Nuciferine

A multiple-targets alkaloid nuciferine overcomes paclitaxel-induced drug resistance in vitro and in vivo

Rui-Ming Liua, Peng Xub, Qi Chena, Sen-ling Fenga, Ying Xiea,⁎

Abstract

Objective: Multidrug resistance (MDR) is the major barrier to the successful treatment of chemotherapy. Compounds from nature products working as MDR sensitizers provided new treatment strategies for chemoresistant cancers patients.
Methods: We investigated the reversal effects of nuciferine (NF), an alkaloid from Nelumbo nucifera and Nymphaea caerulea, on the paclitaxel (PTX) resistance ABCB1-overexpressing cancer in vitro and in vivo, and explored the underlying mechanism by evaluating drug sensitivity, cell cycle perturbations, intracellular accumulation, function and protein expression of efflux transporters as well as molecular signaling involved in governing transporters expression and development of MDR in cancer.
Results: NF overcomes the resistance of chemotherapeutic agents included PTX, doxorubicin (DOX), docetaxel, and daunorubicin to HCT-8/T and A549/T cancer cells. Notably, NF suppressed the colony formation of MDR cells in vitro and the tumor growth in A549/T xenograft mice in vivo, which demonstrated a very strong synergetic cytotoxic effect between NF and PTX as combination index (CI) (CI<0.1) indicated. Furthermore, NF increased the intracellular accumulation of P-gp substrates included DOX and Rho123 in the MDR cells and inhibited verapamil-stimulated ATPase activity. Mechanistically, inhibition of PI3K/AKT/ERK pathways by NF suppressed the activation of Nrf2 and HIF-1α, and further reduced the expression of P-gp and BCRP, contributing to the sensitizing effects of NF against MDR in cancer. Conclusion: This novel finding provides a promising treatment strategy for overcoming MDR and improving the efficiency of chemotherapy by using a multiple-targets MDR sensitizer NF. Keywords: Nuciferine Multidrug resistance Multiple-targets PI3K/AKT/ Nrf2/ HIF-1α P-gp BCRP Introduction Currently, chemotherapy in single or combination remains the most commonly used therapeutic approach and markedly improved the survival of cancers patients. However, 90% of treatment failure with chemotherapy are related to multiple-drug resistance (MDR) (Mansoori et al., 2017; Pluchino et al., 2012) In the past 30 years, three generations of inhibitors of ABC transporters especially for P-glycoprotein (P-gp/ABCB1), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs), were discovered to overcome MDR (Choi and Yu, 2014). Because the most common mechanism of MDR is the overexpression of ABC transporters which actively pump numerous chemotherapeutic drugs out of the cancer cells. However, most clinical trials of ABC transporter inhibitors were terminated due to the limited effects based on the efflux transporters as well as obvious side effects (Tamaki et al., 2011). Obviously, MDR of cancer is a complex issue with different mechanisms. The well-known features of MDR cancer cells included modification of drug transport, alterations of drug metabolism, and mutation of drug targets (Krishna and Mayer, 2000). Recent studies revealed the key roles of PI3K/AKT pathways (Huang and Hung, 2009; Wong et al., 2006) and MAPK/ERK pathways (Abrams et al., 2010; SmorodinskyAtias et al., 2020) to chemotherapy resistance. Moreover, increasing evidence indicates that the increased expression of nuclear factor erythroid 2-related factor 2 (Nrf2), and hypoxia-inducible factor-1alpha (HIF-1α) in chemo-resistance tumor provide a growth advantage for cancer cells and are required for the regulatory expression of ABC transporters such as P-gp and BCRP (Kung et al., 2016; Rojo de la Vega et al., 2018). Therefore, there is an ardent need for novel MDR reversal agents that target on more than one of these pathways. Compounds from natural products hold the important potential in new drug discovery because of their multi-targets