Background
Human pancreatic ductal adenocarcinoma (PDAC) is a highly malignant and lethal digestive tumor. PDAC currently ranks the fourth leading cause of cancer-related death worldwide with a dismal 5-year survival rate, which is less than 5% [
1]. Despite the fact that some effective treatments are available, cancer death rates due to PDAC continue to rise unabated. One of the major obstacles in PDAC treatment is the occurrence of resistance to the gemcitabine (GEM)-based chemotherapy [
2]. GEM, a cytotoxic nucleoside analog, has been widely used as the standard first-line treatment for advanced PDAC [
2,
3], but chemoresistance often occurs clinically. Hence, it is imperative to identify effective adjuvants to enhance the chemosensitivity of GEM in PDAC.
Nuclear factor-erythroid factor 2-related factor 2 (NFE2L2, Nrf2) is an important transcription factor that regulates the antioxidant response by eliciting the expression of genes bearing an antioxidant response element (ARE) in their regulatory regions [
4]. The activity of Nrf2 is primarily governed by its physical and functional interaction with the cytosolic repressor Kelch-like ECH-associated protein 1 (Keap1), which facilitates the ubiquitination and subsequent proteasomal degradation of Nrf2 in the cytoplasm via the Cullin 3 ubiquitin ligase complex under normal condition [
5]. Following oxidative stress, Nrf2 is released from Keap1, then translocates into the nucleus where it activates ARE-mediated Nrf2 downstream genes like Phase II metabolizing-detoxifying and antioxidant defense enzymes such as NADP(H): quinone oxidoreductase (NQO1), heme oxygenase-1 (HO-1), γ-glutamylcysteine synthetase modifier subunit (γ-GCSm) and Aldo–keto-reductase (AKR1B10) [
6‐
9]. In cancer cells, Nrf2 can also stimulate the multidrug-resistance pretein-1 (MRP1) and multidrug-resistance protein-5 (MRP5) which promote chemoresistance [
10‐
12]. Growing evidence indicates that aberrant Nrf2 signaling is frequently found in multiple cancers including PDAC, and is linked to tumor progression and poor prognosis. Activation of Nrf2 is also correlated with chemotherapy drug resistance in PDAC cells [
13‐
17]. Patients with relatively lower expression level of Nrf2 were found to be more sensitive to chemotherapy [
18]. Moreover, Nrf2 deletion in the KPC mice caused a decrease in the formation of precancerous lesions and slowed down the development of invasive PDAC [
13,
19]. Therefore, Nrf2 has been considered as a therapeutic target for PDAC prevention and therapy, and the Nrf2 suppression could be exploited for augmentation of efficacy of PDAC therapeutics. In this regard, it is urgently needed to identify agents that could suppress the Nrf2 activity, and to develop them into adjuvant therapy for the GEM-based chemotherapy for PDAC.
Brucein D (BD) is a quassinoid originally isolated from Chinese herb
Bruceae Fructus (Ya-Dan-Zi in Chinese), which has been used in clinical practice to treat inflammation, malaria, warts and cancers [
20]. Previously, we have found BD to have anti-cancer activity in several cancer types such as PDAC, lung cancer and hepatocellular carcinoma via inducing apoptosis, autophagy and oxidative stress through modulating the reactive oxygen species (ROS)/mitogen-activated protein kinase (MAPK) signaling pathway [
21‐
23]. However, data about the combined effects of BD and GEM remains scarce. The present study aimed to explore whether BD could enhance the chemosensitivity of GEM in PDAC, and to elucidate the underlying molecular mechanisms focusing on Nrf2 pathway. By utilizing human PDAC cell lines, we have confirmed that BD efficiently inhibited cell proliferation and enhanced chemosensitivity of GEM in PDAC cells via modulating Nrf2 and its downstream target genes. Using a genetically engineered mouse model of pancreatic cancer, i.e.,
Krastm4Tyj Trp53tm1Brn Tg (Pdx1-cre/Esr1*) #Dam/J (KPC tamoxifen-inducible) mouse that can generate PDAC spontaneously, we have verified the anti-PDAC effect of BD in vivo. Our findings unambiguously indicate that BD is a promising adjuvant therapy to augment the chemosensitivity of GEM in PDAC, and the action mechanism involves mitigation of the aberrant Nrf2 expression.
