Hernandezine, a Bisbenzylisoquinoline Alkaloid with Selective

Article pubs.acs.org/jnp

Hernandezine, a Bisbenzylisoquinoline Alkaloid with Selective Inhibitory Activity against Multidrug-Resistance-Linked ATP-Binding Cassette Drug Transporter ABCB1 Sung-Han Hsiao,† Yu-Jen Lu,‡ Chun-Chiao Yang,† Wei-Cherng Tuo,† Yan-Qing Li,§ Yang-Hui Huang,⊥ Chia-Hung Hsieh,∥,□ Tai-Ho Hung,¶,# and Chung-Pu Wu*,†,‡,§,⊥ †

Graduate Institute of Biomedical Sciences, §Department of Physiology and Pharmacology, ⊥Molecular Medicine Research Center, and ¶Department of Chinese Medicine, College of Medicine, Chang Gung University, Tao-Yuan, Taiwan ‡ Department of Neurosurgery, Chang Gung Memorial Hospital, Tao-Yuan, Taiwan # Department of Obstetrics and Gynecology, Taipei Chang Gung Memorial Hospital, Taipei, Taiwan ∥ Graduate Institute of Basic Medical Science and □Department of Medical Research, China Medical University Hospital, Taichung, Taiwan ABSTRACT: The overexpression of ATP-binding cassette (ABC) drug transporter ABCB1 (P-glycoprotein, MDR1) is the most studied mechanism of multidrug resistance (MDR), which remains a major obstacle in clinical cancer chemotherapy. Consequently, resensitizing MDR cancer cells by inhibiting the efflux function of ABCB1 has been considered as a potential strategy to overcome ABCB1-mediated MDR in cancer patients. However, the task of developing a suitable modulator of ABCB1 has been hindered mostly by the lack of selectivity and high intrinsic toxicity of candidate compounds. Considering the wide range of diversity and relatively nontoxic nature of natural products, developing a potential modulator of ABCB1 from natural sources is particularly valuable. Through screening of a large collection of purified bioactive natural products, hernandezine was identified as a potent and selective reversing agent for ABCB1-mediated MDR in cancer cells. Experimental data demonstrated that the bisbenzylisoquinoline alkaloid hernandezine is selective for ABCB1, effectively inhibits the transport function of ABCB1, and enhances drug-induced apoptosis in cancer cells. More importantly, hernandezine significantly resensitizes ABCB1-overexpressing cancer cells to multiple chemotherapeutic drugs at nontoxic, nanomolar concentrations. Collectively, these findings reveal that hernandezine has great potential to be further developed into a novel reversal agent for combination therapy in MDR cancer patients.

T

(MRP1; ABCC1) and ABCG2 (BCRP; MXR) were subsequently discovered and are capable of reducing the effect of chemotherapy.2 Furthermore, based on the localization of ABCB1 in human tissues, its transport function can significantly affect the adsorption, distribution, metabolism, elimination, and toxicity of many types of anticancer drugs.7 Consequently, the overexpression of ABCB1 can lead to the development of MDR phenotype and treatment failure.2 The concept of “chemosensitization” is to introduce a compound at a nontoxic concentration that can selectively modulate the function and/or expression of a particular drug transporter such as ABCB1 and elevate drug accumulation in MDR cancer cells.2 Among the various strategies that have been explored, direct and transient inhibition of the transport function of ABCB1 is currently the most efficient way to overcome MDR in ABCB1-overexpressing cancer cells.8 For

he development of multidrug resistance (MDR) is a major obstacle in cancer chemotherapy. After a prolonged treatment with conventional chemotherapeutic agents, cancer cells can spontaneously become insensitive to drugs that are structurally or mechanically unrelated.1 Although multiple mechanisms are known to contribute to an MDR phenotype, the overexpression of some members of the ATP-binding cassette (ABC) protein family is thought to be the major contributor to the development of MDR in cancer cells.2 ABCB1 (P-glycoprotein/MDR1) is the first human ABC protein discovered to confer resistance to anticancer drugs.3,4 Composed of two transmembrane domains and two nucleotide-binding domains, ABCB1 utilizes energy derived from ATP hydrolysis to actively efflux anticancer drugs out of cancer cells, reducing their intracellular concentration and cytotoxicity.5 ABCB1 is capable of transporting a wide variety of clinically active anticancer agents, including the majority of conventional anticancer drugs5 and many protein kinase inhibitors.6 Other ABC transporters such as the multidrug resistant protein 1 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: July 13, 2016