activity (Abdallah et al., 2015; Elh et al., 2014). In this study, we want to find phytocompounds as novel potential chemosensitizers and explore the underlying mechanisms. Nuciferine (NF, PubChem CID: 10146), an aromatic ring-containing alkaloid from Nymphaea caerulea and Nelumbo nucifera (Fig. 1A), were selected based on the ability to reversing resistance to paclitaxel in the primary screening test carried out with hundreds of compounds from natural products. A variety of biological effects including anti-inflammation (Zhang et al., 2018; Chen et al., 2018), anti-cancer (Li et al., 2019), anti-oxidation (Shu et al., 2019) as well as anti-tumor effects (Kang et al., 2017) has been reported for NF. Moreover, Zhou et al. (2019) shown that NF sensitized pancreatic cancer cells to gemcitabine via 3-hydroxy – 3 – methylglutaryl - CoA reductase (HMGCR) down-regulation. However, whether NF overcomes the drug resistance to the first-line chemotherapeutic agents such as paclitaxel (PTX), doxorubicin (DOX), docetaxel (TXT) and daunorubicin (DAU) as well as the complex underlying mechanisms are still unknown. Materials and methods Chemicals and reagents Nuciferine (C19H21NO2, molecular weight 295.38, Pka 7.87 ± 0.20, purity 98% and Log P = 3.4), paclitaxel (purity 99%), docetaxel (purity 99%), doxorubicin (purity 98%), quinidine (QND, purity 98%), verapamil (Vera, purity 98%), 5-fluorouracil (5-FU, purity 99%), daunorubicin (purity 97%) and dimethylsulfoxide (DMSO) were purchased from Dalian Meilun Biology Technology Co., Ltd and stored in -40 °C. The NF structure and purity were confirmed by LC-MS in our lab. LY294002 (purity 99.95%) and IGF-1 (purity 97%) was purchased from MedChem Express (New Jersey, USA). RNase A, leupeptin, aprotinin, phenylmethyl sulfonyl fluoride, Rho123 (purity 85%), Triton X-100, propidium iodide (PI) and other chemicals were purchased from SigmaAldrich (St. Louis, MO). ERK 1/2 and actin antibodies were purchased from Santa Cruz Biotechnology, USA; P-gp antibodies were purchased from Calbiochem; Nrf2 antibodies were purchased from Abcam, Hong Kong; other antibodies including GAPDH, HIF-1α, AKT, p-AKT, and pERK1/2 were purchased from Cell Signaling Technology, Inc. Cell lines and cell culture Human colorectal adenocarcinoma HCT-8 and its drug-resistant cell line HCT-8/T were kindly provided by Professor Zhi-Hong Jiang (Macau University of Science and Technology, Macau). Human nonsmall cell lung cancer (NSCLC) A549 and its ABCB1-overexpressing drug-resistant cell line A549/T were purchased from KeyGen Biotech Co., Ltd. (Nanjing, China). Cells were cultivated in RPMI 1640 medium supplement with 10% FBS and 1% penicillin and streptomycin (GIBCO, Scotland) at 37 °C under a humidified atmosphere of 5% CO2. To maintain drug resistance, HCT-8/T (at passage numbers about 11-21) and A549/T cells (at passage numbers about 9-18) were continuous exposure of 0.94 μM and 0.24 μM paclitaxel, respectively. The mean IC50 values for PTX and DOX were 201-fold and 17.15fold higher in resistant HCT-8/T cells than that of sensitive HCT-8 cells (passage numbers 8-17), suggesting successfully PTX-induced drug-resistant in HCT-8/T (Supplementary Fig. 1A, and 1C). Similarly, as shown in Supplementary Fig. 1B and 1D, A549/T cells were resistant to PTX and DOX compared to its parental cells A549 (passage numbers about 6-17). Cell viability assay and colony formation assay Cell viability was assessed by sulforhodamine B (SRB) assay as previously described (Ma et al., 2015b). Briefly, cells treated with the NF (48, 24, and 4 μM) and varying concentrations of PTX for 48 h to test whether this combination can enhance the growth inhibition of MDR cancer cells. After fixing with trichloroacetic acid, staining with SRB in the acetic acid solution for 30 min and wash, plates were incubated with 10 mM Tris buffer to solubilize the protein-bound SRB. The absorbance was measured using a plate reader (Spectra