Resveratrol Regulates Colorectal Cancer Cell Invasion by Modulation of Focal Adhesion Molecules

Resveratrol, a safe and multi-targeted agent, has been associated with suppression of survival, proliferation and metastasis of cancer, however, the underlying mechanisms for its anti-cancer activity, particularly on cellular signaling during cancer cell migration still remain poorly understood. We investigated the invasion response of two human colorectal cancer (CRC) cells (HCT116 and SW480) to resveratrol and studied the effect of specific pharmacological inhibitors, cytochalasin D (CytD) and focal adhesion kinase-inhibitor (FAK-I) on FAK, cell viability and migration in CRC. We found that resveratrol altered cell phenotype of both CRC cells, reduced cell viability and the results were comparable to FAK-I and CytD. These effects of resveratrol were associated with marked Sirt1 up-regulation, FAK down-regulation, inhibition of focal adhesion and potentiation of effects by combinatorial treatment of resveratrol and inhibitors. Interestingly, inhibition of FAK with FAK-I or treatment with CytD suppressed resveratrol-induced Sirt1 up-regulation and markedly down-regulated FAK expression. Resveratrol or combination treatment with inhibitors significantly activated caspase-3 and potentiated apoptosis. Moreover, resveratrol suppressed invasion and colony forming capacity, cell proliferation, β1-Integrin expression and activation of FAK of cells in alginate tumor microenvironment, similar to FAK-I or CytD. Finally, we demonstrated that resveratrol, FAK-I or CytD inhibited activation of NF-κB, suppressed NF-κB-dependent gene end-products involved in invasion, metastasis, and apoptosis; and these effects of resveratrol were potentiated by combination treatment with FAK-I or CytD. Our data illustrated that the anti-invasion effect of resveratrol by inhibition of FAK activity has a potential beneficial role in disease prevention and therapeutic management of CRC.


Introduction
As the third most prevalent cancer in the world, colorectal cancer affects more than half a million people per annum [1] constituting about 10% of all estimated cancer deaths [2]. Although a long-term decline in colorectal cancer death has been observed since the early 1980s, mainly related to enhanced screening and improved treatment methods, colorectal cancer remains a clinical challenge as~50% of patients develop chemoresistance to current chemotherapeutic drugs, as well as recurrence and with methanol, rinsed with PBS and incubated with bovine serum mary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at r, washed three times with PBS/BSA followed by incubation with y antibodies for 1.5 h and finally washed again three times with aqua performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to ere covered with fluoromount and examined under a fluorescent t, Germany). In FAK immunolabeled cultures the number of focal d by scoring 800-1000 cells from 10 different microscopic fields. One of ts is shown. Values were compared to the control, and statistically are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).
Cells ic changes induced by two different FAK inhibitors (PF-562271 and 480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear . Cells were seeded on glass plates at 80-90% confluency and treated of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, completion of treatment, the cells were fixed with methanol for 10 min, cubated with DAPI solution for 10 min in the dark. Labeled cells were to remove the excess DAPI stain and evaluated under fluorescence t, Germany). Quantification of apoptotic cells was performed by 0 different microscopic fields. Result from one of three independent res. Values were compared to the control, and statistically significant ated by an asterisk ( ); p < 0.01 by two asterisks ( ).
tor, CytD, resveratrol and their combination on the proliferation and and SW480 CRC cells was determined by the 5-diphenyltetrazolium bromide (MTT) uptake method as described ells (5000 per well in triplicate in a 96-well plate) were treated with , different concentrations of CytD (0.1, 1, 2, and 4 μg/mL), different 62271: 0.01, 0.1, 1, and 10 μM) each or co-treated with 5 μM resveratrol ns of either CytD or FAK-I for 12 h. Subsequently, 100 μL of modified ithout phenol red, without ascorbic acid and only 3% FBS) and 10 μL s added to each well and the plate was incubated for 4 h at 37 °C. lubilization solution (10% Triton X-100/acidic isopropanol) was added ted overnight at 37 °C. Metabolically active cells were evaluated by y at 550 nm (OD550) using a 96-well multi-scanner plate ELISA reader unich, Germany). Data were derived from at least three independent alysis was done to obtain the final values. cell cultivation in alginate culture has been previously described by HCT116 and SW480 CRC cells (1 × 10 6 /mL) were resuspended in l) and added dropwise into a 100 mM CaCl2 solution at ambient alginate beads polymerized after 10 min forming a round and stable ate beads were washed three times with 0.15 M NaCl solution and ium (3% FBS) before starting treatment. apacity, HCT116 and SW480 CRC cells were cultured in alginate beads either left untreated, or treated with 5 μM resveratrol alone, different ); p < 0.01 by two asterisks ( utrients 2017, 9, 1073 4 of 21 nvestigation, cells were fixed with methanol, rinsed with PBS and incubated with bovine serum lbumin (BSA) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at °C in a humidfied chamber, washed three times with PBS/BSA followed by incubation with hodamine-coupled secondary antibodies for 1.5 h and finally washed again three times with aqua est. Counter staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to isualize cell nuclei. Slides were covered with fluoromount and examined under a fluorescent icroscope (Leica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal dhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of hree independent experiments is shown. Values were compared to the control, and statistically ignificant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

. DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and F-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear taining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated ith different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 0, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, ashed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were ashed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence icroscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by coring 800-1000 cells from 10 different microscopic fields. Result from one of three independent xperiments is shown in figures. Values were compared to the control, and statistically significant alues with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

. Cell Proliferation Assay
The effect of FAK-inhibitor, CytD, resveratrol and their combination on the proliferation and iability of HCT116 and SW480 CRC cells was determined by the -(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method as described reviously [36]. Briefly, the cells (5000 per well in triplicate in a 96-well plate) were treated with ither 5 μM resveratrol alone, different concentrations of CytD (0.1, 1, 2, and 4 μg/mL), different oncentrations of FAK-I (PF-562271: 0.01, 0.1, 1, and 10 μM) each or co-treated with 5 μM resveratrol nd the indicated concentrations of either CytD or FAK-I for 12 h. Subsequently, 100 μL of modified ell culture medium (DMEM without phenol red, without ascorbic acid and only 3% FBS) and 10 μL TT solution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 °C. inally, 100 μL of the MTT solubilization solution (10% Triton X-100/acidic isopropanol) was added er well, and the cells incubated overnight at 37 °C. Metabolically active cells were evaluated by easuring the Optical Density at 550 nm (OD550) using a 96-well multi-scanner plate ELISA reader Bio-Rad Laboratories Inc. Munich, Germany). Data were derived from at least three independent xperiments, and statistical analysis was done to obtain the final values.

.7. Alginate Culture
A detailed description of cell cultivation in alginate culture has been previously described by ur group [26,30,37]. Briefly, HCT116 and SW480 CRC cells (1 × 10 6 /mL) were resuspended in lginate (2% in 0.15 M NaCl) and added dropwise into a 100 mM CaCl2 solution at ambient emperature (AT) where the alginate beads polymerized after 10 min forming a round and stable ead. Subsequently, the alginate beads were washed three times with 0.15 M NaCl solution and wice with serum-starved medium (3% FBS) before starting treatment.

