Research ArticleExperimental Studies
Open Access

Recombinant Thrombomodulin Has Anti-tumor Effects and Enhances the Effects of Gemcitabine for Pancreatic Cancer Through G-protein Coupled Receptor 15

KENEI FURUKAWA, JIANHUA LING, YICHEN SUN, YU LU, JIE FU, RUI MARUKUCHI and PAUL J. CHIAO
Anticancer Research October 2022, 42 (10) 4679-4687; DOI: https://doi.org/10.21873/anticanres.15972

Abstract

Background/Aim: Thrombomodulin™ has cytoprotective and anti-inflammatory function by interacting with G-protein coupled receptor 15 (GPR15). Recombinant TM (rTM), which comprises the extracellular regions of TM, is approved for treatment of disseminated intravascular coagulation. We investigated the anti-tumor effect of rTM for pancreatic ductal adenocarcinoma (PDAC) through GPR15. Materials and Methods: We evaluated the expression of GPR15 in human PDAC cell lines and the anti-tumor effect and signals of rTM in vitro and in vivo. To test whether GPR15 would be responsible for the inhibition of cell proliferation by rTM, we evaluated the cell viability of the GPR15 knockdown cells treated with rTM using GPR15-targeting siRNA. Results: We identified PDAC cell lines with GPR15 expression and discovered that rTM inhibited tumor growth and enhanced the effects of gemcitabine (GEM) for the PDAC cell line in a GPR15-dependent manner. Furthermore, we showed that rTM inhibited nuclear factor-kappaB (NF-Embedded ImageB) and extracellular signal-regulated kinase (ERK) activation through interactions with GPR15. Conclusion: We demonstrated that rTM had anti-tumor effect and enhancement of cytotoxic effect of GEM for PDAC cells by inhibiting NF-Embedded ImageB and ERK activation via GPR15 and suggest that rTM is a potential therapeutic option for PDAC.

Key Words:

Pancreatic ductal adenocarcinoma (PDAC) is one of the most serious human digestive cancers with rapid tumor growth and high potential for distant metastasis leading to very poor prognosis (1). Despite recent improvements, current treatments mainly based on surgery and chemotherapy have a limited impact on patients’ prognosis (2, 3). Therefore, new therapeutic approaches for PDAC are needed.

Thrombomodulin (TM), a type I-glycosylated membrane protein composed of 5 distinct domains including an N-terminal lectin-like domain, a six-tandem epidermal growth factor-like domain, an O-glycosylation site-rich domain, a transmembrane domain and a cytoplasmic tail, is constitutively expressed on vascular endothelial cells (4, 5). TM acts as an anticoagulant by binding to thrombin and activating protein C (6) and is also implicated in mediating anti-inflammatory functions through activated protein C and lectin-like domain (7-10).

Recombinant human soluble TM (rTM), which consists of the extracellular domain of TM, was approved for the treatment of disseminated intravascular coagulation (DIC) in Japan in 2008 (11). Also, an international phase IIb clinical trial in patients with sepsis and suspected DIC evaluated safety and efficacy supporting further application of rTM (12).

Pan et al. demonstrated that TM binds to G-protein coupled receptor 15 (GPR15) via its epidermal growth factor (EGF)-like domain and exerts cytoprotective and anti-inflammatory functions through inhibition of nuclear factor-kappaB (NF-Embedded ImageB) and extracellular signal-regulated kinase (ERK) activation in lymphocytes and vascular endothelial cells (13-15). GPR15, an orphan receptor member of the G-protein coupled receptor family, was initially found as a coreceptor for the human immunodeficiency virus (16). Our study has shown that constitutive activation of NF-Embedded ImageB played a key role in the aggressive behavior of PDAC (17, 18). Therefore, we hypothesized that rTM would have anti-tumor effects in PDAC by inhibiting NF-Embedded ImageB and ERK activation through GPR15.

Materials and Methods

Cell lines and reagents. The human pancreatic ductal epithelial cell line (HPNE)was obtained from the laboratory of Dr. James W. Freeman at the University of Texas Health Science Center at San Antonio (19). Human pancreatic cancer cell lines PANC-1, MiaPaCa-2, AsPc-1 and BxPC-3 were purchased from the American Type Culture Collection (Manassas, VA, USA). PATC43, 50, 53, 66 and 153LM, which were established from a patient-derived tumor xenograft program (20, 21), were provided by Dr Jason B. Fleming (MD Anderson Cancer Center, Houston, TX, USA). PANC-1 and MiaPaCa-2 were maintained as monolayer cultures in Dulbecco’s modified Eagle’s medium (Caisson Labs, Inc, Smithfield, UT, USA) containing 10% fetal bovine serum and penicillin (100 IU/ml) and streptomycin (100 μg/ml). AsPc-1, BxPC-3, PATC43, 50, 53, 66 and 153LM were maintained as monolayer cultures in RPMI 1640 with L-glutamine (Mediatech, Inc, Manassas, VA, USA) containing 10% fetal bovine serum and penicillin (100 IU/ml) and streptomycin (100 μg/ml). The cells were cultured in an atmosphere of 5% carbon dioxide at 37°C.