A

DOI: 10.1021/acs.jnatprod.6b00597 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Figure 1. Hernandezine inhibits the transport function of ABCB1, but not ABCC1 or ABCG2. (A) Chemical structure of hernandezine. The accumulation of fluorescent calcein in ABCB1-transfected MDR19-HEK293 (B, right panel) or in ABCC1-transfected MRP1-HEK293 (C, right panel) or in parental HEK293 (B and C, left panel) cells, as well as the accumulation of pheophorbide A (PhA) in HEK293 (D, left panel) and ABCG2-transfected R482-HEK293 (D, right panel) cells was measured in the absence (solid lines) or presence of 1 μM hernandezine (shaded, solid lines) or a reference inhibitor (dotted lines) of ABCB1 (3 μM tariquidar), ABCC1 (25 μM MK-571), or ABCG2 (5 μM FTC). Cells were analyzed immediately by flow cytometry as described in the Experimental Section. Representative histograms and immunoblots of ABCB1 (B, inset), ABCC1 (C, inset), or ABCG2 (D, inset) in total cell lysate protein (10 μg) from pcDNA-HEK293, MDR19-HEK293, MRP1-HEK293, and R482-HEK293 cells of three independent experiments are shown. (E) Concentration-dependent inhibition of ABCB1-mediated calcein-AM efflux (open circles) or ABCC1-mediated calcein efflux (filled circles) or ABCG2-mediated PhA efflux (open squares) by hernandezine in MDR19-HEK293, MRP1HEK293, or R482-HEK293 cells. The values represent mean ± SEM from at least three independent experiments.

decades, the task of finding a suitable modulator of ABCB1 has been hindered mostly by the complex pharmacokinetics and the high intrinsic toxicity of candidate compounds.2,9 A large number of clinically active drugs including immunosuppressants, channel blockers, and tyrosine kinase inhibitors have all been tested for their ability to reverse ABCB1-mediated MDR. However, since these drugs were not originally designed to interact with ABCB1, higher drug concentrations were required to inhibit the function of ABCB1, causing unfavorable side effects and adverse drug−drug interactions.9,10 Moreover, the difficulty in finding an ideal modulator can also be associated with the lack of specificity. For instance, tariquidar, the most potent third-generation inhibitor of ABCB1 that has been used for years as the reference inhibitor, was later identified as a substrate of ABCG2.11,12 As a result, none of the thirdgeneration MDR modulators have been approved to treat patients with MDR tumors.13,14 Currently, discovering fourthgeneration modulators of ABCB1 through the high-throughput

screening (HTS) approach is under way.13 The term “fourthgeneration modulators” refers to compounds originating from natural sources, which offer the widest range of diversity and novel chemical scaffolds and, more importantly, are usually low in toxicity and much better tolerated in the human body.13 It was reported that approximately 80% of cancer patients use natural products in combination with anticancer drugs for the potential benefits in chemoprevention and inhibition of cancer progression.15 Many nontoxic natural products or metabolites, including various extracts and active components ranging from plants and fungi to marine organisms, have already been investigated to overcome MDR in cancer cells with some success.13 Unfortunately, although a large number of natural product modulators of ABCB1 have been identified in recent years, most of them lack the combination of potency and selectivity.9,13 This prompted us to search for a potent and selective modulator of ABCB1 deriving from natural sources. B



Article

Table 1. Effect of Hernandezine on ABC Transporter-Mediated Drug Resistance in HEK293 Cells Transfected with ABCB1, ABCG2, or ABCC1 IC50 (nM)a treatment

concentration (nM)

doxorubicin + hernandezine + hernandezine + hernandezine + hernandezine + tariquidar

50 100 200 500 1000

pcDNA-HEK293 (parental)

MDR19-HEK293 (resistant)

