Octa-Substituted Anionic Porphyrins: Topoisomerase I Inhibition and

Jul 30, 2008 - The results implied that compounds 4, 6, and 8 are potential inhibitors to Topo I, which might be one of the important factors inducing...
0 downloads 0 Views 671KB Size
Download PDF
Bioconjugate Chem. 2008, 19, 1535–1542

1535

Octa-Substituted Anionic Porphyrins: Topoisomerase I Inhibition and Tumor Cell Apoptosis Induction Baoping Zhai,†,‡ Li Shuai,†,‡ Li Yang,‡ Xiaocheng Weng,‡ Lin Wu,‡ Shaoru Wang,‡ Tian Tian,‡ Xiaojun Wu,‡ Xiang Zhou,*,‡,§ and Congyi Zheng§ College of Chemistry and Molecular Sciences, State Key Laboratory of Virology, and School of Life Science, Wuhan University, Hubei, Wuhan 430072, PR China. Received December 17, 2007; Revised Manuscript Received March 30, 2008

β-Octabromo-meso-tetra(4-carboxyl)phenyl porphyrin 6 and β-octaphenyl-meso-tetra(4-carboxyl)phenyl porphyrin 8 were synthesized and fully characterized by 1H NMR, UV, and HRMS. Their cytotoxicities to tumor cells were tested using MTT assays. One kind of tumor cell apoptosis induced by these anionic porphyrins under irradiation was examined by flow cytometric analysis. The inhibition of Topo I (Topoisomerase I) indicates that Topo I preferentially binds to the synthesized compounds, thus blocking the interaction between Topo I and DNA. The results implied that compounds 4, 6, and 8 are potential inhibitors to Topo I, which might be one of the important factors inducing apoptosis of tumor cells.

INTRODUCTION Porphyrins have found wide application in catalysis, materials, and medicine (1–6) because of their physical, chemical, and biological properties. When porphyrin molecules are modified by the use of peripheral substitutents, they could show very interesting functions due to the change in electronic and steric properties (7–9). Therefore, the substitution at β-positions is of extraordinary importance. It has been found that substitutents at the β-positions of porphyrins exert much larger steric and electronic effects on the porphyrin ring than substiutents at the meso-aryl positions. What’s more, β-substitutents can induce the porphyrin ring into a nonplanar conformation, which might mimic control of the the biological properties in tetrapyrrole systems like the photosynthetic centers, vitamin B12 and P-450 (10–12). Since Fiel and collaborators studied the interaction between porphyrins and nucleic acid (13), a variety of porphyrins have been synthesized and their abilities to bind to DNA have been confirmed (14–16). Several kinds of porphyrins have been found to induce cell apoptosis (17). Nevertheless, so far most reported works concerning either the interaction between porphyrins and DNA or the induction of tumor cell apoptosis are chiefly concentrated on cation porphyrins such as TMPyP (tetra-(4-Nmethylpyridium)porphyrin). In fact, as is proven in previous work, negatively charged porphyrins are more membrane permeable than cation porphyrins, which makes the porphyrin molecules biologically more active (18). Recently, Vicente and co-workers (19) have reported the cellular uptake and animal toxicity of a anionic porphyrin, tetra(carboranylphenyl)-tetrabenzoporphyrin. Their results suggest that anionic porphyrins might have more favorable abilities to interact with tumor cells. In this paper, we have designed and synthesized a series of * Corresponding author. Prof. Xiang Zhou, Tel: (086) 027-61056559. Fax: (086) 027-87663380. E-mail: [email protected]. † Both authors contributed equally to this work. ‡ College of Chemistry and Molecular Sciences, State Key Laboratory of Virology. § State Key Laboratory, School of Life Science. 1 Abbreviations: Topo I, Topoisomerase I; TMPyP, tetra-(4-Nmethylpyridium) porphyrin; CPT, camptothecin; CT DNA, calf thymus DNA.

anionic porphyrins with β-octabromo or β-octaphenyl substitution; meanwhile, carboxyl groups are introduced onto the mesoaryl positions. Some initial studies on the inducement of tumor cell apoptosis by the synthesized compounds have also been carried out. The results reveal that the inhibition of topoisomerase I activity by the synthesized compounds might be one of the key points to tumor cell apoptosis, which is in accord with the reported fact that DNA topoisomerases are responsible for cell death via the apoptosis pathway (20, 21).

EXPERIMENTAL PROCEDURES Synthesis of Test Compounds. Desired compounds were synthesized according to the procedures shown in Scheme 1. Compound 5 was synthesized with reference to Day’s report (8). The synthesis of β-octasubstituted-meso-tetra-(4-carboxyl)phenyl porphyrin 6 and 8 was involved in Suzuki coupling chemistry starting from β-octabromoporphyrin. Porphyrins 3 (22) and 4 (23) were obtained from mixing methyl 4-formylbenzoate 1 or 4-carboxybenzaldehyde 2 and pyrrole in refluxing propionic acid with yields of 22.4% and 10.5%, respectively. β-Octabromo-substituted porphyrin 5 was prepared from porphyrin 3. Porphyrins 6 and 8 were traced by HPLC (Supporting Information, Figure S1). Synthesis of β-octabromo-meso-tetrakis (4-carboxyphenyl)porphyrin methyl ester (5). Porphyrin 3 (400 mg, 0.47 mmol) was dissolved in 150 mL of chloroform followed by the addition of Cu(OAc)2 · H2O (760 mg, 3.81 mmol, 8 equiv). The reaction mixture was stirred at reflux for 30 min. After complete conversion to copper porphyrin as detected by TLC, 4 mL liquid Br2 was added to the reaction mixture directly. The solution was continually stirred for about 1 day at room temperature. Aqueous solution of sodium thiosulfate was added to quench the reaction. After removal of the excessive bromine, the reaction mixture was washed by H2O several times, and then 30 mL of perchloric acid was added to the solution. The reaction mixture was stirred for about 12 h for demetallization. The organic layer was separated and washed with aqueous and a solution of sodium bicarbonate and water, and dried over anhydrous Na2SO4. The organic mixture was rotary-evaporated to give a solid. The crude product was purified by column chromatography on silica gel using chloroform/ethyl acetate 60:1 as eluent to give porphyrin 5 (478 mg, 68.4% yield). 1H NMR

