Thermostable Direct Hemolysin Downregulates Human Colon Carcinoma Cell Proliferation with the Involvement of E-Cadherin, and β-Catenin/Tcf-4 Signaling

BACKGROUND: Colon cancers are the frequent causes of cancer mortality worldwide. Recently bacterial toxins have received marked attention as promising approaches in the treatment of colon cancer. Thermostable direct hemolysin (TDH) secreted by Vibrio parahaemolyticus causes influx of extracellular calcium with the subsequent rise in intracellular calcium level in intestinal epithelial cells and it is known that calcium has antiproliferative activity against colon cancer. KEY RESULTS: In the present study it has been shown that TDH, a well-known traditional virulent factor inhibits proliferation of human colon carcinoma cells through the involvement of CaSR in its mechanism. TDH treatment does not induce DNA fragmentation, nor causes the release of lactate dehydrogenase. Therefore, apoptosis and cytotoxicity are not contributing to the TDH-mediated reduction of proliferation rate, and hence the reduction appears to be caused by decrease in cell proliferation. The elevation of E-cadherin, a cell adhesion molecule and suppression of β-catenin, a proto-oncogene have been observed in presence of CaSR agonists whereas reverse effect has been seen in presence of CaSR antagonist as well as si-RNA in TDH treated cells. TDH also triggers a significant reduction of Cyclin-D and cdk2, two important cell cycle regulatory proteins along with an up regulation of cell cycle inhibitory protein p27(Kip1) in presence of CaSR agonists. CONCLUSION: Therefore TDH can downregulate colonic carcinoma cell proliferation and involves CaSR in its mechanism of action. The downregulation occurs mainly through the involvement of E-cadherin-β-catenin mediated pathway and the inhibition of cell cycle regulators as well as upregulation of cell cycle inhibitors

Thermostable Direct Hemolysin Downregulates Human Colon Carcinoma Cell Proliferation With the Involvement of E-Cadherin, and β-Catenin/Tcf-4 Signaling

Background: Colon cancers are the frequent causes of cancer mortality worldwide. Recently bacterial toxins have received marked attention as promising approaches in the treatment of colon cancer. Thermostable direct hemolysin (TDH) secreted by Vibrio parahaemolyticus causes influx of extracellular calcium with the subsequent rise in intracellular calcium level in intestinal epithelial cells and it is known that calcium has antiproliferative activity against colon cancer. Key Results: In the present study it has been shown that TDH, a well-known traditional virulent factor inhibits proliferation of human colon carcinoma cells through the involvement of CaSR in its mechanism. TDH treatment does not induce DNA fragmentation, nor causes the release of lactate dehydrogenase. Therefore, apoptosis and cytotoxicity are not contributing to the TDH-mediated reduction of proliferation rate, and hence the reduction appears to be caused by decrease in cell proliferation. The elevation of E-cadherin, a cell adhesion molecule and suppression of β-catenin, a proto-oncogene have been observed in presence of CaSR agonists whereas reverse effect has been seen in presence of CaSR antagonist as well as si-RNA in TDH treated cells. TDH also triggers a significant reduction of Cyclin-D and cdk2, two important cell cycle regulatory proteins along with an up regulation of cell cycle inhibitory protein p27Kip1 in presence of CaSR agonists. Conclusion: Therefore TDH can downregulate colonic carcinoma cell proliferation and involves CaSR in its mechanism of action. The downregulation occurs mainly through the involvement of E-cadherin-β-catenin mediated pathway and the inhibition of cell cycle regulators as well as upregulation of cell cycle inhibitors. © 2011 Chowdhury et al

TDH induces cytosolic free Ca²⁺ level.

<p>Time dependent study of cytosolic free Ca²⁺ rise in COLO-205 cells after treatment with 10 µg/ml TDH. MFI, mean fluorescence intensities (arbitrary units) are measured by flow cytometry in the control (untreated) cells where 200 nM (▴) and 1 mM [Ca²⁺]_o (○) is present and in TDH treated cells in presence of 200 nM [Ca²⁺]_o (Δ); 1 mM [Ca²⁺]_o (◊); 1 mM GdCl₃ and 200 nM [Ca²⁺]_o (□); 40 nM CaSR si-RNA and 200 nM [Ca²⁺]_o (*); 1 mM EGTA (•). Data are obtained from four independent experiments.</p

Role of TDH on cdk2 expression in presence of CaSR agonist and antagonist in COLO-205 cells by Immunoblotting.

<p>(a) Representative western blot analysis of cdk2expression Data represent the mean ± SEM (<i>n</i> = 3). (b) Densitometric analysis of immunoreactive bands of cdk2 of COLO-205 cell. After western blot, immunoreactive bands are photographed and then images are digitized and analyzed. Immunoreactive bands are quantitated and expressed as the ratio of each band density to the internal control (β-actin) band density. Bar 1- TDH untreated in presence of 200 nM [Ca²⁺]_o. Bar 2- TDH untreated in presence of 1 mM [Ca²⁺]_o. Bar 3-TDH treated in the presence of 200 nM [Ca²⁺]_o. Bar 4- TDH treated in the presence of 1 mM GdCl₃ and 200 µM [Ca²⁺]_o. Bar 5- TDH treated in presence of 1 mM [Ca²⁺]_o. Bar 6- TDH treated in the presence of 40 nM CaSR si-RNA and 200 nM [Ca²⁺]_o.</p

Role of TDH on Cyclin D expression in presence of CaSR agonist and antagonist in COLO-205 cells by immunoblot analysis.