. Invasion (Migration) Assay
To investigate invasion capacity, HCT116 and SW480 CRC cells were cultured in alginate beads s described above. Cells were either left untreated, or treated with 5 μM resveratrol alone, different Nutrients 2017, 9,1073 4 of 21 investigation, cells were fixed with methanol, rinsed with PBS and incubated with bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at 4 °C in a humidfied chamber, washed three times with PBS/BSA followed by incubation with rhodamine-coupled secondary antibodies for 1.5 h and finally washed again three times with aqua dest. Counter staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to visualize cell nuclei. Slides were covered with fluoromount and examined under a fluorescent microscope (Leica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal adhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of three independent experiments is shown. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by scoring 800-1000 cells from 10 different microscopic fields. Result from one of three independent experiments is shown in figures. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

Cell Proliferation Assay
The effect of FAK-inhibitor, CytD, resveratrol and their combination on the proliferation and viability of HCT116 and SW480 CRC cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method as described previously [36]. Briefly, the cells (5000 per well in triplicate in a 96-well plate) were treated with either 5 μM resveratrol alone, different concentrations of CytD (0.1, 1, 2, and 4 μg/mL), different concentrations of FAK-I (PF-562271: 0.01, 0.1, 1, and 10 μM) each or co-treated with 5 μM resveratrol and the indicated concentrations of either CytD or FAK-I for 12 h. Subsequently, 100 μL of modified cell culture medium (DMEM without phenol red, without ascorbic acid and only 3% FBS) and 10 μL MTT solution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 °C. Finally, 100 μL of the MTT solubilization solution (10% Triton X-100/acidic isopropanol) was added per well, and the cells incubated overnight at 37 °C. Metabolically active cells were evaluated by measuring the Optical Density at 550 nm (OD550) using a 96-well multi-scanner plate ELISA reader (Bio-Rad Laboratories Inc. Munich, Germany). Data were derived from at least three independent experiments, and statistical analysis was done to obtain the final values.

Alginate Culture
A detailed description of cell cultivation in alginate culture has been previously described by our group [26,30,37]. Briefly, HCT116 and SW480 CRC cells (1 × 10 6 /mL) were resuspended in alginate (2% in 0.15 M NaCl) and added dropwise into a 100 mM CaCl2 solution at ambient temperature (AT) where the alginate beads polymerized after 10 min forming a round and stable bead. Subsequently, the alginate beads were washed three times with 0.15 M NaCl solution and twice with serum-starved medium (3% FBS) before starting treatment.

Invasion (Migration) Assay
To investigate invasion capacity, HCT116 and SW480 CRC cells were cultured in alginate beads as described above. Cells were either left untreated, or treated with 5 μM resveratrol alone, different ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 µM; and PF-573228: 0.1, 1, 10, 20, and 50 µM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by scoring 800-1000 cells from 10 different microscopic fields. Result from one of three independent experiments is shown in figures. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( s 2017, 9,1073 4 of 21 gation, cells were fixed with methanol, rinsed with PBS and incubated with bovine serum in (BSA) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at n a humidfied chamber, washed three times with PBS/BSA followed by incubation with ine-coupled secondary antibodies for 1.5 h and finally washed again three times with aqua Counter staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to ze cell nuclei. Slides were covered with fluoromount and examined under a fluorescent cope (Leica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal on points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of ndependent experiments is shown. Values were compared to the control, and statistically cant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

PI Staining of Apoptotic Cells
examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and 228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear g assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated ifferent concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, d twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were d repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence cope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by 800-1000 cells from 10 different microscopic fields. Result from one of three independent ments is shown in figures. Values were compared to the control, and statistically significant with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).
ll Proliferation Assay e effect of FAK-inhibitor, CytD, resveratrol and their combination on the proliferation and ty of HCT116 and SW480 CRC cells was determined by the imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method as described usly [36]. Briefly, the cells (5000 per well in triplicate in a 96-well plate) were treated with 5 μM resveratrol alone, different concentrations of CytD (0.1, 1, 2, and 4 μg/mL), different trations of FAK-I (PF-562271: 0.01, 0.1, 1, and 10 μM) each or co-treated with 5 μM resveratrol e indicated concentrations of either CytD or FAK-I for 12 h. Subsequently, 100 μL of modified lture medium (DMEM without phenol red, without ascorbic acid and only 3% FBS) and 10 μL olution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 °C. , 100 μL of the MTT solubilization solution (10% Triton X-100/acidic isopropanol) was added ll, and the cells incubated overnight at 37 °C. Metabolically active cells were evaluated by ring the Optical Density at 550 nm (OD550) using a 96-well multi-scanner plate ELISA reader ad Laboratories Inc. Munich, Germany). Data were derived from at least three independent ments, and statistical analysis was done to obtain the final values.

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by scoring 800-1000 cells from 10 different microscopic fields. Result from one of three independent experiments is shown in figures. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

Cell Proliferation Assay
The effect of FAK-inhibitor, CytD, resveratrol and their combination on the proliferation and viability of HCT116 and SW480 CRC cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method as described previously [36]. Briefly, the cells (5000 per well in triplicate in a 96-well plate) were treated with either 5 μM resveratrol alone, different concentrations of CytD (0.1, 1, 2, and 4 μg/mL), different concentrations of FAK-I (PF-562271: 0.01, 0.1, 1, and 10 μM) each or co-treated with 5 μM resveratrol and the indicated concentrations of either CytD or FAK-I for 12 h. Subsequently, 100 μL of modified cell culture medium (DMEM without phenol red, without ascorbic acid and only 3% FBS) and 10 μL MTT solution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 °C. Finally, 100 μL of the MTT solubilization solution (10% Triton X-100/acidic isopropanol) was added per well, and the cells incubated overnight at 37 °C. Metabolically active cells were evaluated by measuring the Optical Density at 550 nm (OD550) using a 96-well multi-scanner plate ELISA reader (Bio-Rad Laboratories Inc. Munich, Germany). Data were derived from at least three independent experiments, and statistical analysis was done to obtain the final values. investigation, cells were fixed with methanol, rinsed with PBS and incubated with bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at 4 °C in a humidfied chamber, washed three times with PBS/BSA followed by incubation with rhodamine-coupled secondary antibodies for 1.5 h and finally washed again three times with aqua dest. Counter staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to visualize cell nuclei. Slides were covered with fluoromount and examined under a fluorescent microscope (Leica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal adhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of three independent experiments is shown. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by scoring 800-1000 cells from 10 different microscopic fields. Result from one of three independent experiments is shown in figures. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

Cell Proliferation Assay
The effect of FAK-inhibitor, CytD, resveratrol and their combination on the proliferation and viability of HCT116 and SW480 CRC cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method as described previously [36]. Briefly, the cells (5000 per well in triplicate in a 96-well plate) were treated with either 5 μM resveratrol alone, different concentrations of CytD (0.1, 1, 2, and 4 μg/mL), different concentrations of FAK-I (PF-562271: 0.01, 0.1, 1, and 10 μM) each or co-treated with 5 μM resveratrol and the indicated concentrations of either CytD or FAK-I for 12 h. Subsequently, 100 μL of modified cell culture medium (DMEM without phenol red, without ascorbic acid and only 3% FBS) and 10 μL MTT solution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 °C. Finally, 100 μL of the MTT solubilization solution (10% Triton X-100/acidic isopropanol) was added per well, and the cells incubated overnight at 37 °C. Metabolically active cells were evaluated by measuring the Optical Density at 550 nm (OD550) using a 96-well multi-scanner plate ELISA reader (Bio-Rad Laboratories Inc. Munich, Germany). Data were derived from at least three independent experiments, and statistical analysis was done to obtain the final values.