10.1021/bc7004686 CCC: $40.75  2008 American Chemical Society Published on Web 07/30/2008

1536 Bioconjugate Chem., Vol. 19, No. 8, 2008

Zhai et al.

Scheme 1a

a (a) Propionic acid, reflux, 3 22.4%, 4 10.5%; (b) (1) Cu(OAc)2, CHCl3, reflux, 0.5 h; (2) Br2, CHCl3, rt, 1 day; (3) HClO4, CHCl3, rt, 12 h, 68.4%; (c) THF, 2 N KOH 3 day, 86.7%; (d) PhB(OH)2, K2CO3, Pd(Ph3P)4, toluene/DMF(v/v 9:1), 95-105 °C, 7 days, 7 52.9%; (e) THF, 2 N KOH 3 day, 92.1%.

(CDCl3, 300 MHz): δ (ppm), 8.47-8.44 (d, J ) 7.8 Hz, 8 H), 8.32-8.30 (d, J ) 8.1 Hz, 8 H), 4.10 (s, 12 H). UV-vis (CHCl3): λmax (nm, log ) ) 370 (4.40), 472 (5.26), 625 (4.10). MALDI-TOF HRMS for C52H30Br8N4O8 [M+ + H] calcd 1478.5527, found 1478.5382. Synthesis of β-octaphenyl-meso-tetrakis(4-carboxyphenyl)porphyrin methyl ester (7). A 100 mL Teflon-stoppered flask was charged with porphyrin 5 (400 mg 0.27 mmol), PhB(OH)2 (656 mg, 5.4 mmol, 20 equiv), (Ph3P)4Pd (96 mg, 0.08 mmol, 0.3 equiv), anhydrous K2CO3 (1500 mg, 10.8 mmol, 40 equiv), DMF (6 mL), and toluene (54 mL). The brown suspension was degassed by the freeze-pump-thaw method (three cycles), and then was heated at 90-100 °C under N2 for 7 days. The reaction mixture was worked up by extracting with 50 mL CHCl3 and washed with water and brine. The organic layer was dried over anhydrous Na2SO4 and rotary-evaporated to dryness. The crude

product was purified by column chromatography on silica gel using chloroform/ethyl acetate 20:1 as eluent to give porphyrin 7 (208 mg, 52.9% yield). 1H NMR (CDCl3, 300 MHz): δ (ppm), 7.63-7.60 (d, J ) 7.2 Hz, 8 H), 7.40-7.37 (d, J ) 9.1 Hz, 8 H), 6.69-6.67 (m, 40 H), 3.99 (s, 12 H). UV-vis (CHCl3): λmax (nm, log ) ) 467 (5.19), 562 (4.03), 611 (3.96). ESI HRMS for C100H70N4O8 [M+ + H] calcd 1455.5266, found 1455.5310. Synthesis of β-octabromo-meso-tetrakis(4-carboxyphenyl)porphyrin (6). Porphyrin 5 (30 mg, 0.02 mmol) was dissolved in 5 mL of THF and 3 mL of a 2 M aqueous solution of potassium hydroxide was added. The solution was stirred in a sealed flask at room temperature, for 3 days. The aqueous phase was collected and the pH lowered, using a 1 M solution of hydrochloric acid, until precipitation of the porphyrin. The precipitate was washed with water and dried under vacuum to

Octa-Substituted Anionic Porphyrins

Bioconjugate Chem., Vol. 19, No. 8, 2008 1537

Table 1. Inhibitory Percentage of Porphyrins 6, 8, and 4 to Tumor Cell Line 7901 (%) Concentration of compounds (µM) 6 8 4

irradiation dark irradiation dark irradiation dark

0.01

0.1

0.5

1

5

10

23.59 -10.67 -2.94 -0.41 19.40 2.09

18.50 -4.70 -0.07 2.42 19.65 3.26

21.09 1.17 -3.04 -0.64 16.23 -0.16

18.54 -3.4 -5.08 1.12 28.87 7.95

23.47 -6.2 -3.74 5.73 69.91 10.66

52.13 4.9 5.22 12.08 95.39 22.26

15

20

33.79 32.64

71.17 31.41 52.00 44.45 99.72 59.11

25

30

50

100

66.80 58.05

96.46 60.38 88.50 69.84 98.96 89.96

99.33 97.54 96.63 95.44 99.76 99.35

99.63 94.13 97.79 95.65 100.5 99.84

The various concentration porphyrins were added, and the cells were irradiated for 30 min by a 50 W high-pressure mercury lamp at a distance of 13 cm after incubation for 4 h.Then, the cells were treated for 72 h and incubated with 5 mg · mL-1 MTT for 4 h. The amount of MTT formazan produced was determined by measuring absorbance at 570 nm (24). All data are represented as the mean values obtained from eight separate cultures, RSD < 0.05.