rTM was purchased from Asahi Kasei Pharma (Tokyo, Japan); it was dissolved in sterile phosphate-buffered saline (PBS) and stored at −20°C until use. Gemcitabine hydrochloride (GEM) was purchased from Selleck Chemicals (Houston, TX); it was dissolved in sterile PBS and stored at −20°C until use.

Western blot analysis. The cells were washed with cold PBS and lysed at 4°C into radioimmunoprecipitation assay buffer (Cell Signaling Technology, Inc, Boston, MA, USA). Each lysate was separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nylon membranes to detect GPR15 (ab8104; Abcam, Cambridge, MA, USA), cleaved PARP (5625; Cell Signaling Technology), cleaved caspase-3 (9664; Cell Signaling Technology), phospho-NF-Embedded ImageB p65 (3033; Cell Signaling Technology), NF-Embedded ImageB p65 (8242; Cell Signaling Technology), phospho-ERK (sc-7383; Santa Cruz Biotechnology, Inc, Santa Cruz, CA, USA), ERK1/2 (4695; Cell Signaling Technology) and β-actin (A5316; Sigma-Aldrich, Saint Louis, MO, USA).

MTT assay. Cells were seeded in 96-well plates (3×103 cells/well). On the next day, cells were treated with rTM or gGEM. Drug-treated cells were analyzed after being incubated for 24, 48, 72, 96 or 120 h. MTT solution (5 mg/ml) was added to the cultures. After being incubated for 90 min, the medium containing MTT was aspirated off and 100 μl of DMSO were added. After being incubated for 30 min, the absorbance was read at 470 nm.

RNA interference. GPR15-targeting small interfering RNA (siRNA) (SASI_Hs01_00126634, NM_005290), synthesized from Sigma-Aldrich was transfected into PATC66 cells with the jetPRIME transfection reagent (Polyplus transfection Inc, Illkirch, France) according to the manufacturer’s protocol. A control siRNA (ThermoFisher Scientific, Waltham, MA, USA) was used as negative control.

Flow cytometry assay. PATC66 cells were incubated with each treatment for 72 h. The cells were washed twice with cold PBS, fixed with 70% ethanol, and stored at −20°C until analysis. After removing ethanol, the cells were washed twice with cold PBS and resuspended in 500 μl of PI/RNase staining buffer (BD Biosciences, San Diego, CA, USA). After the cells were incubated at room temperature for 15 min in the dark, the cell cycle was analyzed by flow cytometry within 1 h.

Colony-formation assay. PATC66 cells were seeded in 6-well dishes (500 cells/well). On the next day, cells were treated with rTM or GEM and incubated for 10 days. The number of colonies that were fixed with 4% formalin for 20 min and stained with 0.1% crystal violet for 20 min was counted.

Subcutaneous xenograft model. Six- to 8-week old female athymic nude mice (NCI-nu) were purchased from the Animal Production Area of the National Cancer Institute-Frederick Cancer Research Facility (Frederick, MD, USA). All mice were housed and treated in accordance with the guidelines of the University of Texas MD Anderson Cancer Center’s Animal Care and Use Committee and maintained in specific pathogen-free conditions. The facilities were approved by the Association for Assessment and Accreditation of Laboratory Animal Care. They meet all current regulations and standards of the US Departments of Agriculture and Health and Human Service and the NIH.

PATC66 cells were harvested in PBS with 50% Matrigel (Corning, Tewksbury, MA, USA). The cells (5×106 cells in 100 μl of PBS) were injected subcutaneously into the back of the mice. At five weeks after implantation, 10 mg/kg i.p. rTM and 25mg/kg i.p. GEM were used to treat the mice weekly. All mice were weighed and tumor volume (1/2 D × d2, where D is the long side and d is the short side of the tumor) was measured weekly. At 4 weeks after treatment, animals were sacrificed, and subcutaneous tumors were excised.