Cell Proliferation Assay
The effect of FAK-inhibitor, CytD, resveratrol and their combination on the proliferation and viability of HCT116 and SW480 CRC cells was determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) uptake method as described previously [36]. Briefly, the cells (5000 per well in triplicate in a 96-well plate) were treated with either 5 µM resveratrol alone, different concentrations of CytD (0.1, 1, 2, and 4 µg/mL), different concentrations of FAK-I (PF-562271: 0.01, 0.1, 1, and 10 µM) each or co-treated with 5 µM resveratrol and the indicated concentrations of either CytD or FAK-I for 12 h. Subsequently, 100 µL of modified cell culture medium (DMEM without phenol red, without ascorbic acid and only 3% FBS) and 10 µL MTT solution (5 mg/mL) was added to each well and the plate was incubated for 4 h at 37 • C. Finally, 100 µL of the MTT solubilization solution (10% Triton X-100/acidic isopropanol) was added per well, and the cells incubated overnight at 37 • C. Metabolically active cells were evaluated by measuring the Optical Density at 550 nm (OD 550 ) using a 96-well multi-scanner plate ELISA reader (Bio-Rad Laboratories Inc. Munich, Germany). Data were derived from at least three independent experiments, and statistical analysis was done to obtain the final values.

Alginate Culture
A detailed description of cell cultivation in alginate culture has been previously described by our group [26,30,37]. Briefly, HCT116 and SW480 CRC cells (1 × 10 6 /mL) were resuspended in alginate (2% in 0.15 M NaCl) and added dropwise into a 100 mM CaCl 2 solution at ambient temperature (AT) where the alginate beads polymerized after 10 min forming a round and stable bead. Subsequently, the alginate beads were washed three times with 0.15 M NaCl solution and twice with serum-starved medium (3% FBS) before starting treatment.

Invasion (Migration) Assay
To investigate invasion capacity, HCT116 and SW480 CRC cells were cultured in alginate beads as described above. Cells were either left untreated, or treated with 5 µM resveratrol alone, different concentrations of CytD (0.1, 1, and 2/mL), FAK-I (PF-562271: 0.01, 0.1, 1, and 10 µM) individually or co-treated with 5 µM resveratrol and the indicated concentrations of either CytD or FAK-I for 28 days. Invasive cells that migrated through the alginate beads and formed adhered colonies on the bottom of the petri dish were fixed with Karnowsky fixative, stained with toluidine blue for 5 min and carefully washed two times with PBS. The number of migrated and positively stained adhered colonies were quantified and evaluated manually by counting all colonies under a light microscope (Axioplan, Zeiss, Germany) and visualized. This assay was repeated every 3 to 4 days until Day 28 of culture. The mean number of colonies in triplicate was calculated, which is reported in each graph. Each experiment was repeated at least three times.

Western Blot Analysis
In another set of experiments, HCT116 and SW480 CRC cells were cultured in alginate beads as described above and were either left untreated, or treated with 5 µM resveratrol alone, 0.1 µg/mL CytD, 10 µM FAK-I (PF-562271) or co-treated with 5 µM resveratrol and either the indicated concentration of CytD or FAK-I for 10 days. Proteins were extracted from the alginate cultures with lysis buffer (50 mM Tris-HCl, pH 7.2, 150 mM NaCl, 1% (v/v) Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% (v/v) aprotinin, 4 µg/mL of pepstatin A, 10 µg/mL of leupeptin, 1 mM phenylmethylsulfonyl fluoride, PMSF) on ice for 30 min, as previously described [38] and total protein concentration was measured with the bicinchonic acid assay system (Uptima, Monlucon, France) using bovine serum albumin as a standard. Subsequently, samples were reduced with 2-mercaptoethanol and equal quantities of protein (500 ng/lane), separated under reducing conditions by SDS-PAGE and transferred onto nitrocellulose membranes using a transblot apparatus (Bio-Rad). After pre-incubation in blocking buffer (5% skim milk powder in PBS, 0.1% Tween 20) for 1 h, membranes were incubated with primary antibodies at 4 • C overnight, washed three times with blocking buffer, and then further incubated with alkaline phosphatase-conjugated secondary antibodies for 2 h at AT. After further washing in 0.1 M Tris, pH 9.5, containing 0.05 M MgCl 2 and 0.1 M NaCl, specific antigen-antibody complexes were detected using nitro blue tetrazolium and 5-bromo-4-chloro-3-indoylphosphate (p-toluidine salt; Pierce). Specific β-actin antibody was used for the internal control to normalize the sample amounts.

Statistical Analysis
All experiments were performed three times as individual experiments with three replicates. For statistical analysis, a Wilcoxon-Mann-Whitney test was applied. Score values for image quality and presence of artifacts were compared for each sequence.
Data were shown as mean values ± SD or SEM, and were compared by one-way or two-way analysis of variance (ANOVA), if the normality test passed (Kolmogorov-Smirnov test). Otherwise, the Kruskal-Wallis ANOVA on ranks was used. A p value of < 0.05 was considered to establish statistically significant differences.

Results
The purpose of this study was to investigate the effect of resveratrol on regulating colorectal cancer cell (CRC) invasion and metastasis through regulation of focal adhesion molecules. We used two well-characterized colorectal cancer cell lines (HCT116 and SW480) in monolayer, as well as 3D alginate tumor microenvironment culture to address this question. The concentration of ethanol or DMSO applied in our study had no suppressive effect on CRC cell viability.