give porphyrin 6 (25 mg, 86.7% yield). 1H NMR ([d6]-DMSO, 300 MHz): δ (ppm), 8.49-8.40 (m, 8 H), 8.37-8.30 (m, 8 H). UV-vis (DMSO): λmax (nm, log ): 489.0 (5.15), 672 (4.28). MALDI-TOF HRMS for C48H22Br8N4O8 [M+] calcd 1422.4911, found 1422.4856. Synthesis of β-octaphenyl-meso-tetrakis (4-carboxyphenyl)porphyrin (8). Porphyrin 7 (36 mg, 0.025 mmol) was dissolved in 5 mL of THF and 3 mL of a 2 M aqueous solution of potassium hydroxide was added. The solution was stirred in a sealed flask at room temperature, for 3 days. The aqueous phase was collected and the pH lowered, using a 1 M solution of hydrochloric acid, until precipitation of the porphyrin. The precipitate was washed with water and dried under vacuum to give porphyrin 8 (32 mg, 92.1% yield). 1H NMR ([d6]-DMSO, 300 MHz): δ (ppm), 8.36 (s, 4 H), 7.41-7.38 (d, J ) 9.0 Hz, 8 H), 7.22-7.19 (d, J ) 9.0 Hz, 8 H), 6.70 (s, 40 H). UV-vis (DMSO): λmax (nm, log ): 450 (5.25), 555 (4.21), 594 (3.98). ESI HRMS for C96H62N4O8 [M+] calcd 1397.4484, found 1397.4437. Cell Incubation and Cell Survival Assay (24). The SGC7901 cells were obtained from China Center for type Culture Collection (CCTCC) and grown in MEM supplemented with 10% FBS (fetal bovine serum), incubated at 37 °C in a 5% CO2, 95% air atmosphere, and were subcultured every two to three days as necessary. The cells (about 3 × 103) were added to a 96-well plate and incubated for 24 h at 37 °C in a 5% CO2 incubator. Then, the as-synthesized porphyrins at various concentrations were added. After incubation for 4 h at 37 °C in a 5% CO2 incubator, the cells were irradiated for 30 min by a 50 W high-pressure mercury lamp at a distance of 13 cm. Then, the cells were allowed to incubate for another 72 h. MTT formazan (10 µL, 5 mg/mL) was added to each well and incubated for another 4 h. The cells were centrifuged at 2000 rpm for 10 min, and then the medium was removed. With addition of 100 µL of DMSO to each well, the values of OD were detected at 570 nm. The cytotoxicity data were presented as IC50 values. Flow Cytometric Determination of Apoptosis (17). The cells were cultured as above. 5 × 104 cells of each well were seeded in a 24-well plate. After 24 h, various concentrations of porphyrins were added and trireplicated. Then, the plate was irradiated for 30 min by a 50 W high-pressure mercury lamp. After 72 h, the cells of each well were harvested by using typsin. After centrifugation and being washed two times with PBS, these cells were fixed in an ice-cold 70% ethanol solution at -20 °C overnight. The 100 µL of suspended single cell solution in PBS were obtained by further centrifugation and washing. Sample wells prepared for the flow cytometry analysis were treated with RNase (1 mg/mL) for 30 min at 37 °C. The mixture was suspended in 1 mL of PBS solution containing propidium iodide PI (100 µg/mL). Then, the cells were incubated for 1 h at room temperature in the dark and analyzed on a flow cytometer (Beckmen Coulter Eltra flow cytometer). The linear DNA content data were analyzed by MultiCycle DNA Content and Cell Analysis software for cell cycle analysis. Cell apoptosis

Figure 1. The histogram drug-induced cell cycle arrest and apoptosis of porphyrin (10 µM) in 7901 cells as measured by flow cytometric analysis. The x-axis is the DNA content and the y-axis is the number of cells with that fluorescence intensity. 6, 8, and 4 represent cells that were treated with 10 µM porphyrins 6, 8, and 4, respectively, for 72 h (O is control) and irradiated by light for 30 min, respectively. The rate of apoptosis: 15.4 ( 0.15%, 9.5 ( 0.25%, and 4.8 ( 0.21%, respectively.

was measured as a sub-G0-G1 hypodiploid peak and indicated with the fluorescence intensity of FITC. Assessment of Intracellular Caspase-3 Activity (25). The CaspGLOW Fluorescein Active Caspase-3 Staining Kit was used to monitor the intracellular caspase-3 activities according to the manufacturer’s recommendations (Biovision, USA). The assay utilizes the caspase-3 inhibitor DEVD-FMK conjugated

1538 Bioconjugate Chem., Vol. 19, No. 8, 2008

Figure 2. Mechanism of human topoisomerase I. * means the τ of this supercoiled DNA is n. ** means the τ of this supercoiled DNA is n - 1.

