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Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells.

Mulvey HE, Chang A, Adler J, Del Tatto M, Perez K, Quesenberry PJ, Chatterjee D - BMC Cancer (2015)

Bottom Line: We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis.Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and The Alpert Medical School of Brown University, Coro West, Suite 5.01, One Hoppin St, Providence, RI, 02903, USA. hillary.e.mulvey@gmail.com.

ABSTRACT

Background: Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype.

Methods: EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB.

Results: This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.

Conclusions: Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus

siRNA mediated Knock Down of 14-3-3 zeta/delta reduces soft agar colony formation. a HCT116 cells were transfected with siRNA to 14-3-3 zeta/delta, or equivalent amount of control siRNA solution. After 48 h incubation with siRNA to 14-3-3 zeta/delta, or equivalent amount of control, HCT116 cells were harvested for soft agar cloning and saved for Western Blot analysis. EVs were harvested from the HCT116 cells transfected with siRNA to 14-3-3 zeta/delta. EVs were resuspended in PBS and co-cultured with 1459 cells. After 7 days of co-culture, soft agar coloning was performed. There were 5 dishes/condition. The data represents the mean +/− s.d. of 3 independent experiments performed in quadruplet. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 transfected with 14-3-3 zeta/delta siRNA, compared to HCT116 transfected with scrambled siRNA as control, *p < 0.00001. Decrease in colony formation of 1459 + HCT116 14-3-3siRNA EV when compared to 1459 + HCT116 scramble siRNA EV was assessed, ** p = 0.0039. b After co-culture, cells were harvested and a portion of the cells were prepared as whole cell lysates for Western blot analysis. Western blot analysis confirmed the reduced expression of 14-3-3 zeta/delta in HCT116 + 14-3-3siRNA compared to HCT116 and HCT116 + scramble siRNA controls. Reduced expression of 14-3-3 zeta/delta was also seen in the co-culture of 1459 + HCT116 14-3-3 siRNA EV as compared to 1459 + HCT116 EV and 1459 + HCT116 scramble siRNA EV controls. c Western blot analysis of 14-3-3 sigma and epsilon protein levels of 1459 cells after co-culture with HCT116 EVs. d Western blot of 14-3-3 zeta/delta protein from vesicles isolated from 3 differential centrifugation preparations. MV = microvesicle fraction; EX = exosome fraction; T = MV and EX fraction
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Fig5: siRNA mediated Knock Down of 14-3-3 zeta/delta reduces soft agar colony formation. a HCT116 cells were transfected with siRNA to 14-3-3 zeta/delta, or equivalent amount of control siRNA solution. After 48 h incubation with siRNA to 14-3-3 zeta/delta, or equivalent amount of control, HCT116 cells were harvested for soft agar cloning and saved for Western Blot analysis. EVs were harvested from the HCT116 cells transfected with siRNA to 14-3-3 zeta/delta. EVs were resuspended in PBS and co-cultured with 1459 cells. After 7 days of co-culture, soft agar coloning was performed. There were 5 dishes/condition. The data represents the mean +/− s.d. of 3 independent experiments performed in quadruplet. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 transfected with 14-3-3 zeta/delta siRNA, compared to HCT116 transfected with scrambled siRNA as control, *p < 0.00001. Decrease in colony formation of 1459 + HCT116 14-3-3siRNA EV when compared to 1459 + HCT116 scramble siRNA EV was assessed, ** p = 0.0039. b After co-culture, cells were harvested and a portion of the cells were prepared as whole cell lysates for Western blot analysis. Western blot analysis confirmed the reduced expression of 14-3-3 zeta/delta in HCT116 + 14-3-3siRNA compared to HCT116 and HCT116 + scramble siRNA controls. Reduced expression of 14-3-3 zeta/delta was also seen in the co-culture of 1459 + HCT116 14-3-3 siRNA EV as compared to 1459 + HCT116 EV and 1459 + HCT116 scramble siRNA EV controls. c Western blot analysis of 14-3-3 sigma and epsilon protein levels of 1459 cells after co-culture with HCT116 EVs. d Western blot of 14-3-3 zeta/delta protein from vesicles isolated from 3 differential centrifugation preparations. MV = microvesicle fraction; EX = exosome fraction; T = MV and EX fraction