Methods
Human tissue specimens
Human PDAC and adjacent normal pancreatic tissue were collected from PDAC patients with informed consent at Queen Elisabeth Hospital, Hong Kong, China. The use of human clinical specimens was approved by the Research Ethics Committee of Kowloon Central / Kowloon East Cluster, Hong Kong, China.
Cell lines and culture conditions
Human PDAC cell lines PANC-1 and Capan-2 were purchased from the American Type Culture Collection (ATCC). Miapaca-2 cell line was a gift of Prof. XU Hong-xi (Shanghai University of Traditional Chinese Medicine, Shanghai, China). All of the cell lines were cultured in Dulbecco’s Modified Eagle medium (DMEM) supplied with 10% fetal bovine serum (FBS) and 10 U/mL penicillin–streptomycin in an incubator at 37 °C and 5% CO2.
Gene expression correlation with stage and survival analysis
The correlation between gene expression and clinical stage was determined using Gene Expression Profiling Integrative Analysis (GEPIA,
http://gepia.cancer-pku.cn) [
24]. The correlation between gene expression and overall survival (OS) was established using the Cox model. Nrf2 with higher expression in the PDAC samples had corresponding lower survival.
Fluorescence-activated cell sorting (FACS) analysis
For the flow cytometry experiment, Annexin V/PI staining kit (BD Pharmingen™, USA) was used to detect the cell apoptosis following the manufacturer’s instruction. Apoptotic cells were counted by a Cytomics™ FC500 flow cytometer (Beckman Coulter, Fullerton, CA, USA). Intracellular ROS concentration was measured by Total ROS Assay Kit (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s manual. All the data generated by flow cytometer were analyzed using the Kaluza software (Beckman Coulter).
Western blot analysis
Proteins were extracted from cell lysates or tumor tissues with lysis buffer, supplemented with a complete protease inhibitor cocktail (Thermo Scientific, USA). The concentrations of the protein extracts were determined by bicinchoninic acid (BCA) test. Protein samples were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. After blocking nonspecific binding with TBS/T (0.1%) containing 5% non-fat milk for 1 h at room temperature (RT), the membranes were then incubated overnight at 4 °C with primary antibody diluted in 3% BSA in TBS/T (0.1%). The membrane was washed with TBS/T four times to remove the unbound antibody and then incubated with the secondary antibody (HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG, 1: 2,500; Cell Signaling Technology) for 1 h at room temperature. Protein bands were visualized with an ECL kit (Invitrogen, Carlsbad, CA, USA). All antibodies and their dilutions used in this experiment were listed in the Additional file 1, Table S1.
Immunofluorescent staining
PDAC cells were plated on coverslips and allowed to adhere overnight and expose to BD (2.5 µM) for 24 h. Then the cells were fixed with 4% paraformaldehyde/PBS, blocked with 5% BSA in PBS and incubated with Nrf2 primary antibody (1:100; Santa Cruz #sc-365949) in PBS containing 3% BSA, followed by Cy3-labeled secondary antibody (Abcam, United Kingdom). Nuclei were stained with DAPI (Santa Cruz, Texas, USA). Images were visualized using an inverted fluorescent microscope (Carl Zeiss, Germany).
Lentivirus transfection of Nrf2 shRNA and stable cell lines
Target cells (Miapaca-2, Capan-2, PANC-1) were plated in 24-well plates at the density of 2 × 104 cells/well with complete growth medium, and then incubated overnight to improve the adherence of cells to the plates. For lentivirus transduction, the cells were infected with either Nrf2 shRNA or control shRNA lentiviral particles (Santa Cruz, sc-37030-V; sc-108080) in serum-free growth medium with 5 μg/mL polybrene at multiplicities of infection (MOI) of 20, 20, 4 separately. The plates were gently shaken and incubated overnight. Then the medium was replaced by fresh DMEM complete medium, and the cells were incubated for additional 48 h to allow the shRNA to reach its maximum effect. After lentivirus transduction, cells were reseeded in 6-well plates and treated with antibiotics puromycin to select stable clones expressing the shRNA. After the first dose, the medium was replaced with the fresh medium containing puromycin (1, 2 and 5 μg/mL) every 2–3 days until resistant colonies can be identified. Stable cell lines were verified by qRT-PCR and western blot.