RRb

± ± ± ± ± ±

504.65 ± 44.94 109.00 ± 29.82*** 75.77 ± 13.10*** 43.93 ± 4.33*** 25.27 ± 1.86*** 24.77 ± 3.36***

96 16 11 8 7 5

5.28 6.99 6.81 5.63 3.85 4.95

0.74 0.84 1.00 0.70 0.66 0.86 IC50 (μM)a

etoposide + hernandezine + MK-571

500 25 000


MRP1-HEK293 (resistant)

0.11 ± 0.03 0.07 ± 0.01 0.06 ± 0.01

38.54 ± 5.62 38.32 ± 5.78 5.22 ± 0.89***

350 547 87

IC50 (nM)a mitoxantrone + hernandezine + FTC

500 3000


R482-HEK293 (resistant)

1.00 ± 0.19 1.21 ± 0.23 0.82 ± 0.17

107.40 ± 13.93 85.08 ± 12.09 5.42 ± 0.97***

107 70 7

a IC50 values are mean ± SD calculated from concentration−response curves obtained from at least three independent experiments. bRR, relative resistance values, were calculated by dividing IC50 values of ABC transporter overexpressing cells by IC50 values of respective parental cells. *p < 0.05; **p < 0.01; ***p < 0.001.

HEK293), human ABCC1 (MRP1-HEK29), or human wildtype ABCG2 (R482-HEK29). The reference inhibitor tariquidar (3 μM), MK-571 (25 μM), or fumitremorgin C (FTC, 5 μM) was used as a positive control to inhibit the function of ABCB1, ABCC1, or ABCG2, respectively. Without affecting the parental HEK293 cells (Figure 1B−D, left panels), hernandezine (1 μM) significantly increased the accumulation of calcein-AM, a known substrate of ABCB1,24 in MDR19HEK293 cells (Figure 1B, right panel). In contrast, hernandezine had no significant effect on the accumulation of calcein in MRP1-HEK293 (Figure 1C, right panel) or pheophorbide A (PhA)25 in R482-HEK293 cells (Figure 1D, right panel). Moreover, ABCB1-mediated transport of calceinAM was selectively inhibited by hernandezine in a concentration-dependent manner, with a calculated IC50 of approximately 300 nM (Figure 1E). Next, the reversal effect of hernandezine on drug resistance mediated by either ABCB1, ABCC1, or ABCG2 was determined in the same cell lines (summarized in Table 1). The relative resistance value (RR) represents the relative resistance of respective MDR cells to a particular anticancer agent in the presence or absence of a reversing agent, which was calculated by dividing the IC50 value of drug-resistant cells by the IC50 value of the respective parental cells in the presence of a particular drug.26 At nontoxic concentrations of 50, 100, 200, and 500 nM, hernandezine substantially sensitized ABCB1-overexpressing MDR19-HEK293 cells to doxorubicin, reducing the RR value from approximately 96 to 16, 11, 8, and 7, respectively. In contrast, hernandezine exhibited no activity against ABCC1-mediated resistance to etoposide or ABCG2mediated resistance to mitoxantrone, which are established substrates of ABCC1 and ABCG2, respectively.2 Of note, hernandezine at these tested concentrations did not affect the IC50 values of doxorubicin, etoposide, or mitoxantrone in parental HEK293 cells as shown in Table 1. Our results suggest that hernandezine resensitizes ABCB1-transfected MDR19HEK293 cells to doxorubicin treatment by selectively

Hernandezine is a biologically active constituent bisbenzylisoquinoline alkaloid of Thalictrum f lavum (Ranunculaceae),16 which has not previously been reported as a selective modulator of ABCB1 to our knowledge, but has been identified as having many biological activities.16−22 Hernandezine has been shown capable of protecting hair cells from aminoglycoside-induced damage,21 inhibiting protein kinase C signaling events in human peripheral blood T cells19 and neuronal nicotinic acetylcholine receptors (nAChRs),20 and blocking non-voltageoperated Ca2+ entry activated by intracellular Ca2+ store depletion induced by thapsigargin in rat glioma C6 cells18 and in human leukemic HL-60 cells.17 Moreover, hernandezine was identified recently as the most effective isoquinoline alkaloid to induce autophagic cell death via direct activation of AMP-activated protein kinase (AMPK).22 In the present study, the experimental results demonstrate that hernandezine selectively inhibits the function of ABCB1 relative to MDRlinked ABC drug transporters ABCC1 and ABCG2. More importantly, hernandezine significantly enhances drug-induced apoptosis and reverses ABCB1-mediated multidrug resistance in cancer cells at nanomolar concentrations.

