NF-кB pathway

Biomedicine & Pharmacotherapy 109 (2019) 563–572

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Ginkgolide B enhances gemcitabine sensitivity in pancreatic cancer cell lines via inhibiting PAFR/NF-кB pathway Changjie Lou, Haibo Lu, Zhigang Ma, Chao Liu, Yanqiao Zhang

T

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Department of Gastrointestinal Medical Oncology, Harbin Medical University Cancer Hospital, Harbin 150000, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Pancreatic cancer Gemcitabine sensitivity Ginkgolide B (GB) Nuclear factor kappa b (NF-кB) Platelet-activating factor receptor (PAFR)

Gemcitabine resistance will occur by time after the initial response in pancreatic cancer. Ginkgolide B (GB), a major terpene lactone component of Ginkgo biloba leaves, is a highly selective and competitive inhibitor for platelet-activating factor (PAF) receptor. In the present study, we evaluated the effect of GB on gemcitabine sensitivity in pancreatic cancer cell lines in vitro and in vivo. Cell viability assay, flow cytometry, dual luciferase reporter assay and tumor xenograft model were used to evaluate cell proliferation, apoptosis, nuclear factor kappa b (NF-кB) activity in vitro and tumor growth in vivo. Western blot, immunohistochemistry (IHC) and immunofluorescence were used to shown different protein expression levels. We found the half maximal inhibitory concentration (IC50) of gemcitabine was significantly downregulated by GB in a dose-dependent manner. Furthermore, GB could suppress cell proliferation, increase cell apoptosis and repress tumor growth when combined with gemcitabine, but had no effect when treated alone. Gemcitabine could upregulate PAFR and phosphorylated NF-кB/p65 expression, and increase NF-кB activity, but this was largely suppressed in combination with GB. GB could suppress PAFR expression in a dose-dependent manner. Knockout of PAFR significantly decreased phosphorylated NF-кB/p65 expression, inhibited NF-кB activity, increased gemcitabine sensitivity and cell apoptosis. Besides, GB had no influence on gemcitabine IC50 in IκBα-SR stably expressed BxPC-3 and CAPAN1. Our results suggested that GB could enhance gemcitabine sensitivity in pancreatic cancer cell lines by suppressing PAFR/NF-кB pathway. Thus GB may have therapeutic potential when used in combination with gemcitabine in pancreatic cancer.

1. Introduction Pancreatic cancer is a devastating malignant disease with a very poor prognosis, highlighted by a median survival of 3–6 months and 5year survival rate less than 5% [1,2]. Despite improvements in surgical techniques and adjuvant medical therapy, these figures have not changed for many years. The low survival rate of pancreatic cancer is attributed to several factors, such as late stage when diagnosed, early recurrence and metastasis, and resistance to chemotherapy and radiotherapy [3]. Chemotherapy is the most important treatment for pancreatic cancer. In a landmark clinical trial in 1997, the median survival of fluorouracil control group was 4.41 months comparing with 5.65 months in gemcitabine chemotherapy group [4]. Since then, gemcitabine has been a standard chemotherapy for locally advanced and metastatic pancreatic cancer.

Gemcitabine (2′, 2′-difluoro-2′-deoxycytidine, dFdC) is a deoxycytidine analog that has been used as a chemotherapeutic agent for many years. There are several mechanisms of gemcitabine, such as inhibition of DNA synthesis, inhibition of enzymes related to deoxynucleotide metabolism and induction of apoptosis through caspase signaling [5–7]. In addition to pancreatic cancer, gemcitabine is also used in many other solid tumors such as breast, ovarian and non-small cell lung cancer, especially when in combination with cisplatin or carboplatin [8,9]. Like many other drugs used in chemotherapy, gemcitabine resistance will occur by time after the initial response. Though mechanisms of gemcitabine resistance are not well understood, it is associated with multiple genetic and epigenetic changes. A variety of studies have demonstrated that molecular pathways such as NF-кB, PI3K/Akt, MAPK and HIF-1а are involved in gemcitabine resistance in vitro and in vivo [10–13].

Abbreviations: GB, Ginkgolide B; PAF, platelet-activating factor; PAFR, platelet-activating factor receptor; PI, propidium iodide; NF-кB, nuclear factor kappa b; IC50, half maximal inhibitory concentration; IκBα-SR, IκB-α super-repressor (SR); PBS, phosphate buffered saline; IHC, immunohistochemistry; PCNA, proliferating cell nuclear antigen ⁎ Corresponding author at: Harbin Medical University Cancer Hospital, No. 150, Haping Road, Nangang District, Harbin City 150000, Heilongjiang Province, China. E-mail address: [email protected] (Y. Zhang). https://doi.org/10.1016/j.biopha.2018.10.084 Received 6 July 2018; Received in revised form 14 October 2018; Accepted 14 October 2018 0753-3322/ © 2018 Published by Elsevier Masson SAS. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

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particles and selected by 1 mg/ml puromycin (Sigma-Aldrich# P8833) for 4 days to generate IκBα-SR stably expressing or PAFR knockout BxPC-3 and CAPAN1.

Nuclear factor kappa b (NF-кB) consists of a heterodimer of various members in the Rel family, such as p50, p52, c-Rel, v-rel, p-65(RelA), and Rel B. It is inactivated in cytoplasm by binding to the inhibitor proteins IκB-α or IκB-β, which block the nuclear translocation of NF-кB. NF-кB is a central component in cellular response to damage, stress and inflammation, therefore it is constitutively activated in many cancers [14,15]. NF-кB has also shown to be activated in pancreatic cancer [16,17]. Dysregulation of NF-кB is associated with gemcitabine resistance [18]. In addition, suppression of NF-кB activity in pancreatic cancer cell lines synergizes with gemcitabine to inhibit tumor growth [19]. Ginkgo biloba (Ginkgoaceae) is an ancient Chinese tree. The extracts of Ginkgo biloba leaves have been used as traditional medicines to treat various diseases in China for centuries. EGb-761 is a patented extract of Ginkgo biloba leaves that has been widely used in stroke treatment. It could attenuate lipopolysaccharide-induced acute lung injury via inhibition of oxidative stress and NF-кB activity [20]. Platelet activating factor (PAF) is a bioactive lipid mediator with a range of physiological and pathological activities, including platelets aggregation, glycogen degradation, reproduction, brain function, blood circulation and as a mediator of inflammation. PAF executes it function by binding with its receptor platelet-activating factor receptor (PAFR). The PAF/PAFR axis plays an important role in cancers including oncogenic transformation, anti-apoptosis and metastasis [21–23]. PAF is reported to promote ovarian cancer cell growth and invasion [24]. Moreover, PAF/PAFR axis has enrolled in cisplatin chemotherapy resistance [25]. Ginkgolide B (GB), a major terpene lactone component of EGb-761, is a highly selective and competitive inhibitor for PAFR, thus make it have many potential use. In this study, we investigated the role of GB in gemcitabine chemotherapy in pancreatic cancer cell lines. Our results demonstrated that GB could enhance gemcitabine sensitivity, inhibit cell proliferation and tumor xenograft growth, and increase cell apoptosis. These effects might correlate with the suppressing of PAFR expression and NF-кB activity by GB.