Histological and immunohistochemical analyses. The tumors were formalin-fixed and paraffin-embedded for immunostaining. Immunohistochemical analyses were conducted according to Vectastain ABC kit (Vector Laboratories, Inc, Burlingame, CA, USA). For antigen retrieval, the sections were subjected to heat in antigen unmasking solution (Vector Laboratories). The sections were incubated at 4°C overnight with the primary human anti-rabbit monoclonal antibody anti-Ki67 (9106; ThermoFisher Scientific), anti-rabbit monoclonal antibody anti-cleaved caspase-3 (9664; Cell Signaling Technology), anti-mouse monoclonal antibody anti-phospho-ERK (sc-7383; Santa Cruz Biotechnology) and anti-rabbit polyclonal antibody anti-phospho-p65 (ab86299; Abcam). DAB peroxidase substrate kit (Vector Laboratories) was used as the final chromogen. The sections were counterstained with hematoxylin. The number of positive cells was counted in 3 random high-power fields at ×40.

Statistical analysis. Non-paired t-test (two-tailed) and repeated measures ANOVA were used for statistical analysis. All p-Values were considered statistically significant when the associated probability was less than 0.05.

Results

Expression of GPR15 in PDAC and effects of rTM on cell proliferation. To determine the expression of GPR15 in PDAC, we examined the expression of GPR15 in HPNE cells and 9 PDAC cell lines. Western blot analysis showed that PATC66 and PATC153LM cells had the high expression of GPR15 (Figure 1). In order to determine the effect of rTM on cell proliferation in PDAC, we evaluated the cell viability of HPDE cells and 9 PDAC cell lines treated by rTM. MTT assay showed that rTM had significant inhibition of cell proliferation for PATC66 and PATC153LM cells (Figure 2).

Figure 1.
Figure 1.

The expression of GPR15 in human pancreatic ductal epithelial cell line (HPNE) and different pancreatic ductal adenocarcinoma (PDAC) cell lines. β-actin is used as a loading control.

Figure 2.
Figure 2.

Effects of rTM on cell proliferation in HPNE cells and different PDAC cell lines by MTT assay repeated 11 times (t-test).

GPR15 mediates rTM-induced inhibition of cell proliferation. To test our hypothesis that GPR15 would be responsible for the inhibition of cell proliferation by rTM in PATC66 cells, we evaluated the cell viability of the GPR15 knockdown and control PATC66 cells treated with rTM. We used GPR15-targeting siRNA to knock down the expression of GPR15. Western blot analysis showed that the expression of GPR15 markedly decreased in the GPR15 knockdown PATC66 cells and MTT assay showed that rTM was not able to inhibit cell proliferation in the GPR15 knockdown PATC66 cells (Figure 3).

Figure 3.
Figure 3.

Effects of rTM on cell proliferation in GPR15 knockdown PATC66 cells by MTT assay (t-test).

Effects of rTM combined with GEM on cell proliferation through GPR15. To determine the effect of rTM combined with GEM on the cell proliferation in PATC66 cells, we evaluated the cell viability of PATC66 cells treated by rTM, GEM or rTM combined with GEM. MTT assay showed that rTM significantly enhanced inhibition of cell proliferation by GEM (Figure 4A). To test our hypothesis that GPR15 would also be responsible for enhancing the inhibition of cell proliferation by GEM in PATC66 cells, we evaluated the cell viability of the GPR15 knockdown and control PATC66 cells treated with GEM or rTM combined with GEM. MTT assay showed that rTM was not able to enhance the inhibition of cell proliferation by GEM in the PATC66 cells when GPR15 was knocked down (Figure 4B).

Figure 4.
Figure 4.

Effects of rTM combined with GEM on cell proliferation in PATC66 cells through GPR15. (A) Cell viability of PATC66 cells treated by rTM, GEM or rTM combined with GEM by MTT assay (ANOVA). (B) Cell viability of GPR15 knockdown PATC66 cells treated by GEM or rTM combined with GEM by MTT assay (t-test).

Effects of rTM and rTM combined with GEM on apoptosis, colony-formation and NF-Embedded ImageB and ERK activity. To determine the effect of rTM and rTM combined with GEM on apoptosis, colony-formation and NF-Embedded ImageB and ERK activity in PATC66 cells, we evaluated apoptosis, the colony number, p65 and ERK of PATC66 cells treated by rTM, GEM or rTM combined with GEM. Cell cycle analysis by flow cytometry showed that rTM significantly increased the sub-G1 population of cells and enhanced the sub-G1 population of cells treated by GEM (Figure 5A). Western blot analysis showed that rTM enhanced GEM-induced apoptosis as demonstrated by PARP and caspase-3 cleavage (Figure 5B). Colony-formation assay showed that rTM significantly decreased the colony number and enhanced inhibition of colony-formation by GEM (Figure 6). Western blot analysis showed that rTM decreased p65 and ERK phosphorylation and inhibited GEM-induced p65 and ERK phosphorylation (Figure 7).

Figure 5.
Figure 5.

Effects of rTM and rTM combined with GEM on apoptosis in PATC66 cells. (A) Cell cycle analysis of PATC66 cells treated by rTM, GEM or rTM combined with GEM (t-test). (B) Western blot analysis for cleaved PARP and caspase3 in PATC66 cells treated by rTM, GEM or rTM combined with GEM.