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RESULTS AND DISCUSSION Hernandezine Selectively Inhibits ABCB1-Mediated Drug Efflux. A high-throughput screening of a compound library consisting of 502 purified bioactive natural products (Enzo Life Sciences Inc.) was conducted according to the method described by Ansbro et al.23 with the intention to identify novel ABCB1-inhibiting compounds derived from natural sources. Hernandezine (Figure 1A) was the novel candidate identified from the screen to exhibit the strongest interaction with human ABCB1 and was selected for subsequent assays. The selectivity of hernandezine was first examined in a short-term drug accumulation assay as described in the Experimental Section. The effect of hernandezine on the accumulation of fluorescent dye substrates was determined in HEK293 cells transfected with either human ABCB1 (MDR19C



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Figure 2. Hernandezine attenuates the function of ABCB1 and reverses ABCB1-mediated MDR in ABCB1-overexpressing human cancer cells. The accumulation of fluorescent calcein in drug-sensitive human epidermal KB-3-1 (A, left panel) and its ABCB1-overexpressing variant KB-V-1 (A, right panel) cancer cell lines or in drug-sensitive human ovarian OVCAR8 (C, left panel) and its ABCB1-overexpressing variant NCI-ADR-RES (C, right panel) cancer cell lines was measured in the absence (solid lines) or presence of 1 μM hernandezine (shaded, solid lines) or 3 μM tariquidar (dotted lines) and analyzed immediately by flow cytometry as described in the Experimental Section. Representative histograms and immunoblots of ABCB1 in total cell lysate protein (10 μg) from KB-3-1 and KB-V-1 (A, inset) or OVCAR8 and NCI-ADR-RES (C, inset) cells of three independent experiments are shown. Cytotoxicity of doxorubicin in (B) KB-V-1 cells and (D) NCI-ADR-RES cancer cells was measured in the absence (open circles) or presence of hernandezine at 50 nM (filled circles), 100 nM (open squares), 200 nM (filled squares), 500 nM (open triangles), or 1 μM tariquidar (filled triangles). Points: means from at least three independent experiments; bars: SEM.

attenuating the function of ABCB1. Considering the overlapping substrate specificity of ABCB1, ABCC1, and ABCG2,2,5 it was surprising to discover that not only did hernandezine inhibit the drug transport and drug resistance mediated by ABCB1, hernandezine was selective for ABCB1 relative to ABCC1 and ABCG2. Hernandezine Resensitizes ABCB1-Overexpressing Cancer Cells to Anticancer Drugs. Knowing that hernandezine is selective for ABCB1 relative to ABCC1 and ABCG2, the effect of hernandezine on ABCB1-mediated drug transport and MDR was evaluated in paired drug-sensitive and ABCB1-overexpressing drug-resistant human cancer cell lines. The human epidermal KB-V-1 and ovarian NCI-ADR-RES cancer cell lines are the ABCB1-overexpressing MDR sublines of KB-3-1 and OVCAR8, which are highly resistant to doxorubicin, colchicine, and vincristine. These drugs are wellestablished ABCB1 substrates with calculated RRs of 34, 55, and 356 in KB-V-1 cells and 46, 59, and 435 in NCI-ADR-RES cells, respectively.2,27 The efflux of calcein-AM and doxorubicin resistance mediated by ABCB1 was completely inhibited and reversed by hernandezine in KB-V-1 (Figure 2A and B) and NCI-ADR-RES cells (Figure 2C and D). Moreover, hernandezine significantly resensitized KB-V-1 and NCI-ADR-RES cancer cells to doxorubicin, colchicine, and vincristine in a concentration-dependent manner as summarized in Table 2. As a positive control, a higher concentration of tariquidar (1 μM) was used to show complete reversal of ABCB1-mediated drug resistance. These results indicate that even at nanomolar concentrations (50−500 nM), hernandezine can be used to