2.3. Drug treatments BxPC-3, CAPAN1, PANC1 and MIA PaCa-2 were plated at a density of 2.5 × 103 cells/well in 96-well plates or 2.0 × 105 cells/well in 6well plates 24 h before initiation of treatment. The cells were treated with: (1) 31.25, 62.5, 125, 250, 500, 1000, 2000 and 4000 μM GB (BxPC-3, CAPAN1, PANC1 and MIA PaCa-2) for 6d in 96-well plates; (2) 0.78, 1.56, 3.13, 6.25, 12.50, 25.00, 50.00, 100.00 and 200.00 nM gemcitabine combined with 25, 100, 400 μM GB or equal volume of PBS (BxPC-3, CAPAN1, PANC1 and MIA PaCa-2) for 6d in 96-well plates; (3) 10 nM gemcitabine for CAPAN1 and 20 nM gemcitabine for BxPC-3 combining with or without 400 μM GB for 48 h or 6d in 6-well plates; (4) 10 nM gemcitabine for CAPAN1 and 20 nM gemcitabine for BxPC-3 combining with 400 μM GB, 100 or 400 μM GB (BxPC-3 and CAPAN1) alone for 6d in 96-well plates. 2.4. Cell viability assay Cell viability was determined according to the operation manual of CellTiter-Glo® Luminescent Cell Viability Assay kit (Promega#G7572). In brief, CellTiter-Glo® Buff ;er and CellTiter-Glo® Substrate were mixed thoroughly to generate CellTiter-Glo® Reagent and stored at −20 °C before use. Then, cells for viability assay and CellTiter-Glo® Reagent were equilibrated to room temperature for 30 min. Add 100ul CellTiterGlo® Reagent per well in 96-well plates and mixed the contents on an orbital shaker to induce cell lysis. Incubate the plates in a dark place at room temperature for 10 min to stabilize luminal signal. Record luminescence to evaluate cell viability. 2.5. Flow cytometry

The human pancreatic cancer cell lines BxPC-3, CAPAN1, PANC1 and MIA PaCa-2 were purchased from the American Type Culture Collection (ATCC, Rockville, MD) and maintained as recommended. Gemcitabine (Selleck#s1714) was dissolved in sterile water at 5 mg/ml and stored at −80 °C. GB (97.4% pure) was provided by Jiangsu Pengyao Pharmaceuticals Inc., China and prepared as a suspension (400 mM) with distilled water and stored at −20 °C.

BxPC-3 and CAPAN1 were treated with gemcitabine (20 nM for BxPC-3 and 10 nM for CAPAN1) combining with or without 400 μM GB for 48 h, then cells were digested with 0.05% trypsin. Cells were centrifuged at 1100 rpm for 3 min and washed with phosphate buffered saline (PBS) for 2 times. 3 × 106 cells were resuspended with 500ul binding buffer. Then 5 u l Annexin V-FITC and 10 u l propidium iodide (PI) (Sigma# APOAF-20TST) were added and incubated at dark for 10 min. After incubation, cells were washed with 1 ml PBS and resuspended in 300ul PBS. Fluorescence of the cells was measured by flow cytometry at 488/530 nm. This experiment was repeated for three times.

2.2. Lentiviral construct and transduction

2.6. Western blot

The PCDH/IκBα lentiviral construct was made by inserting the coding sequence of IKKA (CHUK ENSG00000213341) into the NheI/ NotI sites of pCDH-CMV-MCS-EF1-PURO vector (System Biosciences# CD500B-1). IκB-α super-repressor (SR) expression plasmid (IκBα-SR) was generated by site-directed mutagenesis at S32 A and S36 A according to the operation manual of QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies# 210518). To knockout PAFR in BxPC-3 and CAPAN1, sgPAFR plasmid was constructed by inserting PAFR sgRNA into lentiCRISPRv2 puro (Addgene # 98290) plasmid as protocol. The PAFR sgRNA sequence is 3′-ATA AGT GAT GAC GCC CAG GA-5′. Lentiviral particles of IκBα-SR and sgPAFR were packaged by transfecting combined with lentiviral packaging vectors δ8.9 and VSVG into HEK293 T using Lipofectamine® 3000 (Life Technologies# L3000015) reagents as protocols. The medium with virus particles was collected at 24 h, 48 h and 72 h after transfection and stored at -80 °C. BxPC-3 and CAPAN1 were infected with IκBα-SR or sgPAFR lentiviral

Cells were lysed in RIPA buffer containing protease inhibitors (Sigma-Aldrich, Carlsbad, CA, USA). Protein concentration was evaluated using BCA protein assay kit (Thermo Scientific, Grand Island, NY, USA). 30ug protein was electrophoresed by 10% SDS-PAGE and transferred onto nitrocellulose membranes, then incubated with specific first antibodies and corresponding second antibodies. The specific first antibodies were list as follows: NF-κB p65 Rabbit mAb #8242, Phospho-NF-κB p65 (Ser536) Rabbit mAb #3033, Phospho-Akt (Ser473) Antibody #9271, Akt Antibody #9272, p44/42 MAPK (Erk1/ 2) Antibody #9102, Phospho-p44/42 MAPK (Erk1/2) (Thr202/Tyr204) Antibody #9101, GAPDH (D16H11) Rabbit mAb #5174, PCNA (D3H8P) XP® Rabbit mAb#13110, Cleaved Caspase-3 (Asp175) (5 A1E) Rabbit mAb#9664 and IKKα Antibody #2682 were purchased from Cell Signaling Technology. Anti-PAFR antibody was purchased from Abcam (#ab226936). The second antibody Anti-rabbit IgG HRP-linked Antibody #7074 was purchased from Cell Signaling Technology.

