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Pancreatic cancer rapidly acquires resistance to chemotherapy resulting in its being difficult to treat. Gemcitabine is the current clinical chemotherapy strategy; however, owing to gemcitabine resistance, it is only able to prolong the life of patients with pancreatic cancer for a limited number of months. Understanding the underlying molecular mechanisms of gemcitabine resistance and selecting a suitable combination of agents for the treatment of pancreatic cancer is required. Astaxanthin (ASX) is able to resensitize gemcitabine-resistant human pancreatic cancer cells (GR-HPCCs) to gemcitabine. ASX was identified to upregulate human equilibrative nucleoside transporter 1 (hENT1) and downregulate ribonucleoside diphosphate reductase (RRM) 1 and 2 to enhance gemcitabine-induced cell death in GR-HPCCs treated with gemcitabine, and also downregulates TWIST1 and ZEB1 to inhibit the gemcitabine-induced epithelial-mesenchymal transition (EMT) phenotype in GR-HPCCs and to mediate hENT1, RRM1 and RRM2. Furthermore, ASX acts through the hypoxia-inducible factor 1α/signal transducer and activator of transcription 3 signaling pathway to mediate TWIST1, ZEB1, hENT1, RRM1 and RRM2, regulating the gemcitabine-induced EMT phenotype and gemcitabine-induced cell death. Co-treatment with ASX and gemcitabine in a tumor xenograft model induced by GR-HPCCs supported the
Pancreatic cancer is a lethal malignancy, with a mortality rate of >90%; it is ranked fourth in terms of cancer-related mortality (
Human equilibrative nucleoside transporter 1 (hENT1) is able to carry pyrimidine nucleosides and purine into cells (
Epithelial-mesenchymal transition (EMT) results in the loss of cell-cell junctions and migratory and invasive mesenchymal cell formation (
Astaxanthin (ASX) is a lipophilic compound, exhibiting antioxidant, anti-inflammatory and immunomodulatory characteristics. The anticancer ability of antioxidants has been a focus of research, particularly the effect of oxidative stress and metabolism. Powerful antioxidants may be novel and effective agents for the treatment of carcinoma. Previous studies have demonstrated that ASX was able to inhibit the proliferation of various types of cancer cell through immunomodulatory and cell communication modulation at gap junctions (
The HPCCs Panc-1 and HTB-79 were purchased from the American Type Culture Collection (Manassas, VA, USA). All cells were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin, at 37°C and 5% CO2. All cell culture regents were purchased from Gibco; Thermo Fisher Scientific, Inc. (Waltham, MA, USA). Panc-1 and HTB-79 cells were exposed to gemcitabine at increasing concentrations from 10 nM to generate gemcitabine-resistant cells. After 2 weeks of adaptation, the concentration was doubled. The final gemcitabine concentration to which the cells were adapted was 640 nM, and the cells were designated GR-Panc-1 cells and GR-HTB-79 cells.
For each sample, Panc-1 and HTB-79 cells were lysed for 30 min in radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) on ice, and the cell debris was centrifuged at 12,000 × g for 8 min at 4°C. The protein concentration was determined using the bicinchoninic acid assay, and 40 µg proteins were separated by SDS-PAGE (10–15% gels) and blotted onto a polyvinylidene fluoride (PVDF) membrane. The PVDF membrane was blocked in a solution of 5% non-fat dried milk in PBST (0.05% Tween-20 in PBS) for 1 h at room temperature, and incubated with antibodies at 4°C for 12 h. The PVDF membrane was washed three times for 10 min in PBST, incubated with secondary antibodies at 37°C for 1 h, and washed again three times for 10 min in PBST. GAPDH was used as a loading control. All western blotting reagents were purchased from Beyotime Institute of Biotechnology. All the antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). The first antibodies were diluted at 1:500, and the secondary antibodies were diluted at 1:5,000. The following antibodies were used: hENT1 (cat. no. sc-48489; polyclonal, goat anti-human), RRM1 (cat. no. sc-22786; monoclonal, rabbit anti-human), RRM2 (cat. no. sc-137174; monoclonal, mouse anti-human), GAPDH (cat. no. sc-293335; monoclonal, mouse anti-human), Twist1 (cat. no. sc-134136, polyclonal, mouse anti-human), ZEB1 (cat. no. sc-517272; monoclonal, mouse anti-human), E-cadherin (cat. no. sc-33743; polyclonal, rabbit anti-human), STAT3 (cat. no. sc-8059; monoclonal, mouse anti-human), α-SMA (cat. no. sc-53142; monoclonal, mouse anti-human), HIF-1α (cat. no. sc-13515; monoclonal, mouse anti-human), mouse IgG (cat. no. sc-516176), rabbit IgG (cat. no. sc-2794) and goat IgG (cat. no. sc-2419).