potentiate the efficacy of various anticancer agents in ABCB1overexpressing MDR cancer cells. Hernandezine Does Not Affect the Expression of ABCB1. In addition to inhibiting the function of ABCB1, reversal of ABCB1-mediated MDR can also be achieved by transient down-regulation of ABCB1 expression.28,29 For that reason, the effect of hernandezine on protein expression of ABCB1 was examined after treating KB-3-1 and KB-V-1 cells with increasing concentrations of hernandezine (0−1 μM) for 72 h, harvesting, and processing the cells for immunoblotting as described in the Experimental Section. As shown in Figure 3, hernandezine did not significantly alter the protein expression level of ABCB1 in KB cancer cells. Hernandezine Enhances Colchicine-Induced Apoptosis in ABCB1-Overexpressing Cancer Cells. Next, the potentiating effect of hernandezine on drug-induced apoptosis in ABCB1-overexpressing cancer cells was examined as described in the Experimental Section. After exposing human KB cancer cells to 500 nM colchicine for 48 h, a considerable increase in apoptotic cells was detected in drug-sensitive KB-31 cells, while a lesser effect was observed in drug-resistant KBV-1 cells (Figure 4B). As shown in Figure 4C, colchicine treatment resulted in a significant increase of apoptotic cell population, from 10−13% basal level to approximately 56% of early and late apoptosis in KB-3-1 cells, in contrast to 19% in KB-V-1 cells. Notably, the percentage of apoptosis induced by colchicine in KB-V-1 cells can be restored from 10% basal level to approximately 47% and 61% apoptotic cell population when the function of ABCB1 was blocked by hernandezine (1 μM) or tariquidar (1 μM), respectively. Of note, the level of D



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Table 2. Chemosensitization Effect of Hernandezine on ABCB1-Mediated Drug Resistance in ABCB1-Overexpressing Human Cancer Cells treatment

concentration (nM)

KB-3-1 (parental)

KB-V-1 (resistant)

RRb

IC50 (μM)a doxorubicin + hernandezine + hernandezine + hernandezine + hernandezine + tariquidar

50 100 200 500 1000

0.15 0.15 0.14 0.13 0.11 0.11

± ± ± ± ± ±

0.04 0.05 0.05 0.04 0.04 0.03

50 100 200 500 1000

8.81 9.77 9.15 9.33 9.29 8.41

± ± ± ± ± ±

3.80 4.33 3.99 4.00 3.96 3.46

5.07 1.42 0.70 0.28 0.10 0.07

± ± ± ± ± ±

0.19 0.12*** 0.05*** 0.04*** 0.02*** 0.04***

34 9 5 2 1 1

IC50 (nM)a colchicine + hernandezine + hernandezine + hernandezine + hernandezine + tariquidar

487.57 ± 30.54 304.03 ± 27.94** 187.84 ± 14.08*** 118.02 ± 9.52*** 32.43 ± 3.72*** 9.05 ± 3.15***

55 31 21 13 3 1

277.68 ± 56.61 204.61 ± 44.42 100.25 ± 26.76** 21.42 ± 4.34** 1.59 ± 0.09** 0.66 ± 0.14**

356 325 169 34 3 1

IC50 (nM)a vincristine + hernandezine + hernandezine + hernandezine + hernandezine + tariquidar

0.78 0.63 0.59 0.63 0.62 0.41

50 100 200 500 1000

± ± ± ± ± ±

0.27 0.19 0.18 0.20 0.19 0.11 IC50 (μM)a

doxorubicin + hernandezine + hernandezine + hernandezine + hernandezine + tariquidar

50 100 200 500 1000

OVCAR-8 (parental)

NCI-ADR-RES (resistant)

± ± ± ± ± ±

5.54 ± 0.60 2.81 ± 0.43** 1.31 ± 0.16*** 0.55 ± 0.08*** 0.23 ± 0.02*** 0.24 ± 0.03***

0.12 0.11 0.12 0.11 0.10 0.08

0.03 0.03 0.03 0.03 0.02 0.02

46 26 11 5 2 3

IC50 (nM)a colchicine + hernandezine + hernandezine + hernandezine + hernandezine + tariquidar

