N-myc downstream regulated gene 1 (NDRG1) promotes metastasis of human scirrhous gastric cancer cells through epithelial mesenchymal transition.

Our recent study demonstrated that higher expression of N-myc downregulated gene 1 (NDRG1) is closely correlated with poor prognosis in gastric cancer patients. In this study, we asked whether NDRG1 has pivotal roles in malignant progression including metastasis of gastric cancer cells. By gene expression microarray analysis expression of NDRG1 showed the higher increase among a total of 3691 up-regulated genes in a highly metastatic gastric cancer cell line (58As1) than their parental low metastatic counterpart (HSC-58). The highly metastatic cell lines showed decreased expression of E-cadherin, together with enhanced expression of vimentin and Snail. This decreased expression of E-cadherin was restored by Snail knockdown in highly metastatic cell lines. We next established stable NDRG1 knockdown cell lines (As1/Sic50 and As1/Sic54) from the highly metastatic cell line, and both of these cell lines showed enhanced expression of E-cadherin and decreased expression of vimentin and Snail. And also, E-cadherin promoter-driven luciferase activity was found to be increased by NDRG1 knockdown in the highly metastatic cell line. NDRG1 knockdown in gastric cancer cell showed suppressed invasion of cancer cells into surround tissues, suppressed metastasis to the peritoneum and decreased ascites accumulation in mice with significantly improved survival rates. This is the first study to demonstrate that NDRG1 plays its pivotal role in the malignant progression of gastric cancer through epithelial mesenchymal transition

Comparison of protein and mRNA expression levels of various factors between low and highly metastatic gastric cancer cell lines.

<p>(A) Western blot analysis of total cell lysates shows protein expression levels of NDRG1, growth factor receptor, EMT-related proteins, Wnt/β-catenin-related proteins, and other factors in HSC-58, 58As1 and 58As9 cells. (B) Comparison of mRNA expression levels of NDRG1, E-cadherin, vimentin, Snail, MMP-1 and β-catenin in HSC-58, 58As1 and 58As9 cells by qRT-PCR analysis. (C) Immunocytochemical analysis of E-cadherin and β-catenin in HSC-58 and 58As9, using specific antibodies against E-cadherin, β-catenin and DAP1. Magnification×200. (D) Western blot analysis shows expression of β-catenin and Snail in nucleus and cytosol fraction. CREB, a nuclear marker, and α-tubulin, a cytosol marker. (E,F) Comparison of luciferase activity driven by E-cadhrin promoter and β-catenin (TopFlash) driven promoter between HSC-58 and its highly metastatic cell lines. The relative promoter activity is presented when normalized by the activity in HSC-58. *p<0.01.</p

Altered expression of EMT-related factors by NDRG1 knockdown in highly metastatic 58As1.

<p>(A) Microarray analysis for the effect of NDRG1 knockdown on expression of genes that are up- or down- regulated in Asl/Sic50 versus As1/Mock3. Relative expression rates are presented on genes belonging to three biological functions. (B) Comparison of protein expression levels of NDRG1, EMT-related proteins, β-catenin, Akt, p-Akt, ERK1/2, p-ERK1/2, GSK-3β, p-GSK-3β and EGFR by western blot analysis with total cell lysate. (C) The mRNA expression of NDRG1, E-cadherin, vimentin, Snail, MMP-1 and β-catenin was determined by qRT-PCR analysis. (D) Comparison of luciferase activity driven by β-catenin (TopFlash) between As1/Mock3 and its NDRG1 knockdowned cell lines. Relative luminescence fold is presented when normalized by the value in As1/Mock3. Each column is average of triplicate trials±SD.</p

Suppression of peritoneal dissemination by NDRG1 knockdown.

<p>(A) Macroscopic images show enlarged peritoneal cavity and metastatic nodules by As1/Mock3 and As1/Sic50. Arrowheads show nodules. (B) Number of metastatic nodules in the mesenterium was 51±16 (As1/Mock3) and 35±14 (As1/Sic50) (<i>p</i> = 0.21), but the As1/Mock3 nodule size was 3–4 times larger than those of As1/Sic50. (C) Comparison of the volume of ascites between As1/Mock3 (3.9±1.0 ml) and As1/Sic50 cells (0.5±0.6 ml) following orthotopic implantation (n = 7) (* <i>p</i><0.01). (D) Survival curves show that survival rate in As1/Sic50 tumor-bearing mice was significantly (* <i>p</i><0.01) longer than that of As1/Mock3 tumor-bearing mice (n = 6). (E) Our hypothetic model how NDRG1 overexpression promotes metastasis including peritoneal dissemination through alteration of EMT by scirrhous gastric cancer cells, possibly through modification of Snail expression.</p

Enhancement of E-cadherin promoter activity by NDRG1 knockdown in highly metastatic gastric cancer cell.

