Benzo[b]tryptanthrin inhibits MDR1, topoisomerase activity, and reverses adriamycin resistance in breast cancer cells.

DOI: 10.1002/cmdc.201500068

Full Papers

Benzo[b]tryptanthrin Inhibits MDR1, Topoisomerase Activity, and Reverses Adriamycin Resistance in Breast Cancer Cells Kyu-Yeon Jun,[a] So-Eun Park,[a] Jing Lu Liang,[b] Yurngdong Jahng,[b] and Youngjoo Kwon*[a] tanthrin induced apoptosis through the cleavage of caspase-3 and PARP in HCT15 colon cancer cells. Additionally, benzo[b]tryptanthrin reversed adriamycin resistance by down-regulation of multidrug resistance protein 1 (MDR1) in adriamycin-resistant MCF7 breast cancer cells (MCF7adr) with more potent inhibitory activity than tryptanthrin. Taken together, derivatization by benzo-annulation of tryptanthrin ameliorated the MDRreversing effect of tryptanthrin and may pave the way to the discovery of a novel potent adjuvant agent for chemotherapy.

Tryptanthrin is an indoloquinazoline alkaloid isolated from indigo. Tryptanthrin and its benzo-annulated derivative, benzo[b]tryptanthrin, inhibit both topoisomerases I (topo I) and II (topo II) and cause cytotoxicity in several human cancer cell lines. From diverse assessment methods, including cleavage complex stabilization, comet, DNA unwinding/intercalation, topo II ATPase inhibition, ATP competition for topo II, and wound-healing assays, we determined that the mode of action of benzo[b]tryptanthrin is as a DNA non-intercalative and ATPcompetitive topo I and II dual catalytic inhibitor. Benzo[b]tryp-

Introduction VEGFR2-mediated ERK1/2 signals.[6] Tryptanthrin ameliorated clinical symptoms and skin lesions of apoptotic dermatitis induced by 2,4-dinitrofluorobenzene through down-regulation of thymic stromal lymphopoietin.[7] Tryptanthrin also showed antitumoral activity by targeting indoleamine 2,3-dioxygenase (IDO-1), and 8-fluorotryptanthrin, a stronger IDO-1 inhibitor than tryptanthrin, potently suppressed tumor growth in Lewis lung cancer tumor-bearing mice.[8] Potent cytocidal effects of tryptanthrin on various human leukemia cells[9, 10] and on azoxymethane-induced intestinal tumors have been reported.[11] In addition, antifungal, antimicrobial, tuberculostatic, COX-2 inhibitory, 5-LOX inhibitory, nitric oxide (NO) synthase inhibitory, and antimalarial activities of tryptanthrin have been reported.[12, 13] Many tryptanthrin derivatives have been developed, and their activities have also been reported; 4-azatryptanthrin and 6-oximotryptanthrin showed stronger antimicrobial activity than tryptanthrin in Escherichia coli through DNA intercalation.[14] 4-Azatryptanthrin functioned as a strong DNA intercalator with a preference for p-stacking with GC over TA DNA sequences.[15] 8-Methyltryptanthrin induced differentiation of P19L6 mouse embryonic carcinoma cells into spontaneously beating cardiomyocyte-like cells through activation of the gene expression of pacemaker channels and gap junctions.[16] Among the numerous reported biological and pharmacological activities of tryptanthrin, no inhibitory activity against topoisomerases (topos) was reported, and some cytocidal anticancer activities of tryptanthrin have been stated without clear explanation of the target.[9–11, 13] Topos are essential enzymes for solving DNA topological problems encountered during DNA transcription, replication, recombination, and chromosome segregation in mitosis following replication. Human topos are grouped into two types,

Tryptanthrin (Figure 1) was first isolated as an indoloquinazoline alkaloid from a culture of the yeast Candida lipolytica,[1] and has been continuously isolated from fungi such as Schizophyllum commune[2] and Leucopaxillus cerealis[3] and from various plants such as Polygonum tinctorium, Isatis tinctoria, Strobi-

Figure 1. Structures of tryptanthrin and benzo[b]tryptanthrin.

lanthes cusia, and Couroupita guaianensis.[4, 5] A plethora of biological and pharmacological activities of tryptanthrin have been reported; tryptanthrin suppressed angiogenesis in a Matrigel plug assay in vivo by reducing the expression of angiogenic factors such as Ang-1, PDGFB, and MMP2, as well as [a] Dr. K.-Y. Jun,+ S.-E. Park,+ Prof. Y. Kwon College of Pharmacy, Graduate School of Pharmaceutical Sciences Ewha Global Top 5, Ewha Womans University Seoul 120-750 (Republic of Korea) E-mail: [email protected] [b] J. L. Liang, Prof. Y. Jahng College of Pharmacy, Yeungnam University Gyeongsan 712-749 (Republic of Korea) [+] These authors contributed equally to this work. Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cmdc.201500068.

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Full Papers topoisomerase I (topo I) and topoisomerase II (topo II).[17, 18] Topo II is different from topo I in that topo II functions as a homodimer to cleave both strands of one DNA double helix (G-segment) simultaneously to let another non-cleaved DNA double helix (T-segment) pass through, and then reseals the cleavage of the G-segment by using ATP hydrolysis with a magnesium ion.[19] There are two types of topo II in humans, topo IIa and IIb. The expression level of topo IIa changes during the cell cycle, where it is the highest in G2/M phase, while that of topo IIb is constant.[20] Overexpression of topo IIa reflects a high mitotic rate, indicating that topo IIa could be a marker of proliferation in cancer cells. Therefore, topos have, for a long time, been attractive targets for medicinal chemists in developing chemotherapeutics. Topo-targeting drugs are considered to be highly active anticancer agents in many different clinical settings.[19] We were interested in derivatization of tryptanthrin, as well as in the effect of benzoannulation on its chemical and biological properties. A series of benzotryptanthrins were previously synthesized and examined for their physicochemical and biological properties focusing on the relationships between the reduction potentials, inhibitory activities against topo I and II, and cytotoxicity in several human cancer cell lines.[21] Among tryptanthrin and its benzoannulated derivatives, the benzotryptanthrins, benzo[b]tryptanthrin (Figure 1) showed the strongest inhibition of topo I and topo II, as well cytotoxicity against HCT15 colon cancer cells, in our previous study.[21] In the current study, the mode of action of benzo[b]tryptanthrin was determined to be a DNA non-intercalative and ATPcompetitive topo I and II dual catalytic inhibitor using diverse assessment methods, including cleavage complex stabilization, comet, DNA unwinding/intercalation, topo II ATPase inhibition, ATP competition for topo II, and wound healing assays, as well as a complementary molecular docking study. We further assessed that benzo[b]tryptanthrin induced apoptosis through cleavage of caspase-3 and PARP in HCT15 colon cancer cells and reversed adriamycin resistance through down-regulation of multidrug resistance protein 1 (MDR1) in adriamycin-resistant MCF7 breast cancer cells (MCF7adr).