to FITC (FITC-DEVD-FMK) as the fluorescent in situ marker. FITC-DEVD-FMK is cell-permeable, nontoxic, and irreversibly binds to activated caspase-3 in apoptotic cells. The FITC label allows for direction of activated caspases in apoptotic cells by flow cytometry. The cells were cultured as above. 2 × 105 cells of each well were seeded in a 6-well plate. After 24 h, various concentrations of porphyrins were added and trireplicated. The cells were irradiated for 30 min by a 50 W high-pressure mercury lamp at a distance of 13 cm after incubation for 4 h at 37 °C in a 5% CO2 incubator. The cells of each well were harvested by using typsin at given time intervals. Aliquots (300 µL) of each treatment were added into microtubes. With the addition of 1 µL of FITC-DEVD-FMK, the tubes were incubated for 0.5 h at 37 °C in an incubator with 5% CO2. Cells were centrifuged at 3000 rpm for 5 min and supernatant was removed. The cells were then resuspended in 0.5 mL of wash buffer, and centrifuged again. For flow cytometric analysis, cells were resuspended in 500 µL of wash buffer. Samples were put on ice and analyzed by flow cytometry (Beckman-Coulter, Eltra flow cytometer) using the FL-1 channel. Measurement of Relaxation Activity Topo I (26). Functional activity of Topo I1 was assayed by relaxation of

Zhai et al.

supercoiled plasmid DNA. Briefly, relaxation of 50 ng of supercoiled DNA pBR322 (0.1 U of Topo I) was performed in 10 µL of Topo I relaxation buffer (20 mM Tris pH 7.5, 0.1 mM Na2EDTA, 10 mM MgCl2, 50 mg/mL acetylated-BSA, 100 mM KCl) at 37 °C for 30 min in the presence or absence of the porphyrins dissolved in DMSO solution. Control groups were either DNA alone or DNA treated with Topo I only. Incubation at 37 °C for time course was terminated by addition of 0.5% (w/v) SDS and proteinase K. The mixtures were run in a 0.8% agarose gel. Gels were stained with ethidium bromide (EB) and visualized by UV and photographed using Vilber Lourmat video system. Measurement of Topo I Mediated DNA Cleavage (27). With addition of excessive enzymes (i.e., 0.3 U of Topo I), topoisomerase I reactions were performed in topoisomerase I relaxation buffer at 37 °C for 30 min. Reactions were terminated with 0.5% (w/v) SDS. After digestion with proteinase K, open circular and linear DNA were separated from intact supercoiled and relaxed forms by agarose gel electrophoresis in the presence of 0.5 mg/mL of EB under the same conditions as for the relaxation assay. CPT was used as the reference drug. Analysis of Binding with Topo I and pBR 322 (28). The supercoiled DNA pBR322 (50 ng) or the Topo I enzyme (0.2 U) was preincubated at 4 °C for 15 min in Topo I relaxation buffer with the indicated concentrations of compound 6. Then, Topo I (0.2 U) or supercoiled DNA pBR322 (50 ng) was added and incubated with compound 6 for 30 min at 37 °C and analyzed on 0.8% agarose gels at 4.5 V/cm. The control shows the electrophoretic mobility of pBR 322 DNA alone. Analysis of Topoisomerase-DNA Binding by Electrophoretic Mobility Shift Assay (EMSA) (28). During analysis of Topo I/DNA binding by EMSA, supercoiled DNA pBR322 was incubated with 0.3 U of topoisomerase I for 1 min at 37

Figure 3. (A) The gradient agarose gel assays of compound 4. (B) The gradient agarose gel assays of compound 6. (C) The gradient agarose gel assays of compound 8. (D) Activity inhibition of Topoisomerase I. Agarose gel electrophores from 1 to 30 min. Relaxed DNA (R) and nicked DNA (N) slowly migrate in the gel for a cell free DNA relaxation assay of a pBR322 DNA treated with Topo I inhibitors as compared to supercoiled DNA (S).

Octa-Substituted Anionic Porphyrins

Bioconjugate Chem., Vol. 19, No. 8, 2008 1539

Figure 4. Mechanism of Topo I inhibition. (A) Representative image of agarose gel electrophoresis of the cleavage assay. pBR322 DNA (50 ng) was incubated in the presence of Topo I (0.1 U) and with or without Topo I inhibitors. Only CPT induced the formation of a slowly migrating complex formed by enzyme, drug, and DNA; whereas drug 6, 8, and 4 were unable to interfere. (B) Prevent binding of Topo I to substrate DNA. EMSAs of Topo I incubated with appropriate DNA are shown. Samples contained pBR322 DNA (50 ng), each inhibitor, and excess of topoisomerase I (0.3 U) to allow the strongest possible DNA shift. (C) Inhibition of Topo I catalyzed pBR 322 DNA relaxation by compound 6. Relaxation of 50 ng of pBR 322 DNA by 0.2 units of human topoisomerase I was incubated with compound 6 for 30 min at 37 °C and analyzed on 0.8% agarose gels. The DNA-substrate (lanes 1 to 6) or the enzyme (lanes 7 to 12) were preincubated at 4 °C for 15 min with the indicated concentrations of compound 6. (D) interaction of drug 6, 8, and 4 with DNA was measured in an unwinding assay. All experiments were repeated three times with similar results.