Resveratrol Alters Phenotype of CRC Cells Similar to FAK-Inhibitor or Cytochalasin D
Since cytoskeletal remodeling is critical for cancer cell invasion and migration [8], we first evaluated whether resveratrol alters colorectal cancer cell morphology cytoskeleton organization. HCT116 and SW480 cells in monolayer cultures were left untreated, treated with 5 µM resveratrol, with 10 µM FAK-I (PF-562271), with 0.1 µg/mL CytD alone, or a combination of 5 µM resveratrol and either 10 µM FAK-I or 0.1 µg/mL CytD for 12 h. As shown in Figure 1, in HCT116 untreated control, cultures evenly distributed cell colonies (arrows) with overall epithelial cell morphology and close cell-to-cell contacts were observed ( Figure 1A). In contrast to HCT116, untreated SW480 cells showed a more mesenchymal morphology, less colony formation and fewer tight cell-to-cell contacts. The SW480 cells demonstrated a flattened shape and numerous microvilli-like cytoplasmic processes ( Figure 1G). However, in both cell lines treatment with resveratrol significantly increased epithelial morphology, enhanced tight cell-to-cell contact and showed that most of the cells developed rounded morphology ( Figure 1B,H). In contrast, in both cell lines treatment with FAK-I or CytD led to rounding of the cells and dissolution of the epithelial cell cluster with clear residues of adhesion molecules visible. Furthermore, the remnants of adhesive contacts and many detached cells were observed ( Figure 1C,E,I,K). Combined treatment with resveratrol markedly increased the observed effects of FAK-I or CytD mono-treatment in HCT116 and SW480 ( Figure 1D,F,J,L). Specially, it was observed that cells maintained attachment better than without resveratrol treatment and more cell clusters were formed.  Figure 1, in HCT116 untreated control, cultures evenly distributed cell colonies (arrows) with overall epithelial cell morphology and close cell-to-cell contacts were observed ( Figure 1A). In contrast to HCT116, untreated SW480 cells showed a more mesenchymal morphology, less colony formation and fewer tight cell-to-cell contacts. The SW480 cells demonstrated a flattened shape and numerous microvilli-like cytoplasmic processes ( Figure 1G). However, in both cell lines treatment with resveratrol significantly increased epithelial morphology, enhanced tight cell-to-cell contact and showed that most of the cells developed rounded morphology ( Figure 1B,H). In contrast, in both cell lines treatment with FAK-I or CytD led to rounding of the cells and dissolution of the epithelial cell cluster with clear residues of adhesion molecules visible. Furthermore, the remnants of adhesive contacts and many detached cells were observed ( Figure 1C,E,I,K). Combined treatment with resveratrol markedly increased the observed effects of FAK-I or CytD mono-treatment in HCT116 and SW480 ( Figure 1D,F,J,L). Specially, it was observed that cells maintained attachment better than without resveratrol treatment and more cell clusters were formed.

Resveratrol Suppresses Cancer Cell Viability Similar to FAK-Inhibitor or Cytochalasin D
We next investigated the effects of FAK-I or CytD and/or resveratrol on cell viability by performing a MTT assay in HCT116 and SW480 cells ( Figure 2). Serum-starved HCT116 and SW480 in monolayer culture were either left untreated, treated with 5 μM resveratrol or different

Resveratrol-Induced Sirt1 Expression Is Mediated by Focal Adhesion Kinase and Cytoskeletal Proteins in CRC Cells Monolayer Culture as Revealed by Immunofluorescence Microscopy
The histone deacetylase Sirt1 is a major subcellular target of resveratrol [39,40] and is a major target for resveratrol-mediated suppression of colorectal cancer cell growth and metastasis [30,41]. Therefore, we investigated the role of FAK or cytoskeletal organization on the resveratrol-induced up-regulation of Sirt1 expression (arrows) and of the possible associated mechanism in colorectal cancer cells. Serum-starved HCT116 and SW480 cultures were left untreated, treated with 5 μM investigation, cells were fixed with methanol, rinsed with PBS and incubated with bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at 4 °C in a humidfied chamber, washed three times with PBS/BSA followed by incubation with rhodamine-coupled secondary antibodies for 1.5 h and finally washed again three times with aqua dest. Counter staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to visualize cell nuclei. Slides were covered with fluoromount and examined under a fluorescent microscope (Leica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal adhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of three independent experiments is shown. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence ) and p < 0.01 are designated by two asterisks ( 1073 4 of 21 cells were fixed with methanol, rinsed with PBS and incubated with bovine serum ) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at idfied chamber, washed three times with PBS/BSA followed by incubation with upled secondary antibodies for 1.5 h and finally washed again three times with aqua staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to nuclei. Slides were covered with fluoromount and examined under a fluorescent eica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal ts was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of dent experiments is shown. Values were compared to the control, and statistically ues with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks (

Resveratrol-Induced Sirt1 Expression Is Mediated by Focal Adhesion Kinase and Cytoskeletal Proteins in CRC Cells Monolayer Culture as Revealed by Immunofluorescence Microscopy
The histone deacetylase Sirt1 is a major subcellular target of resveratrol [39,40] and is a major target for resveratrol-mediated suppression of colorectal cancer cell growth and metastasis [30,41]. Therefore, we investigated the role of FAK or cytoskeletal organization on the resveratrol-induced up-regulation of Sirt1 expression (arrows) and of the possible associated mechanism in colorectal cancer cells. Serum-starved HCT116 and SW480 cultures were left untreated, treated with 5 µM resveratrol or treated with resveratrol (5 µM), and co-treated with 10 µM FAK-I (PF-562271) or with 0.1 µg/mL CytD for 12 h and subjected to immunofluorescence labeling with primary antibodies for Sirt1 and rhodamine-coupled secondary antibodies. Counterstaining was performed with DAPI to visualize the cell nuclei. As shown in Figure 3B, resveratrol alone significantly upregulated the expression of Sirt1 protein and nuclear localization compared to the controls ( Figure 3A). However, the resveratrol-induced expression of Sirt1 protein in the nuclei of HCT116 was significantly decreased by incubation with FAK-I and CytD ( Figure 3C,D), indicating that FAK and cytoskeletal signaling proteins, at least in part, are some of the main target proteins of resveratrol during the resveratrol-induced anti-tumor effect in CRC cells. resveratrol or treated with resveratrol (5 μM), and co-treated with 10 μM FAK-I (PF-562271) or with 0.1 μg/mL CytD for 12 h and subjected to immunofluorescence labeling with primary antibodies for Sirt1 and rhodamine-coupled secondary antibodies. Counterstaining was performed with DAPI to visualize the cell nuclei. As shown in Figure 3B, resveratrol alone significantly upregulated the expression of Sirt1 protein and nuclear localization compared to the controls ( Figure 3A). However, the resveratrol-induced expression of Sirt1 protein in the nuclei of HCT116 was significantly decreased by incubation with FAK-I and CytD ( Figure 3C,D), indicating that FAK and cytoskeletal signaling proteins, at least in part, are some of the main target proteins of resveratrol during the resveratrol-induced anti-tumor effect in CRC cells.

Resveratrol-Induced Formation of Focal Adhesions in CRC Cells Are Inhibited by FAK-Inhibitor or Cytochalasin D
FAK has been shown to be involved in cancer cell survival and invasion [18] and on the other hand resveratrol has been shown to suppress cancer cell migration/invasion [26] and inhibition of phosphorylation of FAK in pancreatic cancer [33][34][35]. To examine the effects of resveratrol on the FAK expression in colorectal cancer cells, serum-starved HCT116 cultures were treated as described above and subjected to immunolabeling with anti-FAK. Strong staining for FAK was observed in untreated controls in HCT116 (Figure 4). In control cell cultures, a distribution of FAK positive labeled focal adhesion points (arrows) was observed on the cytoplasmic membranes ( Figure 4I(A), arrows and inset). Contrary to this, resveratrol reduced FAK labeling to a basal level, but interestingly, induced formation of focal adhesion clusters, which were strongly positively stained for FAK ( Figure 4I(B), arrows and inset). The combination treatment with resveratrol and with FAK has been shown to be involved in cancer cell survival and invasion [18] and on the other hand resveratrol has been shown to suppress cancer cell migration/invasion [26] and inhibition of phosphorylation of FAK in pancreatic cancer [33][34][35]. To examine the effects of resveratrol on the FAK expression in colorectal cancer cells, serum-starved HCT116 cultures were treated as described above and subjected to immunolabeling with anti-FAK. Strong staining for FAK was observed in untreated controls in HCT116 (Figure 4). In control cell cultures, a distribution of FAK positive labeled focal adhesion points (arrows) was observed on the cytoplasmic membranes ( Figure 4I(A), arrows and inset). Contrary to this, resveratrol reduced FAK labeling to a basal level, but interestingly, induced formation of focal adhesion clusters, which were strongly positively stained for FAK ( Figure 4I(B), arrows and inset). The combination treatment with resveratrol and with FAK-I or CytD markedly reduced FAK staining in both HCT116 ( Figure 4I(C)). Quantification of focal adhesion points confirmed that resveratrol, FAK-I, CytD or combination treatment markedly decreased the number of focal adhesion clusters compared to untreated control cultures in HCT116 ( Figure 4II).