MAX 250; Molecular Devices, Sunnyvale, CA) at 515 nm according to the manufacture institutions. The reversal fold of MDR was calculated by dividing the IC50 of combination treatment to that of PTX alone. To estimate the proliferation ability, the colony formation assay was carried out by treating HCT-8/T cells with 0.94 μM PTX alone or combine them with varying concentrations of NF for 8 days (Feng et al., 2020; Ma et al., 2015a). After stained with 0.4% crystal violet at room temperature for 30 min, cells were washed twice with PBS to remove excess dye and were counted for the colony formation. Xenograft model Experiments involving animals were approved by the Animal Care and Use Committee of the Guangzhou Sun Yat-sen University. To build up the xenograft model, A549/T cells were injected subcutaneously at the flank near the armpits of four-week-old female BALB/c nude mice (Feng et al., 2019). After tumor size approximately reached 100 mm3 (day 0), mice were randomized into five groups (n = 6 per group): vehicle (control); PTX (10 mg/kg); NF (7.5 mg/kg); PTX with NF (10 mg/kg PTX and 7.5 mg/kg NF); PTX with verapamil (10 mg/kg PTX and 10 mg/kg Vera). The vehicle used to deliver the NF and PTX was mixture of cremophor EL/ethanol/saline (5% / 5% / 90%). The mice were treated via intraperitoneal injection (i.p.) every 3 days for a total of 27 days. The tumor volume was measured every 2 days. Tumor volume was estimated according to the formula: (length × width2 /2). At the end of the experiments, mice were sacrificed. The blood samples were harvested and stored in -80 °C for further analysis. Tumor tissues were excised and weighed immediately before store. Cell-cycle and apoptosis analysis Cell cycle analysis was performed using ethanol-fixed cells stained with propidium iodide (PI) solution (50 µg/ml PI and 200 µg/ml RNase A in PBS) (Ma et al., 2015b). Apoptotic cells were assessed following the manufacturer's protocol (Sigma-Aldrich). In brief, stained the apoptosis cells with Annexin V-FITC and propidium iodide (PI). The mixture was incubated for 15 min at room temperature (25 °C) in the dark and analyzed by FACS using Aria Ⅲ flow cytometer. Drug combination assay The Chou-Talalay method is a mathematical equation used to evaluate the effects of multiple drug interactions (Chou, 2006). The Combination Index (CI), as a measure of synergy (< 1), additivity (=1) or antagonism (> 1) was calculated by the Chou-Talalay method for quantitatively depict drug combinations, based on the median effect analysis (Chou, 2010). In brief, drug-resistant HCT-8/T cells (6 × 103 cells/wells) were seeded in flat bottomed 96-well plates and conditioned with the two-fold serial diluted mixture of NF (IC50=109.65 μM) and PTX (IC50=4.16 μM) as single drugs or combination exposure. After 48 h, the SRB assay was performed and percent survival was calculated as the above description. Data were analyzed using CalcuSyn software V.2.1 (BIOSOFT). Synergy is further subdivided into synergism (CI = 0.3-0.7), strong synergism (CI = 0.1-0.3), and very strong synergism (CI < 0.1) (Chou, 2006). Intracellular accumulation of doxorubicin and Rhodamin123 For fluorescence microscopy monitor, 5×105 cells/well were seeded in six-well cover slips one day (Ma et al., 2015a). Cells were incubated with DOX (5 μM), or Rho123 (1 μM) alone or combine with NF (48 μM) for 3 h. After wash twice with ice-cold PBS, cells were fixed in 4 wt% formaldehyde and stained with 1μg/ml blue-fluorescent DAPI (1 mg/ ml; Invitrogen D1306) for 30 min. One drop of fluorescent preservation solution (fluorsave reagent, CALBIOCHEM) was added before observation. Imaging captured for comparing the intracellular accumulation of DOX and Rho123 using Fluorescence Microscopy (Leica DM2500, Leica, German). For flow cytometry analysis, HCT-8 and HCT-8/T cells were stained with Rho123 (1 μM) or DOX (5 μM) and co-incubated with or without NF (48 μM) or