50 100 200 500 1000

27.26 22.11 24.53 22.75 20.45 23.73

± ± ± ± ± ±

9.96 8.19 8.90 8.46 8.14 9.18

1607.50 1009.50 571.36 223.72 79.31 24.97

± ± ± ± ± ±

497.42 311.22 177.93* 52.69** 30.16** 10.04**

3714.80 3220.21 2660.50 969.58 115.02 18.51

± ± ± ± ± ±

383.58 264.94 296.59* 106.54*** 42.48*** 6.02***

59 46 23 10 4 1

IC50 (nM)a vincristine + hernandezine + hernandezine + hernandezine + hernandezine + tariquidar

50 100 200 500 1000

8.53 6.38 6.14 4.94 6.02 5.18

± ± ± ± ± ±

1.95 1.99 1.72 1.18 1.76 1.90

435 505 433 196 19 4

a IC50 values are mean ± SD calculated from concentration−response curves obtained from at least three independent experiments. bRR, relative resistance values were calculated by dividing IC50 values of ABC transporter overexpressing cells by IC50 values of respective parental cells. *p < 0.05; **p < 0.01; ***p < 0.001.

examined in order to gain insight into the interaction between hernandezine and ABCB1. As shown in Figure 5, hernandezine stimulated the ATPase activity of ABCB1 in a concentrationdependent manner, producing a maximum stimulation of approximately 7-fold of the basal level, and the concentration required for 50% maximal stimulation was approximately 20 nM. These data confirm the interaction between hernandezine and ABCB1 at the ATPase active site in a manner similar to many established modulators of ABCB1.6,30,32 One such example is the calcium channel blocker verapamil, which inhibits the transport activity of ABCB1 while stimulating the ATPase activity of ABCB1.33 Of note, the calculated Ki value

apoptosis in both KB cell lines was not affected by hernandezine or tariquidar alone (Figure 4A). These results further support the notion that hernandezine enhances the sensitivity of ABCB1-overexpressing cancer cells to anticancer drugs by inhibiting the function of ABCB1. Hernandezine Stimulates ATPase Activity of ABCB1. As ABCB1-mediated substrate transport is coupled to ATP hydrolysis30 and most MDR modulators discovered to date are high-affinity competitive substrates of ABCB1, capable of stimulating ABCB1 ATPase activity in a concentrationdependent or biphasic manner,9,31 the effect of hernandezine on vanadate (Vi)-sensitive ATPase activity of ABCB1 was E



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Figure 3. Hernandezine has no significant effect on ABCB1 protein expression in human KB epidermal cancer cells. (A) Immunoblot detection of human ABCB1 and (B) quantification of total lysate protein (10 μg) from KB-3-1 and KB-V-1 cells treated with increasing concentrations of hernandezine for 72 h as described previously.43 αTubulin was used as loading control. Values are presented as mean ± SEM calculated from three independent experiments.

for hernandezine was approximately 20 nM, indicating that ABCB1 interacted with ABCB1 with high affinity. It is worth noting that benzylisoquinoline alkaloid tetrandrine has previously been reported to reverse ABCB1-mediated drug resistance34−37 and to down-regulate ABCB1 expression in human K56238 and Caco-2 cells.39 Although considerably higher concentrations of tetrandrine (2.5−6.5 μM) were required to reverse ABCB1-mediated resistance to vincristine,34 paclitaxel,35 and doxorubicin,36,37 these results support the notion that isoquinoline alkaloids are effective modulators of ABCB1. In conclusion, hernandezine was identified as the most promising candidate modulator of ABCB1 through screening of a large collection of purified bioactive natural products. This study provides evidence that hernandezine selectively inhibits the function of ABCB1, enhances drug-induced apoptosis, and resensitizes ABCB1-overexpressing cancer cells to chemotherapeutic drugs at nontoxic, nanomolar concentrations. Experimental results suggest that the bisbenzylisoquinoline alkaloid hernandezine has great potential to be further developed into a promising reversal agent for the treatment of MDR in ABCB1-overexpressing cancers.