Each well of a 96-well plate was inoculated with 104 Panc-1 or HTB-79 cells and cells were allowed to attach overnight, prior to treatment with drugs for 24 h. The medium was removed and the cells were washed three times with PBS, prior to the addition of 90 µl Dulbecco's modified Eagle's medium (DMEM) and 10 µl Cell Counting kit-8 reagent (Beyotime Institute of Biotechnology) to each well. Cells were incubated at 37°C for 1 h before determining the optical density at 450 nm using a microplate reader.
Treated Panc-1 or HTB-79 cells suspensions were obtained and a 1/9 volume of 0.4% (w/v) trypan blue solution was added. The number of total cells and dead cells (those that did not exclude the dye) were determined, and the total death rate was calculated as (number of dead cells/number of total cells) ×100%.
Transient transfection of GR-Panc-1 cells with small interfering RNAs (siRNAs) and plasmids was performed using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.), the siRNAs were purchased from Shanghai Genechem Co., Ltd. (Shanghai, China), and the sequences of siRNAs were as follows: Control, 5′-UUCUCCGAACGUGUCACGUTT−3′; hENT1, 5′-AUGACAUUGUUGAAGAUGGCA-3′; ZEB1, 5′-GGACUGAAGUCAGGUAAGGCA-3′; Twist1, 5′-AAACAUUUGUUUUAAGGAGAA-3′; STAT3, 5′-GCUAAGUCAGCUUCAUUGAGU−3′; and HIF-1α, 5′-GCAUUGCCAUCAGUCACGCUA-3′. The con-pcDNA3.1, RRM1-pcDNA3.1 and RRM2-pcDNA3.1 plasmids were purchased from Shanghai Genechem Co., Ltd. The 6-wells plates were used for siRNA or plasmid transfection assays. For the each well, 500 ng siRNAs or 2 µg plasmids were added to 10 µl Lipofectamine 2000 according to the manufacturer's protocol, and were incubated with GR-Panc-1 cells for 48 h. The siRNAs or plasmids were then removed, and GR-Panc-1 cells were treated with drugs for an additional 24 h. Subsequently, the cells were collected for western blot analysis, as aforementioned.
A BioCoat Matrigel invasion chamber system (Corning Incorporated, Corning, NY, USA) was used to assay cell invasion. Using a Transwell plate, the lower chamber was filled with culture medium without cells, and the upper chamber was filled with cell suspension and medium containing 10% FBS. The Transwell plate was incubated at 37°C for 24 h. Cells that adhered to the upper chamber surface were removed, and the cells that adhered to the lower chamber surface were stained with 4% paraformaldehyde for 15 min, rinsed with water and dried. The 0.5% crystal violet was extracted with 50% ethanol containing 0.1 M sodium citrate, and the absorbance at 600 nm was determined.
