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Combining TGF-β1 knockdown and miR200c administration to optimize antitumor efficacy of B16F10/GPI-IL-21 vaccine.

Wang X, Zhao F, He X, Wang J, Zhang Y, Zhang H, Ni Y, Sun J, Wang X, Dou J - Oncotarget (2015)

Bottom Line: Our study aimed at optimizing the antitumor effects of the B16F10/glycosylphosphatidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine on melanoma bearing mice by combining the TGF-β1 knockdown and the administration of miR200c agomir.The later combination showed that, when compared with the mice in the control group that received no vaccination, vaccinated mice significantly increased NK and CTL activities, enhanced levels of IFN-γ, and reduced expression of TGF-β1, N-cadherin, Vimentin, Gli1/2, P-Smad2/3 and others involved in promoting expression of EMT-related molecules in tumor areas, and inhibited the melanoma metastasis in lungs and lymph nodes.Altogether, our findings demonstrate that this synergistic anti-cancer regimen effectively induces strong immune response and diminishes the melanoma progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogenic Biology and Immunology, School of Medicine & Collaborative Innovation Center of Suzhou Nano Science and Technology, Southeast University, Nanjing, China.

ABSTRACT
TGF-β1 secreted abundantly by tumors cells as well as present in the local microenvironment promotes neoplasm invasion and metastasis by triggering the epithelial to mesenchymal transition (EMT). MiR200c has been shown to suppress EMT and to regulate the cellular epithelial and interstitial state conversion, whereas the tumor vaccines are intended to specifically initiate or amplify a host response against evolving tumor cells. Our study aimed at optimizing the antitumor effects of the B16F10/glycosylphosphatidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine on melanoma bearing mice by combining the TGF-β1 knockdown and the administration of miR200c agomir. The mice were subcutaneously vaccinated with inactivated B16F10/GPI-IL-21 vaccine and challenged by B16F10 cells transfected with shTGF-β1 (B16F10/shTGF-β1 cells) or B16F10/shTGF-β1 cells with the administration of miR200c agomir. The later combination showed that, when compared with the mice in the control group that received no vaccination, vaccinated mice significantly increased NK and CTL activities, enhanced levels of IFN-γ, and reduced expression of TGF-β1, N-cadherin, Vimentin, Gli1/2, P-Smad2/3 and others involved in promoting expression of EMT-related molecules in tumor areas, and inhibited the melanoma metastasis in lungs and lymph nodes. Altogether, our findings demonstrate that this synergistic anti-cancer regimen effectively induces strong immune response and diminishes the melanoma progression.

No MeSH data available.


Related in: MedlinePlus

Observation of cell biological property in B16F10/shTGF-β1 cellsA, C and E. The images of colony forming rates, of the cellular wound areas, and of the invasive cells were taken from B16F10/WT, B16F10/Scramble, and the B16F10/shTGF-β1 cells, respectively. B, D and F. Statistical analyses of the colony forming rates, the cellular wound areas, and the invasive cells per high power field (HPF, ×400) respectively. The data are represented as mean +/− SEM (n=6); refer to the statistical differences as shown.
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Figure 2: Observation of cell biological property in B16F10/shTGF-β1 cellsA, C and E. The images of colony forming rates, of the cellular wound areas, and of the invasive cells were taken from B16F10/WT, B16F10/Scramble, and the B16F10/shTGF-β1 cells, respectively. B, D and F. Statistical analyses of the colony forming rates, the cellular wound areas, and the invasive cells per high power field (HPF, ×400) respectively. The data are represented as mean +/− SEM (n=6); refer to the statistical differences as shown.

Mentions: To evaluate the influence of the TGF-β1 knockdown on the B16F10 cells, we first developed an expression vector-based small hairpin RNA to target TGF-β1 (shTGF-β1) and then transfected it into the B16F10 cells. In terms of the intercellular regulation of TGF-β1, we found that the TGF-β1 expression was significantly decreased in the B16F10/shTGF-β1 cells compared with the B16F10 cells and the B16F10/Scramble cells (Figure 1A and 1B). The EMT associated molecular expression of E-cadherin and Smad 7 was obviously increased whereas the expression of N-cadherin, Vimentin, Gli1, Gli2, P-Smad2, and P-Smad3 such as those involved in the EMT-related molecules were markedly decreased in the B16F10/shTGF-β1 cells in comparison with the B16F10 cells and the B16F10/Scramble cells (Figure 1). Next, we analyzed the properties of the B16F10/shTGF-β1 cells by observation of the clone formation, and cellular invasive and metastatic ability in vitro. The down-regulation of the TGF-β1 expression in the B16F10/shTGF-β1 cells significantly reduced the colony forming rates and the cellular invasive and metastatic ability in comparison with the B16F10 cells and the B16F10/Scramble cells; the comparisons are shown in Figure 2A, 2C and 2E, respectively. The statistical analysis results in Figure 2B, 2D and 2F show a significant differences between the B16F10/shTGF-β1 cells and the B16F10 cells or the B16F10/Scramble cells. These results suggested that the shTGF-β1 transfected B16F10 cells demonstrated the potential for inhibiting cellular proliferation potential and EMT property.