Figure 6.
Figure 6.

Effects of rTM and rTM combined with GEM on colony-formation in PATC66 cells by colony-formation assay (t-test).

Figure 7.
Figure 7.

Effects of rTM and rTM combined with GEM on NF-Embedded ImageB and ERK activity in PATC66 cells. Western blot analysis for the phosphorylation of p65 and ERK in PATC66 cells treated by rTM, GEM or rTM combined with GEM.

To test our hypothesis that GPR15 would be responsible for the inhibition of NF-Embedded ImageB and ERK activity by rTM in PATC66 cells, we evaluated p65 and ERK of the GPR15 knockdown and control PATC66 cells treated by rTM, GEM or rTM combined with GEM. Western blot analysis showed that rTM was not able to inhibit GEM-induced p65 and ERK phosphorylation in the GPR15 knockdown PATC66 cells (Figure 8).

Figure 8.
Figure 8.

Effects of rTM combined with GEM on NF-Embedded ImageB and ERK activity in GPR15 knockdown PATC66 cells. Western blot analysis for the phosphorylation of p65 and ERK in GPR15 knockdown PATC66 cells treated by rTM, GEM or rTM combined with GEM.

Effects of rTM and rTM combined with GEM on tumor growth. To determine the effect of rTM and rTM combined with GEM on tumor growth in PATC66 cells, we evaluated the tumor growth of PATC66 cells in a subcutaneous xenograft mouse model. Nude mice were subcutaneously injected with PATC66 cells and randomly assigned to four groups to be treated as indicated. rTM group, intraperitoneally injected rTM (10mg/kg) once a week, GEM group, intraperitoneally injected GEM (25mg/kg) once a week, combination group, intraperitoneally injected rTM (10mg/kg) and GEM (25mg/kg) once a week and control group, intraperitoneally injected the equal amount of vehicle once a week. The tumor growth in rTM group was significant slower than those of control group and the tumor growth in combination group was significant slower than those of GEM group (Figure 9).

Figure 9.
Figure 9.

Effects of rTM and rTM combined with GEM on tumor growth (n=4) in PATC66 cells analyzed by repeated ANOVA.

To determine the effect of rTM and rTM combined with GEM on the expressions of Ki67, caspase-3, NF-Embedded ImageB and ERK in the tumors, we carried out immunohistochemical analyses. Ki67 positive cells in the combination group were significantly less than those of GEM group. Cleaved caspase-3 positive cells in the combination group were significantly more than those of GEM group. Activated p65 positive cells in the combination group were significantly less than those of the GEM group. Phospho-ERK positive cells in the combination group were significantly less than those of the GEM group (Figure 10).

Figure 10.
Figure 10.

Immunohistochemical analysis of the tumors treated by rTM, GEM or rTM combined with GEM that were stained with antibodies to Ki67 (A), cleaved caspase-3 (B), phospho-p65 (C) and phospho-ERK (D). The bar graphs showing the number of positive cells counted in 3 random high-power fields at x40 (t-test).

Discussion

GPR15 is a member of the G-protein coupled receptor family which regulates NF-Embedded ImageB and mitogen activated protein kinase (MAPK) signaling pathways through G-protein coupled receptor kinases (22) and has crucial roles in tumor growth and metastasis (23). We demonstrated that in the presence of GPR15, rTM decreased activation of NF-Embedded ImageB and ERK, which is consistent with the inhibition of cell proliferation for PATC66 cells. GPR15 is an orphan G protein-coupled receptor that is found lymphocytes and mediates homing of T cells to the lamina propria in the colon and alleviates inflammatory bowel disease (24). Suply et al. reported that a natural ligand for GPR15 (GPR15L) was detected in some epithelia such as colon, skin and cervix and encoded by the gene C10ORF99 (25). C10ORF99 was proposed to encode a secreted factor which inhibits colon cancer cell growth (26). Also, we found that the anti-tumor effect and enhancement of the cytotoxic effect of GEM by rTM depended on expression of GPR15, which suggests that GPR15 is probably a cell surface receptor to rTM. Ligands for GPR15 such as GPR15L and rTM could be a potential therapeutic target for cancer. However, modulation of receptor signaling is not shown in the present study. Further experiments are required to clarify the function of GPR15 in PDAC cells.

We reported that rTM suppressed tumor growth of pancreatic cancer through the inhibition of thrombin-induced protease activate receptor 1 and NF-Embedded ImageB activation (27). TM has multiple functions, which indicates that rTM could have multiple mechanisms of anti-tumor effects. In addition to GRP15, Kuo et al. showed that rTM domain 1 suppressed tumor angiogenesis and growth by binding to Lewis Y antigen on epidermal growth factor receptor (EGFR) and inhibiting EGFR activation (28). Also, Kuo et al. identified fibroblast growth factor receptor (FGFR)-I as a possible receptor for TM (29). rTM is widely used for patients with poor performance status and organ function due to DIC, with minimal adverse effects (12). Therefore, rTM has a potential to become a new therapeutic option for cancer patients.