A total of 30 male 6-week-old BALB/c nude mice (weighing 18–22 g) were purchased from the Institute of Zoology (Chinese Academy of Sciences, Beijing, China). All animal experiments were performed according to the guidelines of the Institutional Animal Care and Use Committee of Institute of Zoology (Chinese Academy of Sciences, Beijing, China). All the animals were fed in a pathogen-free environment, at 24–28°C. Ventilation was required 10–20 times per hour, relative humidity was 50–60%, the light/dark cycle was natural circadian light, the food was sterilized by irradiation, and the water contained bacitracin (4 g/l) and neomycin (4 g/l). Each BALB/c nude mouse was subcutaneously inoculated with 5×106 Panc-1 or GR-Panc-1 cells into the right and left hind footpads. At 2 days after inoculation, the mice inoculated with Panc-1 cells were treated with 10 mg/kg gemcitabine 3 times/day by intraperitoneal injection, and the mice inoculated with GR-Panc-1 cells were treated with 10 mg/kg gemcitabine or co-treated with 500 mg/kg ASX and 10 mg/kg gemcitabine 3 times/day by intraperitoneal injection, with the injection of ASX occurring 2 h before that of gemcitabine. ASX and gemcitabine were dissolved in saline. Tumor volumes were determined weekly.
Tumors were immersed in 4% paraformaldehyde for 24 h and dehydrated in 30% sucrose solutions, prior to paraffin-embedding and cutting into sections (10 µm). The sections were treated using an
Results are presented as the mean ± standard deviation of triplicate experiments and were performed with SPSS 17.0 (SPSS, Inc., Chicago, IL, USA). Two-way analysis of variance, followed by Bonferroni post hoc testing, was used to compare different groups. The unpaired Student t-test or Mann-Whitney U test was used to compare two means. P<0.05 was considered to indicate a statistically significant difference.
Using increasing gemcitabine concentrations starting at 10 nM to induce Panc-1 and HTB-79 GR-HPCCs, designated GR-Panc-1 and GR-HTB-79. Panc-1 and HTB-79 cells were treated with various concentrations of gemcitabine; cell viability was identified to decrease with increasing gemcitabine concentration, and the cell death ratio was identified to increase with increasing gemcitabine concentration (
It has been demonstrated previously that hENT1, RRM1 and RRM2 serve important roles in the anticancer efficiency of gemcitabine (
To investigate whether ASX affects the EMT phenotype which was established to investigate gemcitabine resistance, the expression epithelial (E-)cadherin, TWIST1, ZEB1 and α-smooth muscle actin (α-SMA) were examined in HPCCs and GR-HPCCs treated with gemcitabine or co-treated with gemcitabine and ASX. It was identified that the expression levels of the mesenchymal cell markers in GR-HPCCs were increased compared with those in HPCCs, and that of the epithelial cell marker was decreased (
It has been demonstrated previously that HIF-1α and STAT3 were mediators of TWIST1 and ZEB1 (
The aforementioned results indicated that ASX is able to stimulate gemcitabine-induced cell death in GR-HPCCs
In the present study, it was identified that ASX is able to selective kill GR-HPCCs by increasing sensitivity to gemcitabine. The results of the present study indicated that GR-HPCCs exhibit an EMT phenotype, and GR-HPCCs exhibit a downregulated hENT1 expression level and upregulated RRM1 and RRM2 expression levels via TWIST1 and ZEB1 mediated by the HIF-1α/STAT3 signaling pathway (
Previous studies investigated that hENT1 was a key mediator of gemcitabine resistance in the clinic (
Using a tumor xenograft model to evaluate the effect of ASX and gemcitabine co-treatment
The results of the present study indicated that HIF-1α/STAT3-TWIST1/ZEB1-EMT was a novel mechanism for gemcitabine resistance in GR-HPCCs. Furthermore, it is suggested that co-treatment with ASX and gemcitabine may be a novel and efficient therapeutic strategy for gemcitabine-resistant pancreatic cancer by targeting hENT1, RRM1 and RRM2, and inhibiting the gemcitabine-induced EMT phenotype.