verapamil in the dark for 6 h (Ma et al., 2015a). After treatment, cells were trypsinized and collected, washed twice with cold PBS and resuspended in 500 μl of ice-cold PBS, evaluated by flow cytometry FACS Aria (BD Biosciences, San Jose, CA). Verapamil (Vera, 50 μM), a known ABCB1 inhibitor, was used as a positive control. ATPase activity The drug-stimulated activity of P-gp ATPase was determined by the luminescent ATP detection kit (Pgp-Glo Assay Kit, Promega, Madison, WI) according to the manufacturers’ recommendation (Ma et al., 2015a). In brief, The stimulating effect (EC50 measurements) on the Pgp ATPase activity by NF were investigated by incubated NF in various concentrations with 0.25 mM Na3VO4 (an ABCB1 ATPase inhibitor), assay buffer, 25 μg recombinant human ABCB1 membranes and 5 mM mgATP at 37 °C for 40 min. The difference in luminescent signal between Na3VO4 -treated samples and samples treated with NF represents the P-gp ATPase activity of NF. Moreover, the inhibitory effects (IC50 measurements) of NF were examined against the verapamil stimulated P-gp ATPase activity by incubated with 200 μM verapamil with NF together. The luminescence of the samples was read with Microplate Detector (infinite M200 PRO, TECAN, Switzerland). Western blot analysis Cells were harvested and rinsed twice with ice-cold PBS buffer and lysed in ice-cold RIPA buffer (1M Tris-HCl, 4% SDS, 20% glycerol, 0.2% 2-mercaptoethanol) containing protease inhibitor for 30 min on ice, then centrifuged 12,000 rpm for 15 min at 4 °C (Ma et al., 2015a). Protein concentrations were determined by BCA assay (Bio-Rad Laboratories, Richmond, CA). Equal amounts of cell lysates were resolved by 10% SDS–PAGE, and subsequently electrophoretic transferred to PVDF membrane (Millipore, Darmstadt, Germany). PVDF membrane blocked with 5% (w/v) nonfat dry milk (Nestle Carnation, New Zealand) in tris-buffered saline containing 0.1% of Tween20 (TBST) for 1 h at room temperature. After immunoblotted with the first antibody in TBST with 1% nonfat dry milk at 4 °C overnight, membranes were washed 5 min with TBST, then incubated with secondary antibodies for 2 h at room temperature and washed three times before detected using the ECL Western blotting analysis system (Thermo Scientific™ Chemiluminescent Substrate, USA). Statistical analysis All experiments were carried out at least 3 times (n = 3) in triplicate and the data were expressed as the mean ± SD unless noted otherwise. Statistical analysis was carried out using Student's t-test or one-way analysis of variance (ANOVA) with Microsoft Excel 2010, and the level of significance was set at a p-value of < 0.05 (*), < 0.01 (**) or < 0.001 (***). Results NF overcomes the drug resistance to chemotherapeutic agents Firstly, we evaluated the toxicity of NF itself to both cell lines. The IC50 values of NF were 104.79 μM and 164.16 μM in HCT-8/T and HCT8, and 105.1 μM and 129.4 μM for A549/T and A549, respectively (Fig. 1). For all testing cell lines, as 80% of the cells survived after given NF at 48 μM, therefore 48 μM was selected as the maximum concentration to test its sensitizing effects to chemotherapy agents. As shown in Fig. 2A and 2C, NF could significantly enhance the cytotoxicity of PTX and DOX against HCT-8/T cells. In Fig. 2A, the addition of NF at 4, 24, and 48 μM significantly decreased the IC50 of PTX with reversal fold of 8.6, 110.96, and 321.8, respectively, in HCT8/T cells. The IC50 of DOX in HCT-8/T cells was decreased 2.3-, 9.5-, 45.7-fold after combination with 4, 24, 48 μM NF, respectively. At the same concentrations, NF did not affect on the IC50 of PTX or DOX in the sensitive HCT-8 (Fig. 2B, Supplementary figure 2). Moreover, as shown in Table 1, NF sensitized HCT-8/T cells to doxorubicin, paclitaxel, daunorubicin, and docetaxel in a dose-dependent manner, indicating NF could reverse the resistance to multiple drugs. In another PTX induced ABCB1-overexpression