100 mm (Figure 2). Etoposide does not affect the catalytic activity of topo II, as its cleavage step is reversible.[23] However, treatment with ellipticine strongly inhibits topo II decatenation. These behaviors are consistent with observations from gel

Results and Discussion

Benzo[b]tryptanthrin behaves as a catalytic inhibitor of topoisomerase II

Figure 2. Benzo[b]tryptanthrin inhibits topo II-induced kDNA decatenation. A) Topo II decatenation assay. Lane M: Marker; Lane D: kDNA only; Lane T: kDNA + topo IIa; Lane 4 ~ 7: kDNA + topo IIa + each compound with designated concentration. B) The percentage of inhibition of each compound on topo II-induced kDNA decatenation is calculated from A).

electrophoresis, as decatenated products are the major product in samples treated with etoposide, while the majority of kDNA remains in ellipticine-treated samples. Benzo[b]tryptanthrin did not inhibit topo II decatenation at 100 mm but did inhibit it at 200 mm, with an inhibition rate of 43.4 %. This result confirms benzo[b]tryptanthrin as a topo II inhibitor.

Benzo[b]tryptanthrin inhibits topo II-mediated kDNA decatenation

The mode of action of benzo[b]tryptanthrin was further explored by conducting several assays, such as cleavage complex stabilization, comet formation, DNA unwinding, ATPase, and ATP competition assays. Topo II inhibitors are characterized as two types: topo poisons and catalytic inhibitors. Topo poisons inhibit topo II by stabilizing transiently formed topo II–DNA complexes and forming undesired truncated linear DNA, thereby causing DNA damage, while topo catalytic inhibitors block the enzyme before DNA cleavage or in the last step of the catalytic cycle after re-ligation.[18, 19, 24] The cleavage complex stabilization assay clearly showed the linear band formation, with etoposide at 100 mm used as a positive control (Figure 3). However, benzo[b]tryptanthrin did not form linear DNA at either 100 or 300 mm, reflecting that it is a topo II catalytic inhibitor.

Previously, we observed that benzo[b]tryptanthrin exhibited the strongest inhibition potency in the topo II relaxation assay and produced the highest level of cytotoxicity among the synthesized compounds.[21] To further confirm the topo II inhibitory activity of benzo[b]tryptanthrin, a kinetoplast DNA (kDNA) decatenation assay was performed. Through the specific catalytic activity of topo II, kDNA forms several decatenated forms of DNA, including nicked (Nck), relaxed (Rel), and supercoiled (SC) DNA. Because of its size, catenated kDNA is unable to enter into agarose gels unless it is decatenated by topo II.[22] Thus, the inhibitory activity of benzo[b]tryptanthrin was compared with that of controls, etoposide and ellipticine, at

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Full Papers Benzo[b]tryptanthrin does not intercalate into DNA Benzo[b]tryptanthrin was shown to be a catalytic inhibitor, according to the above results. To distinguish between DNA intercalators and non-intercalators, a DNA unwinding assay was carried out. Amsacrine (m-AMSA) is a typical DNA intercalator, and when it is incubated with relaxed plasmid DNA and then removed by washing, DNA supercoiling occurs.[26] Figure 4 A shows an agarose gel from the DNA unwinding assay. m-AMSA lanes show the supercoiled DNA increasing as the concentration increases. However, the benzo[b]tryptanthrin lanes show no supercoiled DNA at 100–1000 mm treatment, demonstrating that benzo[b]tryptanthrin did not intercalate into DNA. In addition, the retardation of pBR322 supercoiled DNA was observed in the migration during gel electrophoresis in the presence of ethidium bromide, a well-known DNA intercalator, at a very low concentration of 1.0 mm,[27] while there was no retardation with treatment of benzo[b]tryptanthrin at the very high concentration of 300 mm (Figure 4 B). The results obtained from topo I and II relaxation inhibition assays[21] and observations from the DNA unwinding/intercalation assay demonstrate that benzo[b]tryptanthrin is a DNA non-intercalative topo I and II dual catalytic inhibitor.

Figure 3. Benzo[b]tryptanthrin is not a topo poison but is a catalytic inhibitor. A) Topo II cleavage complex stabilization assay. Benzo[b]tryptanthrin did not stabilize a transient cleavage complex of topo II–DNA. Etoposide generated a linear DNA band, boxed with the dotted line, as etoposide stabilized the cleavage complex and prevented the cleaved DNA from re-ligation. B) Comet assay. Benzo[b]tryptanthrin did not induce DNA damage in HCT15 cells. Images of control (non-treated), etoposide (topo IIa poison), and benzo[b]tryptanthrin-treated-HCT15 cells. Etoposide-treated HCT15 cells only showed comet formation. C) Graphical representation of the selected comet lengths of untreated and treated HCT15 cells in pixels with corresponding concentration. Columns and error bars indicate mean SD (n = 50). ***p < 0.001 significantly different from the value of control.

