtubule disassembly (9,10). Therefore, we examined whether apoptosis was similarly induced by pironetin in these resistant cell lines and their parental cell lines. In slow- growing H69 cells, mitotic arrests and sub-G1 peaks in flow cytometric analysis were significantly observed 72 h after pironetin (30 nM) treatment (Figure 6) and signals of cleaved caspase-3 in the immunoblot assay were detected following 30 nM and 100 nM pironetin to the same degree in vindesine- and paclitaxel-resistant cells and the H69 cells (Figure 7). These results suggest that pironetin exhibits antitumor activity against these resistant cell lines via apoptosis induction, as in the parental H69 cells. In human leukemic K562 and doxorubicin-resistant cells (K562/ADM), mitotic arrest and sub-G1 peaks were similarly observed (Figure 8) and signals of cleaved caspase-3 were also detected following 30 nM and 100 nM pironetin treatment (Figure 7). Taken together, pironetin inhibited the growth of vindesine- and paclitaxel-resitant H69 cells to the same degree as in the parental cells, and also the growth of the multidrug- Figure 5. Western blot analysis of P-glycoprotein. The cell lysates (1 Ìg) digested by lysis buffer were subjected to 4-20% SDS-PAGE and stained resistant K562 cells as well as parental K562 cells. Using with anti-P-glycoprotein antibody, after being transferred to a PVDF the same concentrations of pironetin (30 nM and 100 membrane. Lane 1: K562, lane 2: K562/ADM, lane 3: H69, lane 4: nM), apoptosis was induced in these cell lines, although H69/VDS and lane 5: H69/Txl.

732 Yoshida et al: Antiproliferating Activity of Pironetin

Figure 6. Apoptosis induction by pironetin in H69, H69/VDS and H69/Txl cells. Cells were treated with 30 nM pironetin for 72 h and stained with propidium iodide. DNA content was analyzed using a flow cytometer. The numbers in the figure indicate the percentage of cells of the sub-G1 peak representative of at least 3 experiments.

their induction times differed due to the difference in their generation times. These results suggested that pironetin could show antitumor activity against a variety of tumors via apoptosis induction.

Discussion

Microtubules consisting of ·- and ‚- are an important target for anticancer drugs. These structural elements interact with a range of structurally diverse natural and synthetic agents. Several tubulin-targeted agents are clinically effective. Paclitaxel is an effective agent against ovarian carcinoma (11), breast carcinoma (12) and non-small cell lung cancer (NSCLC) (13, 14). Vindesine is also effective against small-cell lung cancer (15) and NSCLC in combination with (16, 17). The hemisynthetic alkaloid was reported to be a less neurotoxic yet microtubule-targeted agent (18) and significantly prolonged overall survival among patients with completely resected NSCLC in combination Figure 7. Apoptosis induction by pironetin in K562 and K562/ADM cells. Cells were treated with 30 nM pironetin for 43 h and stained with with cisplatin (19). propidium iodide. DNA content was analyzed using a flow cytometer. However, one of the major obstacles to the Values in the figure represents the percentage of cells of the sub-G1 peak chemotherapy of cancer is the development of drug representative of at least 3 experiments.

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Figure 8. Caspase -3 activation by pironetin in H69, H69/VDS, H69/Txl, K562 and K562/ADM cells. Cells were treated with or without 30 nM and 100 nM pironetin for the periods of time indicated in Figures 6 and 7. Staurosporin treated HL-60 cells were used as positive control for caspase-3 activation. Western blot analysis was performed with total cellular extracts (10 Ìg/lane) using polyclonal antibody to caspase-3.

resistance during treatment. Acquired resistance to typical multidrug-resistant phenotype in its resistance paclitaxel and vindesine and multidrug resistance were pattern to anticancer drugs and intracellular accumulation found in chemotherapy. Resistance to vinca and of . In their reverse transcription polymerase paclitaxel might be mainly due to gene mutation (20); chain reaction (RT-PCR) analysis, MDR-1 mRNA qualitative and quantitative changes of ‚-tubulin expression was not detected in either resistant cell line. expression were also reported (21-23). Multidrug These data were consistent with our data of P-glycoprotein resistance has also been found by amplification of the gene in Western blot analysis. On the other hand, qualitative for a 170 kDa membrane glycoprotein (P-glycoprotein), and quantitative changes in tubulin were found in both whose apparent function is to pump various hydrophobic H69/VDS and H69/Txl cell lines (3, 4). It is not clear agents (including paclitaxel, vinca alkaloids, doxorubicin, whether they are responsible for their mechanism of and ) out of the cell (24, 25). Both resistant cell resistance. The results suggested that the binding sites of lines H69/VDS and H69/Txl used in this study showed vindesine and paclitaxel in ‚-tubulin were structurally cross-resistance to paclitaxel and vindesine. However, modified, but further work is necessary to determine the Ohta et al. (3) reported that H69/VDS did not show a cause of resistance of H69/VDS and H69/Txl.

734 Yoshida et al: Antiproliferating Activity of Pironetin

The paclitaxel site has been accurately identified in the 3 Ohta S, Nishio K, Kubo S, Nishio M, Ohmori T, Takahashi T structure of tubulin protofilaments in Zn-sheets (26). The and Saijo N: Characterisation of a vindesine-resistant human colchicine site has also been identified in the structure of ‚- small cell lung cancer cell line. Br J Cancer 68: 74-79, 1993. 4 Ohta S, Nishio K, Kubota N, Ohmori T, Funayama Y, Ohira T, tubulin with the stathmin-like domain (SLD) of RB3 (27). Nakajima H, Adachi M and Saijo N: Characterization of a In addition to the binding sites, the interface of ·- and ‚- taxol-resistant human small cell lung cancer cell line. Jpn J tubulins may be also important in antitumor action. The Cancer Res 85: 290-297, 1994. colchicine binding site to ‚-tubulin exists at the interface 5 Tsuruo T, Iida-Saito H, Kawabata H, Oh-hara T, Hamada H with ·-tubulin (27). introduces a wedge at the and Utakoji T: Characteristics of resistance to adriamycin in interface of the two tubulin molecules and thus interferes human myelogenous K562 resistant to adriamycin and with tubulin assembly (28). Pironetin inhibits vinblastine in isolated clones. Jpn J Cancer Res 77: 682-692, 1986. binding to ‚-tubulin, suggesting that Lys352 on ·-tubulin 6 Yoshida M, Feng W, Saijo N and Ikekawa T: Antitumor activity of daphnane-type diterpene gnidimacrin isolated from Stellera locates close to vinblastine binding site on ‚-tubulin (2, 10). chamaejasme L. Int J Cancer 66: 268-273, 1996. Therefore, further work is necessary to elucidate the three- 7 Vindelov LL, Christensen IJ and Nissen NI: A detergent-trypsin dimensional structure of the tubulin complex with these method for the preparation of nuclei for flow cytometric DNA agents, including pironetin. analysis. Cytometry 3: 323-327, 1983. Pironetin not only has a unique structure containing only 8 Yoshida M, Feng W, Nishio K, Takahashi M, Heike Y, Saijo N, one pyran residue and an alkyl chain, which is simpler than Wakasugi H and Ikekawa T: Antitumor action of the PKC the structures of other M- phase inhibitors such as paclitaxel activator gnidimacrin through cdk2 inhibition. Int J Cancer 94: 348-352, 2001. and vindesine, but also has the unique charcteristic of 9 Kondoh M, Usui T, Kobayashi S, Tsuchiya K, Nishikawa K, binding covalently to ·-tubulin. 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