NSCLC cell line A549/T and its parental sensitive cells A549, we observed similar reversal effects of NF to PTX and other chemotherapeutic drugs (Table 1). Specifically, treatment with 4, 24, and 48 μM NF reduced the IC50 of PTX by 13.01-, 50.4-, and 151.2- fold, respectively (Fig. 2D), in A549/T cells. However, there was no change for the IC50 values of PTX in the parental sensitive A549 cells treated with NF (Supplementary figure 2). And NF only slightly enhanced the cytotoxicity of 5-fluorouracil (5-FU) who is not the substrates of P-gp. Furthermore, colony formation assays were used to evaluate the long-term reversal effects of NF on PTX-resistant cancer cells. Completely inhibition of colony formation was achieved for NF at 1.26 μM in HCT-8/T cells, while no inhibition was observed after given 48 μM NF alone without PTX (Fig. 2E). These results demonstrated that NF sensitized MDR cancer cells to chemotherapeutic drugs. NF boosts the apoptosis and arrests MDR cells in G2/M-phase To further understand the reversal effects of NF, we next investigated the apoptosis of MDR cancer cells after the treatment of NF via double staining with PI and annexin V-FITC. The quantitative analysis by flow cytometry showed that treatment of NF (4, 24, and 48 μM) significantly enhanced the apoptosis comparing with PTX alone (p < 0.001, Fig. 3A), while 48 μM NF alone did not show any toxicity. Notably, treatment with NF at only 4 μM could boost the apoptosis induced by PTX (0.94 μM) to a similar degree as that of 4.2 μM PTX (IC50 of PTX in HCT-8/T cells). We then tested whether the treatment of NF affects the cell cycle progression in HCT8/T cells using flow cytometric analysis. As shown in Fig. 3B, about 59.70% of PTX resistance HCT-8/T cells cultured with PTX (0.94 μM) were in the G0/G1 phase, and only 16.40% of cells were in the G2/M phase. However, these distributions were obviously shifted to 7.74% of cells in the G0/G1 phase, and 79.46% of cells in the G2/M phase after added 48 μM NF. However, the treatment of NF alone at 48 μM had no effect on cell cycle distribution of HCT-8/T. Synergistic effects evaluation using combination index The combination index (CI) analysis was carried out to evaluate the synergistic effect for NF and PTX in a quantitative manner. As shown in the Table 2, the CI values calculated at 50% (ED50) and 90% (ED90) based on the cytotoxicity for the combination of NF and PTX were 0.064 and 0.001, respectively, indicating a very strong synergistic cytotoxic effect in HCT-8/T cells. Moreover, the quantitative diagnosis graphics for the synergistic effect between NF and PTX were shown in the Supplementary figure 3.With CalcuSyn simulation, an ED50 calculated was 99.6 μM for NF or 6.03 μM for PTX in HCT-8/T cells. However, co-treated with 3.79 μM NF, the ED50 of PTX was reduced to 0.16 μM which was a 40-fold lower than that of PTX alone (Table 2). NF abrogated PTX resistance in vivo We evaluated the MDR sensitizing effects of NF in vivo with an A549/T human xenograft model in BALB/c-nu/nu mice. Treatment with PTX at 10 mg/kg did not suppress the tumor growth by compared with the vehicle group, indicating the tumor resistance to PTX (10 mg/ kg). Notably, mice treated with NF (7.5 mg/kg) and PTX (10 mg/kg) together exhibited significant inhibition on the tumor growth (~65%) and tumor size (~50%) as shown in Fig. 4 A and D, which is more effective than that of the verapamil (the positive control drug) group. Moreover, there was no obvious loss of body weight for mice co-treated of NF and PTX (Fig. 4 B), suggesting no significant toxicity induced by NF. And NF administration alone at a dosage of 7.5 mg/kg did not result in an obvious suppression of tumor growth. These in vivo results further suggested that NF is a potential MDR sensitizer without significant toxicity. NF promotes the accumulation of DOX and Rho123 To explore whether NF overcomes MDR via the inhibition of ABC