Apoptosis DOI 10.1007/s10495-014-0998-8
ORIGINAL PAPER
Reversal of multidrug resistance in vitro and in vivo by 5-N-formylardeemin, a new ardeemin derivative Xuelian Zheng • Daoxia Li • Chen Zhao • Qiong Wang Hao Song • Yong Qin • Linchuan Liao • Lin Zhang • Yong Lin • Xia Wang
•
Ó Springer Science+Business Media New York 2014
Abstract Because multidrug resistance (MDR) is a serious impediment to the use of chemotherapy in treating cancer patients, great efforts have been made to search for effective MDR-reversing agents. We have developed a brand new synthetic ardeemin derivative, 5-N-formylardeemin, and investigated the activity of which in reversing MDR in MDR cancer cell lines derived from human breast cancer (MCF-7-R) or lung cancer (A549-R). 5-N-formylardeemin strongly enhanced the anti-cancer efficacy of doxorubicin, vincristine through potentiation of apoptosis in both MCF-7-R and A549-R at relatively noncytotoxic concentrations in vitro. Mechanistic studies showed that 5-N-formylardeemin inhibited the expression of MDR-1 (P-gp) and increased the intracellular accumulation of cytotoxic drugs in the MDR cells, suggesting that 5-Nformylardeemin reverses MDR activities through inhibiting Electronic supplementary material The online version of this article (doi:10.1007/s10495-014-0998-8) contains supplementary material, which is available to authorized users. X. Zheng Q. Wang L. Zhang Y. Lin X. Wang (&) Laboratory of Molecular and Translational Medicine, Key Laboratory of Birth Defects and Related Diseases of Women and Children (Sichuan University) of Ministry of Education, Department of Obstetrics and Gynecology, West China Second University Hospital, Sichuan University, Chengdu 610041, China e-mail:
[email protected] D. Li C. Zhao L. Liao Department of Forensic Analytical Toxicology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041, China H. Song Y. Qin Department of Chemistry of Medicinal Natural Products, West China School of Pharmacy, Sichuan University, Chengdu 610041, China
MDR-1 expression. Interestingly, 5-N-formylardeemin also sensitized the parent wild-type cancer cells toward these chemotherapeutic agents to various extents. Importantly, in vivo studies demonstrated that 5-N-formylardeemin significantly improved the therapeutic effects of doxorubicin in nude mice bearing A549-R xenografts, which was associated with reduced expression of MDR-1 protein level and increased apoptosis in tumor tissues. These results underscore 5-N-formylardeemin as a potential sensitizer for chemotherapy against multidrug resistant cancers. Keywords Multidrug resistance Chemotherapy 5-N-Formylardeemin Apoptosis Cancer xenograft
Introduction Multidrug resistance (MDR) remains a main hurdle of chemotherapy in the treatment of cancer patients. Cancer cells acquired MDR phenotype are cross-resistant to a variety of chemotherapeutic drugs that have unrelated structures and different mechanisms of action. Although the underlying mechanisms are multifactorial and complicated, MDR is usually associated with the overexpression of ATP-binding cassette (ABC) transporters, such as MDR1 (also known as P-glycoprotein, P-gp), breast cancer resistance proteins (BCRP), and multidrug resistance associated proteins (MRPs) on cell membrane [1–3]. These ABC transporters actively pump cytotoxic drugs out of cell, resulting in decreased intracellular concentrations of the drugs. It is intriguing how these transporters have a remarkable ability to interact with, and to transport, such a wide variety of anticancer compounds. The hydrophobic vacuum cleaner model, which proposes that various hydrophobic compounds are bound indiscriminately by