To confirm this result, the comet assay was performed for benzo[b]tryptanthrin. The comet assay is an effective tool to evaluate the toxicity of the drug by measuring the extent of DNA damage in cells.[25] DNA damage was quantified by measuring the comet tails of a single cell using LMAgarose gel electrophoresis after treatment of HCT15 cells with etoposide or benzo[b]tryptanthrin. The comet tail occurs when the enzyme– DNA cleavage complex is stabilized by the drug and DNA fragments have accumulated in cells. Etoposide significantly induced the formation of DNA tails at a concentration of 10 mm (Figure 3). In contrast, benzo[b]tryptanthrin did not form comet tails at a much higher concentration of 50 mm, which coincided with the result of the cleavage complex assay. These results confirm that benzo[b]tryptanthrin is a topo II catalytic inhibitor.


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Figure 4. Benzo[b]tryptanthrin functions without intercalating or unwinding of DNA. A) DNA unwinding assay. Reaction mixtures (10 mL) consisting of 1 mL supercoiled DNA pHOT1 (100 ng mL 1), 1 mL 10 topo I-reaction buffer, 1 mL topo I (4 units) and 6 mL DDW were incubated at 37 8C for 30 min. After incubation, 1 mL of each investigational compound solution was added to a final concentration of 100, 200, 500, and 1000 mm, respectively, and incubated at 37 8C for 30 min. Reactions were terminated by 1 mL topo I-stop buffer. Then, the reaction mixture was resolved on 1 % agarose gels at 15 V for 14–15 h. After electrophoresis, gels were stained in TAE buffer with ethidium bromide for 5 min and visualized using an AlphaImager. B) DNA intercalation assay. Negatively supercoiled DNA pBR322 and 1 mL (125 ng) of each compound stock solution were mixed with DDW to be the designated final concentrations in a total reaction volume of 10 mL. Reaction mixtures were incubated at 37 8C for 20 min and then resolved on 1 % agarose gels at 20 vol per cm for 12–16 h. After electrophoresis, gels were stained in TAE buffer with ethidium bromide for 30 min and visualized using an Alpha Imager.

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Full Papers Benzo[b]tryptanthrin potentially binds to the ATP-binding pocket of the ATPase domain of topo II, leading to topo II catalytic inhibition Another possible mode of action of topo II catalytic inhibitors is targeting the ATPase domain. Topo II ATPase assay and ATP competition assays were performed to study the effects of benzo[b]tryptanthrin on the ATPase domain of topo II. Novobiocin is known to block the ATP-binding site of topo II,[28] and the experimental results showed that novobiocin inhibited ATPase activity in a dose-dependent manner (Table 1). Ben-

Table 1. Compound inhibitory activities against topo IIa ATPase activity. C [mm]

Inhibition [%][a]

Novobiocin

400 300 200

60.23 2.52 45.86 3.83 38.54 0.78

Benzo[b]tryptanthrin

400 200

47.24 2.93 30.66 2.92

Compd

[a] Data are the mean SD obtained from three different experiments performed in triplicate.

zo[b]tryptanthrin also inhibited ATPase activity by 30.7 % and 47.2 % at 200 and 400 mm, respectively, which is slightly lower than the inhibitory activity of novobiocin. Benzo[b]tryptanthrin was further analyzed using an ATP competition assay. The ATP concentrations were set at 1 and 1.5 mm, and the benzo[b]tryptanthrin concentration was increased from 50 to 500 mm (Figure 5 A, B). At 1 mm ATP, the inhibition percentage of benzo[b]tryptanthrin at 20 and 100 mm was 1.2 and 78.1 %, respectively. When the concentration of ATP was increased to 1.5 mm, the inhibition percentage of benzo[b]tryptanthrin treated at 100 mm decreased to 16.5 %. However, the topo II inhibitory activity of etoposide, a well-known topo II poison, did not change as the ATP concentration increased, which was attributed to a different mechanism for stabilizing the topo II cleavage complex. This suggests that benzo[b]tryptanthrin competes with ATP for the ATP-binding site of the topo II ATPase domain. Docking studies were carried out for benzo[b]tryptanthrin and tryptanthrin with the ATPase domain of human topo IIa (PDB ID: 1ZXM) to examine the mode of action at the molecular level. Analysis of these results showed that both of the compounds fit into the ATP-binding site (Figure 5 C, D). Benzo[b]tryptanthrin formed van der Waals interactions with Asn 91, Ala 92, Asn 95, Arg 98, Asn 120, Ile 125, Ile 141, Phe 142, Ser 149, Thr 215, and Ile 217. Tryptanthrin also bound to the topo II ATPase domain and overlaid well with benzo[b]tryptanthrin. The only difference between the two is one more van der Waals interaction between Ile 141 and the additional benzene ring of benzo[b]tryptanthrin. Therefore, benzo[b]tryptanthrin has a stronger interaction with the ATPase domain than does tryptanthrin. The position of the benzo[b]tryptanthrin ring was compared with AMPPNP, a non-hydrolyzable ATP analogue that possess the same binding affinity as ATP

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Figure 5. The topo II inhibitory activity of benzo[b]tryptanthrin is attributed to its binding to the ATP-binding pocket of topo II in competition mode with ATP. A) Topo II ATP competition assay. Benzo[b]tryptanthrin blocked topo II in an ATP-dependent manner. B) Graphical representation of the topo II ATP competition assay from three different experiments. Each data represents mean SD C) Molecular docking study of benzo[b]tryptanthrin. Benzo[b]tryptanthrin potentially binds to the ATP-binding pocket in the ATPbinding domain of topo IIa. The secondary structure of the ATP-binding domain of topo IIa is shown as a ribbon diagram, with benzo[b]tryptanthrin shown as sticks colored according to atom type (carbon, magenta; oxygen, red; nitrogen, blue). D) Magnified view of the topo IIa ATP-binding domain. The residues of the topo IIa ATP-binding domain that have van der Waals contact with benzo[b]tryptanthrin and tryptanthrin are shown as sticks. The stick representation of the complex is colored by the atom type (benzo[b]tryptanthrin, cyan; tryptanthrin, magenta; ATP-binding pocket channel, green). E) Overlay view of AMPPNP and benzo[b]tryptanthrin in the topo IIa ATP-binding pocket.

toward the ATP-binding site.[28] The center pyrrolidinone ring of the benzo[b]tryptanthrin overlaps well with the purine ring of AMPPNP (Figure 5 E). The oxygen of the carbonyl group on the pyrrolidinone forms a hydrogen bond with Asn 120, which is the key residue involved in hydrogen bonding with the adenine ring of AMPPNP. AMPPNP has stronger interactions with the ATPase domain through hydrogen bonding interactions with the sugar and phosphate backbone. Therefore, modifica4



Full Papers tion of the benzo[b]tryptanthrin by adding a substituent to the pyrimidine ring that can participate in hydrogen bonding can be suggested based on the docking results. Taken together, the topo II inhibitory activity of benzo[b]tryptanthrin is attributed to its binding to the ATP-binding pocket of topo II in competition with ATP.