Specific Inhibitors against FAK Lead to Increased Apoptosis in CRC Cells
To investigate the effects of two different FAK inhibitors (PF-562271 and PF-573228) on the growth rate inhibition and the levels of apoptosis on colorectal cancer cells, HCT116 and SW480 were treated with different concentrations of FAK-I (0.1, 1, 10, 20 and 50 μM) for 12 h and a nuclear staining assay with DAPI (4, 6-Diamidino-2-phenylindole, Sigma) was performed. This fluorescence-based staining method reveals apoptotic bodies containing nuclear fragmentation and chromatin condensation in apoptotic cells. The number of apoptotic nuclei was quantified by scoring 800-1000 cells from 10 different microscopic fields. As shown in Figure 5, the number of apoptotic In FAK immunolabeled cultures the number of focal adhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of three independent experiments is shown. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by In FAK immunolabeled cultures the number of focal adhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of three independent experiments is shown. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by ). Magnification 400×; bar = 30 nm.

Specific Inhibitors against FAK Lead to Increased Apoptosis in CRC Cells
To investigate the effects of two different FAK inhibitors (PF-562271 and PF-573228) on the growth rate inhibition and the levels of apoptosis on colorectal cancer cells, HCT116 and SW480 were treated with different concentrations of FAK-I (0.1, 1, 10, 20 and 50 µM) for 12 h and a nuclear staining assay with DAPI (4, 6-Diamidino-2-phenylindole, Sigma) was performed. This fluorescence-based staining method reveals apoptotic bodies containing nuclear fragmentation and chromatin condensation in apoptotic cells. The number of apoptotic nuclei was quantified by scoring 800-1000 cells from 10 different microscopic fields. As shown in Figure 5, the number of apoptotic nuclei in control cultures of both HCT116 and SW480 ranged from 8-15%. The number of apoptotic nuclei markedly increased in both cell lines by FAK inhibitor treatment. A significant dose-dependent effect was observed in HCT116 treated with 0.1, 1, 10, 20 and 50 µM PF-562271 with an apoptotic increase of 23%, 39%, 46%, 82% and 98%, as well as with 0.1, 1, 10, 20 and 50 µM PF-573228 with an apoptotic increase of 30%, 38%, 41%, 79% and 96%, respectively ( Figure 5A). In SW480 cells, a low concentration of 0.1 µM PF-562271 or PF-573228 resulted in 15% and 13% apoptotic cells, respectively, similar to untreated controls. Contrary to this, a significant dose-dependent increase in apoptosis was observed in SW480 cells treated with 1, 10, 20 and 50 µM PF-562271 with an apoptotic increase of 40%, 69%, 97% and 100%, as well as with 1, 10, 20 and 50 µM PF-573228 with an apoptotic increase of 38%, 65%, 95% and 100%, respectively ( Figure 5B). These results demonstrate that FAK-I shows similar levels of apoptotic induction in both cell lines. SW480 cells, a low concentration of 0.1 μM PF-562271 or PF-573228 resulted in 15% and 13% apoptotic cells, respectively, similar to untreated controls. Contrary to this, a significant dose-dependent increase in apoptosis was observed in SW480 cells treated with 1, 10, 20 and 50 μM PF-562271 with an apoptotic increase of 40%, 69%, 97% and 100%, as well as with 1, 10, 20 and 50 μM PF-573228 with an apoptotic increase of 38%, 65%, 95% and 100%, respectively ( Figure 5B). These results demonstrate that FAK-I shows similar levels of apoptotic induction in both cell lines.

Resveratrol Potentiates FAK-Inhibitor-and Cytochalasin D-Induced Activation of Caspase-3 in CRC Cells Monolayer Culture as Revealed by Immunofluorescence Microscopy
To examine the mechanisms by which resveratrol and/or FAK-I or CytD inhibit cell viability and induce apoptosis of CRC cells, we analyzed and compared their effect on the rate of the activation levels of caspase-3, which induces the apoptotic pathway in cells [42,43]. Therefore, the amount of apoptosis induced by resveratrol, FAK-I, CytD and their combined treatment in HCT116 ( Figure 6I) was confirmed by investigating the activation of caspase-3 with immunofluorescent labeling and induction of morphological nuclear changes with DAPI nuclear staining (arrows) ( Figure 6II). In untreated control cultures of HCT116 ( Figure 6I(A)) only small amounts of caspase-3 positive stained cells (arrows) could be detected. In contrast to this, treatment with resveratrol,

Resveratrol Potentiates FAK-Inhibitor-and Cytochalasin D-Induced Activation of Caspase-3 in CRC Cells Monolayer Culture as Revealed by Immunofluorescence Microscopy
To examine the mechanisms by which resveratrol and/or FAK-I or CytD inhibit cell viability and induce apoptosis of CRC cells, we analyzed and compared their effect on the rate of the activation levels of caspase-3, which induces the apoptotic pathway in cells [42,43]. Therefore, the amount of apoptosis induced by resveratrol, FAK-I, CytD and their combined treatment in HCT116 ( Figure 6I) was confirmed by investigating the activation of caspase-3 with immunofluorescent labeling and induction of morphological nuclear changes with DAPI nuclear staining (arrows) ( Figure 6II). In untreated control cultures of HCT116 ( Figure 6I(A)) only small amounts of caspase-3 positive stained cells (arrows) could be detected. In contrast to this, treatment with resveratrol, markedly induced caspase-3 expression in colorectal cancer cells ( Figure 6I(B)). Interestingly, a significant increase of caspase-3 was observed in the combined treatment of resveratrol with either FAK-I or CytD ( Figure 6I(C,D)).
Nutrients 2017, 9, 1073 11 of 21 Nuclear staining revealed that untreated control HCT116 ( Figure 6II) cell cultures displayed an overall normal nuclear size with around 12% of apoptotic nuclei in the cell culture. In contrast, in cells treated with resveratrol, a significant increase of 59% apoptotic cells in HCT116 with nuclear morphological changes such as pyknosis and chromatin condensation was observed. Significant dose-dependent increases in apoptosis was observed in HCT116 treated with 10 μM FAK-I (PF-562271), with an apoptotic rate of 70%, and with 0.1 μg/mL CytD, with an apoptotic rate of 56% ( Figure 6II). Furthermore, a significant increase in fragmented nuclei and apoptotic features was observed by combined treatment of resveratrol and either 10 μM FAK-I (51%) or 0.1 μg/mL CytD (94%) compared to the treatment of only resveratrol, FAK-I or CytD.