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MDR-1 due to their hydrophobicity for efflux, may partly explain this phenomenon [4]. Thus, a number of natural and synthetic compounds with various structures have been developed to block the function of P-gp for reversing the MDR phenotype [5]. These compounds include calcium channel blockers (e.g., verapamil, nifedipine), quinolines (e.g., quinidine, chloroquine), calmodulin antagonists (e.g., chlorpromazine, trifluoperazine), steroids (e.g., progesterone, tamoxifen), and immunosuppressive drugs (e.g., rapamycin, cyclosporine). However, most of these modulators are unfortunately poor P-gp inhibitors that have either low MDR reversal efficacy or unacceptable toxicity, preventing their clinical application [6, 7]. Thus, it is important to search for new and more effective MDR-reversing agents. An early study showed that extracts of the fungus Aspergillus fischeri (var. brasiliensis) exerted the activity to reverse MDR in human cancer cell lines [8]. Isolation of the active components from the extracts led to discovery of three structurally related compounds named ardeemin, 15bb-hydroxy-5-N-acetylardeemin, and 5-N-acetylardeemin. These compounds belong to the natural products family of ‘‘reverse prenyl’’ hexahydropyrroloindole alkaloids, with the presence of a ‘‘reverse prenyl’’ (a,a-dimethallyl) group at the junction of rings B and C of the cyclized tryptophan. Among the three compounds, 5-Nacetylardeemin is the major and most active constituent in the A. fischeri extracts for MDR reversal. 5-N-acetylardeemin is able to potentiate the anticancer activity of vinblastine, doxorubicin or paclitaxel in multidrug resistant human tumor cells in vitro and in mice bearing tumors in vivo [8, 9]. Mechanistic studies showed that reversal of MDR by 5-N-acetylardeemin is mainly through the direct inhibition of the P-glycoprotein-mediated drug efflux [10]. Because of considerable difficulty in isolation and purification of ardeemins from fungus fermentation mixture, chemical synthesis serves as a better recourse to access significant quantities of these compounds. A series of ardeemins and its derivatives and analogues have been synthesized and their MDR reversing activities were systematically compared [10–15], however, a clinical applicable ardeemin derivative has not been found. In this study, we synthesized a new ardeemin derivative, 5-N-formylardeemin, and investigated its MDR reversing activity. The results show that 5-N-formylardeemin strongly enhanced the anti-cancer efficacy of several anticancer drugs including vincristine and doxorubicin in MDR cancer cell lines derived from human breast cancer (MCF-7-R) or lung cancer (A549-R). The MDR reversing activity of 5-Nformylardeemin is associated with inhibiting MDR-1 expression, which increase the intracellular accumulation of cytotoxic drugs in the MDR cells. Importantly, 5-Nformylardeemin strongly enhanced the anti-cancer efficacy of doxorubicin in mice bearing A549-R xenograft. These
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Fig. 1 Structures of ardeemin, 5-N-acetylardeemin, and 5-Nformylardeemin
results strongly suggest 5-N-formylardeemin as a potential chemotherapy sensitizer for reversing MDR in cancer cells.
Materials and methods Reagents 5-N-Formylardeemin was synthesized in a related way as described [13], which has a similar structure as the natural occurring compound 5-N-acetylardeemin except substituting the 5-N-acetyl group with 5-N-formyl group (Fig. 1). Antibodies against active caspase-3 and poly (ADP-ribose) polymerase (PARP) were from BD bioscience (San Diego, CA). Antibodies against Mdr-1 and b-actin were from Santa Cruz Biotechnology (Santa Cruz, CA) and Protein Tech Group (Chicago, IL), respectively. Vincristine and doxorubicin were purchased from Sigma (St. Louis, MO). Cell culture and in vitro cytotoxicity assay The non-small cell lung cancer cell line A549 and breast cancer cell line MCF-7 were purchased from American Type Culture Collection (ATCC, Manassas, VA) and grown in RPMI 1640 and DMEM, respectively, supplemented with 10 % fetal bovine serum (Hyclone), 100 units/ mL penicillin, and 100 lg/mL streptomycin under standard incubation condition (37 °C, 5 % CO2). The cell lines with MDR phenotype (MCF-7-R and A549-R), which are crossresistant to vincristine and overexpress MDR-1 (Table 1 and data not shown), were established by a multistep selection with exposure to increasing concentrations of doxorubicin up to 20 lM, The MDR cell lines were maintained in culture medium with doxorubicin (2 lM) and were cultured in medium without doxorubicin 1 week prior to experiments. Cell death was detected quantitatively
Apoptosis Table 1 Sensitivity of A549 and MCF-7 cells and their MDR cells to doxorubicin and vincristine Cell line