Mentions: Repression of 14-3-3 zeta via siRNA knock-down in HCT116 cells was used to observe the putative role of 14-3-3 zeta in EV-mediated promotion of a malignant phenotype in non-malignant colon epithelial cells. HCT116 cells were transfected with 14-3-3 zeta siRNA or scramble siRNA for 48 h. After transfection, the cells were grown for 7 days before harvesting. Media was collected for EV isolation and cells were harvested for protein lysates and soft agar assay. Western blot analysis confirmed reduced expression of 14-3-3 in HCT116 cells transfected with siRNA to 14-3-3 zeta, as compared to both HCT116 and HCT116 + scrambled siRNA controls (Fig. 5b). Non-malignant 1459 cells were co-cultured with each of the three types of vesicles for a period of 7 days. After 7 days, cells were harvested for protein lysates and soft agar assay. HCT116 cells with a reduced expression of 14-3-3 zeta showed a significant decrease in colony formation, compared to HCT116 cells transfected with control scramble siRNA (p < 0.00001) (Fig. 5a). Additionally, the co-culture of 1459 and vesicles isolated from HCT116 + 14-3-3 siRNA cells showed a significant decrease in colony formation, compared to co-cultures with vesicles isolated from HCT116 + scramble siRNA control (p = 0.0078) (Fig. 5a). Western blot analysis confirmed the decrease in endogenous 14-3-3 zeta levels (Fig. 5b). These results indicate that 14-3-3 zeta may be responsible, in part, for conferring the malignant phenotype via EVs in HCT116 cells.Fig. 5


Extracellular vesicle-mediated phenotype switching in malignant and non-malignant colon cells.

Mulvey HE, Chang A, Adler J, Del Tatto M, Perez K, Quesenberry PJ, Chatterjee D - BMC Cancer (2015)

siRNA mediated Knock Down of 14-3-3 zeta/delta reduces soft agar colony formation. a HCT116 cells were transfected with siRNA to 14-3-3 zeta/delta, or equivalent amount of control siRNA solution. After 48 h incubation with siRNA to 14-3-3 zeta/delta, or equivalent amount of control, HCT116 cells were harvested for soft agar cloning and saved for Western Blot analysis. EVs were harvested from the HCT116 cells transfected with siRNA to 14-3-3 zeta/delta. EVs were resuspended in PBS and co-cultured with 1459 cells. After 7 days of co-culture, soft agar coloning was performed. There were 5 dishes/condition. The data represents the mean +/− s.d. of 3 independent experiments performed in quadruplet. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 transfected with 14-3-3 zeta/delta siRNA, compared to HCT116 transfected with scrambled siRNA as control, *p < 0.00001. Decrease in colony formation of 1459 + HCT116 14-3-3siRNA EV when compared to 1459 + HCT116 scramble siRNA EV was assessed, ** p = 0.0039. b After co-culture, cells were harvested and a portion of the cells were prepared as whole cell lysates for Western blot analysis. Western blot analysis confirmed the reduced expression of 14-3-3 zeta/delta in HCT116 + 14-3-3siRNA compared to HCT116 and HCT116 + scramble siRNA controls. Reduced expression of 14-3-3 zeta/delta was also seen in the co-culture of 1459 + HCT116 14-3-3 siRNA EV as compared to 1459 + HCT116 EV and 1459 + HCT116 scramble siRNA EV controls. c Western blot analysis of 14-3-3 sigma and epsilon protein levels of 1459 cells after co-culture with HCT116 EVs. d Western blot of 14-3-3 zeta/delta protein from vesicles isolated from 3 differential centrifugation preparations. MV = microvesicle fraction; EX = exosome fraction; T = MV and EX fraction
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Related In: Results  -  Collection