transporters, intracellular accumulation assay was performed with two fluorescent substrates of P-gp included Rho 123 and DOX using a fluorescence microscope and flow cytometric analysis. The intracellular amounts of DOX and Rho123 were measured in the presence or absence of NF in A549/T cells and compared with that of parental A549 cells. As shown in Fig. 5 A and 5C, overexpression of P-gp in drug-resistant A549/T cells resulted in a significant decrease in the intracellular concentrations of DOX and Rho123 compared with that of sensitive A549 cells. However, 48 μM NF or 50 μM verapamil (positive control) significantly increased the accumulation of DOX and Rho123 in A549/T cells, but not in the parental sensitive cells, indicating NF could inhibit the efflux function of P-gp transporter. Moreover, the similar effects for enhancing the intracellular drug concentration by NF were observed in HCT-8/T using flow cytometry analysis. After NF treatment, the fluorescence of DOX and Rho123 increased about 22.3-fold and 43.5-fold in HCT-8/T, respectively. All these results suggested that NF increased the intracellular accumulation of anti-cancer drugs via inhibiting the efflux activity of ABC transporters, leading to reversal effects in MDR cancer cells. NF modulates the P-gp ATPase activity P-gp is an ATP-driven multidrug efflux transporter. Therefore, we monitored the nature of the interaction between NF and transporter using an in vitro P-gp-ATPase assay. As shown in Fig. 6A, NF simulate basal ATPase activity in a dose-dependent manner with EC50 of 39.85 μM, suggesting that NF interacts specifically with P-gp at the binding site as a substrate of ABCB1. Moreover, we also noticed the inhibition effects of NF on the stimulated ATPase activity by 200 μM Verapamil with an IC50 value of 41.13 μM (Fig. 6B). Although NF is a substrate of P-gp transporter, our data showed that it also acted as a P-gp inhibitor, because it competed with other substrates for binding to the P-gp ATPase and therefore inhibited the P-gp efflux transport. NF decreases the P-gp and BCRP expression To gain more insight into the mechanisms of NF in reversing MDR, we examined the expression levels of the three ABC transporters related to MDR as well as the effects of NF. Compared with the parental sensitive cancer cells, both HCT-8/T and A549/T cell lines express higher levels of P-gp, MRP2, and BCRP (Fig. 7A and 7B). Treatment of NF markedly decreased the protein expression of P-gp and BCRP in a dosedependent manner, but not for MRP2. Several recent studies found that Nrf2 and HIF-1α are the key transcriptional factors that regulate P-gp and/or BCRP expression patterns in chemo-resistant cancer cells (Roncuzzi et al., 2014; Sadeghi et al., 2018; Singh et al., 2010). Here, we found that Nrf2 and HIF-1α were significantly higher in resistant cancer cells compared with sensitive ones. Notably, NF treatment significantly reduced the expression of Nrf2 and HIF-1α in a dose-dependent manner, which is consistent with the expressions of P-gp and BCRP2, indicating Nrf2 and HIF-1α were associated with the activity of NF. Moreover, gene expression of P-gp (MDR1), BCRP, Nrf2 and HIF-1α but not MRP2 were reduced after the treatment of NF as shown in Supplementary Fig. 4, which further confirmed the WB results. Inhibition of PI3K/AKT and MAPK/ERK signaling pathway Enhanced PI3K/Akt and MAPK/ERK signaling pathways were responsible for the development of MDR in tumors (Garcia et al., 2009; Katayama et al., 2007; Wang et al., 2016; Zhang et al., 2020). We noticed the upregulated expression of phosphorylation of AKT (p-AKT) and ERK (p-ERK) in both HCT-8/T and A549/T cells than their parental cells (Fig. 7C, 7D), whereas there was no apparent change for the total amount of AKT and ERK. Interestingly, treatment with HCT-8/T and A549/T cells with NF for 48h significantly decreased the p-AKT and pERK levels. To investigate whether suppression