Benzo[b]tryptanthrin induces apoptosis through activation of caspase-3 and PARP The effect of benzo[b]tryptanthrin on cell migration and proliferation was monitored by a wound healing assay in both HEK293 human embryonic kidney and HCT15 colon cancer cells. Previously, it was reported that benzo[b]tryptanthrin showed the strongest cytotoxicity against HCT15 cells (6.15 0.23 mm) among the tested cancer cell lines;[21] therefore, HCT15 cells were used for the wound healing assay. Also, HEK293 is often used in the test for cell migration.[29] For these reasons, HEK293 and HCT15 cells were chosen and treated with 5, 10, and 20 mm of benzo[b]tryptanthrin after a wound was made. In untreated control cells, the gap was narrowed after 12 h, and the wound was completely covered after 24 h in HEK293 cells (Figure 6 A). A similar result for HCT15 cells was Figure 7. Benzo[b]tryptanthrin induced apoptosis through activation of caspase-3 and PARP in HCT15 cells. Cleaved caspase-3 and cleaved PARP were detected in HCT15 cells when treated with A) diverse concentrations of benzo[b]tryptanthrin (0, 15, 20, 25, and 30 mm) for 24 h or B) 25 mm benzo[b]tryptanthrin for diverse times (0, 8, 12, 24, and 30 h). C) and D) The levels of proteins in A) and B) were normalized to b-actin. Contents of benzo[b]tryptanthrin-treated or -untreated cells (control) are depicted as histograms. Values represent the mean SD obtained from three different experiments. **p < 0.01, ***p < 0.001 significantly different from the value of control.

dicate that benzo[b]tryptanthrin induced apoptosis in HCT15 colon cancer cells.

Figure 6. Wound healing assay. Benzo[b]tryptanthrin reduced cell migration and proliferation in a dose-dependent manner. Cells were cultured in sixwell plates until reaching 90 % confluence. A clean wound in a mono layer of cells across the center of the well was created with a sterile micro tip. After 4 h of starvation, the cells were exposed to benzo[b]tryptanthrin (0, 5, 10, or 20 mm) for 24 h or 48 h and allowed to migrate into the medium. The wound was assessed by a fluorescence inverted microscope with 5 magnification at different time points (0, 12, 24, and 48 h), respectively. A) HEK293 human embryonic kidney cells. B) HCT15 human colon cancer cells. Gray lines indicate the original wound edges.

Suppression of MDR1 gene expression and protein synthesis in benzo[b]tryptanthrin-treated MCF7adr cells Tryptanthrin was reported to have MDR-reversing effects through down-regulation of the MDR1 gene expression and protein synthesis.[31–33] Benzo[b]tryptanthrin has one more benzene ring attached to tryptanthrin (Figure 1). According to the previously reported results, benzo-annulation did not change the topo I inhibition activity, but noticeably enhanced the topo II inhibitory activity and cytotoxicity against HCT15 cells.[21] Therefore, we speculated whether benzo[b]tryptanthrin was able to inhibit MDR1 more effectively than tryptanthrin. We first evaluated the effect of the two compounds adriamycin and verapamil on the growth of MCF7 and MCF7adr cells (Table 2). Cells were treated with compounds for 72 h, and a cell viability assay was performed. As expected, the difference in IC50 values for adriamycin was large between MCF7 wild-type (2.63 0.99) and MCF7adr cells (> 50). In contrast, tryptanthrin and benzo[b]tryptanthrin showed more potent cytotoxicity in MCF7adr cells (10.01 0.030 and 9.82 0.16, re-

observed (Figure 6 B). The migration and proliferation of cells were inhibited in a concentration-dependent manner, as shown by retardation of the gap wound. Additionally, the apoptotic markers, cleaved caspase-3 and cleaved PARP,[30] were monitored by Western blot analysis of HCT15 cells treated with 15, 20, 25, and 30 mm of benzo[b]tryptanthrin for 24 h (Figure 7 A, C). The cleaved caspase-3 (17 and 19 kDa) and cleaved PARP (89 kDa) increased in a concentration-dependent manner. Also, the concentration of benzo[b]tryptanthrin was fixed at 25 mm for different treatment times of 8, 12, 24, and 30 h. Figures 7 B, D show that cleaved caspase-3 and cleaved PARP increased in a time-dependent manner. These results inChemMedChem 0000, 00, 0 – 0

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Full Papers Table 2. Cell proliferation inhibitory activities of tryptanthrin and benzo[b]tryptanthrin against MCF7 and MCFadr cells. IC50 [mm][a]

Compd Adriamycin Verapamil Tryptanthrin Benzo[b]tryptanthrin

MCF7

MCF7adr

2.63 0.99 > 50 15.51 0.58 12.07 0.27

> 50 > 50 10.01 0.03 9.82 0.16


spectively) than in MCF7 cells (15.51 0.58 and 12.07 0.27, respectively) (Table 2). To evaluate the MDR1-reversing effect of benzo[b]tryptanthrin, we co-treated MCF7adr cells with adriamycin and benzo[b]tryptanthrin. Verapamil was used as a positive control for adriamycin sensitization, because it is a wellknown MDR1 substrate and down-regulates MDR1 transport activity by blocking its efflux pump.[31, 34] Other than the effects of adriamycin (3 mm) on MCF7, treatment of verapamil (10 mm), tryptanthrin (5 mm), and benzo[b]tryptanthrin (5 mm) did not exhibit severe cytotoxicity in both MCF7 and MCF7adr cells at the concentrations used (Table 3). The same concentrations of Figure 8. Benzo[b]tryptanthrin down-regulated MDR1 mRNA and MDR1 protein in MCF7adr cells. After MCF7adr cells were treated with 5 mm tryptanthrin and 5 mm benzo[b]tryptanthrin for 72 h, A) MDR1 mRNA was monitored by semi-quantitative RT-PCR and B) the level of MDR1 protein was examined by Western analysis. The negative control was MDR1 in MCF7 cells. The contents of compound-treated or -untreated cells (control) are depicted as histograms. Values represent the mean SD obtained from three different experiments. *p < 0.05 and ***p < 0.001 significantly different from the value of control. C, control; T, tryptanthrin; B, benzo[b]tryptanthrin.