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Fig5: siRNA mediated Knock Down of 14-3-3 zeta/delta reduces soft agar colony formation. a HCT116 cells were transfected with siRNA to 14-3-3 zeta/delta, or equivalent amount of control siRNA solution. After 48 h incubation with siRNA to 14-3-3 zeta/delta, or equivalent amount of control, HCT116 cells were harvested for soft agar cloning and saved for Western Blot analysis. EVs were harvested from the HCT116 cells transfected with siRNA to 14-3-3 zeta/delta. EVs were resuspended in PBS and co-cultured with 1459 cells. After 7 days of co-culture, soft agar coloning was performed. There were 5 dishes/condition. The data represents the mean +/− s.d. of 3 independent experiments performed in quadruplet. A paired t-test was performed to analyze the decrease in soft agar colony formation of HCT116 transfected with 14-3-3 zeta/delta siRNA, compared to HCT116 transfected with scrambled siRNA as control, *p < 0.00001. Decrease in colony formation of 1459 + HCT116 14-3-3siRNA EV when compared to 1459 + HCT116 scramble siRNA EV was assessed, ** p = 0.0039. b After co-culture, cells were harvested and a portion of the cells were prepared as whole cell lysates for Western blot analysis. Western blot analysis confirmed the reduced expression of 14-3-3 zeta/delta in HCT116 + 14-3-3siRNA compared to HCT116 and HCT116 + scramble siRNA controls. Reduced expression of 14-3-3 zeta/delta was also seen in the co-culture of 1459 + HCT116 14-3-3 siRNA EV as compared to 1459 + HCT116 EV and 1459 + HCT116 scramble siRNA EV controls. c Western blot analysis of 14-3-3 sigma and epsilon protein levels of 1459 cells after co-culture with HCT116 EVs. d Western blot of 14-3-3 zeta/delta protein from vesicles isolated from 3 differential centrifugation preparations. MV = microvesicle fraction; EX = exosome fraction; T = MV and EX fraction
Mentions: Repression of 14-3-3 zeta via siRNA knock-down in HCT116 cells was used to observe the putative role of 14-3-3 zeta in EV-mediated promotion of a malignant phenotype in non-malignant colon epithelial cells. HCT116 cells were transfected with 14-3-3 zeta siRNA or scramble siRNA for 48 h. After transfection, the cells were grown for 7 days before harvesting. Media was collected for EV isolation and cells were harvested for protein lysates and soft agar assay. Western blot analysis confirmed reduced expression of 14-3-3 in HCT116 cells transfected with siRNA to 14-3-3 zeta, as compared to both HCT116 and HCT116 + scrambled siRNA controls (Fig. 5b). Non-malignant 1459 cells were co-cultured with each of the three types of vesicles for a period of 7 days. After 7 days, cells were harvested for protein lysates and soft agar assay. HCT116 cells with a reduced expression of 14-3-3 zeta showed a significant decrease in colony formation, compared to HCT116 cells transfected with control scramble siRNA (p < 0.00001) (Fig. 5a). Additionally, the co-culture of 1459 and vesicles isolated from HCT116 + 14-3-3 siRNA cells showed a significant decrease in colony formation, compared to co-cultures with vesicles isolated from HCT116 + scramble siRNA control (p = 0.0078) (Fig. 5a). Western blot analysis confirmed the decrease in endogenous 14-3-3 zeta levels (Fig. 5b). These results indicate that 14-3-3 zeta may be responsible, in part, for conferring the malignant phenotype via EVs in HCT116 cells.Fig. 5

Bottom Line: We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis.Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Rhode Island Hospital and The Alpert Medical School of Brown University, Coro West, Suite 5.01, One Hoppin St, Providence, RI, 02903, USA. hillary.e.mulvey@gmail.com.

ABSTRACT

Background: Extracellular vesicles (EVs) are secreted from many cells, carrying cargoes including proteins and nucleic acids. Research has shown that EVs play a role in a variety of biological processes including immunity, bone formation and recently they have been implicated in promotion of a metastatic phenotype.

Methods: EVs were isolated from HCT116 colon cancer cells, 1459 non-malignant colon fibroblast cells, and tumor and normal colon tissue from a patient sample. Co-cultures were performed with 1459 cells and malignant vesicles, as well as HCT116 cells and non-malignant vesicles. Malignant phenotype was measured using soft agar colony formation assay. Co-cultures were also analyzed for protein levels using mass spectrometry. The importance of 14-3-3 zeta/delta in transfer of malignant phenotype was explored using siRNA. Additionally, luciferase reporter assay was used to measure the transcriptional activity of NF-κB.

Results: This study demonstrates the ability of EVs derived from malignant colon cancer cell line and malignant patient tissue to induce the malignant phenotype in non-malignant colon cells. Similarly, EVs derived from non-malignant colon cell lines and normal patient tissue reversed the malignant phenotype of HCT116 cells. Cells expressing an EV-induced malignant phenotype showed increased transcriptional activity of NF-κB which was inhibited by the NF--κB inhibitor, BAY117082. We also demonstrate that knock down of 14-3-3 zeta/delta reduced anchorage-independent growth of HCT116 cells and 1459 cells co-cultured with HCT derived EVs.

Conclusions: Evidence of EV-mediated induction of malignant phenotype, and reversal of malignant phenotype, provides rational basis for further study of the role of EVs in tumorigenesis. Identification of 14-3-3 zeta/delta as up-regulated in malignancy suggests its potential as a putative drug target for the treatment of colorectal cancer.

No MeSH data available.


Related in: MedlinePlus