PI3K/AKT and ERK pathways by NF lead to the downregulation of P-gp and BCRP, PI3K/AKT signaling activator IGF-1 and PI3K/AKT inhibitor LY294002 were used to activate or suppress the PI3K/AKT pathways. Western blot (WB) analysis (Fig. 8A) illustrated that LY294002 suppressed the p-AKT as well as the Nrf2, HIF-1α, P-gp, and BCRP expression. In contrast, activation of PI3k/AKT as shown with the promoted phosphorylation of AKT by IGF1 companies simultaneously with the increased expressions of Nrf2, HIF-1α, P-gp and BCRP. Moreover, IGF-1 counteracted the modulation effects of NF as well as LY294002. These data suggested that downregulating P-gp and BCRP by NF was associated with suppression of PI3K/AKT, Nrf2, and HIF-1α signaling pathways. persisted a stronger resistance to PTX as shown with cell viability (Fig. 8B). As expected, LY294002 at 2 μM overcome the resistance to the PTX in A549/T cells with a reversal fold of 37.7. However, the addition of IGF-1 could significantly block the sensitizing effects of NF and LY294002, which was consistent with the WB results. Together, these data indicated that P-gp and BCRP overexpression in PTX-resistant HCT-8/T and A549/T cells were positively regulated by the PI3K/AKT pathway, and inhibition of PI3K/AKT pathway by NF leading to suppression of Nrf2, and HIF-1α as well as their downstream targets P-gp and BCRP mainly contributed to its MDR sensitizing effects. Discussion Overcoming the MDR is the major challenge in treating cancer patients, because chemo-resistance causes disease relapse and metastasis. Current strategies to overcome tumor resistance mainly focused on the inhibitors of ATP binding cassette transporter such as P-gp, MRP2, and BCRP (Choi and Yu, 2014). However, limited or no benefits observed in clinical trials so far for the tested chemosensitizers which also associated with toxicity and/ or unwanted drug-drug interactions to cancer patients. Therefore, new effective MDR sensitizers without side effects will be valuable to drug resistant patients with cancer. Herein, for the first time, we showed that NF sensitized PTX-resistant P-gp overexpressing A549/T and HCT8/T cells to the chemotherapeutic agents included PTX, DOX, docetaxel (TXT) and daunorubicin (DAU) in vitro. Notably, NF (7.5 mg/kg) co-treatment with PTX (10 mg/kg) significantly inhibited the tumor growth, which is stronger than that of verapamil (10 mg/kg). Because NF could inhibit the P-gp function, and suppressed the expression of P-gp and BCRP, leading to the enhanced intracellular concentration of drugs. Although the mechanisms for overexpression of these transporters in MDR cancers are still unclear, nuclear factors HIF-1α and Nrf2 have been reported to regulate the expressions of ABC transporters (Comerford et al., 2002; Krishnamurthy and Schuetz, 2006). In addition, recent studies reveal that stable overexpression of Nrf2 and HIF-1α resulted in enhanced resistance of cancer cells to multiple chemotherapeutic drugs (Wang et al., 2008; No et al., 2014). Consistent with the literature, we noticed that there were significantly higher levels of Nrf2 and HIF-1α in two resistant cancer cells than that of the sensitive cells. NF could significantly reduce the expression of HIF-1α and Nrf2 in a dose-dependent manner, which may contribute to its suppressing effects on P-gp and BCRP. Moreover, growing evidence suggests activation of MAPK/ERK (Lee et al., 2007; Persons et al., 1999) and PI3K/AKT (Hayakawa et al., 2000; Mabuchi et al., 2002; Ohta et al., 2006; Hu et al., 2002) signaling pathways play an important role in chemo-resistance. Both pathways were reported to be involved in the regulation of P-gp and/or BCRP gene expression as well as the expression of HIF-1α (He et al., 2016) and Nrf2 (Krajka-Kuźniak et al., 2017), respectively. Therefore, we examined the activation of ERK and AKT and observed the increased phosphorylation of AKT and ERK in A549/T and HCT-8/T