Resveratrol Potentiates FAK-Inhibitor-and Cytochalasin D-Inhibited Invasion of CRC Cells in 3D Alginate Tumor Microenvironment Culture
To investigate the effect of resveratrol, FAK-I and CytD on cell motility and invasion capacity through the 3D tumor microenvironment, HCT116 and SW480 cells were cultured in an

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and 573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear ning assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated h different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, shed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were shed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence roscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by ring 800-1000 cells from 10 different microscopic fields. Result from one of three independent ); p < 0.01 by two asterisks ( investigation, cells were fixed with methanol, rinsed with PBS and incubated with bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at 4 °C in a humidfied chamber, washed three times with PBS/BSA followed by incubation with rhodamine-coupled secondary antibodies for 1.5 h and finally washed again three times with aqua dest. Counter staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to visualize cell nuclei. Slides were covered with fluoromount and examined under a fluorescent microscope (Leica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal adhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of three independent experiments is shown. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by scoring 800-1000 cells from 10 different microscopic fields. Result from one of three independent investigation, cells were fixed with methanol, rinsed with PBS and incubated with bovine serum albumin (BSA) for 30 min. Primary antibodies were diluted 1:50 in PBS/BSA, incubated overnight at 4 °C in a humidfied chamber, washed three times with PBS/BSA followed by incubation with rhodamine-coupled secondary antibodies for 1.5 h and finally washed again three times with aqua dest. Counter staining was performed with DAPI (4,6-diamidino-2-phenylindole, Sigma) to visualize cell nuclei. Slides were covered with fluoromount and examined under a fluorescent microscope (Leica, Darmstadt, Germany). In FAK immunolabeled cultures the number of focal adhesion points was quantified by scoring 800-1000 cells from 10 different microscopic fields. One of three independent experiments is shown. Values were compared to the control, and statistically significant values with p < 0.05 are designated by an asterisk ( ); p < 0.01 by two asterisks ( ).

DAPI Staining of Apoptotic Cells
To examine the apoptotic changes induced by two different FAK inhibitors (PF-562271 and PF-573228) in HCT116 and SW480 CRC cells, DAPI (4,6-diamidino-2-phenylindole, Sigma) nuclear staining assay was performed. Cells were seeded on glass plates at 80-90% confluency and treated with different concentrations of FAK-I (PF-562271: 0.1, 1, 10, 20, and 50 μM; and PF-573228: 0.1, 1, 10, 20, and 50 μM) for 12 h. After completion of treatment, the cells were fixed with methanol for 10 min, washed twice with PBS, and incubated with DAPI solution for 10 min in the dark. Labeled cells were washed repeatedly with PBS to remove the excess DAPI stain and evaluated under fluorescence microscope (Leica, Darmstadt, Germany). Quantification of apoptotic cells was performed by scoring 800-1000 cells from 10 different microscopic fields. Result from one of three independent ). Magnification 400×; bar = 30 nm.
Apoptosis induction by caspase-3 activation was confirmed by examining morphological nuclear changes indicative of apoptosis with DAPI nuclear staining ( Figure 6II). The number of apoptotic nuclei was quantified by scoring 800-1000 cells from 10 different microscopic fields. Nuclear staining revealed that untreated control HCT116 ( Figure 6II) cell cultures displayed an overall normal nuclear size with around 12% of apoptotic nuclei in the cell culture. In contrast, in cells treated with resveratrol, a significant increase of 59% apoptotic cells in HCT116 with nuclear morphological changes such as pyknosis and chromatin condensation was observed. Significant dose-dependent increases in apoptosis was observed in HCT116 treated with 10 µM FAK-I (PF-562271), with an apoptotic rate of 70%, and with 0.1 µg/mL CytD, with an apoptotic rate of 56% ( Figure 6II). Furthermore, a significant increase in fragmented nuclei and apoptotic features was observed by combined treatment of resveratrol and either 10 µM FAK-I (51%) or 0.1 µg/mL CytD (94%) compared to the treatment of only resveratrol, FAK-I or CytD.

Resveratrol Potentiates FAK-Inhibitor-and Cytochalasin D-Inhibited Invasion of CRC Cells in 3D Alginate Tumor Microenvironment Culture
To investigate the effect of resveratrol, FAK-I and CytD on cell motility and invasion capacity through the 3D tumor microenvironment, HCT116 and SW480 cells were cultured in an alginate-based tumor microenvironment, treated as described in detail in material and methods and the capacity of migration and invasion was determined through the evaluation of the colony formation with toluidine blue staining after 10 days ( Figure 7).

Resveratrol-Mediated Anti-Tumorigenic Effects Are Potentiated by Inhibition of FAK and Cytoskeletal Proteins
Next, to investigate the underlying mechanism of FAK signaling and cytoskeletal organization on the resveratrol-induced anti-carcinogenic effects in colorectal cancer cells, immunoblotting was performed. HCT116 ( Figure 8) cells were cultured in tumor microenvironment alginate beads as described in materials and methods and either left untreated, or were treated with 5 µM resveratrol alone, 0.1 µg/mL CytD, 10 µM FAK-I (PF-562271) or co-treated with 5 µM resveratrol and either the indicated concentration of CytD or FAK-I for 10 days.

Resveratrol-Mediated Anti-Tumorigenic Effects Are Potentiated by Inhibition of FAK and Cytoskeletal Proteins
Next, to investigate the underlying mechanism of FAK signaling and cytoskeletal organization on the resveratrol-induced anti-carcinogenic effects in colorectal cancer cells, immunoblotting was performed. HCT116 ( Figure 8) cells were cultured in tumor microenvironment alginate beads as described in materials and methods and either left untreated, or were treated with 5 μM resveratrol alone, 0.1 μg/mL CytD, 10 μM FAK-I (PF-562271) or co-treated with 5 μM resveratrol and either the indicated concentration of CytD or FAK-I for 10 days.  Destabilization of actin cytoskeleton by CytD or by inhibition of FAK with FAK inhibitor (PF-562271) markedly suppressed resveratrol-induced Sirt1 up-regulation in HCT116. Further, marked Ki67 suppression was observed in CytD, FAK-I or the combinational treatment ( Figure 8A,C). These results highlight that, at least in part, FAK has a functional role in resveratrol-Sirt1-mediated anti-tumorigenic signaling in CRC cells.