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Figure 4. Hernandezine enhances drug-induced apoptosis in ABCB1overexpressing human epidermal KB-V-1 cancer cells. KB-3-1 (top panels) and KB-V-1 (lower panels) cells were isolated and analyzed 48 h after treatment with colchicine alone or in combination with hernandezine or tariquidar. KB cells were treated with either DMSO (left panels), 1 μM hernandezine (middle panels), or 1 μM tariquidar (right panels) in the (A) absence or (B) presence of 500 nM colchicine. Representative histograms of three independent experiments are shown. Apoptotic cells were quantified by flow cytometry after staining with annexin V-FITC and PI. Live cells (annexin V− PI−) appear in the lower left quadrant; early apoptotic cells (annexin V+ PI−) appear in the lower right quadrant; necrotic or late apoptotic cells (annexin V+ PI+) appear in the upper right quadrant. Cells in the upper right quadrant were confirmed by fluorescence microscopy to be late apoptotic and not necrotic cells. (C) Quantification of colchicineinduced apoptosis in human KB cells. Values are presented as mean ± SEM calculated from three independent experiments. *p < 0.05; **p < 0.01; ***p < 0.001 versus the same treatment without hernandezine or tariquidar.

EXPERIMENTAL SECTION

Chemicals and Cell Culture. Hernandezine (purity >95% as determined by HPLC) was purchased from International Laboratory USA (South San Francisco, CA, USA). The Enzo Natural Product Library (502 purified compounds) was purchased from Enzo Life Sciences Inc. (Farmingdale, NY, USA). Cell Counting Kit-8 (CCK-8), MTT dye, calcein-AM, doxorubicin, colchicine, vincristine, and other chemicals were purchased from Sigma (St. Louis, MO, USA), unless stated otherwise. Tariquidar was purchased from MedKoo Biosciences Inc. (Chapel Hill, NC, USA). Dulbecco’s modified Eagle medium (DMEM), Iscove’s modified Dulbecco’s medium (IMDM), fetal calf serum (FCS), trypsin-EDTA, penicillin, streptomycin, and PBS were purchased from Gibco, Invitrogen (Carlsbad, CA, USA). Annexin V:FITC apoptosis detection kit was purchased from BD Pharmingen

(San Diego, CA, USA). The human KB-3-1 epidermal carcinoma cell line and its drug-selected ABCB1-overexpressing variant KB-V-1 F



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upper right dot-plot quadrant (PS-positive and PI-positive) have leaky membranes and can be either necrotic or late apoptotic.44 ATPase Assay of ABCB1. The hernandezine-stimulated vanadatesensitive ATPase activity of ABCB1 was recorded by using the PgpGlo assay system (Promega, Fitchburg, WI, USA) according to the manufacturer’s instructions and determined based on end point Pi assay as described previously.8,45 Immunoblotting. Cells were treated with increasing concentrations of hernandezine for 72 h before harvesting. Crude membrane protein was prepared and subjected to electrophoresis on a 7.5% SDSpolyacrylamide gel and transferred onto a nitrocellulose membrane. Each blot was then incubated in blocking buffer containing 5% (w/v) milk powder in 0.1% TBS-Tween (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.1% Tween-20) for an hour prior to the addition of the ABCB1-specific primary antibody C219, ABCC1-specific primary antibody MRPm5, ABCG2-specific primary antibody BXP-21, or antiα-tubulin primary antibody for the detection of ABCB1, ABCC1, ABCG2, or tubulin as positive control. The secondary antibodies used were horseradish peroxidase-conjugated goat anti-mouse IgG and antirabbit IgG. Signals were detected as described previously.26,40,46 Statistical Analysis. GraphPad Prism (La Jolla, CA, USA) and KaleidaGraph (Reading, PA, USA) software products were used to plot the curves and perform statistical analysis. Data are presented as mean ± SEM, whereas IC50 values were calculated as mean ± SD from at least three independent experiments. Differences between any mean values were analyzed by two-sided Student’s t test, and results were considered statistically significant at P < 0.05.