2. Material and methods 2.1. Cell culture and chemicals

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Fig. 1. GB enhanced gemcitabine sensitivity and cell growth in pancreatic cancer cell lines. (A) CAPAN1, BxPC-3, PANC1 and MIA PaCa-2 were exposed to 0, 31.25, 62.5, 125, 250, 500, 1000, 2000 and 4000 μM GB for 6d and assayed for cell viability. (B) CAPAN1, BxPC-3, PANC1 and MIA PaCa-2 were exposed to 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 nM gemcitabine combined with 25, 100, 400 μM GB or equal volume of PBS (Control group) for 6d and assayed for cell viability. (C–D) CAPAN1 and BxPC-3 were cultured with normal medium (Control group), 100 μM GB, 400 μM GB, Gemcitabine (10 nM for CAPAN1 and 20 nM for BxPC-3), Gemcitabine + 100 μM GB and Gemcitabine + 400 μM GB for 6d and assayed for cell viability every other day. Data were presented as mean ± SD with at least 3 independent experiments. Compared with Control group, *P＜0.05, compared with Gemcitabine group, #P＜0.05.

2.7. Dual luciferase reporter assay

2.9. Immunohistochemistry (IHC)

The Lenti-NF-κB-luc luciferase reporter plasmid was purchased from Beyotime (#D2206). Lenti-Ubiquitin-Renilla-Luc was constructed from pRL-SV40-N (Beyotime#D2762) plasmid as renilla luciferase control. BxPC-3 or CAPAN1 was simultaneously infected with Lenti-NF-κB-luc and Lenti-Ubiquitin-Renilla-Luc virus mixed with 8ug/ml polybrene overnight to develop cells stably expressing the NF-κB luciferase reporter and renilla luciferase control [26]. NF-кB luciferase reporter activities were measured by Dual Luciferase reporter assay system (Promega#E1910) according to the manufacturer’s instructions. Reporter activities were normalized to Renilla luciferase values.

Tumor xenograft samples of CAPAN1 were fixed in formalin, embedded in paraffin and sectioned at 5 μm thick. Antigen retrieval were done in citrate buffer and washed by PBS. Tissue sections were blocked with 10% goat serum (Sigma, St. Louis, MO, USA) for 1 h at room temperature and incubate with primary antibody overnight at 4℃. The anti-rabbit second antibody was diluted (1:300) and incubated at room temperature for 1 h. All slides were incubated in avidin biotin peroxidase complex (Sigma, St. Louis, MO, USA) diluted 1:300 in PBS for 30 min at 37℃. Primary antibody Ki-67 (D2H10) Rabbit mAb (IHC Specific) #9027 (1:400 diluted), NF-κB p65 Rabbit mAb #8242 (1:800 diluted), and Cleaved Caspase-3 (Asp175) (5 A1E) Rabbit mAb#9664 (1:1000 diluted) were purchased from Cell Signaling Technology. AntiPAFR antibody (ab226936, 1:200) was purchased from Abcam.

2.8. Tumor Xenograft model All animal experiments were approved by Animal Care and Experimental Committee of Harbin Medical University Cancer Hospital. CAPAN1 (2 × 106 cells) were subcutaneous injected into six-weeks old nude mice for two weeks before visible tumor (6 mm in each dimension) achieved. Then the mice were randomly divided into the following group (n = 6 each): (1) Placebo group received equal volume of PBS; (2) Gemcitabine group received 100 mg/kg gemcitabine; (3) Gemcitabine + GB group received 100 mg/kg gemcitabine combining with 40 mg/kg GB; (4) GB group received 40 mg/kg GB. Drugs were given intraperitoneally every three day for 4 weeks. Tumor volume were monitored every three day by calipers to calculate tumor volumes according to the formula (length × width2)/2.

2.10. Immunofluorescence Cells were fixed with 4% paraformaldehyde for 30 min, then permeabilized with 0.5% Triton X-100 in PBS. After blocking with 1% gelatin in PBS for 1 h, cells were incubated with primary antibody AntiPAFR antibody (Abcam#ab226936, 1:50) and NF-κB p65 Rabbit mAb (Cell signaling#8242, 1:100) at 4 °C overnight. Incubated with Alexa Flour-conjugated secondary antibodies (Invitrogen, 1: 1000) and 5 μg/ ml DAPI (Sigma-Aldrich) at room temperature for 30 min. Then cells were washed with TBST thoroughly and mounted in a fluorescence microscope (DMI3000; Leica, Allendale, NJ, USA). 565

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effect of GB on cell apoptosis of gemcitabine treated cells, BxPC-3 and CAPAN1 were treated with gemcitabine or gemcitabine + 400 μM GB for 48 h, then stained with Annexin V-FITC and Propidium iodide (PI) for flow cytometry. As shown in Fig. 2, the percentage of apoptosis cell in Gemcitabine +400 μM GB group of BxPC-3 and CAPAN1 (20.5% and 31.1%) was significantly greater than Gemcitabine group (12.6% and 15.3%) (p＜0.05). This indicated that GB promoted cell apoptosis in BxPC-3 and CAPAN1 when combined with gemcitabine. Proliferating cell nuclear antigen (PCNA) is the marker of cell proliferation, and cleaved caspase-3 is the maker of cell apoptosis. As shown in Fig. 3A and B, PCNA was significantly downregulated by gemcitabine, and this effect was enhanced by combining with 100 or 400 μM GB. In contrast, cleaved caspase-3 was strongly upregulated by gemcitabine, and this effect was also enhanced by combining with 100 or 400 μM GB. These results further proved that cell proliferation of BxPC-3 and CAPAN1 was suppressed by GB while cell apoptosis increased when combining with gemcitabine.

