Anti-cancer effects of HS1002, a novel peptide, by dual targeting the GnRH receptor and human telomerase reverse transcriptase (hTERT) in prostate cancer cells

국문 초록 (Abstract)

HS1002, 전립선암, 인간 텔로머레이즈 역전사 효소, GnRH 수용체, 면역 반응

다국어 초록 (Multilingual Abstract)

Human telomerase reverse transcriptase (hTERT) is overexpressed in most human cancers. hTERT is considered an important target for cancer therapy. In the present study, a new peptide, HS1002, was synthesized with a sequence of pGlu-His-Trp-Ser-Tyr-Arg-Leu-Arg-Phe-Ile-Pro-NHEt based on the amino acid sequences of gonadotropin-releasing hormone (GnRH) and hTERT. The aim of this study was to evaluate the anticancer activity of HS1002 and test its dual-targeting effects on the GnRH receptor (GnRHR) and hTERT in prostate cancer cells. Upon comparison with different prostate cancer cell lines, HS1002 exhibited the highest cytotoxicity against LNCaP cells. The hTERT expression correlated with telomerase activity in LNCaP cells was significantly suppressed by HS1002, resulting in a reduction of proliferation, metastasis, and an increase in apoptosis. Additionally, HS1002 suppressed c-Myc, AKT, and ERK pathways in LNCaP cells. In the tumor-bearing nude mice model, HS1002 significantly inhibited tumor growth and downregulated the expression of hTERT and Ki-67 in tumor tissue, as evidenced by western blot and immunohistochemistry (IHC) analyses, respectively. Moreover, repeated subcutaneous injections of HS1002 resulted in a reduction of both serum testosterone levels and seminal vesicle weight. HS1002 also increased cytosolic calcium influx and cAMP response element (CRE)-luciferase activity in GnRHR-overexpressing HEK293 cells compared to mock-transfected transfected cells. Furthermore, potent cytotoxicity in LNCaP cells was exhibited by HS1002/interleukin (IL)-2-pretreated peripheral blood mononuclear cells (PBMC). In addition, a flow-cytometric analysis confirmed an elevation in the production of granzyme B and interferon (IFN)-γ in CD8+ T cells in the MC38 syngeneic mouse model by HS1002. These data indicate that the dual targeting of GnRHR and hTERT by HS1002 suppresses prostate cancer cell growth and induces anti-cancer immunity. Therefore, HS1002 might be used as a novel therapeutic agent for prostate cancer.

목차 (Table of Contents)

• 1. Introduction 1
• 2. Impact of hTERT inhibition by HS1002 on prostate cancer 11
• 2.1 Materials and Methods 11
• 2.1.1 Reagents 11
• 2.1.2 Cell culture and cell lines 12
• 2.1.3 Prostate tissue microarray and IHC 12
• 2.1.4 Cytotoxicity assay 13
• 2.1.5 Clonogenic assay 13
• 2.1.6 Western blot analysis 14
• 2.1.7 qRT-PCR analysis 14
• 2.1.8 Telomerase activity and telomere length quantification 15
• 2.1.9 Annexin V-FITC binding assay 16
• 2.1.10 IncuCyte ZOOM analysis 16
• 2.1.11 Transwell invasion assay 17
• 2.1.12 Caspase-3/7 activity assay 17
• 2.1.13 c-Myc transcriptional activity 18
• 2.1.14 Tumor xenograft model 19
• 2.1.15 TUNEL assay 19
• 2.1.16 Statistical analysis 20
• 2.2 Result 21
• 2.2.1 IHC analysis of tissue microarray 21
• 2.2.2 Peptide design for targeting GnRHR and hTERT 24
• 2.2.3 Cytotoxic activity of peptides in prostate cancer cells 26
• 2.2.4 Effect of HS1002 on hTERT expression in prostate cancer cells 30
• 2.2.5 In vitro anti-cancer effect of HS1002 35
• 2.2.6 In vivo anti-cancer effect of HS1002 47
• 3. The functional role of HS1002 as a GnRHR ligand 57
• 3.1 Materials and Methods 57
• 3.1.1 Reagents 57
• 3.1.2 Establishment of GnRHR-overexpressing HEK293 cells 57
• 3.1.3 siRNA transfection 58
• 3.1.4 Western blot analysis 58
• 3.1.5 Intracellular calcium flux assay 59
• 3.1.6 CRE-luciferase reporter assay 59
• 3.1.7 Measurement of serum testosterone level 60
• 3.1.8 Statistical analysis 60
• 3.2 Result 61
• 3.2.1 In vivo effect of HS1002 on serum testosterone level 61
• 3.2.2 Activation of GnRHR signaling by HS1002 64
• 3.2.3 Effect of HS1002 on GnRHR-knockdown LNCaP cells 69
• 4. Effect of HS1002 on the anti-tumor immune response 74
• 4.1 Materials and Methods 74
• 4.1.1 Reagents 74
• 4.1.2 Cell culture and cell lines 74
• 4.1.3 IFN-γ ELISpot assay 75
• 4.1.4 PBMC-mediated cytotoxicity and cytokine secretion 75
• 4.1.5 Syngeneic mouse tumor model 76
• 4.1.6 TUNEL assay 77
• 4.1.7 Tumor digestion and flow cytometry 77
• 4.1.8 Library preparation, sequencing, and data analysis 78
• 4.1.9 Statistical analysis 79
• 4.2 Result 80
• 4.2.1 Evaluation of peptide-specific IFN-γ production 80
• 4.2.2 Cytotoxicity of peptide-pretreated PBMC against cancer cells 82
• 4.2.3 Inhibition of tumor growth by HS1002 in a syngeneic model 88
• 4.2.4 Effect of HS1002 on tumor-infiltrating leukocytes in tumors 92
• 4.2.5 RNA-Seq gene expression profiling in MC38 tumors 96
• 5. Discussion 99
• 6. Conclusion 113
• 7. References 117