Homeworks academic service


Effects of wnt 3a and wnt 5a on proliferation of hek293 cell

Department of Pathology, No. China Published online on: This is an open access article distributed under the terms of Creative Commons Attribution License.

Abstract Wnt proteins are a group of secreted signaling proteins, which function to regulate cell fate and pattern formation during embryogenesis. Altered expression of Wnt5a has been implicated in human carcinogenesis and tumor progression. A previous study identified that Wnt5a is overexpressed in human pancreatic cancer tissues, and that upregulated expression of Wnt5a promotes tumor cell migration and invasion.

Effects of Wnt proteins on cell proliferation and apoptosis in HEK293 cells.

Western blot analysis was used to analyze the expression of Wnt signaling molecules. By contrast, knockdown of the expression of Wnt5a inhibited cell growth and promoted apoptosis of the pancreatic cancer cells. These results suggested that Wnt5a is involved in the modulation of pancreatic cancer cell proliferation, and that Wnt5a may be a potential target for pancreatic cancer therapy.

Introduction Pancreatic cancer is a significant worldwide health problem, with an estimated 277,000 new cases, and a cancer-associated mortality rate of 266,000 annually worldwide 1. Thus, there is a significant and urgent requirement to elucidate the molecular mechanisms that mediate pancreatic cancer development and progression, in order to effectively control and prevent this life-threatening disease. The development of pancreatic cancer, as with other types of cancer in the gastrointestinal tract, involves multiple genetic alterations, including oncogene activation and tumor suppressor gene dysfunction.

Over the past three decades, a large body of evidence has accumulated regarding the molecular alterations associated with the development of pancreatic cancer 4 — 6.

  • We demonstrated that Wnt3a and Wnt5a can promote proliferation of HEK293 cells and inhibit serum starvation-induced apoptosis, which implies that Wnt3a and Wnt5a can maintain the survival of HEK293 cells under stress, and also provide a novel insight into the role of Wnt3a and Wnt5a and their related signalings in carcinogenesis;
  • Succinate dehydrogenase SDH with its subunit SDHA is a key component of the citric acid cycle, which is involved in complex II of the mitochondrial electron transport chain and is responsible for transferring electrons from succinate to ubiquinone;
  • Abstract The Wnt signaling pathway has been associated with many essential cell processes.

These disparate observations have coalesced somewhat into an improved understanding of this disease, however, further investigation is required to provide early cancer identification and novel treatment strategies for pancreatic cancer.

The present study focused on Wnt signaling proteins in pancreatic cancer. Wnt consists of a large family of secreted lipid-modified glycoproteins, which regulate numerous processes in animal development 7 and normal cell processes, including cell growth, differentiation, apoptosis, migration and cell polarity 8.

Each member of the Wnt family of proteins exhibits unique expression patterns and has distinct functions in embryonic development and normal cell homeostasis 9. Based on their ability to induce transformation of the mouse mammary epithelial cell line, Wnt proteins are divided into two functional groups, canonical and noncanonical Wnt 10.

  • Flow cytometric analysis of apoptosis Tumor cell apoptosis was detected using flow cytometry using an Annexin V-fluorescein isothiocyanate FITC apoptosis detection kit cat;
  • For example, Yoon et al;
  • The source of these cancer-associated Wnts is often from the cancer cells themselves, although stromal cells including fibroblasts and hematopoietic cells also produce multiple Wnts Luga et al;
  • Briefly, tissue sections were deparaffinized and hydrated in xylene ShengGong Biotechnology Co;
  • The proportion of positive tumor cells was scored.

Wnt5a is classified as a member of the noncanonical Wnt family, is crucial for embryonic development and has been implicated in several human diseases 11 — 13. Previous studies have demonstrated that Wnt5a is important in the progression of various types of human cancer 14 — 16 ; however, the particular signaling and physiological function of the Wnt5a protein in pancreatic cancer remains to be elucidated.

Our previous study identified that Wnt5a protein is expressed at high levels in human pancreatic cancer tissues, and that upregulation of Wnt5a promotes epithelial-to-mesenchymal transition and metastasis of pancreatic cancer cells 17. On the basis of these data, the aim of the present study was to characterize the role of Wnt5a in the proliferation of pancreatic cancer cells, and to elucidate the signaling events of the Wnt5a protein.

The medium was replaced every third day. Stable clones were confirmed by immunoblot analysis. The membranes were probed with the following primary antibodies: Rabbit polyclonal anti-Wnt5a 1: The goat polyclonal anti-rabbit 1: The intensity of each band was measured by densitometric analysis using the Quantity One software Bio-Rad Laboratories, Inc. The optical density was then measured using a spectrophotometer Ultrospec K; Biochrom, Ltd.