2.11. Statistical analysis All data was shown as mean ± standard deviation (x ± s). Statistical analysis was performed by SPSS 20.0 software (IBM Corp, Armonk, New York, USA). The difference between two groups was analyzed by paired sample t-test. The difference of multiple groups was analyzed by one-way analysis. P＜0.05 was considered statistically significant. IC50 of gemcitabine in each group was analyzed using nonlinear regression model of GraphPad Prism 6 (GraphPad Software, La Jolla, CA,USA). 3. Results 3.1. Ginkgolide B enhanced gemcitabine sensitivity in pancreatic cancer cell lines In this study, we first tried to determine a suitable concentration range of GB which would have no apparent cytotoxicity on pancreatic cancer cells. Thus, a dose response of GB on cell viability of BxPC-3, Capan-1, PANC-1 and MIA PaCa-2 was analyzed. The suitable concentration range of GB was defined as a cell viability greater than 90% comparing with parental cells when exposed to GB for six days. As shown in Fig. 1A, the suitable concentration range of GB was 0–500 μM. Next, a low, middle and high dose of GB (25, 100 and 400 μM) were selected to evaluate the effect of GB on gemcitabine sensitivity. BxPC-3, Capan-1, PANC-1 and MIA PaCa-2 were exposed to a series concentrations of gemcitabine (0–200 nM) combining with GB (25, 100 or 400 μM) for six days, then cell viability was measured. The IC50 of gemcitabine was evaluated by GraphPad Prism 6. As shown in Fig. 1B and Table 1, IC50 of gemcitabine in 100 and 400 μM GB treated group of all four cell lines was significantly decreased comparing with Control group (p＜0.05). With regard to 25 μM GB treated group, IC50 of gemcitabine in BxPC-3 and Capan-1 decreased (p＜0.05) while no statistic difference was seen in PANC-1 and MIA PaCa-2 comparing with control group (p＞0.05). Nevertheless, gemcitabine IC50 of 25 μM GB treated group in PANC1 and MIA PaCa-2 was inclined to decrease. Thus, we could conclude that GB attenuated gemcitabine resistance in these cell lines.

3.3. Ginkgolide B suppressed PAFR expression and NF-кB activity in gemcitabine treated BxPC-3 and CAPAN1 Many studies demonstrated that PAF could induced IκBα phosphorylation and NF-κB activation [27–29]. GB is a highly competitive antagonist of PAFR, so we speculated that GB increased gemcitabine sensitivity in BxPC-3 and CAPAN1 through antagonizing PAFR and subsequently suppressing NF-κB activity. In order to prove this, BxPC-3 and CAPAN1 were exposed to gemcitabine (50 nM) combining with or without 100 or 400 μM GB for 24 h, then cell lysates were collected for western blot. We found that the protein expression of PAFR was induced by gemcitabine treatment but suppressed when combined with GB (Fig. 3A, B). To further prove this, CAPAN1 cells were treated with a range concentrations of gemcitabine (0, 12.5, 50 and 200 nM) and evaluated for PAFR expression. Our results proved that Gemcitabine induced PFAR expression in CAPAN1 in a dose-dependent manner (Fig. 3C). In contrast, as a highly competitive antagonist of PAFR, GB suppressed PAFR expression in CAPAN1 also in a dose-dependent manner (Fig. 3C). In previous studies, NF-кB activiation was proved to confer gemcitabine resistance and modulation of this activity by inhibitors or genetic approaches would potentiate gemcitabine sensitivity [16–18]. As shown in Fig. 3A and B, we found that Gemcitabine alone showed a strong induction of phosphorylated NF-кB/p65 (p-p65) in CAPAN1 and BxPC-3, but this effect was abolished when combined with 100 or 400 μM GB. These results made us speculate that GB enhanced gemcitabine sensitivity by suppressing NF-кB activity in BxPC-3 and CAPAN1. In order to prove this, a dose response of gemcitabine combining with or without GB (100 or 400 μM) on NF-кB activity was measured by luciferase reporter assay. As shown in Fig. 3D and E, gemcitabine induced a dose-dependent activation of NF-кB in BxPC-3 and CAPAN1, but apparently suppressed by GB treatment. Immunofluorescence localization of NF-кB/p65 in CAPAN1 cells showed that gemcitabine treatment induced nuclear accumulation of NF-кB/p65, but this was suppressed by combining treatment with 400 μM GB, further proved that GB suppressed NF-кB activity in gemcitabine treated CAPAN1 cells (Fig. 3F). In conclusion, our results demonstrated that GB treatment suppressed PAFR expression and NF-кB activity in gemcitabine treated CAPAN1 and BxPC-3 cells.

3.2. Ginkgolide B suppressed proliferation and increased apoptosis of gemcitabine treated BxPC-3 and CAPAN1 Gemcitabine is a deoxycytidine analog which can hinder cell proliferation and induce cell apoptosis by inhibiting DNA synthesis. Thus we speculated that GB could enhance gemcitabine sensitivity by antagonizing these effects. To evaluate the effect of GB on cell proliferation of gemcitabine treated cells, BxPC-3 and CAPAN1 were treated with gemcitabine, 400 μM GB or gemcitabine combining with 400 μM GB for six days in 96-wel plates, then cell viability was assayed every other day. A decreased cell proliferation of BxPC-3 and CAPAN1 was seen in Gemcitabine +400 μM GB group comparing with Gemcitabine group (p＜0.05) (Fig. 1C, D). As a comparison, there was no significant difference of cell proliferation between 400 μM GB group and Control group. Thus, we could conclude that GB suppressed cell proliferation of BxPC-3 and CAPAN1 when combined with gemcitabine. To evaluate the Table 1 IC50 of gemcitabine in pancreatic cell lines treated with different concentrations of GB. Group

CAPAN1

BxPC-3

PANC1

MIA PaCa-2

Control 25uM GB 100uM GB 400uM GB

18.12 7.948* 6.63* 5.28*

31.63 13.74* 9.3* 7.87*

8.32 6.69 5.2* 3.98*

20.98 19.05 13.48* 11.33*

3.4. Knockout of PAFR significantly inhibited NF-кB activity and increased gemcitabine sensitivity in BxPC-3 and CAPAN1 We evaluated the protein expression of PAFR by immunofluorescence in BxPC-3 cells, and found that GB dramatically decreased while gemcitabine increased the fluorescence signal of PAFR comparing with parental untreated BxPC-3 cells (Fig. 4A). These results indicated that GB suppressed while gemcitabine increased PAFR expression in BxPC-3 cells. To further analyze the role of PAFR in gemcitabine

Comparing with Control group, *p＜0.05. 566

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Fig. 2. GB increased cell apoptosis of BxPC-3 and CAPAN1 when combined with gemcitabine. CAPAN1 (A) and BxPC-3 (C) were treated with normal medium (Parental group), 400 μM GB, Gemcitabine (10 nM for CAPAN1 and 20 nM for BxPC-3) or Gemcitabine + 400 μM GB for 48 h, then cells were digested and stained with Annexin V-FITC and PI for flow cytometry. The apoptosis cells (Annexin V-FITC positive subset) of CAPAN1 (B) and BxPC-3 (D) were evaluated. Data was presented as mean ± SD with at least 3 independent experiments. Compared with Control group, *P＜0.05, compared with Gemcitabine group, #P＜0.05.