°C. SDS denaturation and proteinase K digestion were omitted. Samples were immediately loaded onto the 0.8% agarose gel with 0.5 mg/mL of ethidium bromide in TBE buffer and separated by electrophoresis for 6 h. Measurement of DNA Intercalation (29, 30). DNA intercalation was measured with the unwinding assay. Supercoiled pBR322 DNA was relaxed with 0.3 U of Topo I at 37 °C for 10 min in Topo I relaxation buffer. To confirm full relaxation of DNA, the sample was terminated with SDS after 10 min. Porphyrins were added and the mixture was allowed to incubate for another 30 min. A parallel experiment ensured that Topo I retained its activity in the presence of the compounds used. The reaction was terminated by addition of 0.5% (w/v) SDS and followed by digestion with proteinase K as described above. Excess porphyrins were removed by extraction with chloroform/ isoamyl alcohol (24:1). Reaction samples were loaded onto the 0.8% agarose gel. At this moment, EB was used as the reference. CT DNA topoisomerase I (Topo I) (20 U/µL) and supercoiled pBR322 plasmid DNA were purchased from Toyobo (Osaka, Japan). Camptothecin (CPT) was purchased from Acros. Human

gastric carcinoma cell line SGC7901 was obtained from China Center for Type Culture Collection (CCTCC).

RESULTS Cell Survival Assay. For initial investigation, we chose tumor cell line 7901 as the target to study the anticancer abilities of the synthesized porphyrins. The effect of the compounds on proliferation of cancer cell lines was determined using the standard MTT assay under irradiation (24). All the stock solutions were dissolved in DMSO and diluted with water, and the final DMSO concentration is less than 0.1%. Results from MTT assay are shown in Table 1. Cell Cycle and Apoptosis analysis. In further research, we investigated the effects of anionic porphyrins on the growth of tumor cells through cell cycle and apoptosis analysis. Normally, there is a cell cycle in the proliferation of cells. If the cell cycle was perturbed and was not successfully repaired by itself, the cell will go to death (necrosis or apoptosis). Apoptosis is a physiological “cell suicide” program, and is the normal pathway for clearance of defective or aged cells in the body (31, 32). It is also the key pathway in animal development (33). The

1540 Bioconjugate Chem., Vol. 19, No. 8, 2008

apoptosis analysis was conducted by flow cytometry assay staining with propidium iodide (Figure 1). Assessment of Intracellular Caspase-3 Activity. Caspase-3 is a member of the cysteine aspartic-acid-specific protease family. As the enzyme takes part in the execution phase of programmed cell death, it plays a key role in early phases of apoptosis in mammalian cells (34). Therefore, the activity of the enzyme is an important biochemical indicator of the apoptosis procedure. We studied the enzymatic activity of caspase-3 in cells incubated with the synthesized compounds using the CaspGLOW Fluorescein Active Caspase-3 Staining Kit. Inhibition of Topoisomerase I Activity. The mechanism of human Topo I (topoisomerase I) has been well-established, as is shown in Figure 2 (35). CPT (camptothecin) binds to the supercoiled DNA*-Topo I covalent intermediate after cleavage and inhibits subsequent religation. It means that CPT can inhibit step 4 in the procedure. Our results indicate that porphyrins 4, 6, and 8 could bind to free Topo I and subsequently inhibit the binding of the free enzyme to the DNA cleavage site, and thus prevent all following steps of the catalytic cycle. Such a hypothesis is supported by CT DNA (calf thymus DNA) Topo I activity in a cell-free system was evaluated with relaxation assay. We tested whether these compounds could inhibit Topo I relaxation activity of a supercoiled DNA pBR322. The experimental data are shown in Figure 3. Figure 3A,B,C are gradient agarose gel assays which show the inhibition of these compounds. The IC50 value for the inhibition of the catalytic activity of Topo I by 6, 8, and 4 were 2.34 µM, 3.40 µM, and 11.75 µM, respectively. Mechanism of Interaction between Topo I and Porphyrins. It is known that Topo I is a single-stranded endonuclease and ligase. CPT, which is used as our control, is known to inhibit ligase without affecting the cleavage step (36, 37). Therefore, CPT shows a slowly migrating complex, which is named a cleavable complex, formed with the enzyme and drug and DNA, As we have found that 4, 6, and 8 could inhibit Topo I activity, we try to understand their working sites of compounds 4, 6, and 8 either as pure catalytic inhibitors or as topoisomerase poisons, stabilizing the cleavable complex (27). Figure 4A shows that CPT could stabilize the Topo I cleavable complex, resulting in the generation of open-circle plasmid DNA (28). In contrast, open-circle DNA was not observed with 4, 6, or 8. These findings encouraged us to consider that 4, 6, and 8 might exert their effect on Topo I before DNA cleavage (29). To verify such a hypothesis, we performed the EMSA (electrophoretic mobility shift assay) experiments between supercoiled pBR322 DNA and Topo I and investigated the impact of these compounds on Topo I. We used CPT as a control, which inhibits the ligase activity without interfering with the binding of the enzyme to DNA. Figure 4B demonstrates that 4, 6, and 8 inhibit the formation of these enzyme-DNA complexes. In contrast, DNA binding of Topo I is not affected by CPT (Figure 4B, lane 6). These observations are inconsistent with the mechanism of CPT, and thus we suggested that drugs could interfere in the binding of Topo I to DNA (30).