DOX Cytotoxicity-IC50 (lM)
A549-WT A549-R MCF-7-WT MCF-7-R
1.750 ± 0.305
VCR Resistance fold 69.67
121.936 ± 2.875 1.412 ± 0.202
Cytotoxicity-IC50
Resistance fold
19.437 ± 0.594 (nM)
626.54
12.178 ± 0.333 (lM) 69.86
98.639 ± 1.781
17.752 ± 0.218 (nM)
312.02
5.539 ± 0.144 (lM)
The IC50 values, which mean concentrations causing 50 % of cell death by in vitro cytotoxicity assay, were determined from 5 to 6 concentrations of each compound and the dose–effect curves were analyzed with SPSS statistics software package. Resistance fold was calculated using the following formula: Resistance fold = IC50 resistant/IC50 parent
by lactate dehydrogenase (LDH) releasing assay using a cytotoxicity detection kit (Promega, Madison, WI) as described previously [16]. All the experiments were repeated three to five times and the average was shown in each figure. The IC50 value was calculated as the concentration of anticancer drug yielding a 50 % rate of cell death, which was calculated using probit regression using SPSS statistics software package. Apoptosis analysis by flow cytometry Apoptosis in cultured cells was measured by flow cytometry using an Annexin V-FITC Apoptosis Detection Kit (Nanjing KeyGen Biotech, Nanjing, China). In brief, after designated treatment, cells were double staining with annexin V-FITC and propidium iodide (PI), and apoptosis was analyzed by flow cytometry (BD bioscience, Franklin Lakes, NJ). Early apoptosis is defined by Annexin V?/PIstaining (Q4) and late apoptosis is defined by Annexin V?/ PI? staining (Q2). Western blot Cell extracts were prepared by lysing cells in M2 buffer [20 mmol/L Tris–HCl (pH 7.6), 0.5 % NP40, 250 mmol/L NaCl, 3 mmol/L EDTA, 3 mmol/L EGTA, 2 mmol/L DTT, 0.5 mmol/L phenylmethylsulfonyl fluoride, 20 mmol/L h-glycerophosphate, 1 mmol/L sodium vanadate, and 1 lg/ mL leupeptin]. Cell extracts (*50 lg) were resolved in SDS-PAGE and analyzed by Western blot. The specific proteins were visualized by enhanced chemiluminescence (Millipore, Billerica, MA) using BIO-RAD Image station. Each experiment was repeated at least three times and representative results were shown. Assessment of doxorubicin content in cells Doxorubicin is intrinsically fluorescent and can be excited at wavelength around 480 nm. Thus doxorubicin content in
cells can be detected through monitoring the cellular doxorubicin fluorescence intensity. For flow cytometric determination of doxorubicin content, MCF-7-R cells treated as indicated in figure legend were washed and suspended in PBS and cellular fluorescence was detected by flow cytometry (BD bioscience, Franklin Lakes, NJ). A minimum of 10,000 cells were analyzed for each histogram generated. For fluorometric determination of cellular doxorubicin content, cells were washed twice with an excess volume of ice-cold PBS and lysed with 1 % sodium dodecyl sulfate (SDS). Fluorescence intensity in supernatant recovered after centrifugation of the cell suspension was measured using a microplate reader (Tecan) at excitation and emission wavelengths of kex = 480 nm and kem = 570 nm, respectively. In vivo xenograft and chemotherapy study Athymic male nude mice (6 weeks old) were obtained from the Animal Center of Sichuan University (Chengdu, China). All procedures involving animals and their care were conducted in accordance with the guidelines of the Institutional Animal Care and Use Committee of Sichuan University. A549-R cells (2 9 106) resuspended in 0.05 mL of PBS were mixed with 0.05 mL of matrigel (BD bioscience, Franklin Lakes, NJ) and then injected subcutaneously into mice. When the xenograft tumors were palpable, the mice were randomly divided into four groups and administrated with the following agents by i.p. injection every 3 days: (a) vehicle control; (b) 1 mg/kg of doxorubicin; (c) 50 mg/kg of 5-N-formylardeemin (d) combination of 1 mg/kg of doxorubicin and 50 mg/kg of 5-N-formylardeemin. Tumor sizes were measured using a micrometer caliper and tumor volume (V) were calculated using the following formula: V = length 9 width2/2. The body weight of the mice were also measured during the course of treatment. At the end of experiment, animals were euthanized and sacrificed. Excised tumors were measured and weighed.