Figure 5. Hernandezine stimulates vanadate (Vi)-sensitive ABCB1 ATPase activity. The effect of 0−1 μM hernandezine on Vi-sensitive ABCB1 ATPase activity was measured as described previously.43 Points: mean from at least three independent experiments; bar: SEM. subline and human OVCAR8 ovarian carcinoma cell line and its ABCB1-overexpressing variant NCI-ADR-RES subline were cultured in DMEM, supplemented with 10% FCS, 2 mM L-glutamine, and 100 units of penicillin/streptomycin/mL. KB-V-1 cells were maintained in media containing 1 mg/mL vinblastine.4 Parental pcDNA3.1-HEK293, MDR19-HEK293 (HEK293 cells transfected with human ABCB1), MRP1-HEK293 (HEK293 cells transfected with human ABCC1), and R482-HEK293 (HEK293 cells transfected with wild-type human ABCG2) cells were cultured in DMEM, supplemented with 10% FCS, 2 mM L-glutamine, 100 units of penicillin/streptomycin/mL, and 2 mg/mL G418.40 Cells were maintained at 37 °C in 5% CO2 humidified air and placed in drug-free medium 7 days prior to assay. MDR cell lines were generous gifts from Dr. Suresh V. Ambudkar (National Cancer Institute, NIH, Bethesda, MD, USA). Fluorescent Drug Accumulation Study. Drug efflux assays were performed using a FACSort flow cytometer equipped with Cell Quest software (Becton-Dickinson, Franklin Lakes, NJ, USA) as described previously.25 Fluorescent substrate calcein-AM was used to monitor ABCC1- and ABCB1-mediated efflux, whereas pheophorbide a (PhA) was used to monitor ABCG2-mediated efflux. Briefly, cells were harvested after trypsinization by centrifugation at 500g and then resuspended in IMDM supplemented with 5% FCS. PhA or calceinAM was added to 3 × 105 cells in 4 mL of IMDM in the presence or absence of hernandezine, ABCB1 reference inhibitor tariquidar, ABCC1 reference inhibitor MK-571, or ABCG2 reference inhibitor FTC. The effect of hernandezine or reference inhibitors on fluorescent drug efflux mediated by ABCB1, ABCC1, or ABCG2 was measured and analyzed according to the method described by Gribar et al.41 Cytotoxicity Study. The CCK-8 and MTT assays were used to determine the sensitivity of cells to a particular agent according to the method described by Ishiyama et al.42 Briefly, cells were plated at a density of 5000 cells per well in 100 μL of culture medium in 96-well plates at 37 °C for 24 h before adding compounds to make a final volume of 200 μL. Cells were incubated for an additional 72 h with various concentrations of drugs before being developed with CCK-8 reagent (for HEK293 and stably transfected HEK293 cell lines) or MTT (for attached cancer cell lines) as described previously.43 For the reversal of cytotoxicity assays, a nontoxic concentration of hernandezine or a reference inhibitor was added to the cytotoxicity assay, and the extent of reversal was then calculated based on the relative resistance values. IC50 values were calculated from fitted concentration−response curves obtained from at least three independent experiments. Apoptosis Assay. In order to determine the apoptotic effect of a particular compound or compound combination in cancer cells, apoptosis assays were performed. Briefly, 1 × 106 cells were treated with indicated regimens for 48 h before being harvested by a series of washing and centrifugation and finally resuspended in FACS buffer containing 1.25 μg/mL annexin V-FITC and 0.1 mg/mL propidium iodide (PI) and incubated for 15 min at room temperature. The labeled cells were analyzed using a FACSort flow cytometer equipped with the Cell Quest software (Becton-Dickinson). Cells in the lower right dot-plot quadrant (PS-positive and PI-negative) were counted as apoptotic and have intact plasma membranes, whereas cells in the

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AUTHOR INFORMATION

Corresponding Author

*E-mail (C.-P. Wu): [email protected]. Tel: + 88632118800, ext 3754. Fax: +886-32118700. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. S. V. Ambudkar (National Cancer Institute, NIH) for generously providing cell lines. This work was supported by funds from the Chang Gung Medical Research Program (CMRPD1D0153), the Ministry of Science and Technology of Taiwan (MOST-105-2320-B-182-018), and a grant to Chang Gung University from the Ministry of Education (EMRPD1F0191).

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