Table 3. Cell growth percentage after treatment with compound alone and co-treatment. Growth [%][a] MCF7 MCF7adr

Compd (concentration) Adriamycin (3 mm) Verapamil (10 mm) Tryptanthrin (5 mm) Benzo[b]tryptanthrin (5 mm) Adriamycin (3 mm) + Verapamil (10 mm) Adriamycin (3 mm) + Tryptanthrin (5 mm) Adriamycin (3 mm) + Benzo[b]tryptanthrin (5 mm)

24.1 3.89 87.8 4.78 94.2 3.50 82.1 4.10 20.9 1.90 10.7 0.40 19.3 1.60

78.2 0.44 82.8 0.49 96.4 0.63 75.3 0.94 18.1 0.39 76.6 0.39 59.0 0.40

(Table 3).[31] The mechanism of the MDR1-reversing effect of benzo[b]tryptanthrin was further studied by observing the MDR1 mRNA and protein levels of MDR1. MDR1 mRNA was evaluated by semi-quantitative RT-PCR (Figure 8 A). MDR1 mRNA was not expressed in MCF7 cells. In MCF7adr cells, MDR1 mRNA was expressed, and treatment with 5 mm benzo[b]tryptanthrin for 72 h down-regulated MDR1 mRNA. MDR1 protein expression was also inhibited by benzo[b]tryptanthrin (Figure 8 B). However, treatment with 5 mm tryptanthrin did not change the level of MDR1 mRNA nor the MDR1 protein, in contrast to results from a previous report.[31] On the contrary, it was reported that tryptanthrin was not a substrate of MDR1 and did not affect the expression of MDR1 but instead was a potent inhibitor of MDR1 in the Caco-2 monolayers.[33] This inconsistency in tryptanthrin activity as an MDR1 inhibitor may be attributed to different experimental designs, such as compound treatment time, concentration, and cell line used. In the present study, benzo[b]tryptanthrin showed better potency than tryptanthrin for reversing adriamycin resistance and down-regulating the level of MDR1 mRNA and protein in MCF7adr cells. This result demonstrates that benzo[b]tryptanthrin may overcome multidrug resistance through a decrease in MDR1 gene levels as well as the down-regulation of MDR1 protein levels under the applied conditions.


compounds were applied for co-treatment with adriamycin in MCF7adr cells to clearly assess their MDR1-reversing effects against adriamycin resistance. Treatment with 3 mm adriamycin alone for 72 h resulted in a cell growth percentage of 24.1 3.9 % relative to control (untreated cells) in MCF7 cells and 78.2 0.44 % in MCF7adr cells. The much lower cell growth inhibition in MCF7adr cells was attributed to overexpression of the MDR1 protein (Figure 8). MDR1 functions as an ATP-dependent efflux pump with broad substrate selectivity. Therefore, overexpression of MDR1 in cancer cells causes multidrug resistance by pumping out drugs.[31–33] In the case of co-treatment with adriamycin and verapamil, the growth percentage decreased from 78.2 % to 18.1 % in MCF7adr cells. Although it was not as effective as verapamil, benzo[b]tryptanthrin decreased the growth percentage to 59.0 %, showing the MDR1reversing effect. However, tryptanthrin did not show an effective MDR1-reversing effect, contrary to previous observations

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Full Papers Conclusions

(FBS; HyClone, USA) and 1 % penicillin–streptomycin (HyClone, USA). HEK293 cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM; HyClone, USA) containing 10 % FBS and 1 % penicillin–streptomycin. Cells were grown in a humidified 5 % CO2 incubator at 37 8C, and the media was changed every 2 to 3 days.

Benzo[b]tryptanthrin functions as a DNA non-intercalative topo I and II dual catalytic inhibitor and especially inhibits topo II by blocking ATP binding and ATP hydrolysis of topo II. In addition, benzo[b]tryptanthrin reversed adriamycin resistance in MDR1-overexpressing breast cancer cells, MCF7adr, through down-regulation of MDR1 gene and protein levels. Topo poisons stabilize the transiently formed DNA–topo covalent complex leading to generation of DNA lesions. Some of the most clinically active chemotherapeutics are topo poisons, including etoposide, doxorubicin, and mitoxantrone.[19b] In general, topo poisons necessitate monitoring when used as medication, due to the severe genetic toxicity. While a topo catalytic inhibitor does not increase the levels of the DNA–topo covalent complex, it induces cancer cell death through the elimination of the essential enzymatic activity.[19] For these reasons, many researchers have focused on developing topo catalytic inhibitors rather than topo poisons. Furthermore, overexpression of MDR1 has been reported in various cancers during medication with anticancer drugs and has become a major source of failure of cancer chemotherapy.[35] Benzo[b]tryptanthrin, possessing multi-anticancer functions such as inhibiting topo I and II catalytic activity and reversing MDR1-mediated adriamycin resistance, can be a novel scaffold for developing a potent topo-targeting drug with less risk of inducing resistance.