DISCUSSION As expected, data in Table 1 show good abilities of porphyrins 6, 8, and 4 to inhibit the growth of the 7901 cell line at low concentrations. Under light irradiation, the IC50 values of 6, 8, and 4 are 13.6 ( 1.5, 18.7 ( 0.7, and 3.7 ( 0.4 µM, respectively; while the values in the absence of light irradiation are 24.6 ( 1.6, 21.6 ( 0.9, and 17.5 ( 1.1 µM. These data indicate that light irradiation has not resulted in significant changes in the IC50 values; i.e., the inhibitory efficiency of 4 and 6 with irradiation are 5 times and 2 times higher than in

Zhai et al. Table 2. Caspase-3 Activity Induced by Compound 6a samples

control 1

control 2

22 h

44 h

96 h

10 µM 20 µM

0% 0%

2.2% 2.2%

9.5% 14.8%

13.6% 27.2%

25.8% 34.6%

a Percentages refer to the ratio of caspase-3-activated cells. Control 1: cells without any compounds or Z-VAD-FMK. Control 2: cells with Z-VAD-FMK but without compounds.

the dark. For compound 6 at a concentration of 10 µM, as seen in Table 1, the proliferation of cells is inhibited to 52% in the light, whereas the inhibitory percentage is only 4.9% in the dark, which shows good photoactivity of this compound. To further elucidate the mechanism of the cytotoxicity of these compounds, their abilities to induce apoptosis are investigated. The apoptosis analysis indicated that compound 6 can significantly induce apoptosis of 7901 cells at a 10 µM concentration, while under the same conditions, compounds 8 and 4 have much slighter effects as compared to 6. From the analysis of the cell cycle, we found that all three compounds can disturb the cell cycle of the 7901 cell line, which is shown with the decreased G0-G1 phase and increased S phase. Obviously, compound 6 has the most significant effect on cell cycle compared to the other two compounds. The G0-G1 phase percentage of 6 decreases from 60.1 ( 0.35% to 48.6 ( 0.40%. The apoptosis of tumor cells 7901 resulting from porphyrins causes the decrease of cells in the G0 phase. This is because the activation of endonucleases following the apoptosis of cells plays a direct and important role in DNA fragmentation, which results in the decrease of cells in the G0 phase (38). The outstanding inhibiting ability of compound 6 might be attributed to the electron-deficient property of the molecule resulting from the strong electron-withdrawing effect of the eight bromine atoms at β-positions. The preliminary results of intracellular caspase-3 activity have shown that cells incubated with 6 exhibits significant caspase-3 activity as compared to the negative control (Table 2). Data in Table 2 also show that the caspase-3 activity increases with prolonged the incubation time. After incubating for 96 h, the percentages of apoptosis cells are 25.8% and 34.6%, respectively, with 10 µM and 20 µM of 6. Another clear trend is that, at the same incubation time, the higher concentration of the compound leads to higher caspase-3 activity. We also examined 8 and 4 with the same method. However, no caspase-3 activity was detected in the cells incubated with these two compounds. Thus, it is concluded that the cell death induced by 6 is via an apoptosis pathway. Mechanism of interaction between Topo I and porphyrins has been studied and experimental data shown in Figure 4A,B give a clear indication that the inhibition of the catalytic activity of Topo I by 4, 6, and 8 is due to the inhibition of the DNA binding of the enzyme. The effect of 4, 6, and 8 could be achieved by intercalation of these compounds into partially relaxed plasmid DNA or by a direct interaction of the drugs with the enzyme itself. To distinguish between these possibilities and further elucidate the mechanism of topoisomerase inhibition, we carried out further experiments with the results shown in Figure 4C,D (25, 30, 31). Upon preincubation of 6 with enzyme, effective inhibition of topoisomerase I-catalyzed pBR 322 DNA relaxation could be obviously observed at 1 µM concentration, whereas in the case when the porphyrin was preincubated with pBR322, 3 µM 6 was needed to observe the DNA relaxation. Results in Figure 4C further indicate that the enzyme preferentially combines with the drug molecule; hence, the interaction of enzyme with DNA is blocked and the enzymatic activity is weakened. The inhibitory effect of 4, 6, and 8 on Topo I-mediated DNA relaxation can be enhanced by preincubation with the enzyme before addition of the DNA plasmid. On the

Octa-Substituted Anionic Porphyrins

other hand, we employed a DNA unwinding assay to assess any possible impact of 4, 6, and 8 on the superhelical state of closed circular DNA. This assay is based on the ability of intercalating compounds to unwind the DNA duplex and thereby change the DNA twist. These drug-induced changes in DNA twist also induce structural tension in the DNA backbone, and this tension can be relieved by topoisomerases (35). On removal of both topoisomerase and intercalating agent, the unwinding effect of the intercalating compound is no longer present and the DNA returns to a supercoiled state. Figure 4D shows that 4, 6, and 8 were unable to modulate DNA unwinding, whereas EB, used as a positive control, did it. These findings therefore indicate that the mechanism through which 4, 6, and 8 inhibit topoisomerases was independent of DNA intercalation. Taken together, these data suggest that 4, 6, and 8 bind directly to Topo I. For porphyrin 6, it has a potent ability to inhibit Topo I comparing with porphyrins 4 and 8. In all, we have synthesized two β-substituted anionic porphyrinsm, and their bioactivities were evaluated. Through MTT and flow cytometric analysis, their abilities to induce cell apoptosis and kill tumor cells were investigated. The β-octabromo porphyrin 6 was preliminarily proven to potentially be a strong anticancer agent. Meanwhile, we found that compound 6 has a stronger ability to inhibit Topo I, and it might be one of the factors that induce tumor cell apoptosis. Further research concerning the mechanism of inducing apoptosis as well as factors affecting the induction are currently in process.