Limitations of this study are that we examined only one PDAC cell line with GPR15 expression and we did not investigate detailed mechanism about interaction between rTM and GPR15 and immunity in tumors even though GPR15 plays a role in the immune system. To overcome these problems, creating various cell lines and lymphocytes with knockout and forced expression of GPR15 is one of our future challenges.

Conclusion

In conclusion, we demonstrated that rTM has anti-tumor effects and enhances the cytotoxic effect of GEM in PDAC cells by inhibiting NF-Embedded ImageB and ERK activation through interacting with GPR15. rTM treatment may be therapeutic application for patients with pancreatic cancer and GPR15 can be a new target for cancer therapy. Furthermore, Furthermore, GPR15 may be a clue to the development of new cancer immunotherapies.

Acknowledgements

This research was supported by The Jikei University Research Fund.

Footnotes

  • Authors’ Contributions

    Kenei Furukawa: Design of the study, collection and analysis of data and drafting of the article. Jianhua Ling: Collection of data. Yichen Sun: Collection of data. Yu Lu: Collection of data. Jie Fu: Collection of data. Rui Marukuchi: Revision of the article. Paul J. Chiao: Revision and final approval of the article.

  • Conflicts of Interest

    The Authors have no conflicts of interest to declare.

  • Received July 11, 2022.
  • Revision received July 28, 2022.
  • Accepted August 6, 2022.

This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).