Resveratrol-Induced Suppression of β1-Integrin and FAK Is Potentiated by FAK Inhibition and Cytochalasin D
It has been shown that FAK participates in integrin mediated signaling functions, thereby enabling cell adhesion signals which stimulate reorganization of the cytoskeleton [11,12], and resveratrol has been shown to decrease the levels of β1-integrins and inhibit phosphorylation of FAK in several cancer cell lines [32][33][34][35]. Therefore next, we investigated the effect of resveratrol on Integrin and FAK activation in CRC cells. HCT116 cells were treated as described above and Western blotting performed with Integrin, FAK and p-FAK antibodies ( Figure 8A,C). As demonstrated in Figure 8, resveratrol treatment markedly suppressed Integrin expression and activation of FAK in CRC cells compared to untreated controls ( Figure 8A,C). Further, treatment with CytD alone or in combination with resveratrol had a suppressive effect on Integrin expression and activation of FAK. Interestingly, marked down-regulation of Integrin expression and FAK phosphorylation was observed in individual treatment with CytD or FAK-I or in co-treatment with resveratrol, but had no such effect on basal, inactivated FAK levels. Taken together, these results support a functional role for Integrin-FAK-mediated signaling and cytoskeletal signaling proteins as target proteins, at least in part of resveratrol mediated anti-tumorigenic effects in CRC cells.

Resveratrol-Potentiates FAK-Inhibitor-and Cytochalasin D-Induced Suppression of NF-κB Activation in CRC Cells
It has been previously shown that resveratrol has a functional role in suppression of the NF-κB signaling pathway in CRC cells [26]. To elucidate whether suppression of NF-κB signaling by resveratrol is modulated by FAK activity or cytoskeletal signaling proteins, HCT116 and SW480 cells were treated as described above and immunoblotting was performed with anti-NF-κB-p50 and anti-NF-κB-p65 ( Figure 8B,D). Immunoblotting analysis demonstrated that resveratrol treatment significantly suppressed activation of NF-κB compared to control cultures ( Figure 8B,D). Further, phosphorylation of NF-κB subunits (p50/p65) was markedly suppressed by CytD or FAK inhibition in HCT116 ( Figure 8B,D). Interestingly, a synergistic effect on suppression of NF-κB activation was observed in the combination treatment with either CytD or FAK inhibitor. Overall, these results indicate that resveratrol-induced suppression of NF-κB activation is at least in part dependent on FAK and cytoskeletal signaling proteins.

Resveratrol-Potentiates FAK-Inhibitor-and Cytochalasin D-Induced Suppression of NF-κB-Dependent Gene End Products Involved in Invasion, Metastasis and Apoptosis
To further characterize the role of FAK and cytoskeletal signaling proteins on resveratrol-mediated suppression of malignancy and metastatic ability in HCT116 cells, protein expression of tumor metastasis associated promoting factors influencing tumor cell invasion, metastasis and apoptosis were investigated. CRC cells were treated as described above and Western blotting was performed for anti-MMP-9, -MMP-13, -CXCR4 and -cleaved-caspase-3 ( Figure 8B,D). Compared to untreated control cultures resveratrol significantly suppressed MMP-9, MMP-13 and CXCR4 and markedly up-regulated activated Caspase-3 in CRC cells (not shown). Interestingly, these effects were significantly stronger in resveratrol mono-treatment compared to CytD, FAK-I or the combinational treatment. These results highlight that resveratrol-induced suppression of malignancy, metastatic ability and induction of apoptosis in CRC cells is, at least in part, mediated by FAK and cytoskeleton signaling proteins.