ACKNOWLEDGMENT Author thanks financial support by the National Science of Foundation of China (No. 20272046, 20425206, 20621502), the Cultivation Fund of the Key Scientific and Technical Innovation Project, the Ministry of Education of China (No. 706040), Open funds supported by The State Key Laboratory of Natural and Biomimetic Drugs of China, State Key Laboratory of Applied Organic Chemistry of China. Supporting Information Available: HPLC as described in the text. This material is available free of charge via the Internet at http://pubs.acs.org.

LITERATURE CITED (1) Bonnett, R., and Martinez, G. (2001) Photobleaching of sensitisers used in photodynamic therapy. Tetrahedron 57, 9513– 9547. (2) Detty, M. R., Gibson, S. L., and Wagner, S. J. (2004) Current clinical and preclinical photosensitizers for use in photodynamic therapy. J. Med.Chem. 47, 3897–3915. (3) Dozzo, P., Koo, M-S., Berger, S., Forter, T. M., and Kahl, S. B. (2005) Synthesis, characterization, and plasma lipoprotein association of a nucleus-targeted boronated porphyrin. J. Med. Chem. 48, 357–359. (4) Nishiyama, N., Stapert, H. R., and and Zhang, G. D. (2003) Light-harvesting ionic dendrimer porphyrins as new photosensitizers for photodynamic therapy. Bioconjugate Chem. 14, 58– 66. (5) Lottner, C., Bart, K.-C., Bernhardt, G., and Brunner, H. (2002) Hematoporphyrin-derived soluble porphyrin-platinum conjugates with combined cytotoxic and phototoxic antitumor activity. J. Med. Chem. 45, 2064–2078. (6) Ochsenbein, P., Ayougou, K., Mandon, D., Fischer, J., Weiss, R., Austin, R. N., Jayaraj, K., Gold, A., Terner, J., and Fajer, J. (1994) Conformational effects on the redox potentials of tetraarylporphyrins halogenated at the β-pyrrole positions. Angew. Chem., Int. Ed. Engl. 33, 348–350.

Bioconjugate Chem., Vol. 19, No. 8, 2008 1541 (7) Nurco, D. J., Medforth, C. J., Forsyth, T. P., Olmstead, M. M., and Smith, K. M. (1996) Conformational flexibility in dodecasubstituted porphyrins. J. Am. Chem. Soc. 118, 10918–10919. (8) Kachadourian, R., Flaherty, M. M., Crumbliss, A. L., Patel, M., and Day, B. J. (2003) Synthesis and in vitro antioxidant properties of manganese (III) β-octabromo-meso-tetrakis(4carboxyphenyl)porphyrin. J. Inorg. Biochem 95, 240–248. (9) Bhyrappa, P., and Krishnan, V. (1991) Octabromotetraphenylporphyrin and its metal derivatives: Electronic structure and electrochemical properties. Inorg. Chem. 30, 239–245. (10) Gust, D., Moore, T. A., and Moore, A. L. (2001) Mimicking photosynthetic solar energy transduction. Acc. Chem. Res. 34, 40–48. (11) Palacio, M., Loire, V., Mansuy-Mouries, G., LeBarch-Ozette, K., Leduc, P., Barkigia, K. M., Fajer, J., Battioni, P., and Mansuy, D. (2000) A new general method for selectiveβ-polynitration of porphyrins; preparation and redox properties of Zn-porphyrins bearing one through to eight β-nitro substituents and X-ray structure of the first Znβ-pernitro porphyrin. J. Chem. Soc.: Chem. Commun. 19, 1907–1908. (12) Grinstaff, M. W., Hill, M. G., Labinger, J. A., and Gray, H. B. (1994) Mechanism of catalytic oxygenation of alkanes by halogenated iron porphyrins. Science 264, 1311–1313. (13) Fiel, R. J., Howard, J. C., Mark, E. H., and Datta-Gupta, N. (1979) Interaction of DNA with a porphyrin ligand: evidence for intercalation. Nucleic Acids Res. 6, 3093–3118. (14) Pasternack, R. F., Brigandi, R. A., Abrams, M. J., Williams, A. P., and Gibbs, E. J. (1990) Interactions of porphyrins and metalloporphyrins with single-stranded poly(dA). Inorg. Chem. 29, 4483–4486. (15) Gibbs, E. J., Maurer, M. C., Zhang, J. H., Reiff, W. M., Hill, D. T, Maliska-Blaskiewicz, M., McKinnie, R. E., Liu, H. Q., and Pasternack, R. F. (1988) Interactions of porphyrins with purified DNA and more highly organized structures. J. Inorg. Biochem. 32, 39–65. (16) Sari, M. A., Battioni, J. P., Dupre, D., and Pecq, J. B. (1990) Interaction of cationic porphyrins with DNA: importance of the number and position of the charges and minimum structural requirements for intercalation. Biochemistry 29, 4205–4215. (17) He, H. P., Zhou, Y., Liang, F., Li, D. Q., Wu, J. J., Yang, L., Zhou, X., Zhang, X. L., and Cao, X. P. (2006) Combination of porphyrins and DNA-alkylation agents: Synthesis and tumor cell apoptosis induction. Bioorg. Med. Chem. 14, 1068–1077. (18) Krishnamurthy, P. C., Du, G., Fukvda, Y., Sun, D., Sampath, J., Mercer, K. E., Wang, J., Sosa-Pineda, B., Murti, K. G., and Schuetz, J. D. (2006) Identification of a mammalian mitochondrial porphyrin transporter. Nature 443, 586–589. (19) Gottumukkala, V., Ongayi, O., Baker, D. G., Lomax, L. G., and Vicente, M. G. H. (2006) Synthesis, cellular uptake and animal toxicity of a tetra(carboranylphenyl)-tetrabenzoporphyrin. Bioorg. Med. Chem. 14, 1871–1879. (20) Lansiaux, A., Laine, W., Baldeyrou, B., Mahieu, C., Wattez, N., Vezin, H., Martinez, F. J., Pineyro, A., and Bailly, C. (2001) DNA topoisomerase II inhibition by peroxisomicine A1 and its radical metabolite induces apoptotic cell death of HL-60 and HL-60/MX2 human leukemia cells. Chem. Res. Toxicol. 14, 16–24. (21) Froelich-Ammon, S. J., and Osheroff, N. (1995) Topoisomerase poisons: harnessing the dark side of enzyme mechanism. J. Biol. Chem. 271, 21429–21432. (22) Naomi, A., Sayo, U., Tomoya, K., Hiroaki, A., and Tadashi, M. (2007) Synthesis of biladienone and bilatrienone by coupled oxidation of tetraarylporphyrins. J. Org. Chem. 72, 5320–5326. (23) Bradshaw, J. E. K., Gillogly, A., Wilson, L. J., Kumar, K., Wan, X. M., Tweedle, M. F., Hernandez, and BryantG. R. G. (1998) New non-ionic water-soluble porphyrins: evaluation of manganese (III) polyhydroxylamide porphyrins as MRI contrast agents. Inorg. Chim. Acta 275-276, 106–116. (24) Alley, M. C., Scudiero, D. A., Monks, A., Hyrsey, M. L., Czerwinski, M. J., Fine, D. L., Abbott, B. J., Mayo, J. G.,