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Fig. 2 5-N-formylardeemin enhanced the cytotoxicity of doxorubicin and vincristine in MDR cancer cells and their parental wild-type cells in vitro. a, b A549-R cells were treated with doxorubicin (DOX, 10 lM) or vincristine (VCR, 0.3 lM) in the absence or presence of increasing concentrations of 5-N-formylardeemin as indicated in the figures. c, d MCF-7-R cells were treated with doxorubicin (10 lM) or vincristine (0.3 lM) in the absence or presence of increasing concentrations of 5-N-formylardeemin as indicated in the figures. e,
f A549-WT cells were treated with doxorubicin (0.5 lM) or vincristine (5 nM) in the absence or presence of increasing concentrations of 5-N-formylardeemin as indicated in the figures. g, h MCF7-WT cells were treated with doxorubicin (0.5 lM) or vincristine (5 nM) in the absence or presence of increasing concentrations of 5-N-formylardeemin as indicated in the figures. The cells were treated for 72 h, and cell death was measured by lactate dehydrogenase (LDH) release assay. Columns mean of three experiments; bars SD
TUNEL assay
Results
Apoptosis in tumor tissues was detected by terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay with the in situ cell death detection kit (Roche Diagnostics, Mannheim, Germany) according to the manufacturer’s instructions. Briefly, paraffin-embedded tumor tissue sections were dewaxed, rehydrated, treated with protease K, and permeabilized. Then the sections were incubated with the terminal deoxynucleotidyl transferase labeling reaction mixture for 60 min at 37 °C. After incubated with the converter-AP (anti-fluorescein antibody conjugated with alkaline phosphatase) for 30 min at 37 °C, the slides were stained with AP-red and haematoxylin for color development. The apoptotic cells (TUNEL-positive cells) in five fields (409) were counted under a microscope and the average number of TUNEL-positive cell per field was calculated.
5-N-Formylardeemin enhanced the cytotoxicity of doxorubicin, vincristine in both MDR cell lines and their parental cell lines in vitro
Statistical analysis Data were expressed as mean ± SD. Statistical significance was determined by paired Student’s t test using SPSS statistics software package. A P \ 0.05 was considered as statistically significant.
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The effects of 5-N-formylardeemin on drug sensitivity were investigated in two MDR cell lines. A549-R cells were treated with 10 lM of doxorubicin in the absence or presence of increasing concentrations of 5-N-formylardeemin. The 5-N-formylardeemin concentrations were not toxic, which caused \10 % cell death in A549-R cells (Fig. 2a). As expected, A549-R cells were resistant to doxorubicin as \5 % cell death induced in the cells treated with 10 lM of doxorubicin alone. However, co-treatment with 5-N-formylardeemin and doxorubicin resulted in a synergistic cytotoxicity in a 5-N-formylardeemin dosedependent manner (Fig. 2a). 5-N-Formylardeemin also similarly enhanced vincristine-induced cytotoxicity in A549-R cells (Fig. 2b). Synergistic effects were seen in MCF-7-R cells treated with doxorubicin or vincristine in combination (Fig. 2c, d). Interestingly, 5-N-formylardeemin also exhibited synergistic effects on doxorubicin and vincristine against the parent wild-type cells that have not acquired MDR (Fig. 2e–h). The IC50 values of
Apoptosis Table 2 Reversal of resistance to doxorubicin and vincristine in A549 and MCF-7 MDR cells by 5-N-formylardeemin
Compound
A549-R Cytotoxicity-IC50 (lM)
DOX (A)
MCF-7-R Ratio
Cytotoxicity-IC50 (lM)
A/B = 5.86
17.386 ± 0.456
D/F = 69.19
0.121 ± 0.007
121.936 ± 2.875
DOX ? F-Ard (5 lM) (B)
20.808 ± 0.962
VCR (D)
12.178 ± 0.333
VCR ? F-Ard (5 lM) (F)