The optical density values of each experiment were plotted to determine the cell viability curve. The experiment was repeated three times.

INTRODUCTION

The assay was performed in triplicate and repeated once. Flow cytometric analysis of apoptosis Tumor cell apoptosis was detected using flow cytometry using an Annexin V-fluorescein isothiocyanate FITC apoptosis detection kit cat. The cell solution was incubated for 15 min at room temperature in the dark.

Each experiment was performed in triplicate and repeated at least three times. The mice were fed with a standard pellet diet and water ad libitum. The present study was approved by the ethics committee of The Second Military Medical University Shanghai, China and was performed in compliance with the regulations of the Administration of Experimental Animals of the State Council in China.

Result Filters

Subsequently, the pancreas was relocated into the abdominal cavity, which was then closed in two layers using 6—0 absorbable vicryl sutures [Anhui Kangning Industrial Group Co. Each group consisted of three animals. When the orthotopic tumors reached a size of 1—2 cm in diameter, the mice were sacrificed by exposure to CO2 followed by cervical dislocation.

A streptavidin-peroxidase-biotin immunohistochemical method was used for immunostaining. Briefly, tissue sections were deparaffinized and hydrated in xylene ShengGong Biotechnology Co.

  1. Rabbit polyclonal anti-Wnt5a 1. For example, Yoon et al.
  2. Previous studies have demonstrated that Wnt5a is important in the progression of various types of human cancer 14 — 16 ; however, the particular signaling and physiological function of the Wnt5a protein in pancreatic cancer remains to be elucidated. On the basis of these data, the aim of the present study was to characterize the role of Wnt5a in the proliferation of pancreatic cancer cells, and to elucidate the signaling events of the Wnt5a protein.
  3. The experiment was repeated three times.
  4. A subsequent cell viability assay showed that the cell density of PANC-1 and BXPC-3 cells was significantly different among the groups following seven days of culture Fig. For example, stromal cells that support the intestinal stem cell niche express at least six different Wnts at the same time Kabiri et al.
  5. Gels were run at 150 V for 1 h and transferred onto nitrocellulose membranes via the iBlot System Life Technologies. In the BrdU assays an increase of the proliferation rate could be detected, which correlated to the applied Wnt3a concentration.

The antigen retrieval step was performed in a steam pressure cooker containing preheated 0. Rabbit polyclonal anti-Wnt5a antibody cat.

International Journal of Cell Biology

The tissue sections were then treated with biotinylated polyclonal anti-rabbit secondary antibodies cat. Immunoreactivity was visualized with diaminobenzidine Sigma-Aldrich.

  1. Altered expression of Wnt5a has been implicated in human carcinogenesis and tumor progression. Cells were treated equally to proliferation analysis experiments and incubated with SDHA antibody as described above.
  2. In the BrdU assays an increase of the proliferation rate could be detected, which correlated to the applied Wnt3a concentration.
  3. Results of the proliferation assays. Epub 2008 Mar 29.

The tissue sections were then counter-stained with hematoxylin ShengGong Biotechnology Co. Wnt5a staining was predominantly located in the cell cytoplasm and nuclei, whereas Ki67 staining in the nuclei. The proportion of positive tumor cells was scored. Differences in the means were determined using Student's t-test or one-way analysis of variance followed by Tukey's post-hoc test.

Results Wnt5a induces pancreatic cancer cell viability and colony formation Stable Wnt5a-overexpressing or Wnt5a siRNA-expressing pancreatic cancer cell lines were established following gene transfection and confirmed using immunoblot analysis Fig.

A subsequent cell viability assay showed that the cell density of PANC-1 and BXPC-3 cells was significantly different among the groups following seven days of culture Fig.

Wnt5a increases the viability of pancreatic cancer cells. GAPDH was used as an internal control.

The experiments were repeated three times. B Monolayer cell culture. Representative images of the morphology of indicated cells following 7 days culture under a phase-contrast microscope. C and D Cell viability assay. The stably transfected cells were grown in a monolayer for up to 7 days and then subjected to a cell viability assay. Furthermore, as shown in Fig. A Colony formation assay. Images of the cells in one of three independent experiments are shown. Representative results of flow cytometric analysis are shown in the top panels.

The bar graphs show the quantitative results of cell apoptosis. Wnt5a induces pancreatic cancer cell xenograft growth in an orthotopic nude mouse model Following the in vitro investigations, the present study investigated the in vivo effects of Wnt5a on tumor growth using an orthotopic mouse model.

In addition, microscopic examination and immunohistochemistry analysis demonstrated that the Wnt5a-overexpressing tumors exhibited increased protein expression levels of Ki67 Fig.