Combining TGF-β1 knockdown and miR200c administration to optimize antitumor efficacy of B16F10/GPI-IL-21 vaccine.

Wang X, Zhao F, He X, Wang J, Zhang Y, Zhang H, Ni Y, Sun J, Wang X, Dou J - Oncotarget (2015)

Observation of cell biological property in B16F10/shTGF-β1 cellsA, C and E. The images of colony forming rates, of the cellular wound areas, and of the invasive cells were taken from B16F10/WT, B16F10/Scramble, and the B16F10/shTGF-β1 cells, respectively. B, D and F. Statistical analyses of the colony forming rates, the cellular wound areas, and the invasive cells per high power field (HPF, ×400) respectively. The data are represented as mean +/− SEM (n=6); refer to the statistical differences as shown.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4494953&req=5

Figure 2: Observation of cell biological property in B16F10/shTGF-β1 cellsA, C and E. The images of colony forming rates, of the cellular wound areas, and of the invasive cells were taken from B16F10/WT, B16F10/Scramble, and the B16F10/shTGF-β1 cells, respectively. B, D and F. Statistical analyses of the colony forming rates, the cellular wound areas, and the invasive cells per high power field (HPF, ×400) respectively. The data are represented as mean +/− SEM (n=6); refer to the statistical differences as shown.
Mentions: To evaluate the influence of the TGF-β1 knockdown on the B16F10 cells, we first developed an expression vector-based small hairpin RNA to target TGF-β1 (shTGF-β1) and then transfected it into the B16F10 cells. In terms of the intercellular regulation of TGF-β1, we found that the TGF-β1 expression was significantly decreased in the B16F10/shTGF-β1 cells compared with the B16F10 cells and the B16F10/Scramble cells (Figure 1A and 1B). The EMT associated molecular expression of E-cadherin and Smad 7 was obviously increased whereas the expression of N-cadherin, Vimentin, Gli1, Gli2, P-Smad2, and P-Smad3 such as those involved in the EMT-related molecules were markedly decreased in the B16F10/shTGF-β1 cells in comparison with the B16F10 cells and the B16F10/Scramble cells (Figure 1). Next, we analyzed the properties of the B16F10/shTGF-β1 cells by observation of the clone formation, and cellular invasive and metastatic ability in vitro. The down-regulation of the TGF-β1 expression in the B16F10/shTGF-β1 cells significantly reduced the colony forming rates and the cellular invasive and metastatic ability in comparison with the B16F10 cells and the B16F10/Scramble cells; the comparisons are shown in Figure 2A, 2C and 2E, respectively. The statistical analysis results in Figure 2B, 2D and 2F show a significant differences between the B16F10/shTGF-β1 cells and the B16F10 cells or the B16F10/Scramble cells. These results suggested that the shTGF-β1 transfected B16F10 cells demonstrated the potential for inhibiting cellular proliferation potential and EMT property.

Bottom Line: Our study aimed at optimizing the antitumor effects of the B16F10/glycosylphosphatidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine on melanoma bearing mice by combining the TGF-β1 knockdown and the administration of miR200c agomir.The later combination showed that, when compared with the mice in the control group that received no vaccination, vaccinated mice significantly increased NK and CTL activities, enhanced levels of IFN-γ, and reduced expression of TGF-β1, N-cadherin, Vimentin, Gli1/2, P-Smad2/3 and others involved in promoting expression of EMT-related molecules in tumor areas, and inhibited the melanoma metastasis in lungs and lymph nodes.Altogether, our findings demonstrate that this synergistic anti-cancer regimen effectively induces strong immune response and diminishes the melanoma progression.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathogenic Biology and Immunology, School of Medicine & Collaborative Innovation Center of Suzhou Nano Science and Technology, Southeast University, Nanjing, China.

ABSTRACT
TGF-β1 secreted abundantly by tumors cells as well as present in the local microenvironment promotes neoplasm invasion and metastasis by triggering the epithelial to mesenchymal transition (EMT). MiR200c has been shown to suppress EMT and to regulate the cellular epithelial and interstitial state conversion, whereas the tumor vaccines are intended to specifically initiate or amplify a host response against evolving tumor cells. Our study aimed at optimizing the antitumor effects of the B16F10/glycosylphosphatidylinositol-interleukin 21 (B16F10/GPI-IL-21) tumor vaccine on melanoma bearing mice by combining the TGF-β1 knockdown and the administration of miR200c agomir. The mice were subcutaneously vaccinated with inactivated B16F10/GPI-IL-21 vaccine and challenged by B16F10 cells transfected with shTGF-β1 (B16F10/shTGF-β1 cells) or B16F10/shTGF-β1 cells with the administration of miR200c agomir. The later combination showed that, when compared with the mice in the control group that received no vaccination, vaccinated mice significantly increased NK and CTL activities, enhanced levels of IFN-γ, and reduced expression of TGF-β1, N-cadherin, Vimentin, Gli1/2, P-Smad2/3 and others involved in promoting expression of EMT-related molecules in tumor areas, and inhibited the melanoma metastasis in lungs and lymph nodes. Altogether, our findings demonstrate that this synergistic anti-cancer regimen effectively induces strong immune response and diminishes the melanoma progression.

No MeSH data available.


Related in: MedlinePlus