doxorubicin or vincristine in A549-R cells and MCF-7-R cells were determined with a fixed dose of 5-N-formylardeemin (at 5 lM). As shown in Table 2, 5-N-formylardeemin sensitized the MDR cells about 5.67- to 5.86-fold in cytotoxic response to doxorubicin and about 45.78- to 69.19-fold to that of vincristine. It is noteworthy that 5-Nformylardeemin had little effect on doxorubicin- or vincristine-induced cell death in untransformed human bronchial epithelial cell lines HBEC-2, which are immortalized by insertion of cyclin-dependent kinase 4 and human telomerase reverse transcriptase (Fig. S1, S2). These results provided strong in vitro evidence supporting that 5-Nformylardeemin effectively sensitizes MDR cancer cells to different chemotherapeutics. 5-N-Formylardeemin enhanced doxorubicin-induced apoptosis in MDR cancer cells To investigate whether the reversal of drug resistance by 5-N-formylardeemin is through potentiation of apoptosis, A549-R cells were treated with doxorubicin in the absence or presence of 5-N-formylardeemin and apoptosis was analyzed by flow cytometric assay. The results showed that both early apoptotic and late apoptotic cells were significantly increased after doxorubicin and 5-N-formylardeemin co-treatment (Fig. 3a). Additionally, the activation of apoptotic pathway in co-treated cells was potentiated as detected by Western blot showing that both the generation of active form of caspase-3 and cleavage of the caspase-3 substrate PARP were increased (Fig. 3b). These results suggest that the enhanced cytotoxicity induced by doxorubicin in the presence of 5-N-formylardeemin likely occurs through potentiation of doxorubicin-induced apoptosis. 5-N-Formylardeemin increased cellular doxorubicin accumulation by inhibiting MDR-1 expression in MDR cells Cell killing has been reported to be closely correlated with the intracellular concentration of doxorubicin in cancer cells, especially in doxorubicin-resistant cell lines that express high levels of MDR-1 [17]. Thus, we investigated
0.176 ± 0.059
Ratio
98.639 ± 1.781 A/B = 5.67
5.539 ± 0.144 D/F = 45.78
whether 5-N-formylardeemin enhances doxorubicininduced cell killing through increasing drug accumulation in the MDR cells. By flow cytometric analysis, 5-Nformylardeemin was found to effectively increase the intracellular concentration of doxorubicin in MCF-7-R cells, which was demonstrated by the rightward shift of the peak of cells co-treated with doxorubicin and 5-N-formylardeemin compared with that of the cells treated with doxorubicin alone (Fig. 4a). Consistently, fluorometric determination of cellular doxorubicin content showed that 5-N-formylardeemin significantly promoted the retention of doxorubicin in A549-R cells, and the intracellular fluorescence intensity increased with the increase of concentrations of doxorubicin (Fig. 4b). When different doses of 5-N-formylardeemin were used, 5-N-formylardeemin exerted the doxorubicin retention effect in a dose-dependent manner (Fig. 4c). Importantly, 5-N-formylardeemin was shown to inhibit the expression of MDR-1 in A549-R cells in a time- and dose-dependent manner (Fig. 4d). Therefore, it is likely that 5-N-formylardeemin enhances chemotherapeutic-induced cytotoxicity through increasing cellular drug accumulation by inhibiting MDR-1 expression in MDR cells. 5-N-Formylardeemin enhanced the anti-cancer activity of doxorubicin in A549-R xenograft mouse model The aforementioned in vitro data prompted us to further examine whether 5-N-formylardeemin is able to enhance the in vivo anticancer efficacy of doxorubicin in a mouse model with A549-R xenografted tumors. As expected, doxorubicin caused only moderate retardation of tumor growth compared with that of the tumors in the vehicle treated animals. A pronounced tumor growth inhibition was seen in mice treated with doxorubicin plus 5-Nformylardeemin (Fig. 5a, b), which was also supported by the results of tumor weight assessed at the end of experiment (Fig. 5c). Thus, the synergistic anticancer effects of combination of doxorubicin and 5-N-formylardeemin were validated in vivo. It was noted that body weight was moderately decreased in the animals co-treated with 5-Nformylardeemin and doxorubicin. This result implies that the combined treatment of 5-N-formylardeemin and
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Fig. 3 5-N-Formylardeemin enhanced doxorubicin-induced apoptosis in cultured MDR cancer cells. a A549-R cells were treated with 5-N-formylardeemin (10 lM) or doxorubicin (10 lM) alone or both for 24 h. The cells were then stained with annexin V and PI followed by flow cytometry analysis. The percentage of cell population in Q2 and Q4 were shown. b A549-R cells were treated with 5-Nformylardeemin (10 lM) or doxorubicin (10 lM) alone or both for indicated times. Caspase-3 and PARP were detected by Western blot. b-Actin was detected as an input control