Discussion
Our data reveal that resveratrol indeed suppresses CRC proliferation and invasion by up-regulation of Sirt1 activity, inhibition of NF-κB-mediated inflammatory pathway and suppression of focal adhesion kinase (FAK) activity, resulting in a loss of focal adhesion molecules, a planar surface of the CRC cells and an increase in apoptosis.
Despite major advances in oncology, colorectal cancer (CRC) remains one of the major causes of cancer-related morbidity and mortality worldwide [1]. Although treatment options range from radiotherapy, surgery and chemotherapy to combination treatment, chemotherapy still offers the best chance to prevent invasion and metastasis. However, most chemotherapies fail to effectively combat metastatic cancer and have high prevalence of recurrence, chemoresistance and are toxic and expensive.
Therefore, new pharmacologically effective and safe therapeutic approaches that may prevent the initial invasion of cancer cells from primary tumors and thus prevent metastasis are urgently sought. Natural compounds such as resveratrol have gained much attention for preventive and therapeutic strategies in cancer treatment including colorectal cancer [44,45]. Resveratrol (3,5,4 -trihydroxy-trans-stilbene), found in more than 70 plant species, has shown to exhibit both chemopreventive and chemotherapeutic potential [20][21][22]. We have shown in this study that resveratrol indeed altered the cell phenotype of two CRC cell lines HCT116 and SW480, by inducing cell cluster formation, encouraging cell attachment and reducing cell viability. Indeed, in a study from our own laboratory we could previously show that resveratrol affects multiple cell adhesion molecules, desmosomes, tight, gap junctions and signaling molecules involved in cell proliferation, invasion and metastasis [26].
Further, we found that treatment of the CRC cell lines with an inhibitor to FAK or with CytD markedly promoted cellular attachment and rather more cell clusters were observed. Indeed, it has been previously shown that cancer cell invasion (migration) is an essential initial step of spontaneous carcinogenesis and metastasis and to understand more detailed this important mechanism may be useful for the treatment/prevention of CRC. Focal adhesion kinase (FAK) is an intracellular tyrosine kinase that regulates cellular adhesion, control of cell-extracellular interactions such as cell spreading, migration, metastasis, motility, cancer stem cell self-renewal and survival in many types of cancers and FAK is overexpressed in a number of tumors including CRC [18]. As FAK is a non-receptor tyrosine kinase that is activated in response to cell adhesion and transduces extracellular signals through tyrosine phosphorylation onto various intracellular molecules in both growth factor dependent and adhesion-dependent manner, it is fundamentally involved in modulating cancer progression and metastasis [8]. Interestingly, in several cancers, including breast and ovarian carcinomas, genetic tumor profiling has revealed both FAK DNA amplification (PTK2 gene at 8q24.3) and elevated FAK mRNA levels [19]. Indeed, activation of FAK has been shown to be high in metastatic aggressive tumors and is correlated with poor clinical outcome [8].
Cytochalasins are a class of fungal metabolites affecting a wide variety of motile functions of eukaryotic cells [46]. CytD has been described to de-stabilize, disorganize and alter the actin cytoskeleton [47,48]. Here we could demonstrate that FAK inhibition and CytD additionally dose-dependently reduced CRC HCT116 and SW480 cell viability, induced cell cluster formation and encouraged cell attachment similar to resveratrol treatment. Indeed, it has been previously shown that resveratrol or FAK-inhibition suppresses cancer cell viability and survival [8,26,30,41]. These observed effects of resveratrol, FAK-inhibition and CytD in HCT116 and SW480 CRC cells highlight the involvement of cytoskeletal signaling molecules in resveratrol-mediated anti-tumorigenic effects.
We have further shown that resveratrol up-regulates Sirt1 expression and suppresses FAK expression in HCT116 and SW480 CRC cells. It has been previously suggested that resveratrol acts as a multi-targeted agent in cancer as it modulates several cellular signaling pathways involved in cancer cell survival, proliferation and metastasis [23][24][25][26]. Indeed, it has been previously shown that the nucleus related NAD + histone deacetylase class III sirtuin 1 (Sirt1), is a major target for resveratrol [40]. Resveratrol provokes a structural conformational change in Sirt1, consequently resulting in an increased enzymatic activity [40]. Moreover, we could demonstrate previously that resveratrol-induced Sirt1 up-regulation is required for the resveratrol-mediated chemopreventive effects in colorectal cancer cells [30]. Further, combinational treatment of resveratrol with FAK-I or CytD suppressed resveratrol-induced Sirt1 activation and FAK. Interestingly, it has been shown that deletion of Sirt1 in bone marrow macrophages (BMMs) increased expression and acetylation of FAK, as well as NF-κB [49]. This highlights a potential role for FAK-Sirt1 signaling pathway in resveratrol-mediated anti-tumorigenic effects of resveratrol in CRC cells.
It has been previously described that inhibition of FAK impairs cancer development in preclinical and clinical trials [8]. Indeed, in our study, we could demonstrate a marked dose-dependent effect of specific FAK inhibitors (PF-562271 or PF-573228) on growth inhibition and induction of apoptosis in HCT116 and SW480 CRC cells. Both specific FAK inhibitors are ATP-competitive, reversible inhibitors of FAK. Indeed, it has been recently shown that inhibition of FAK, with specific FAK inhibitors, enhances chemosensitivity and suppresses invasion of cancer cells such as lung and breast carcinoma [50,51]. This underlines the important aspect of active FAK-signaling for the interaction between the CRC cells and their surrounding tumor microenvironment to control and regulate cancer progression and metastasis. It is well known that activation of caspase-3 induces the apoptotic pathway in cells [42,43]. We further demonstrated that indeed resveratrol alone activated caspase-3 and potentiated nuclear signs of apoptosis in CRC cells and this was markedly enhanced by combinational treatment of resveratrol with FAK-I or CytD. Furthermore, here we assessed a potential relationship between resveratrol and FAK-signaling pathway and investigated the combined potential to enhance and stimulate apoptosis in CRC cells. Interestingly, it has been recently shown that inhibition of FAK and CXCR4 and simultaneous treatment of lung carcinoma cells with doxorubicin potentiated their anti-tumorigenic effects [51]. These results highlight that FAK and cytoskeletal signaling molecules are, at least in part, one of the main target proteins for resveratrol-mediated anti-tumorigenic effects in CRC cells.
Several reports from our and other laboratories have reported that three dimensional culture models provide excellent in vitro culture conditions compared to two dimensional models, as they more adequately represent physiological conditions that exist in vivo [37,52,53] Indeed, recent reports from our laboratory have shown that alginate bead culture provides an excellent tumor microenvironment were the CRC cells can proliferate, form spheroids, migrate through the alginate matrix and finally form metastases [30,37]. Our data in this study indicate that resveratrol suppressed invasion (migration) and colony forming capacity of HCT116 and SW480 CRC cells in alginate bead tumor microenvironment culture, similar to FAK inhibitor or CytD. This is consistent with previous reports, which showed that resveratrol inhibits angiogenesis, proliferation and metastasis in different kinds of tumors [54][55][56]. Additionally, the anti-invasive and anti-metastases effect of resveratrol was potentiated in the combinational treatment of FAK-I and CytD. Cytoskeleton reorganization and remodeling is a critical step for cancer cell invasion and metastasis. In line with our results, FAK inhibition in a murine model with PF-562271 resulted in decreased tumor cell proliferation, migration and tumor-associated macrophages and fibroblasts [57]. The data presented here now underline the importance of participation of FAK and cytoskeletal signaling proteins in resveratrol mediated anti-invasive and anti-metastasis properties in CRC cells.
Further, we also found in alginate tumor microenvironment cultures of CRC cells that resveratrol-induced up-regulation of Sirt1, blocked CRC cell proliferation and concomitantly suppressed Integrin and activation of FAK. In contrast to this, FAK inhibitor or CytD alone did not up-regulate Sirt1 expression, but merely suppressed Integrin expression and activation of FAK. Resveratrol has been previously shown to suppress migration of cancer cells by modulation of integrin activity [31] and to decrease the levels of cell adhesion proteins and suppress expression of epithelial-to-mesenchymal-transition associated mediators α5β1 integrins and hyaluronic acid in ovarian cancer cell lines [32]. Interestingly, resveratrol has been further shown to decrease the levels of β1-integrins and at the same time inhibit phosphorylation of FAK in several cancer cell lines [32][33][34][35]. Further, it has been shown that FAK participates in integrin-mediated signaling functions, thereby enabling cell adhesion signals that stimulate reorganization of the cytoskeleton [11,12]. Indeed, it has been suggested that overexpression of β4-Integrins and FAK in colorectal cancer lead to subsequent FAK activation, essential for early steps of tumorigenesis [58]. Furthermore, we also found that resveratrol, FAK-I or CytD inhibited activation of NF-κB, suppressing NF-κB-dependent gene end-products involved in invasion (MMPs), metastasis (CXCR4) and activating those involved in apoptosis (cleavage of caspase-3). Interestingly, these observed effects of resveratrol were significantly potentiated by combinational treatment with either FAK-I or CytD. It has been previously reported that resveratrol inhibits IκB-kinase-β-mediated NF-κB activation and it is a potent natural activator of Sirt1 [27][28][29]. Indeed, in CRC cells, resveratrol-induced Sirt1 has a functional role in inhibiting activation of the NF-κB signaling pathway [26,30], wereby Sirt1 deacetylates the NF-κB-p65 subunit and thus attenuates NF-κB-mediated gene transcription and inflammation pathways [59]. Further, it has been shown that deletion of Sirt1 increased expression and acetylation of FAK, as well as NF-κB [49]. Moreover, it has been observed that FAK, besides its canonical roles as a cytoplasmic kinase downstream of integrin and growth factor receptor signaling, can also serve as a co-transcriptional regulator that alters a gene transcriptional activity by shuttling from focal adhesions to the nucleus to directly convey extracellular signals [60]. Further, FAK contains NF-κB and p53 binding sites, which regulate induction and inhibition of FAK transcription [61]. Transcription of FAK is regulated by binding of NF-κB to the Ptk2 gene promoter and activation of NF-κB by TNF-α increases FAK transcriptional activation [61]. These results highlight the potential pharmacological use of resveratrol-induced Sirt1 pathway in targeting FAK and NF-κB in chronic inflammatory diseases including cancer.

Conclusions
In conclusion, for the first time, we describe herein that the anti-cancerogenic mechanism of resveratrol is mediated, at least partially, by the inhibition of FAK in CRC cells. This results in suppression of early steps of cancerogenesis, with increased apoptosis and suppression of migration and metastasis in cancer cells (Figure 9). In addition, we could further demonstrate that FAK-inhibition suppresses resveratrol-induced Sirt1 activation. As Sirt1 is an important molecular target of resveratrol, it seems that the anti-cancerogenic effect induced by resveratrol-Sirt1-activation in CRC cells is mediated at least in part by the FAK signaling pathway. Indeed, it has been reported that resveratrol is apparently rapidly metabolized in vivo [62]. Although further investigations are needed to understand more on the signaling mechanism of resveratrol, our findings suggest that a molecular signaling pathway relationship between resveratrol-induced Sirt1 and FAK activation, which might be a novel therapeutic target for crucial regulation of cell proliferation, migration and metastases in colon cancer.