Kinetoplast DNA relaxation assay: The topo II specificity of the compound was determined by a Kinetoplast DNA (kDNA) decatenation assay.[38] The assay was performed in a total reaction volume of 10 mL containing 10 mm Tris-HCl (pH 7.9), 50 mm NaCl, 50 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, 15 mg mL 1 bovine serum albumin (BSA), 1 mm ATP, and 50 ng kDNA (TopoGEN, USA). Compounds were then added at the desired concentrations with a 1 % total content of DMSO. Then, 3 units of topo IIa (USB, USA) were added to initiate the reaction and incubated at 37 8C for 30 min. Stop solution (2 mL; 5 % SDS, 25 % ficoll, and 0.05 % bromophenol blue) was added to terminate the reaction, and then 20 mg Proteinase K (Roche, USA) was added at 55 8C over 30 min. Samples were loaded onto a 1.2 % agarose gel containing 0.5 mg mL 1 ethidium bromide in TAE buffer and electrophoresed at 20 V for 3 h. The result was visualized and quantified by AlphaImager (Alpha Innotech Corporation). Ellipticine was used as the positive control. Cleavage complex stabilization assay: First, 125 ng of supercoiled DNA pBR322 (Fermentas, USA) was mixed with 3 units of topo IIa (USB, USA) in topo II reaction buffer (10 mm tris-HCl (pH 7.9) containing 50 mm NaCl, 50 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, 15 mg mL 1 BSA, and 1 mm ATP) to obtain a total reaction volume of 10 mL, and the reaction mixture was incubated at room temperature for 5 min. Then, the compound was added and incubated at 37 8C for 20 min. The reaction was terminated by adding 1 mL of stop solution (10 % SDS) and incubating at 37 8C for an additional 5 min. Proteinase K (20 mg) was added to digest the protein during incubation at 45 8C for 30 min. After adding the loading buffer, the samples were heated for 2 min at 70 8C. The samples were loaded onto a 0.8 % agarose gel containing 0.5 mg mL 1 ethidium bromide in TAE buffer, and electrophoresis was carried out at 20 V for 6 h.

Experimental Section Chemistry Benzo[5,6]indolo[2,1-b]quinazoline-6,14-dione (benzo[b]tryptanthrin) was prepared as previously published.[19] The 1H NMR spectra of the compound are in consensus with the published data (Supporting Information).

Comet assay: The comet assay is a simple method for evaluating the DNA damage in cells. The Alkaline Comet Assay kit (Trevigen, USA) was used following the provided protocols. HCT15 cells were seeded in a six-well plate (1 105 cells per well) and incubated at 37 8C. The next day, 10 mm of benzo[b]tryptanthrin or etoposide was added to serum-free media for 24 h. An 8 mL aliquot of cells was combined with 80 mL molten low-melting agarose at 37 8C, and the mixture was pipetted onto the slides and placed at 4 8C in the dark for 40 min. The slides were then immersed in cold lysis buffer (4 8C) for 30 min at 4 8C in the dark. Subsequently, the slides were immersed in unwinding buffer (200 mm NaOH, 1 mm EDTA) for 30 min at room temperature. The slides were then electrophoresed in alkaline electrophoresis buffer (200 mm NaOH, 1 mm EDTA, pH > 13) at 1 V cm 1 for 15–30 min. The slides were washed by immersing them in distilled water twice and in 70 % EtOH once, followed by drying at 42 8C for 40 min. The cells were stained with diluted SYBR Green at 4 8C for 5 min in the dark and analyzed using a Zeiss HBO100 microscope illumination system (Carl Zeiss, Germany) and image analysis Komet software (Komet 5.5, Kinetic Imaging Ltd., UK).

The log P of benzo[b]tryptanthrin was 3.27, which was estimated by Crippen’s fragmentation[36] and Viswanadhan’s fragmentation.[37] A 10 mm stock solution of benzo[b]tryptanthrin was prepared with dimethyl sulfoxide (DMSO; Sigma–Aldrich, USA) and diluted to the indicated experimental concentrations in media or experimental buffer. There was no solubility problem for the experimental conditions executed. Etoposide, ellipticine, adriamycin, novobiocin, and verapamil were purchased from Sigma–Aldrich (USA).

Biological methods Cell culture: The primary antibodies for caspase-3, PARP, MDR1, and b-actin, and anti-IgG secondary antibody, were purchased from Cell Signaling Technology, Inc. (USA). HCT15 (human colon cancer), HEK293 (human kidney embryonic), T47D (human breast cancer), MCF-7 (human breast cancer), and DU145 (human prostate cancer) cell lines were purchased from the Korean Cell Line Bank (KCLB, Seoul, Korea). MCF-7adr (adriamycin resistance-induced human breast cancer) and DLD1 (human colon cancer) cell lines were provided by Professors H. J. Lee and Y. S. Lee (Ewha Womans University, Korea). HCT15, T47D, DU145, DLD1, MCF-7, and MCF-7adr cells were cultured in Roswell Park Memorial Institute Medium (RPMI 1640; HyClone, USA) containing 10 % fetal bovine serum


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DNA unwinding assay: The DNA-unwinding capacity of benzo[b]tryptanthrin was analyzed using a DNA unwinding kit (TopoGEN, Port Orange, FL, USA), according to the manufacturer’s instructions. Briefly, 100 ng plasmid (pHOT1) was treated with 4 units of topo I (TopoGEN, USA) in a total of 10 mL of reaction buffer (10 mm Tris-HCl, pH 7.9, containing 1 mm EDTA, 1.5 m NaCl, 1 % BSA, 1 mm

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Full Papers spermidine, and 50 % glycerol) for 30 min at 37 8C. Relaxed plasmids were then incubated in the presence of various test compounds at 37 8C for an additional 30 min. m-AMSA (100, 200, 500, and 1000 mm) was used as a positive control for DNA unwinding activity. At the end of the incubation, 1 mL of stop solution (5 % sarcosyl, 0.0025 % bromophenol blue, and 25 % glycerol) was added to terminate the reactions. The samples were resolved on 1 % agarose gels at 15 V cm 1 for 12–15 h. After electrophoresis, gels were stained in TAE buffer with ethidium bromide for 30 min and visualized using an AlphaImager.

centrations of compounds. Compounds dissolved in DMSO were diluted with serum-free media and treated alone or in combination with cells at designated concentrations (Table 3). Then, cells were incubated at 37 8C in a 5 % CO2 incubator for 72 h, followed by addition of 5 mL of cell counting kit-8 solution (Dojindo, Japan) to each well and additional incubation for 4 h under the same conditions. The absorbance of each well was then measured with an automatic ELISA reader system (Bio-Rad 3550) at a wavelength of 450 nm. To determine IC50 values, absorbance readings at 450 nm were fitted to a four-parameter logistic equation using TableCurve (Systat Software, Inc., USA).