cells than that of parental A549 and HCT-8 cells. Moreover, NF dose-dependently reduced phosphorylation levels of AKT and ERK in the presence of PTX. We further confirmed the regulation role of PI3K/AKT pathway on the expression of Nrf2, HIF-1α, P-gp, and BCRP by using AKT inhibitor LY294002 and PI3K/AKT signaling activator IGF-1. These results not only help to elucidate the multiple-targets mechanisms for the reversal effect of NF, but also helpful for explaining the complex molecular signaling and pathways involved in the acquired MDR during cancer chemotherapy. There is cross-talk between reactive oxygen species (ROS) and PI3K/AKT and ERK pathways as well as the activation of Nrf2 and HIF‑1. Additionally, ROS exert central roles in cancer cell survival and also play an important role in MDR (Cen et al., 2016). Considering the effects of NF on blocking the PI3K/AKT and ERK signaling pathways as well as reducing the Nrf2 and HIF-1α levels, ROS may be the critical target associated with the sensitizing effects of NF against the resistance to multiple chemotherapeutic drugs, which needed further study. NF was the major alkaloid present in lotus leaves (Ye et al., 2014), which have low toxicity with LD50 at 240 mg/kg for mouse and 282 mg/kg for rat and high bioavailability of 69.56% after oral administration in rats (Xie et al., 2010), indicating a new potential drug as MDR sensitizer. Although we found NF works on multiple targets related to the development of MDR, the functional group of this alkaloid is still unclear. Based on the docking results by the Surflex-dock module embedded in Tripos Sybyl X 2.0 (St. Louis, USA) to the crystal structure of P-gp in complex with QZ59-RRR (PDB ID: 4M2S), nuciferine formed an H-bond with residue Gln721, and strong π-π and π‒δ interactions with residues Phe979, Phe332, and Phe724 as well as van der Waals interactions and hydrophobic interactions with residues of Ile336, Phe728, and Tyr306 as shown in supplementary figure 5A. Moreover, it was found that the key hydrogen bond is lost after the methoxyl group is replaced by NH2 or Cl, leading the docking scores decreased from 5.46 to 3.86 and 3.72, respectively (supplementary figure 5B). Therefore, the methoxyl group is very important for the binding between nuciferine and P-gp. Although numerous genes and pathways contribute to the development of MDR in cancer, however, natural compounds hold the important potential in new drug discovery because of their multi-targets activity (Dinić et al., 2015; Wu et al., 2011). They can modulate more than two MDR mechanisms such as the efflux transporters such as P-gp, 3 times as described in Method, and presented are representative images. BCRP and/or MRP2 (Wu et al., 2011), PI3K/AKT/ Nrf2 and HIF-1α pathways, as well as ROS levels (Guo et al., 2017), but not affect the bioavailability of co-administered chemotherapeutic agents. Moreover, phytocompounds come from natural products that have been used for more than thousands of years with good safety, compatibility and therapeutic potential (Mehta and Dhapte, 2016; Mehta et al., 2017; Mehta et al., 2015). All these characters indicated that it would be worthwhile to test natural compounds like NF as adjuvant chemotherapy to overcome drug resistance. In conclusion, this study provided the first evidence that NF is a novel and potent multiple targets MDR sensitizer. References Abdallah, H.M., Al-Abd, A.M., El-Dine, R.S., El-Halawany, A.M., 2015. P-glycoprotein inhibitors of natural origin as potential tumor chemo-sensitizers: a review. J. Adv. Abrams, S.L., Steelman, L.S., Shelton, J.G., Wong, E.W.T., Chappell, W.H., Bäsecke, J.Res. 6, 45–62. ,Stivala, F., Donia, M., Nicoletti, F., Libra, M., Martelli, A.M., McCubrey, J.A., 2010. Cen, J., Zhang, L., Liu, F., Zhang, F., Ji, B.-S., 2016. Long-term alteration of reactivtargeted therapy. Cell Cycle 9, 1781–1791. 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