Fig. 3. GB suppressed PAFR expression and NF-кB activity in gemcitabine treated BxPC-3 and CAPAN1. (A–B) CAPAN1 and BxPC-3 were cultured with normal medium plus with equal volume sterile water (Vehicle group), 50 nM gemcitabine + 0 μM GB (0 μM GB group), 50 nM gemcitabine + 100 μM GB (100 μM group) and 50 nM gemcitabine + 400 μM GB (400 μM group) for 24 h, then cell lysates were collected for western blot. (C) CAPAN1 cells were treated with 0, 12.5, 50 or 200 nM gemcitabine for 24 h, or 0, 25, 100 or 200 μM GB for 24 h, then cell lysates were collected for western blot analysis of PAFR expression. (D–E) CAPAN1 and BxPC-3 were exposed to 0, 12.5, 50 or 200 nM gemcitabine combining with 100 μM or 400 μM GB, then NF-кB activity was evaluated by luciferase reporter assay. (F) immunofluorescence staining of NF-кB p65 (Green) in 50 nM gemcitabine or 50 nM gemcitabine + 400 μM GB treated CAPAN1 cells. DAPI (Blue) was used to stain the nucleus of CAPAN1 cells. Data was presented as mean ± SD with at least 3 independent experiments. Compared with Control group, *P＜0.05.

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Fig. 4. Knockout of PAFR significantly inhibited NF-кB activity, increased gemcitabine sensitivity and cell apoptosis in BxPC-3 and CAPAN1. (A) immunofluorescence staining of PAFR (Green) in parental, 400 μM GB or 50 nM gemcitabine treated BxPC-3 cells. DAPI (Blue) was used to stain the nucleus of BxPC-3 cells. (B) Protein lysates of parental, 400 μM GB treated for 24 h or sgPAFR transduced CAPAN1 and BxPC-3 were collected. The protein expression of PAFR, NF-кB/p65 (p65), phosphorylated NF-кB/p65 (p-p65) were analyzed by Western blot. GAPDH was used as internal control. (C) CAPAN1 and BxPC-3 were treated with 400 μM GB for 24 h (400 μM GB group) or stably transduced with sgPAFR, then evaluated for NF-кB activity by luciferase reporter assay. (D) Parental or sgPAFR transduced CAPAN1 and BxPC-3 cells were exposed to 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 nM gemcitabine combined with or without 400 μM GB for 6 d and assayed for cell viability. (E) Parental or sgPAFR transduced CAPAN1 cells were treated with or without 50 nM gemcitabine for 48 h, then stained with Annexin VFITC and PI for flow cytometry. The apoptosis cells (Annexin V-FITC positive subset) of CAPAN1 were evaluated. Data was presented as mean ± SD with at least 3 independent experiments. Compared with Parental group, *P＜0.05.

and increased gemcitabine sensitivity in BxPC-3 and CAPAN1.

sensitivity, the RNA-guided CRISPR-Cas9 nuclease system was used to knock out PAFR in BxPC-3 and CAPAN1. In our study, BxPC-3 and CAPAN1 were infected with sgPAFR lentiviral particles overnight and selected by 1 mg/ml puromycin for 4 days to generate PAFR knockout BxPC-3 and CAPAN1. As shown in Fig. 4B, PAFR was successfully depleted by sgPAFR lentiviral particles in BxPC-3 and CAPAN1 comparing with parental cells. After treating with 400 μM GB for 24 h, PAFR protein expression in BxPC-3 and CAPAN1 was significantly downregulated, too (Fig. 4B). Moreover, phosphorylated NF-кB/p65 (p-p65) was downregulated by PAFR knockout, showing a same pattern compared with GB treatment. NF-кB activity of PAFR knockout or 400 μM GB treated BxPC-3 and CAPAN1 was analyzed by luciferase reporter assay. As shown in Fig. 4C, PAFR knocked out apparently suppressed NF-кB activity comparing with parental cells, and this effect was similar with GB. These results indicated that PAFR could induce NF-кB activation, corresponding with previous reports [27–29]. We also evaluated the influence of PAFR knockout on gemcitabine sensitivity in CAPAN1 and BxPC-3. As shown in Fig. 4D, gemcitabine IC50 in PAFR knockout CAPAN1 (3.75 nM) and BxPC-3 (4.02 nM) was significantly decreased comparing with parental CAPAN1 (18.11 nM) and BxPC-3 (31.62 nM) controls, and this effect was similar with GB treatment. PAFR knockout in CAPAN1 also increased gemcitabine induced cell apoptosis. As shown in Fig. 4E, after treating with gemcitabine for 48 h, the percentage of apoptosis cells was apparently increased in PAFR knockout CAPAN1 comparing with parental cells. In conclusion, our results demonstrated that PAFR knockout significantly inhibited NF-кB activity