ATPase assay: The DNA-dependent ATPase activity of human topo IIa was monitored using a malachite green phosphate assay kit (BioAssay Systems, USA) according to the previous method with a slight modification.[39, 40] In a 96-well plate, reaction buffer (10 mm Tris-HCl, pH 7.9, 50 mm NaCl, 50 mm KCl, 5 mm MgCl2, 0.1 mm EDTA, and 15 mg mL 1 BSA), supercoiled DNA pBR322 (100 ng), ATP (250 mm), compound at the desired concentration, and 4 units of topo IIa were mixed and incubated at 37 8C for 1 h. Working reagent (20 mL) from the malachite green phosphate assay kit was added to each well, and the reactions were incubated for an additional 30 min at 37 8C. The absorbance of the reaction solution was read at 620 nm using a VERSAmax microplate reader (Molecular Devices, USA). The OD value was determined by the amount of inorganic phosphate complexed with the malachite green and acidic molybdate. The inorganic phosphate is the product released from ATP hydrolysis performed by human topo IIa. The blank OD value was < 0.1, showing that no inorganic phosphate existed before ATP hydrolysis. The percent inhibition of ATP hydrolysis was calculated by setting the control without the compound as 0 % inhibition. Novobiocin, a topo II-catalyzed ATP hydrolysis inhibitor, was used as the positive control.[28]

Western blot analysis: HCT15 cells were grown in 60 mm tissue culture dishes at a density of 1 106 cells until reaching 80 % confluence. The cells were then treated with benzo[b]tryptanthrin at increasing concentrations (0, 15, 20, 25, and 30 mm) for 24 h. For the time-dependent analysis, cells were treated with 25 mm of benzo[b]tryptanthrin for increasing times (0, 8, 12, 24, and 30 h). Cells were lysed in lysis buffer solution containing 50 mm Tris-HCl (pH 7.4), 300 mm NaCl, 1 % Triton X-100, 10 % glycerol, 1.5 mm MgCl2, 1 mm CaCl2, 1 mm phenylmethanesulfonyl fluoride (PMSF), and 1 % protease inhibitor cocktail. Protein concentrations of soluble extracts of lysates were then determined using a BCATM Protein Assay Kit (Pierce, USA), and a total of 70 mg protein per sample was loaded on a 8 % polyacrylamide gel, which was then transferred to a poly vinylidene fluoride membrane (Millipore Corporation, USA) at 200 mA for 1–2 h. Next, membranes were blocked with 5 % skim milk in Tris-buffered saline containing 0.1 % tween 20 (TBST) for 1 h at room temperature. After washing the membranes with TBST every 20 min for three times, they were incubated with primary antibodies diluted at a ratio of 1:1000 in TBST with 5 % skim milk at 4 8C overnight. The next day, the membranes were washed with TBST every 20 min for three times and were then incubated with diluted secondary anti-rabbit IgG horseradish peroxidase antibody at a ratio of 1:2000 of for 2 h at room temperature. Membranes were washed three times with TBST and detected with ECL western blotting detection reagent (Animal Genetics, Inc., Korea).[42] Western blot images were analyzed using Multi-Gauge Software (Fuji Photo Film Co., Ltd., Japan).

ATP competition assay: The reaction mixture was prepared by adding solutions as follows: 1 mL supercoiled DNA pBR322 (100 ng mL 1), 1 mL 10 reaction buffer (100 mm tris-HCl, pH 7.9, containing 500 mm NaCl, 500 mm KCl, 50 mm MgCl2, 1 mm EDTA and 150 mg mL 1 BSA), 1 mL ATP (10 mm or 15 mm), 1 mL topo IIa (0.4 units, USB, USA), 0.5 mL of each investigational compound solution, and the required amount of double-distilled water (DDW) to obtain a final reaction volume of 10 mL. The tested concentrations of benzo[b]tryptanthrin were 5, 10, 20, 50, 100, 200, and 500 mm. The reaction mixtures were then incubated at 37 8C for 1 h. Reactions were terminated by addition of 2 mL topo IIa stop buffer, and the reaction mixtures were then resolved on 0.8 % agarose gels at 60 V for 1 h. After electrophoresis, gels were stained in TAE buffer with ethidium bromide for 15 min and visualized using an AlphaImager. Etoposide was used as the positive control.

Semi-quantitative RT-PCR analysis: MCF7 and MCF7adr cells were seeded in 60 mm tissue culture dishes at a density of 1 106 cells until reaching 80 % confluence, followed by treatment with each compound at 5 mm for 72 h. Total RNA was extracted from untreated- (control) and treated cells using the QuickGene SP kit (FujiFilm, Japan), according to the manufacturer’s protocol. RNA was quantified by absorbance spectroscopy. cDNA was synthesized from the extracted RNA using the following reaction: 4 mL of 5 RT buffer, 2 mL of 2.5 mm dNTPs, 2 mL of random primer (0.1 mg mL 1), 0.5 mL of RNase inhibitor (Promega, USA), 0.25 mL of M-MLV reverse transcriptase (Promega, USA), and 2.0 mg of extracted RNA. The reaction mixture was incubated at 25 8C for 5 min, followed by incubation at 42 8C for 1 h and an additional incubation at 70 8C for 15 min. The synthesized cDNA was stored at 20 8C until needed. Each synthesized cDNA was amplified using PCR with the following PCR cocktail: addition of 5 mL of 10 reaction buffer, 3 mL of 10 mm dNTPs, 1.25 U of Taq polymerase, 2 mL of cDNA, and 0.5 mL of each of forward/reverse primers to a final reaction volume of 50 mL. PCR was performed on a thermal cycler (PerkinElmer, USA) with an initial denaturation at 94 8C for 4 min, followed by 28 cycles with denaturation at 94 8C for 45 s, annealing at 48 8C for 45 s, extension for 72 8C for 45 s, and a final extension at 70 8C for 10 min. The primer sequences used for PCR were adapted from published results: MDR1 forward 5’-CCCATCATTGCAATAGCAGG-3’