<p>(A) Comparison of protein expression levels of E-cadherin, β-catenin, NDRG1 and vimentin when transiently treated with Snail siRNA for 0, 48 and 96 hr by western blot analysis in HSC-58 cell. (B) E-cadherin promoter-driven luciferase activity in the absence or presence of Snail expression in HSC-58 and BxPC-3 cells. E-cadherin-luc was transfected with or without pcDNA3-Snail, and the luciferase activity was measured. Each column is average of triplicate trials (*<i>p</i><0.05). (C) Comparison of E-cadherin promoter-driven luciferase activity (E-cadherin-luc) in As1/Mock3, As1/Sic50 and As1/Sic54. Each column is average of triplicate trials (*<i>p</i><0.05). (D) The effect of CT99021 on protein expression of NDRG1 and various EMT-related molecules by Western blot analysis. 58Asl cells were treated with indicated doses of the drug for 24 hr. (E) The effect of β-catenin knockdown by its siRNAs on expression of E-cadherin. HSC-58 cells were transfected with siRNAs for 24 hr, and total cell lysates were analyzed by Western blot analysis.</p

Biological properties of low and highly metastatic gastric cancer cell lines <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Comparison of cell proliferation rates <i>in vitro</i>. Cells were seeded on day 0 (5×10⁴ cells/dish) and proliferation was measured in RPMI 1640 containing 10% FBS. Doubling times were 34, 27, and 23 hr for HSC-58 (black), 58As1 (gray) and 58As9 cells (white), respectively. (B) Morphology of gastric cancer cell line <i>in vitro</i>. HSC-58 and 58As9 cell growth was visible as attached layers with fibroblastic morphology. 58As1 cells showed suspension-type cell growth with round morphology. (C) Tumor growth of gastric cancer cell lines at day 14 and 28. HSC-58 (black), 58As1 (light gray), and 58As9 (dark gray) were subcutaneously implanted with 1×10⁷ cells at day 0. 58As1 or 58As9 cells showed significantly (*<i>p</i><0.01) higher tumor growth rates than HSC-58 cells. (D, E) Peritoneal dissemination and ascites formation <i>in vivo</i>. Typical figures show high and low peritoneal dissemination and ascites accumulation at day 55 after inoculation of 1×10⁶ cells into peritoneal cavity. Each mouse showed ascites accumulation of 0.7–2.5 ml and 5–12 nodules on the mesenterium following 58As1 inoculation, but this was not apparent with HSC-58 cells. N.D., not detectable. (F) Microarray analysis on expression of genes that are up- or down- regulated in highly metastatic cell line, 58Asl, as compared with HSC-58. Relative expression rates are presented on genes belonging to three biological functions.</p

Efficacy of Low-Level Laser Therapy for Oral Mucositis in Hematologic Patients Undergoing Transplantation: A Single-Arm Prospective Study

Oral mucositis significantly affects the quality of life in hematologic cancer patients undergoing hematopoietic stem cell transplantation. Despite global evidence supporting the efficacy of low-level laser therapy (LLLT) for mucositis prevention, its clinical adoption in Japan is limited. This study aimed to fill this gap by evaluating the safety and efficacy of LLLT in a Japanese patient population. In a single-group, non-blinded, exploratory trial, we compared 21 LLLT-treated patients against a historical control of 96 patients. The primary endpoint was the incidence of Grade ≥ 2 mucositis, based on NCI-CTCAE ver. 4.0. The LLLT group showed a significantly lower incidence of Grade ≥ 2 mucositis (23.8%) compared to the control group (64.6%) (p = 0.0006). Furthermore, Grade ≥ 2 mucositis correlated with increased oral dryness and longer hospital stays. Our study confirms the efficacy of LLLT in reducing the onset of severe oral mucositis among Japanese hematologic cancer patients, advocating for its clinical introduction as a preventive measure in Japan