3.5. Ginkgolide B had no influence on gemcitabine sensitivity in NF-кB inactivated BxPC-3 and CAPAN1 NF-кB activation is suppressed in cell cytoplasm by binding to inhibitor protein IкB-α or IкB-β. Phosphorylation of IкB-α or IкB-β leads to degradation of them by 26S proteosome and activation of NF-кB. IкBα super-repressor (SR) protein that mutated at phosphorylation sites S32 A and S36 A is unable to be phosphorylated, therefore remains bound to NF-кB and prevents its activation. In our study, IкB-α superrepressor (SR) expression plasmid was constructed by QuikChange Lightning Site-Directed Mutagenesis kit (Agilent Technologies# 210518) from PCDH/IкB-α expression plasmid. Then, IкBα-SR was stably expressed in BxPC-3 and CAPAN1, and phosphorylated NF-кB/ p65 (p-p65) was significantly decreased (Fig. 5A). NF-кB activity was analyzed by luciferase reporter assay. As shown in Fig. 5B, NF-кB activity was significantly downregulated by IкBa-SR comparing with parental cells. To further investigate the role of NF-кB in gemcitabine resistance, IкBa-SR stably expressing BxPC-3 and CAPAN1 were treated with a series concentration of gemcitabine combining with or without 400 μM GB for six days, then cell viability was evaluated. As shown in Fig. 5C, there was no significant difference of gemcitabine IC50 between Control group and 400 μM GB group in IкBa-SR stably expressing BxPC-3 and CAPAN1. This indicated that GB had no influence on gemcitabine sensitivity in NF-кB inactivated BxPC-3 and CAPAN1. 568

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Fig. 5. GB had no influence on gemcitabine sensitivity in IкBα-SR stably expressed BxPC-3 and CAPAN1. (A) The expression of IкBα, NF-кB/p65 (p65) and phosphorylated NF-кB/p65 (p-p65) in IкBα-SR transduced BxPC-3 and CAPAN1 was evaluated by western blot. GAPDH was used as internal control. (B). NF-кB activity in parental or IкBα-SR transduced BxPC-3 and CAPAN1 was evaluated by luciferase reporter assay. (C) IкBα-SR transduced CAPAN1 and BxPC-3 were exposed to 0, 0.78, 1.56, 3.13, 6.25, 12.5, 25, 50, 100 and 200 nM gemcitabine combined with 400 μM GB or equal volume of PBS (Control group) for 6 d and assayed for cell viability. (D) Parental or IкBα-SR transduced CAPAN1 cells were treated with or without 50 nM gemcitabine for 48 h, then stained with Annexin V-FITC and PI for flow cytometry. The apoptosis cells (Annexin V-FITC positive subset) of CAPAN1 were evaluated. Data was presented as mean ± SD with at least 3 independent experiments. Compared with Parental group, *P＜0.05.

PAFR, phosphorylated NF-кB/p65 (p-p65), ki67 and cleaved caspase-3 in CAPAN1 xenograft tumors was used evaluated PAFR expression, NFкB activity, cell proliferation and apoptosis. As shown in Fig. 6C and D, PAFR positive cells was increased in gemcitabine treated mice comparing with Placebo group, but GB treatment dramatically decreased PAFR positive cells in 400 μM GB group or Gemcitabine +400 μM GB group. Gemcitabine treatment significantly increased phosphorylated NF-кB/p65 (p-p65) positive cells in Gemcitabine group compared with Placebo group or 400 μM GB group, but phosphorylated NF-кB/p65 (pp65) positive cells was decreased in Gemcitabine + 400 μM GB group comparing with Gemcitabine group. This indicated that GB could suppress NF-кB activity induced by gemcitabine in CAPAN1 xenograft tumors. IHC staining of ki67 suggested that ki67 positive cells were significantly decreased in Gemcitabine group comparing with Placebo group or 400 μM GB group, and this was enhanced by combining with 400 μM GB. Besides, Cleaved Caspase-3 positive cell in Gemcitabine group was apparently increased comparing with Placebo group or 400 μM GB group, this was also enhanced by combining with 400 μM GB. In conclusion, GB could potentiate gemcitabine sensitivity, suppress cell proliferation and increase cell apoptosis in CAPAN1 xenograft tumors when combined with gemcitabine, and these effects might correlate with downregulation of PAFR and phosphorylated NF-кB/ p65.

These results further proved that GB potentiated gemcitabine sensitivity in BxPC-3 and CAPAN1 by suppressing NF-кB activity. Besides, gemcitabine IC50 was significantly decreased in IкBa-SR stably expressing BxPC-3 (8.89 nM) and CAPAN1 (8.93 nM) comparing with parental BxPC-3 (31.62 nM) and CAPAN1 (18.11 nM), indicating that inhibition of NF-кB activity could increase gemcitabine sensitivity in some degree. Furthermore, inhibition of NF-кB activity by IкBa-SR also increased gemcitabine induced cell apoptosis. As shown in Fig. 5D, after treating with gemcitabine for 48 h, the percentage of apoptosis cells was apparently increased in IкBa-SR stably expressing CAPAN1 comparing with parental cells. In conclusion, these results indicated that NF-кB activity was necessary for GB to increase gemcitabine sensitivity and inhibition of NF-кB activity increased gemcitabine induced cell apoptosis. 3.6. Ginkgolide B potentiated gemcitabine sensitivity in CAPAN1 xenograft tumors To evaluate the effect of GB on gemcitabine resistance in vivo, nude mice xenograft model of CAPAN1 was built. CAPAN1 (2 × 106 cells) were subcutaneous injected into six-weeks old nude mice for two weeks before visible tumor (6 mm in each dimension) achieved. Then mice in each group were treated with gemcitabine (100 mg/kg), 40 mg/kg GB, gemcitabine (100 mg/kg) +40 mg/kg GB or equal volume of PBS intraperitoneally for four weeks. As shown in Fig. 6A and B, tumor growth was significantly suppressed by gemcitabine treatment, and this effect was strengthened by combining with GB. The mean tumor volume of Gemcitabine + 40 mg/kg GB group (330 ± 39 mm3) was obvious smaller than Gemcitabine group (503 ± 52 mm3) or Placebo group (752 ± 86 mm3) (p＜0.05). This indicated that GB potentiated gemcitabine sensitivity in CAPAN1 xenograft tumors in vivo. IHC staining of

4. Discussion Pancreatic cancer is a highly lethal disease characterized by dense stroma and deficient vascularization, which makes it difficult to penetrate by many drugs and immune system. In fact, the extensive desmoplastic reaction of pancreatic cancer makes up 90% of the tumor volume, thus leading to poor drug delivery and intrinsic 569