Cell migration assay: HEK293 and HCT15 cells were cultured in sixwell plates to 90 % confluence. A clean wound in the cell monolayer was made across the center of the well with a sterile micro tip. After starvation with low-serum medium (1 % FBS in medium) for 4 h, cells were exposed to the compound at either 5, 10, or 15 mm and allowed to migrate into the medium. The wound was assessed by microscopy at 5 magnification at different time points (0, 12, 24, and 48 h). MCF7 and MCF7adr viability assay: Cells were seeded in 96-well plates at a density of 1 104 cells per well and incubated overnight in 0.1 mL of RPMI 1640 supplemented with 10 % FBS (HyClone, USA) in an incubator at 37 8C in 5 % CO2 atmosphere.[41] Before treatment with compounds, the cells were stabilized in serum-free media for 4 h. Then, the culture medium in each well was exchanged with 0.1 mL aliquots of medium containing varying con-

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Full Papers and reverse 5’GTTCAAACTTCTGCTCCTGA-3’ primers; GAPDH forward 5’-AGTCAACGGATTTGGTCGTA-3’ and reverse 5’-GGAACATGTAAACCATGTAG-3’ primers.[31] PCR products were electrophoresed on a 2 % agarose gel to analyze the amplification level of each gene. After electrophoresis, gels were stained in TAE buffer containing ethidium bromide for 30 min and visualized using an AlphaImager.

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Molecular docking studies The coordinates for the ATP-binding domain of human topo IIa were retrieved from the Protein Data Bank (PDB ID: 1ZXM).[43] The water molecules were removed, and the hydrogen atoms were added to the original crystal structure. The magnesium ion was assigned a charge of + 0.9.[44] The coordinate file for the structure of benzo[b]tryptanthrin was constructed by Sybyl 8.0 and minimized energetically using a Tripos force field with Gasteiger–Hckel charges. The receptor and ligand file were prepared according to the original publication protocols.[45] Gasteiger charges computed by ADT (AutoDock tools) were used on the atoms for the ligand. Docking was carried out with simulations using the Lamarckian Genetic Algorithm. Default search parameters were used, except for a population size of 270 000 and 50 docking runs. Finally, the docking was analyzed by clustering the orientations lying within 2.0 of the root-mean square deviation (RMSD) tolerance of each other to see the most favorable docking mode.

Acknowledgements This research was supported by the Basic Science Research Program through the National Research Foundation of Korea, (NRF) funded by the Ministry of Education, Science and Technology (NRF-2013R1A1A2060408). K.-Y.J. was supported by RP-Grant 2013 of Ewha Womans University. Keywords: adriamycin · ATP competitive benzo[b]tryptanthrin · MDR1 · topoisomerases

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Received: February 13, 2015 Published online on && &&, 0000

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FULL PAPERS K.-Y. Jun, S.-E. Park, J. L. Liang, Y. Jahng, Y. Kwon*

Double threat! Benzo[b]tryptanthrin is a topo I and II dual catalytic inhibitor. Benzo[b]tryptanthrin was found to induce apoptosis through cleavage of caspase-3 and PARP in HCT15 colon cancer cells and to reverse adriamycin resistance by down-regulating multidrug resistance protein 1 (MDR1) in adriamycin-resistant MCF7 breast cancer cells (MCF7adr).

&& – && Benzo[b]tryptanthrin Inhibits MDR1, Topoisomerase Activity, and Reverses Adriamycin Resistance in Breast Cancer Cells

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ADR cells via downregulation of MDR1.

Germacrone reverses Adriamycin resistance through cell apoptosis in multidrug-resistant breast cancer cells.

Recombinant human interleukin 24 reverses Adriamycin resistance in a human breast cancer cell line.

P-glycoprotein.

Nitric oxide inhibits ATPase activity and induces resistance to topoisomerase II-poisons in human MCF-7 breast tumor cells.

Nitric oxide inhibits topoisomerase II activity and induces resistance to topoisomerase II-poisons in human tumor cells.

Knockdown of microRNA-127 reverses adriamycin resistance via cell cycle arrest and apoptosis sensitization in adriamycin-resistant human glioma cells.

Nanolipoparticles-mediated MDR1 siRNA delivery reduces doxorubicin resistance in breast cancer cells and silences MDR1 expression in xenograft model of human breast cancer.

Identification of proteins responsible for adriamycin resistance in breast cancer cells using proteomics analysis.

Inhibition of KLF4 by Statins Reverses Adriamycin-Induced Metastasis and Cancer Stemness in Osteosarcoma Cells.

Resveratrol reverses multidrug resistance in human breast cancer doxorubicin-resistant cells.

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Letter: Adriamycin in advanced breast cancer.

Letter: Adriamycin in advanced breast cancer.

β-arrestin 2 is associated with multidrug resistance in breast cancer cells through regulating MDR1 gene expression.

ABCB1 (MDR1) induction defines a common resistance mechanism in paclitaxel- and olaparib-resistant ovarian cancer cells.

Hyaluronidase enhances the activity of adriamycin in breast cancer models in vitro and in vivo.

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β-catenin signaling and exerts anticancer activity in breast cancer cells.

Reversal of adriamycin resistance by lonidamine in a human breast cancer cell line.

β-catenin activity and reverses the osteogenic transformation of vascular smooth muscle cells.

Combination of siRNA-directed gene silencing with epigallocatechin-3-gallate (EGCG) reverses drug resistance in human breast cancer cells.

ABCG2 gene amplification and expression in esophageal cancer cells with acquired adriamycin resistance.

Cancer: Multifunctional nanodevice reverses drug resistance.