References

  1. ↵
    1. Ferlay J,
    2. Soerjomataram I,
    3. Dikshit R,
    4. Eser S,
    5. Mathers C,
    6. Rebelo M,
    7. Parkin DM,
    8. Forman D and
    9. Bray F
    : Cancer incidence and mortality worldwide: sources, methods and major patterns in GLOBOCAN 2012. Int J Cancer 136(5): E359-E386, 2015. PMID: 25220842. DOI: 10.1002/ijc.29210
    OpenUrlCrossRefPubMed
  2. ↵
    1. Conroy T,
    2. Desseigne F,
    3. Ychou M,
    4. Bouché O,
    5. Guimbaud R,
    6. Bécouarn Y,
    7. Adenis A,
    8. Raoul JL,
    9. Gourgou-Bourgade S,
    10. de la Fouchardière C,
    11. Bennouna J,
    12. Bachet JB,
    13. Khemissa-Akouz F,
    14. Péré-Vergé D,
    15. Delbaldo C,
    16. Assenat E,
    17. Chauffert B,
    18. Michel P,
    19. Montoto-Grillot C,
    20. Ducreux M, Groupe Tumeurs Digestives of Unicancer. and PRODIGE Intergroup
    : FOLFIRINOX versus gemcitabine for metastatic pancreatic cancer. N Engl J Med 364(19): 1817-1825, 2011. PMID: 21561347. DOI: 10.1056/NEJMoa1011923
    OpenUrlCrossRefPubMed
  3. ↵
    1. Michelakos T,
    2. Pergolini I,
    3. Castillo CF,
    4. Honselmann KC,
    5. Cai L,
    6. Deshpande V,
    7. Wo JY,
    8. Ryan DP,
    9. Allen JN,
    10. Blaszkowsky LS,
    11. Clark JW,
    12. Murphy JE,
    13. Nipp RD,
    14. Parikh A,
    15. Qadan M,
    16. Warshaw AL,
    17. Hong TS,
    18. Lillemoe KD and
    19. Ferrone CR
    : Predictors of resectability and survival in patients with borderline and locally advanced pancreatic cancer who underwent neoadjuvant treatment with FOLFIRINOX. Ann Surg 269(4): 733-740, 2019. PMID: 29227344. DOI: 10.1097/SLA.0000000000002600
    OpenUrlCrossRefPubMed
  4. ↵
    1. Suzuki K,
    2. Kusumoto H,
    3. Deyashiki Y,
    4. Nishioka J,
    5. Maruyama I,
    6. Zushi M,
    7. Kawahara S,
    8. Honda G,
    9. Yamamoto S and
    10. Horiguchi S
    : Structure and expression of human thrombomodulin, a thrombin receptor on endothelium acting as a cofactor for protein C activation. EMBO J 6(7): 1891-1897, 1987. PMID: 2820710. DOI: 10.1002/j.1460-2075.1987.tb02448.x
    OpenUrlCrossRefPubMed
  5. ↵
    1. Sadler JE
    : Thrombomodulin structure and function. Thromb Haemost 78(1): 392-395, 1997. PMID: 9198185.
    OpenUrlPubMed
  6. ↵
    1. Dahlbäck B and
    2. Villoutreix BO
    : The anticoagulant protein C pathway. FEBS Lett 579(15): 3310-3316, 2005. PMID: 15943976. DOI: 10.1016/j.febslet.2005.03.001
    OpenUrlCrossRefPubMed
  7. ↵
    1. White B,
    2. Schmidt M,
    3. Murphy C,
    4. Livingstone W,
    5. O’Toole D,
    6. Lawler M,
    7. O’Neill L,
    8. Kelleher D,
    9. Schwarz HP and
    10. Smith OP
    : Activated protein C inhibits lipopolysaccharide-induced nuclear translocation of nuclear factor kappaB (NF-kappaB) and tumour necrosis factor alpha (TNF-alpha) production in the THP-1 monocytic cell line. Br J Haematol 110(1): 130-134, 2000. PMID: 10930989. DOI: 10.1046/j.1365-2141.2000.02128.x
    OpenUrlCrossRefPubMed
    1. Cao C,
    2. Gao Y,
    3. Li Y,
    4. Antalis TM,
    5. Castellino FJ and
    6. Zhang L
    : The efficacy of activated protein C in murine endotoxemia is dependent on integrin CD11b. J Clin Invest 120(6): 1971-1980, 2010. PMID: 20458145. DOI: 10.1172/JCI40380
    OpenUrlCrossRefPubMed
    1. Conway EM,
    2. Van de Wouwer M,
    3. Pollefeyt S,
    4. Jurk K,
    5. Van Aken H,
    6. De Vriese A,
    7. Weitz JI,
    8. Weiler H,
    9. Hellings PW,
    10. Schaeffer P,
    11. Herbert JM,
    12. Collen D and
    13. Theilmeier G
    : The lectin-like domain of thrombomodulin confers protection from neutrophil-mediated tissue damage by suppressing adhesion molecule expression via nuclear factor kappaB and mitogen-activated protein kinase pathways. J Exp Med 196(5): 565-577, 2002. PMID: 12208873. DOI: 10.1084/jem.20020077
    OpenUrlAbstract/FREE Full Text
  10. ↵
    1. Abeyama K,
    2. Stern DM,
    3. Ito Y,
    4. Kawahara K,
    5. Yoshimoto Y,
    6. Tanaka M,
    7. Uchimura T,
    8. Ida N,
    9. Yamazaki Y,
    10. Yamada S,
    11. Yamamoto Y,
    12. Yamamoto H,
    13. Iino S,
    14. Taniguchi N and
    15. Maruyama I
    : The N-terminal domain of thrombomodulin sequesters high-mobility group-B1 protein, a novel antiinflammatory mechanism. J Clin Invest 115(5): 1267-1274, 2005. PMID: 15841214. DOI: 10.1172/JCI22782