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Fig. 6. GB suppressed CAPAN1 xenograft tumors growth in nude mice. CAPAN1 (2 × 106 cells) were subcutaneous injected into six-weeks old nude mice for two weeks, then mice were received equal volume of PBS (Placebo group), 100 mg/kg gemcitabine (Gemcitabine group), 40 mg/kg (GB group) GB and 100 mg/kg gemcitabine combining with 40 mg/kg GB (Gemcitabine + GB group) every three day for 4 weeks. Mice with tumors were taken photos 4 weeks after treatment (A), and tumor volume was measured every three day (B). IHC staining of PAFR, phosphorylated NF-кB/p65 (p-p65), ki67 and Cleaved Caspase-3 in CAPAN1 xenograft tumors was shown in (C). (D) Quantification of PAFR, p-p65, Ki67 and cleaved caspase-3 staining. Arrows were pointed to apoptosis cells in each group. Scale bars = 50 μm. Data was presented as mean ± SD. Compared with Placebo group, *P＜0.05, compared with Gemcitabine group, #P＜0.05.

biloba leaves extracts, on gemcitabine sensitivity in pancreatic cancer cell lines in vitro and in vivo. We are aimed to explore if GB could increase gemcitabine sensitivity in pancreatic cell lines, and furthermore, to study the exact mechanism of that. Ginkgo biloba L. (Ginkgoaceae) is a popular herb used in traditional Chinese medicine for thousands of years. In current practice, ginkgo is mainly used to stop pain, calm wheezing, and treat hypertension, coronary artery disease and cerebrovascular disease [33]. The biological activities and pharmacological effects of Ginkgo biloba extracts include free radical scavenging, anti-inflammation, anti-tumor, anti-aging, and cardioprotective properties [20,34]. We are aimed to find more potential use of GB in our study. Pancreatic cancer cell lines BxPC-3, CAPAN1, PANC1 and MIA PaCa-2 are reported to be gemcitabine resistance [16,26]. Treatment for 24 h with low dose gemcitabine led to a strong apoptosis in pancreatic cancer cell line PT45-P1 and T3M4 but not in BxPC-3, CAPAN1, PANC1 and MIA PaCa-2. In this study, these pancreatic cancer cell lines were used to evaluate the effect of GB on gemcitabine sensitivity. We have found that the IC50 of gemcitabine was significantly downregulated by GB in a dose-dependent manner. In BxPC-3 and CAPAN1, GB had a great influence on gemcitabine IC50 even in a low dose (25 μM). However, this effect was largely diminished in PANC1 and MIA PaCa-2. Only middle or high dose of GB (100 or

chemoresistance [30]. It is not known exactly why gemcitabine is effective against pancreatic cancer while other chemotherapy drugs are not, but like many other drugs used in cancer chemotherapy, resistance to gemcitabine will occur in pancreatic cancer within several weeks of treatment [31]. Chemoresistance to gemcitabine may be intrinsic or acquired by individual patients during drug treatment cycles, and several mechanisms have been reported to correlate with gemcitabine resistance [32]. Among them, signaling pathways involved in cell growth, differentiation, proliferation and apoptosis such as NF-кB, PI3K/AKT, MAPK and HIF-1a pathway have been reported, but as the process are complex, the precise roles and consequences of them can be difficult to understand [10–13]. Although there is only modest overall effect of gemcitabine on median survival of pancreatic cancer patients (5. 6 months for gemcitabine vs. 4.4 months for 5-FU based regiments), the clinical benefit of gemcitabine is more profound with a reported 23.8% response rate compared with 4.8% for 5-FU based therapies [4]. Though the initially sensitive tumors become resistant to gemcitabine within several weeks of treatment, it represents a time window of opportunity for pancreatic cancer patients if researchers deciphered the molecular basis of gemcitabine resistance and developed novel strategies to increase the potency of this chemotherapy. In this study, we studied the effect of GB, a major terpene lactone component of Ginkgo

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400 μM) had sufficient influence on gemcitabine IC50 in PANC1 and MIA PaCa-2. Though, we could conclude that GB enhanced gemcitabine sensitivity in a dose-dependent manner in pancreatic cancer cell lines. The most important action mechanism of gemcitabine is inhibition of DNA synthesis and induction of apoptosis through caspase signaling. As a prodrug, gemcitabine must be metabolized to active triphosphate form. When the active triphosphate form of gemcitabine is incorporated into DNA, a single deoxynucleotide is incorporated afterwards. Then DNA synthesis is stopped and cell proliferation is suppressed [35]. Gemcitabine can induce cell apoptosis through caspase signaling [36]. Gemcitabine activates p38 mitogen-activated protein kinase (MAPK) to trigger apoptosis in response to cellular stress in tumor cells, but not in normal cells [37]. In order to explain why GB could increase gemcitabine sensitivity in BxPC-3 and CAPAN1, cell proliferation and apoptosis was analyzed by cell viability assay, flow cytometry and western blot. Our results indicated that GB could suppress cell proliferation and increase cell apoptosis in BxPC-3 and CAPAN1 in vitro when combined with gemcitabine, but had no effect when treated alone. To explore the effect of GB on gemcitabine sensitivity in vivo, the tumor xenograft model of CAPAN1 was built. Gemcitabine significantly suppressed tumor growth of CAPAN1, and this effect was enhanced by combining with GB. In summary, these results indicated that GB could potentiate the effect of gemcitabine on cell proliferation, apoptosis and tumor xenograft growth, but had no effect when treated alone. PAF, an alkylphospholipid produced by a variety of cells throughout the body, is one of the most potent lipid mediators known [38]. PAF executes it function by binding with platelet activating factor receptor (PAFR). GB, one of terpene lactone components of Ginkgo biloba leaves extracts, is the most potent antagonist of PAFR [39,40]. By antagonizing PAFR, GB is able to inhibit PAF-induced cascade effect in platelet aggregation, inflammatory response and other biological activities [41,42]. In our study, we found that gemcitabine induced a dose-dependent increasing of PAFR expression, while GB suppressed PAFR expression also in a dose-dependent manner. We knocked out PAFR in CAPAN1 and BxPC-3 using a sgRNA targeting PAFR and found that PAFR knockout significantly decreased gemcitabine IC50 and increased cell apoptosis induced by gemcitabine. These results suggested that GB might increase gemcitabine sensitivity through downregulation of PAFR. In previous reports, PAF was shown to induce NF-кB activation through a G protein-coupled pathway [43]. Besides, PAF could induce phosphorylation of IκBα [27]. Moreover, activation of PAFR could repress tumor necrosis factor (TNF) α induced cell apoptosis through NFкB pathway [28]. In our study, we found that PAFR knockout could repress NF-кB activity and phosphorylation of NF-кB/p65 in BxPC-3 and CAPAN1 as the same as GB. Thus we speculated that GB might increase gemcitabine sensitivity through PAFR/ NF-кB pathway. As previous studies reported, NF-кB plays an important role in gemcitabine resistance. It is upregulated in response to gemcitabine treatment in pancreatic cancer, colon cancer and breast cancer [16,44]. Gemcitabine upregulates NF-кB in a dose-dependent manner, and inhibition of NF-кB pathway in pancreatic cancer cells attenuate gemcitabine resistance at various concentrations [45,46]. Knockdown of p65 subunit of NF-кB synergizes with gemcitabine to suppress tumor growth in vivo and increase apoptosis in pancreatic cancer cell lines [47]. Inhibition of gemcitabine-induced activation of NF-кB in pancreatic cancer cells by nafamostat mesilate (NF-кB inhibitor) potentiates sensitivity to gemcitabine by suppressing IкBа phosphorylation [48]. Moreover, GB functions as a determinant constituent of Ginkgolides in alleviating lipopolysaccharide-induced lung injury by suppressing NFкB activity [49]. In our study, we found that gemcitabine increased phosphorylation of NF-кB/p65 but suppressed by combining with GB. Besides, gemcitabine induced NF-кB activity in a dose-dependent manner in BxPC-3 and CAPAN1 as previous studies described [16], and this effect was largely suppressed when combining with GB. GB was shown to inhibit expression of inflammatory proteins by blocking NFкB translocation [50]. In our study, we found that gemcitabine induced