doxorubicin may have some toxicity in mice with the regime tested, which may be related to drug doses and frequency. This issue should be clarified in future studies including pharmacological and histopathological evaluation and biochemical analysis in animals. 5-N-Formylardeemin suppressed MDR-1 expression and enhanced doxorubicin-induced apoptosis in xenografted A549-R tumors Apoptosis in xenografted A549-R tumors was determined by TUNEL assay. While only a few TUNEL-positive cells (apoptotic cells) were seen in doxorubicin- or 5-Nformylardeemin-treated tumors, the number of TUNELpositive cells in the tumor tissues from animals receiving doxorubicin and 5-N-formylardeemin co-treatment was significantly increased (Fig. 6a, b). In addition, 5-Nformylardeemin treatment significantly down-regulated the expression of MDR-1 in tumor tissues (Fig. 6c), which is consistent with the in vitro data (Fig. 4d). These results suggest that the in vivo synergistic anticancer effects of combination of doxorubicin and 5-N-formylardeemin are likely through MDR-1 suppression and apoptosis potentiation.
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Fig. 4 5-N-Formylardeemin increased cellular doxorubicin concentration and inhibited MDR-1 expression in MDR cancer cells. a MCF7-R cells were pretreated with 10 lM of 5-N-formylardeemin for 16 h or left untreated, followed by doxorubicin treatment (10 lM) for another 4 h, and cellular fluorescence was analyzed by flow cytometry. The histogram overlays showed the results of treated cells (2, doxorubicin, 3, doxorubicin and 5-N-formylardeemin) compared with untreated cells (1). b A549-R cells were pretreated with 10 lM of 5-N-formylardeemin for 16 h or left untreated, followed by treatment with increasing concentration of doxorubicin (10–160 lM) for another 4 h. c A549-R cells were pretreated with increasing concentration of 5-N-formylardeemin (1.25–20 lM) for 16 h or left untreated, followed by treatment with 20 lM of doxorubicin for another 4 h. Fluorescence intensity in cell lysate was measured using a microplate reader. The mean fluorescence intensity (columns) and SD (bars) of three experiments were shown in b and c. d A549-R cells were treated with 10 lM of 5-Nformylardeemin for indicated times (upper panel) or increasing concentration of 5-N-formylardeemin (2.5–20 lM) for 16 h (lower panel). The expression of MDR-1 was detected by Western blot. bActin was detected as an input control
Discussion In our attempts in screening for ardeemin derivatives that are able to reverse drug resistance in MDR cancer cell lines, 5-N-formylardeemin was found to be potent in enhancement of the toxicity of vincristine and doxorubicin in cultured MCF-7-R and A549-R cells and potentiation of the anti-cancer activity of doxorubicin in mice bearing A549-R xenograft tumors. Importantly, 5-N-formylardeemin inhibited the expression of MDR-1 and increased the intracellular concentration of doxorubicin in the MDR cancer cells, which suggests that this compound may serve as a potential MDR-reversing agent. While it is a crucial to find MDR-reversing agents for anticancer chemotherapy, the discovery of the MDR reversal activity of natural ardeemins is intriguing. As the major and most active constituent isolated from A. fischeri fermentation mixture, 5-N-acetylardeemin could
Apoptosis
Fig. 5 5-N-Formylardeemin enhanced the antitumor activity of doxorubicin in A549-R xenograft in vivo. a Athymic male nude mice were injected s.c. with A549-R cells (2 9 106) for the development of xenograft tumors. The mice were randomly divided into four groups for treatment: vehicle control; 1 mg/kg doxorubicin; 50 mg/kg of 5-N-formylardeemin; combination of 1 mg/kg of doxorubicin and 50 mg/kg of 5-N-formylardeemin. The volume of tumors was measured and the mean tumor size of each group was shown. b, c Xenograft tumors were excised and then weighed. Columns, mean of tumor weight in each group, bars, SD. **P \ 0.01. d Body weight of the mice was measured during the course of treatment. The result was presented as mean ± SD