Measurement of Nrf2 protein stability and ubiquitination
Cells were treated with BD (1.5 µM) and with MG132 (20 µM). After treatment for a given time, the cells were harvested for the western blot analysis of the Nrf2 protein level. The half-time of Nrf2 in cells was detected by cycloheximide (CHX)-chase analysis. Cells were co-treated with or without BD (1.5 µM) with CHX (25 µM). Total cell extracts were prepared at indicated time points following treatment with CHX, and then cell extracts were detected by western blot. For analysis of Nrf2 ubiquitination, cells were treated with or without BD (1.5 µM) for the indicated time periods (0–48 h), and the level of ubiquitination was detected by western blot using an anti-ubiquitin antibody.
Molecular docking of BD on human Keap-1 and Nrf2 complex
SwissDock (URL:
www.swissdock.ch) was used to perform the molecular docking analysis of BD on Keap-1: Nrf2 complex. The 3D structure of BD was downloaded from ZINC webpage with the number ZINC8221322 (UCSF; San Francisco, CA, USA). Crystal structure of Keap-1: Nrf2 (PDB ID:2FLU) was downloaded from RCSB PDB Bank (
http://www.pdb.org). The docking results were analyzed using UCSF Chimera 1.11.1 (RVBI, UCSF; San Francisco, CA, USA). Ligand binding results with negative △G values were regarded as having an affinity in the binding between BD and Keap-1: Nrf2. The number of possible hydrogen bonds and the bond lengths were determined by the Find H-Bond tool in UCSF Chimera. All docking procedures were performed using Windows 10.
PDAC mouse models
Six-week-old female BALB/c nude mice were obtained from the Laboratory Animal Services Centre, The Chinese University of Hong Kong (CUHK). Genetically engineered transgenic mouse model (GEMM), which has the mixed background Krastm4Tyj Trp53tm1Brn Tg (Pdx1-cre/Esr1*) #Dam/J (KPC tamoxifen-inducible), was purchased from the Jackson laboratory (Stock number: 032429) (Bar Harbor, Maine, USA). PCR was applied for the genotyping of the transgenic mice (genotyped for the presence of KRAS, P53, and Cre). The primer sequences used for the genotyping of transgenic mice were presented in the Additional file 1: Table S2. All animals were kept in a pathogen-free environment with free access to food and water. All animal experiments were conducted according to the ethical policies and procedures approved by the Animal Experimentation Ethics Committee of CUHK (Ref. No.: 19/079/NSF and 20/169/MIS).
For the orthotopic implantation, Capan-2, control non-specific shRNA (shControl) or Nrf2-targeting shRNA (shNrf2)-transfected Miapaca-2 cells (1.5 × 106 cells/100 μL) were injected into the pancreatic tail of each nude mouse. The mice were randomly divided into four groups (8 mice per group) 1 week after tumor implantation (day 1): Vehicle control (0.5% DMSO, daily, i.g) group, BD (2 mg/kg, daily, i.g.) group, GEM (20 mg/kg twice a week, i.g.) group, and BD (2 mg/kg, daily, i.g.) plus GEM (20 mg/kg twice a week, i.g.) group. At the end of drug treatment, the mice were sacrificed and the tumor tissues harvested for western blot and immunohistochemistry analysis. Tumor volumes were measured using a vernier caliper and calculated using the following formula: [(shortest diameter)2 × (longest diameter) / 2].