    OpenUrlCrossRefPubMed
  11. ↵
    1. Saito H,
    2. Maruyama I,
    3. Shimazaki S,
    4. Yamamoto Y,
    5. Aikawa N,
    6. Ohno R,
    7. Hirayama A,
    8. Matsuda T,
    9. Asakura H,
    10. Nakashima M and
    11. Aoki N
    : Efficacy and safety of recombinant human soluble thrombomodulin (ART-123) in disseminated intravascular coagulation: results of a phase III, randomized, double-blind clinical trial. J Thromb Haemost 5(1): 31-41, 2007. PMID: 17059423. DOI: 10.1111/j.1538-7836.2006.02267.x
    OpenUrlCrossRefPubMed
  12. ↵
    1. Vincent JL,
    2. Ramesh MK,
    3. Ernest D,
    4. LaRosa SP,
    5. Pachl J,
    6. Aikawa N,
    7. Hoste E,
    8. Levy H,
    9. Hirman J,
    10. Levi M,
    11. Daga M,
    12. Kutsogiannis DJ,
    13. Crowther M,
    14. Bernard GR,
    15. Devriendt J,
    16. Puigserver JV,
    17. Blanzaco DU,
    18. Esmon CT,
    19. Parrillo JE,
    20. Guzzi L,
    21. Henderson SJ,
    22. Pothirat C,
    23. Mehta P,
    24. Fareed J,
    25. Talwar D,
    26. Tsuruta K,
    27. Gorelick KJ,
    28. Osawa Y and
    29. Kaul I
    : A randomized, double-blind, placebo-controlled, Phase 2b study to evaluate the safety and efficacy of recombinant human soluble thrombomodulin, ART-123, in patients with sepsis and suspected disseminated intravascular coagulation. Crit Care Med 41(9): 2069-2079, 2013. PMID: 23979365. DOI: 10.1097/CCM.0b013e31828e9b03
    OpenUrlCrossRefPubMed
  13. ↵
    1. Pan B,
    2. Wang X,
    3. Kojima S,
    4. Nishioka C,
    5. Yokoyama A,
    6. Honda G,
    7. Xu K and
    8. Ikezoe T
    : The fifth epidermal growth factor-like region of thrombomodulin alleviates murine graft-versus-host disease in a G-protein coupled receptor 15 dependent manner. Biol Blood Marrow Transplant 23(5): 746-756, 2017. PMID: 28167153. DOI: 10.1016/j.bbmt.2017.02.001
    OpenUrlCrossRefPubMed
    1. Pan B,
    2. Wang X,
    3. Nishioka C,
    4. Honda G,
    5. Yokoyama A,
    6. Zeng L,
    7. Xu K and
    8. Ikezoe T
    : G-protein coupled receptor 15 mediates angiogenesis and cytoprotective function of thrombomodulin. Sci Rep 7(1): 692, 2017. PMID: 28386128. DOI: 10.1038/s41598-017-00781-w
    OpenUrlCrossRefPubMed
  15. ↵
    1. Pan B,
    2. Wang X,
    3. Kojima S,
    4. Nishioka C,
    5. Yokoyama A,
    6. Honda G,
    7. Xu K and
    8. Ikezoe T
    : The fifth epidermal growth factor like region of thrombomodulin alleviates LPS-induced sepsis through interacting with GPR15. Thromb Haemost 117(3): 570-579, 2017. PMID: 28078348. DOI: 10.1160/TH16-10-0762
    OpenUrlCrossRefPubMed
  16. ↵
    1. Deng HK,
    2. Unutmaz D,
    3. KewalRamani VN and
    4. Littman DR
    : Expression cloning of new receptors used by simian and human immunodeficiency viruses. Nature 388(6639): 296-300, 1997. PMID: 9230441. DOI: 10.1038/40894
    OpenUrlCrossRefPubMed
  17. ↵
    1. Wang W,
    2. Abbruzzese JL,
    3. Evans DB,
    4. Larry L,
    5. Cleary KR and
    6. Chiao PJ
    : The nuclear factor-kappa B RelA transcription factor is constitutively activated in human pancreatic adenocarcinoma cells. Clin Cancer Res 5(1): 119-127, 1999. PMID: 9918209.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Ling J,
    2. Kang Y,
    3. Zhao R,
    4. Xia Q,
    5. Lee DF,
    6. Chang Z,
    7. Li J,
    8. Peng B,
    9. Fleming JB,
    10. Wang H,
    11. Liu J,
    12. Lemischka IR,
    13. Hung MC and
    14. Chiao PJ
    : KrasG12D-induced IKK2/β/NF-κB activation by IL-1α and p62 feedforward loops is required for development of pancreatic ductal adenocarcinoma. Cancer Cell 21(1): 105-120, 2012. PMID: 22264792. DOI: 10.1016/j.ccr.2011.12.006
    OpenUrlCrossRefPubMed
  19. ↵
    1. Lee KM,
    2. Nguyen C,
    3. Ulrich AB,
    4. Pour PM and
    5. Ouellette MM
    : Immortalization with telomerase of the Nestin-positive cells of the human pancreas. Biochem Biophys Res Commun 301(4): 1038-1044, 2003. PMID: 12589817. DOI: 10.1016/s0006-291x(03)00086-x
    OpenUrlCrossRefPubMed
  20. ↵
    1. Kim MP,
    2. Evans DB,
    3. Wang H,
    4. Abbruzzese JL,
    5. Fleming JB and
    6. Gallick GE