astaxanthin
epithelial-to-mesenchymal transition
gemcitabine-resistant human pancreatic cancer cells
human pancreatic cancer cells
human equilibrative nucleoside transporter 1
ribonucleoside diphosphate reductase
ASX enhances gemcitabine-induced pancreatic cancer cell death. (A) HPCCs and GR-HPCCs were treated with various concentrations of gemcitabine for 24 h. (B) GR-HPCCs were pretreated with 0.1% DMSO or 200 µM ASX for 2 h, and then treated with various concentrations of gemcitabine for 24 h. (C) GR-HPCCs were pretreated with various concentrations of ASX for 2 h, and then treated with 1 µM gemcitabine or 0.1% DMSO for 24 h. Cell viability was determined using the Cell Counting kit-8. The cell death ratio was determined using a trypan blue assay. *P<0.05; **P<0.01; ***P<0.005. ASX, astaxanthin; HPCCs, human pancreatic cancer cells; GR-HPCCs, gemcitabine-resistant HPCCs; DMSO, dimethyl sulfoxide.
ASX upregulates hENT1 and downregulates RRM1 and RRM2 expression in GR-HPCCs, and increases gemcitabine-induced GR-HPCC death through hENT1, RRM1 and RRM2 signaling. (A) HPCCs and GR-HPCCs were treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. ASX was able to restore the decrease in hENT1 protein expression and restore the RRM1 and RRM2 protein expression levels in GR-HPCCs. (B) GR-Panc-1 cells were transfected with control siRNA or hENT1-siRNA, and treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. The cell death ratio was analyzed using a trypan blue assay. GR-Panc-1 cells were transfected with (C) RRM1 plasmid (RRM1-pcDNA3.1) or (D) RRM2 plasmid (RRM2-pcDNA3.1) or control plasmid (Con-pcDNA3.1), and treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. The cell death ratio was determined using a trypan blue assay. *P<0.05; **P<0.01; ***P<0.005. ASX, astaxanthin; hENT1, human equilibrative nucleoside transporter 1; RRM, ribonucleoside diphosphate reductase; GR-HPCCs, gemcitabine-resistant HPCCs; HPCCs, human pancreatic cancer cells; DMSO, dimethyl sulfoxide.
ASX suppresses the EMT phenotype of GR-HPCCs through TWIST1 and ZEB1 signaling, and recover the activation of TWIST1 and ZEB1 in GR-HPCCs. (A) HPCCs and GR-HPCCs were treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. (B) GR-Panc-1 cells were transfected with control siRNA (Con-siRNA) or TWIST1-siRNA. (C) The cell death ratio was determined using a trypan blue assay. (D) GR-Panc-1 cells were transfected with control siRNA (Con-siRNA) or ZEB1-siRNA. (E) The cell death ratio was determined using a trypan blue assay. (F) ASX suppresses the invasive ability of GR-HPCCs. HPCCs and GR-HPCCs were treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. **P<0.01; ***P<0.005. ASX, astaxanthin; EMT, epithelial-mesenchymal transition; GR-HPCCs, gemcitabine-resistant HPCCs; HPCCs, human pancreatic cancer cells; siRNA, small interfering RNA; E-cadherin, endothelial cadherin; α-SMA, α-smooth muscle actin; hENT1, human equilibrative nucleoside transporter 1; RRM, ribonucleoside diphosphate reductase.
ASX resensitizes GR-HPCCs to gemcitabine-induced cell death through the HIF-1α/STAT3 signaling pathway. (A) HPCCs and GR-HPCCs were treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. (B) GR-Panc-1 was transfected with control siRNA (Con-siRNA) or STAT3-siRNA, and treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. (C) The cell death ratio was determined using a trypan blue assay. (D) GR-Panc-1 was transfected with control siRNA (Con-siRNA) or HIF-1α-siRNA, and treated with 1 µM gemcitabine alone or co-treated with 200 µM ASX (2 h pretreatment) and 1 µM gemcitabine for 24 h. (E) The cell death ratio was determined using a trypan blue assay. **P<0.01; **P<0.01. ASX, astaxanthin; GR-HPCCs, gemcitabine-resistant HPCCs; HIF-1α, hypoxia-inducible factor 1α; HPCCs, human pancreatic cancer cells; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3; hENT1, human equilibrative nucleoside transporter 1; RRM, ribonucleoside diphosphate reductase.
ASX inhibits the growth of gemcitabine-resistant pancreatic tumors