1542 Bioconjugate Chem., Vol. 19, No. 8, 2008 Shoemaker, R. H., and Boyd, M. R. (1988) Feasibility of drug screening with panels of human tumor cell lines using a microculture tetrazolium assay. Cancer Res. 48, 589–601. (25) Yu, D. Y., Matsuya, Y., Zhao, Q. L., Ahmed, K., Wei, Z. L., Nemoto, H., and T., Kondo. (2007) Enhancement of hyperthermia-induced apoptosis by a new synthesized class of furan-fused tetracyclic compounds. Apoptosis 12, 1523–1532. (26) Raspaglio, G., Ferlini, C., Mozzetti, S., Prislei, S., Gallo, D., Das, N., and Scambia, G. (2005) Thiocolchicine dimers: a novel class of topoisomerase-I inhibitors. Biochem. Pharmacol. 69, 113–121. (27) Y, Pommier. (2006) Topoisomerase I inhibitors: camptothecins and beyond. Nat. ReV. Cancer 6, 789–802. (28) Hsiang, Y. H., Hertzberg, R., Hecht, S., and Liu, L. F. (1985) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. J. Biol. Chem. 260, 14873–14878. (29) Habermeyer, M., Fritz, J., Barthelmes, H. U., Christensen, M. O., Larsen, M. K., Boege, F., and Marko, D. (2005) Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase I. Chem. Res. Toxicol. 18, 1395–1404. (30) Boege, F., Straub, T., Kehr, A., Boesenberg, C., Christiansen, K., Andersen, A., Jakob, F., and Kohrle, J. (1996) Selected novel flavones inhibit the DNA binding or the DNA religation step of eukaryotic topoisomerase I. J. Biol. Chem. 271, 2262–2270. (31) Alam, J. J. (2003) Apoptosis: target for novel drugs. Trends Biotechnol. 21, 479–483.

Zhai et al. (32) Wylli, A. H. (1980) Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 284, 555–556. (33) Huang, Z. (2002) The chemical biology of apoptosis: Exploring protein-protein interactions and the life and death of cells with small molecules. Chem. Biol. 9, 1059–1072. (34) Datta, R., Kojima, H., Yoshida, K., and Kufe, D. (1997) Caspase-3-mediated cleavage of protein kinase C θ in induction of apoptosis. J. Biol. Chem. 272, 20317–20320. (35) Stewart, L., Redinbo, M. R., Qiu, X. Y., Hol, W. G. J., and Champoux, J. J. (1998) A model for the mechanism of human topoisomerase I. Science 279, 1534–1541. (36) Syrovets, T., Buchele, B., Gedig, E., Slupsky, J. R., and Simmet, T. (2000) Acetyl-boswellic acids are novel catalytic inhibitors of human topoisomerases I and II alpha. Mol. Pharm. 58, 71–81. (37) Raspaglio, G., Ferlini, C., Mozzetti, S., Prislei, S., Gallo, D., Das, N., and Scambia, G. (2005) Thiocolchicine dimers: a novel class of topoisomerase-I inhibitors. Biochem. Pharmacol. 69, 113–121. (38) Moosavi, M. S., Yazdanparast, R., and Lotfi, A. (2006) GTP induces S-phase cell-cycle arrest and inhibits DNA synthesis in K562 cells but not in normal human peripheral lymphocytes. J. Biochem. Mol. Biol. 39, 492–501. BC7004686