For the GEMM of PDAC, the KPC mice were received with the intraperitoneal injection of tamoxifen (1.5 mg/mouse) for 5 consecutive days at 4 weeks of age to induce metastatic pancreatic carcinoma. Tamoxifen was diluted in corn oil (Sigma-Aldrich) to afford the injection solution. The KPC mice were randomly divided into 4 groups (7 mice per group): Vehicle control group (0.5% DMSO, i.g.), BD (2 mg/kg, once daily, i.g.) group, GEM (20 mg/kg twice weekly, i.g.) group, BD (2 mg/kg, once daily, i.g.) plus GEM (20 mg/kg twice a weekly, i.g.) group. BD, GEM or vehicle were given to the KPC mice for 4 consecutive weeks. At the end of drug treatment, blood samples (500 µL) were collected from the mice under anaesthesia, and then the mice were sacrificed by cervical dislocation. Serum was collected after centrifugation at 2,000 xg for 15 min in a refrigerated centrifuge. The liver and renal toxicities of BD were determined by measuring the levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and creatinine using respective activity assay kits (Nanjing Jiancheng Bioengineering Institute, China).
Histopathological and immunohistochemical analysis
Tissues for histopathological analysis were fixed in 4% paraformaldehyde (PFA) at 4 °C overnight before paraffin embedding. All sections used for histological analysis were cut into 5 μm thickness using a microtome. Histological characterization and consequent scoring of neoplastic lesion on hematoxylin and eosin (H&E)-stained sections of pancreas were performed. A registered pathologist provided supervision and confirmation for this work. Masson’s trichrome staining was performed to determine the collagen deposition using the Trichrome stain (Masson) Kit (HT15-1KT, Sigma-Aldrich, Heatherhouse, United Kingdom) according to the manufacturer’s instructions. The percentages of the stained area were calculated using Image J software.
For immunohistochemical analysis, paraffin sections were de-waxed with xylene, and re-hydrated with gradient ethanol before they were stained with antibodies against Ki-67 (1:100), Nrf2 (1:100) and NQO1 (1:100). All sections were photographed using inverted fluorescence microscope (Carl Zeiss, Germany). The proportion of IHC-positive cells was determined in the randomly selected microscopic fields.
Statistical analysis
Data were presented as the mean ± SD. Student’s t-test was used for comparison between two different groups, and one-way analysis of variance (ANOVA) was used for multiple comparisons. A two-tailed value of p < 0.05 was considered statistically significant. Statistical analyses were carried out using GraphPad Prism 8.0 (Version 8, GraphPad Software, Inc., CA, USA).
Discussion
Drug resistance continues to be one of the biggest challenges in cancer treatment, and exists across many types of cancer and chemotherapeutic regimens, including chemotherapy, targeted therapy and immunotherapy. Both intrinsic and acquired factors are involved in drug resistance. Overcoming drug resistance to improve chemosensitivity is a goal that has been pursued on many fronts, including basic science to uncover the underlying fundamental biological mechanisms and clinical trials testing new treatment strategies [
2,
25‐
27]. Previous studies have reported that Nrf2 over-activity is a cardinal molecular mechanism of chemoresistance, and Nrf2 inhibition can reverse the drug resistance in many cancer types including PDAC [
28‐
34]. Nrf2 also modulates a number of genes which control endogenous antioxidant protection and detoxification of ROS. The following genes including NQO1, HO-1, γ-GCSm, AKR1B10, MRP1 and MRP5 are known to be the important targets of Nrf2, and they are all involved in drug resistance in PDAC [
35,
36]. Congruent with previous studies, we found that Nrf2 was highly expressed in tumor tissues of PDAC patients and predict poor prognosis, and silencing Nrf2 could markedly augment the sensitivity of GEM in PDAC. Our results unequivocally indicate that Nrf2 is a critical target for PDAC treatment, and inhibition of Nrf2 would therefore constitute an attractive strategy to sensitize PDAC cells to chemotherapeutic agents.