Fig. 6 5-N-Formylardeemin enhanced doxorubicin-induced apoptosis and suppressed MDR-1 expression in A549-R xenograft tumors. a In Situ TUNEL staining in tumor tissues. Representative images for each group were shown. b TUNEL-positive cells (apoptotic cells) were counted in five fields (409) for tumor tissues from each group and average TUNEL-positive cell numbers per field were shown as mean ± SD. **P \ 0.01. c Expressions of MDR-1 in tumor tissues from each group were detected by Western blot and b-actin was measured as an input control
significantly increase the therapeutic activity of doxorubicin in B6D2F1 mice inoculated with wild-type and doxorubicin-resistant P388 tumor cells as well as in nude mice bearing human MX-1 mammary carcinoma xenografts [10]. Another worth noting property of 5-N-
acetylardeemin is its low toxicity in mice, as quantities of 5-N-acetylardeemin as high as 300 mg/kg per day for 3 days or 150 mg/kg per day for 8 days were well tolerated by mice. However, verapamil is far more toxic; 150 mg/kg of which per day for 3 days resulted in 40 % lethality to mice [10]. The potent MDR-reversing activity of the ardeemin coupled with its low level of toxicity aroused great interest in the study of ardeemin analogues for reversing drug insensitivity and led to the finding of several members of ardeemin family with different extent of MDR reversal activity. The detailed assessment of the structure–activity relationships of these compounds showed that the pyrazinoquinazoline DEF portion of 5-Nacetylardeemin is the pharmacophoric moiety [18]. This conclusion was in agreement with the potent MDRmodulating activity of the simplified ardeemin analogue AV-200 that contains the DEF portion of the natural compound 5-N-acetylardeemin [11]. The ‘‘reverse prenyl’’ (a,a-dimethallyl) group at the junction of rings B and C also seems be important for the activity of ardeemins. In this report, 5-N-formylardeemin bearing formyl substituent at 5-N site exerted comparable MDR-reversing activity as 5-N-acetylardeemin in MDR cancer cells, which is consistent with previous report showing that the 5-N-acetyl group is not essential for reversal power of ardeemins [15]. Importantly, 5-N-formylardeemin significantly increased the therapeutic activity of doxorubicin in mice bearing A549-R xenograft without markedly increasing overall systemic toxicity. The exact mechanisms of MDR-modulating action of ardeemins in the MDR cells have not been well elucidated. Previously, it was reported that 5-N-acetylardeemin and its analogues reversed MDR likely through inhibiting MDR-1mediated drug efflux. As expected, we found that 5-Nformylardeemin effectively increased cellular doxorubicin concentration in both A549-R and MCF-7-R cells. Furthermore, 5-N-formylardeemin inhibited MDR-1 expression in MDR cancer cells both in vitro and in vivo. These data indicate that the downregulation of the expression of MDR-1 by 5-N-formylardeemin may account for the inhibition of MDR1 function and increasing of doxorubicin concentration in the MDR-1 expressing MDR cancer cells. However, the detailed mechanisms are warranted for future study. It is noted that 5-N-formylardeemin also potentiated the anticancer effects of doxorubicin and vincristine against the wild-type cancer cells that have not acquired MDR. This may be due to the suppression of the basal MDR-1 expression in these cells. Alternatively, a MDR-1-independent mechanism of 5-N-formylardeemin may be involved, which remains to be elucidated. In summary, our studies show that 5-N-formylardeemin, a brand new analogue of ardeemins, suppresses MDR expression both in vitro and in vivo, substantiating this
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compound as a potential sensitizer for chemotherapy against multidrug resistant cancers. Acknowledgments This study was supported by grants from National Natural Science Foundation of China (81172111 and 81372377) and by Program for New Century Excellent Talents in University (NCET-11-0349) from Chinese Ministry of Education and also partly supported by Program for Changjiang Scholars and Innovative Research Team in University from Chinese Ministry of Education (IRT0935). Conflict of interest of interest.
The authors declare that they have no conflict
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