    : Generation of orthotopic and heterotopic human pancreatic cancer xenografts in immunodeficient mice. Nat Protoc 4(11): 1670-1680, 2009. PMID: 19876027. DOI: 10.1038/nprot.2009.171
    OpenUrlCrossRefPubMed
  21. ↵
    1. Kang Y,
    2. Zhang R,
    3. Suzuki R,
    4. Li SQ,
    5. Roife D,
    6. Truty MJ,
    7. Chatterjee D,
    8. Thomas RM,
    9. Cardwell J,
    10. Wang Y,
    11. Wang H,
    12. Katz MH and
    13. Fleming JB
    : Two-dimensional culture of human pancreatic adenocarcinoma cells results in an irreversible transition from epithelial to mesenchymal phenotype. Lab Invest 95(2): 207-222, 2015. PMID: 25485535. DOI: 10.1038/labinvest.2014.143
    OpenUrlCrossRefPubMed
  22. ↵
    1. Packiriswamy N and
    2. Parameswaran N
    : G-protein-coupled receptor kinases in inflammation and disease. Genes Immun 16(6): 367-377, 2015. PMID: 26226012. DOI: 10.1038/gene.2015.26
    OpenUrlCrossRefPubMed
  23. ↵
    1. Dorsam RT and
    2. Gutkind JS
    : G-protein-coupled receptors and cancer. Nat Rev Cancer 7(2): 79-94, 2007. PMID: 17251915. DOI: 10.1038/nrc2069
    OpenUrlCrossRefPubMed
  24. ↵
    1. Kim SV,
    2. Xiang WV,
    3. Kwak C,
    4. Yang Y,
    5. Lin XW,
    6. Ota M,
    7. Sarpel U,
    8. Rifkin DB,
    9. Xu R and
    10. Littman DR
    : GPR15-mediated homing controls immune homeostasis in the large intestine mucosa. Science 340(6139): 1456-1459, 2013. PMID: 23661644. DOI: 10.1126/science.1237013
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Suply T,
    2. Hannedouche S,
    3. Carte N,
    4. Li J,
    5. Grosshans B,
    6. Schaefer M,
    7. Raad L,
    8. Beck V,
    9. Vidal S,
    10. Hiou-Feige A,
    11. Beluch N,
    12. Barbieri S,
    13. Wirsching J,
    14. Lageyre N,
    15. Hillger F,
    16. Debon C,
    17. Dawson J,
    18. Smith P,
    19. Lannoy V,
    20. Detheux M,
    21. Bitsch F,
    22. Falchetto R,
    23. Bouwmeester T,
    24. Porter J,
    25. Baumgarten B,
    26. Mansfield K,
    27. Carballido JM,
    28. Seuwen K and
    29. Bassilana F
    : A natural ligand for the orphan receptor GPR15 modulates lymphocyte recruitment to epithelia. Sci Signal 10(496): eaal0180, 2017. PMID: 28900043. DOI: 10.1126/scisignal.aal0180
    OpenUrlAbstract/FREE Full Text
  26. ↵
    1. Pan W,
    2. Cheng Y,
    3. Zhang H,
    4. Liu B,
    5. Mo X,
    6. Li T,
    7. Li L,
    8. Cheng X,
    9. Zhang L,
    10. Ji J,
    11. Wang P and
    12. Han W
    : CSBF/C10orf99, a novel potential cytokine, inhibits colon cancer cell growth through inducing G1 arrest. Sci Rep 4: 6812, 2014. PMID: 25351403. DOI: 10.1038/srep06812
    OpenUrlCrossRefPubMed
  27. ↵
    1. Shirai Y,
    2. Uwagawa T,
    3. Shiba H,
    4. Shimada Y,
    5. Horiuchi T,
    6. Saito N,
    7. Furukawa K,
    8. Ohashi T and
    9. Yanaga K
    : Recombinant thrombomodulin suppresses tumor growth of pancreatic cancer by blocking thrombin-induced PAR1 and NF-κB activation. Surgery 161(6): 1675-1682, 2017. PMID: 28094003. DOI: 10.1016/j.surg.2016.12.008
    OpenUrlCrossRefPubMed
  28. ↵
    1. Kuo CH,
    2. Chen PK,
    3. Chang BI,
    4. Sung MC,
    5. Shi CS,
    6. Lee JS,
    7. Chang CF,
    8. Shi GY and
    9. Wu HL
    : The recombinant lectin-like domain of thrombomodulin inhibits angiogenesis through interaction with Lewis Y antigen. Blood 119(5): 1302-1313, 2012. PMID: 22101897. DOI: 10.1182/blood-2011-08-376038
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Kuo CH,
    2. Sung MC,
    3. Chen PK,
    4. Chang BI,
    5. Lee FT,
    6. Cho CF,
    7. Hsieh TT,
    8. Huang YC,
    9. Li YH,
    10. Shi GY,
    11. Luo CY and
    12. Wu HL
    : FGFR1 mediates recombinant thrombomodulin domain-induced angiogenesis. Cardiovasc Res 105(1): 107-117, 2015. PMID: 25388665. DOI: 10.1093/cvr/cvu239
    OpenUrlCrossRefPubMed
Back to top

In this issue

Print
Download PDF
Article Alerts
Email Article
Citation Tools
Reprints and Permissions
Recombinant Thrombomodulin Has Anti-tumor Effects and Enhances the Effects of Gemcitabine for Pancreatic Cancer Through G-protein Coupled Receptor 15
KENEI FURUKAWA, JIANHUA LING, YICHEN SUN, YU LU, JIE FU, RUI MARUKUCHI, PAUL J. CHIAO
Anticancer Research Oct 2022, 42 (10) 4679-4687; DOI: 10.21873/anticanres.15972
Twitter logo Facebook logo Mendeley logo

Jump to section

Keywords