Several natural compounds have been shown to act as Nrf2 inhibitors and used as chemosensitizers in different types of cancer. Brusatol, a quassinoid originally isolated from
Bruceae Fructus, a Chinese herbal medicine, potently reduces the protein level of Nrf2 in A549 cells, and sensitizes these cells to cisplatin and other chemotherapeutic drugs [
37]. Digoxin was reported to sensitize GEM-resistant PDAC cells to GEM by inhibiting Nrf2 through suppressing the PI3K/Akt signaling pathway [
38]. Wogonin, an O-methylated flavone isolated from
Scutellariae Radix, was able to reduce the Nrf2 activity by suppressing the PI3K/Akt and Stat3/NF-κB signaling pathways and reverse chemoresistance [
39,
40]. Our previous study revealed that BD could induce apoptosis in PDAC cells via modulating the activation of p38-mitogen activated protein kinase [
21].
In the present study, we evaluated the anti-cancer effects of BD combined with GEM in PDAC cells. Our results revealed that BD could significantly improve the chemosensitivity of GEM. We also evaluated the therapeutic efficacy of the combination of BD and GEM in PDAC using KPC mice, a genetically engineered mouse model of PDAC widely used in PDAC research. Among which the
KRAS gene encodes a protein that plays an essential role in cell signaling in normal tissue, through its activity as an ‘on/off’ switch for many signal transduction pathways, particularly those regulating cell division. Activation of mutations in pancreatic tumorigenesis causes
Kras to be constitutively active, subsequently renders cells to grow and divide in an uncontrolled manner. p53, a tumor suppressor protein, stops cells from dividing too fast, and causes the damaged or mutated cells that might otherwise become tumorigenic to undergo apoptosis. It is well-known that p53 is often mutated in pancreatic tumors, meaning that mutated cells do not undergo apoptosis, thereby resulting in the occurrence of unregulated cell division. Because KPC mice carry mutations in genes which accurately mimics both the genetic and histologic changes of human PDAC, this model is therefore ideal for testing the efficacy of novel therapeutics [
41,
42]. We found that BD could also enhance the chemosensitivity of GEM on KPC mice. Our findings revealed that treatment with combination of BD and GEM resulted in fewer cancer incidents, reduced number and size of macroscopic tumors, as compared with BD or GEM alone treatment. Biochemical and histological analyses showed that BD, GEM and their combination treatment did not affect the serum AST, ALT and creatinine levels and no obvious toxicity was observed in liver and kidney tissues of KPC mice, indicating that BD plus GEM treatment has good in vivo safety profile.
We subsequently tested BD for its ability to inhibit Nrf2 and to sensitize PDAC cells to apoptosis and PDAC tumors to GEM-based chemotherapy. As expected, BD exerted significant inhibitory effect on Nrf2 activity in a dose-dependent manner in PDAC cell lines. We also found that BD treatment did not alter Nrf2 mRNA level in PDAC cell lines but regulated and decreased Nrf2 protein levels by promoting its degradation. As proteasome activation contributes to anti-apoptotic effect of Nrf2 in tumor cells, not only the cellular response to anticancer drugs depends on phase II enzymes and detoxification genes expression, but also the responsiveness to death ligands would be affected by Nrf2 inhibition [
43‐
45]. Accordingly, Nrf2-dependent resistance to anticancer drugs was significantly abrogated in all cell lines when pretreated with BD. Besides, the cellular ubiquitin level was increased after BD treatment and the BD-induced degradation of Nrf2 was effectively abolished by proteasome inhibitor MG-132. Our results amply indicate that the BD-induced Nrf2 downregulation involves the ubiquitin–proteasome-dependent pathway. Moreover, lentivirus depletion of Nrf2 together with BD treatment markedly sensitized anti-PDAC effect of GEM in both in vitro and in vivo pancreatic models.
Our present study has unambiguously demonstrated that BD, a naturally occurring quassinoid, is a potent inhibitor of Nrf2. Mechanistically, BD inhibits Nrf2 signaling via promoting degradation of Nrf2 protein and suppressing its downstream genes, thereby enhancing the chemosensitivity of GEM (Fig.
9g). Our experimental data strongly indicate that BD is worthy of being developed as a chemotherapeutic adjuvant for the treatment of PDAC, especially for those patients with aberrantly high Nrf2 expression.
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