<p>(a) Representative western blot analysis of Cyclin D expression. Data represent the mean ± SEM (<i>n</i> = 3). (b) Densitometric analysis of immunoreactive bands of Cyclin D of COLO-205 cell. After western blot, immunoreactive bands are photographed and then images are digitized and analyzed. Immunoreactive bands are quantitated and expressed as the ratio of each band density to the internal control (β-actin) band density. Bar 1- TDH treated in the presence of 40 nM CaSR si-RNA and 200 nM [Ca²⁺]_o. Bar 2- TDH treated in presence of 200 nM [Ca²⁺]_o. Bar 3- TDH treated in the presence of 1 mM GdCl₃ and 200 nM [Ca²⁺]_o. Bar 4- TDH treated in the presence of 1 mM [Ca²⁺]_o. Bar 5- TDH untreated in the presence of 1 mM [Ca²⁺]_o. Bar 6- TDH untreated in the presence of 200 nM [Ca²⁺]_o.</p

Role of TDH on β-catenin expression in presence of CaSR agonist and antagonist in COLO-205 cells by immunoblot analysis.

<p>(a) Representative western blot analysis of β-catenin expression. Data represent mean ± S.E.M. of three independent experiments, p<0.05. (b) Densitometric analysis of immunoreactive bands of β-catenin of COLO-205 cell. After western blot, immunoreactive bands are photographed and then images are digitized and analyzed. Immunoreactive bands are quantitated and expressed as the ratio of each band density to the internal control (β-actin) band density. Bar 1- TDH treated in presence of 200 nM [Ca²⁺]_o. Bar 2- TDH treated in the presence of 1 mM [Ca²⁺]_o. Bar 3- TDH treated in the presence of 1 mM GdCl₃ and 200 nM [Ca²⁺]_o. Bar 4- TDH untreated in the presence of 1 mM [Ca²⁺]_o. Bar 5- TDH untreated in the presence of 200 nM [Ca²⁺]_o. Bar 6- TDH treated in the presence of 40 nM CaSR si-RNA and 200 nM [Ca²⁺]_o.</p

SDS-PAGE and western blot analysis of Thermostable Direct Hemolysin (TDH).

<p>(a) Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showing the purification of the TDH of <i>Vibrio parahaemolyticus</i>. Lane1- Protein molecular weight marker (Fermentus). Lane2- Purified TDH (23 kDa). (b) Western blot analysis of purified 23 kDa TDH with anti-TDH antibody, produced by immunization of mice with purified TDH of <i>Vibrio parahaemolyticus</i>.</p

Expression of CaSR mRNA and presence of CaSR protein in COLO- 205 cells.

<p>(a) RT-PCR product of amplified calcium sensing receptor (CaSR) cDNA. Lane1- Amplified product of CaSR cDNA (3253 bp) in si-RNA transfected COLO-205 cells. Lane2- DNA molecular weight marker (Bangalore Genei). Lane3- Amplified product of CaSR cDNA (3253 bp) in non-transfected COLO-205 cells. (b) Immunocytochemical study showing the predominance of CaSR protein at the plasma membrane in [i] si-RNA transfected COLO-205 cells and [ii] non-transfected COLO-205 cells. The data shown are representative of three independent experiments. (c) Immunoblot analysis of the expression of CaSR in COLO-205. COLO-205 cell lysates were separated by SDS-PAGE, transferred to the membrane and finally probed with monoclonal antibody to CaSR. Lane 1, cells without CaSR si-RNA; Lane 2, cells in presence of scramble si-RNA (as negative control); and Lane 3, cells with CaSR si-RNA. β-Actin was used as loading control. The data shown are representative of three independent experiments.</p

Role of TDH on E-cadherin expression in presence of CaSR agonist and antagonist in COLO-205 cells by Western Blot analysis.

<p>(a) Representative western blot analysis of E-cadherin expression. Data represent mean ± S.E.M. of three independent experiments, p<0.05. (b) Densitometric analysis of immunoreactive bands of E-cadherin of COLO-205 cell. After western blot, immunoreactive bands are photographed and then images are digitized and analyzed. Immunoreactive bands are quantitated and expressed as the ratio of each band density to the internal control (β-actin) band density. Bar 1- TDH untreated in presence of 200 nM [Ca²⁺]_o and 40 nM CaSR siRNA. Bar 2- TDH treated in presence of 200 nM [Ca²⁺]_o and 40 nM CaSR siRNA. Bar 3- TDH untreated in presence of 200 nM [Ca²⁺]_o. Bar 4- TDH untreated in the presence of 1 mM [Ca²⁺]_o. Bar 5- TDH treated in the presence of 1 mM GdCl₃ and 200 nM [Ca²⁺]_o. Bar 6- TDH treated in presence of 1 mM [Ca²⁺]_o. Bar 7- TDH treated in the presence of 200 nM [Ca²⁺]_o.</p