nuclear accumulation of NF-кB/p65 but this was suppressed by combining with GB in CAPAN1. These results indicated GB could suppress NF-кB activity induced by gemcitabine. To further verify this, IκBα-SR, a non-phosphorylation form of IκBα, was transduced into BxPC-3 and CAPAN1. Stably expressing of IκBα-SR in BxPC-3 and CAPAN1 successfully inhibited NF-кB activity. As we expected, we found that GB had no influence on gemcitabine IC50 in IκBα-SR stably expressed BxPC-3 and CAPAN1, indicated that NF-кB activity was necessary for GB to increase gemcitabine sensitivity. Furthermore, we found that IC50 of gemcitabine in IκBα-SR stably expressing BxPC-3 and CAPAN1 was significantly decreased comparing with parental BxPC-3 (31.63 nM for BxPC-3 vs. 8.89 nM for IκBα-SR BxPC-3) and CAPAN1 (18.12 nM for CAPAN1 vs. 8.93 for IκBα-SR CAPAN1). Moreover, inhibition of NF-кB activity by IкBa-SR also increased gemcitabine induced cell apoptosis, suggesting the important role of NF-кB in gemcitabine sensitivity. 5. Conclusion In summary, our data suggested that GB could enhance gemcitabine sensitivity in pancreatic cancer cell lines BxPC-3, Capan-1, PANC-1 and MIA PaCa-2, and potentiate the effect of gemcitabine on cell proliferation, apoptosis and tumor xenograft growth, but had no effect when treated alone. GB suppressed PAFR expression and NF-кB activity induced by gemcitabine in BxPC-3 and CAPAN1, which was critical for gemcitabine sensitivity. Our findings suggest that GB may have therapeutic potential when used in combination with gemcitabine in pancreatic cancer. Conflict of interests The authors declare that they have no conflict of interests. Funding This study was supported by The National Natural Science Foundation of China (grant numbers: 81502587, 81200570, 81703000); Heilongjiang Provincial Natural Science Foundation Youth Science Foundation of China (grant number: QC2013C092) and Funding for post doctoral funds in Heilongjiang of China (grant number: LRB14-562). References [1] D.P. Ryan, T.S. Hong, N. Bardeesy, Pancreatic adenocarcinoma, N. Engl. J. Med. 371 (2014) 1039–1049. [2] M. Quaresma, M.P. Coleman, B. Rachet, 40-year trends in an index of survival for all cancers combined and survival adjusted for age and sex for each cancer in England and Wales, 1971–2011: a population-based study, Lancet 385 (2015) 1206–1218. [3] Terumi Kamisawa, Laura D. Wood, Takao Itoi, Kyoichi Takaori, Pancreatic cancer, Lancet 388 (2016) 73–85. [4] H.A. Burris 3rd, M.J. Moore, J. Andersen, M.R. Green, M.L. Rothenberg, M.R. Modiano, M.C. Cripps, R.K. Portenoy, A.M. Storniolo, P. Tarassoff, et al., Improvements in survival and clinical benefit with gemcitabine as first-line therapy for patients with advanced pancreas cancer: a randomized trial, J. Clin. Oncol. 15 (1987) 2403–2413. [5] P. Huang, S. Chubb, L.W. Hertel, G.B. Grindey, W. Plunkett, Action of 2’,2’ -difluorodeoxycytidine on DNA synthesis, Cancer Res. 51 (1991) 6110–6117. [6] V. Heinemann, Y.Z. Xu, S. Chubb, A. Sen, L.W. Hertel, G.B. Grindey, W. Plunkett, Cellular elimination of 20,20-difluorodeoxycytidine 50-triphosphate: a mechanism of self-potentiation, Cancer Res. 52 (1992) 533–539. [7] N.M. Chandler, J.J. Canete, M.P. Caller, Caspase-3 drives apoptosis in pancreatic cancer cells after treatment with gemcitabine, J. Gastrointest. Surg. 8 (2004) 1072–1078. [8] R.A. Nagourney, M. Flam, J. Link, S. Hager, J. Blitzer, W. Lyons, B.L. Sommers, S. Evans, Carboplatin plus gemcitabine repeating doublet therapy in recurrent breast cancer, Clin. Breast Cancer 8 (2008) 432–435. [9] M. Reck, J. Von Pawel, P. Zatloukal, R. Ramlau, V. Gorbounova, V. Hirsh, N. Leighl, J. Mezger, V. Archer, N. Moore, C. Manegold, Phase III trial of cisplatin plus gemcitabine with either placebo or bevacizumab as first-line therapy for nonsquamous non-small-cell lung cancer, Avail. J. Clin. Oncol. 27 (2009) 1227–1234. [10] P.O. Simon Jr, J.E. McDunn, H. Kashiwagi, K. Chang, P.S. Goedegebuure,

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