Biomedical subjects

Vijayalakshmi Thamilselvan

Publications and source records attributed to Vijayalakshmi Thamilselvan.

11 recordsLinked to original sources

PubMed2006

Paxillin modulates squamous cancer cell adhesion and is important in pressure-augmented adhesion.

Paxillin is an adapter protein regulating signaling and focal adhesion assembly that has been linked to malignant potential in many malignancies. Overexpression of paxillin has been noted in aggressive tumors. Integrin-mediated binding through the focal adhesion complex is important in metastatic adhesion and is upregulated by extracellular pressure in malignant colonocytes through FAK and Src activation. Neither head and neck cancers nor paxillin have been studied in this regard. We hypothesized that paxillin would play a role in modulating squamous cancer adhesion both at baseline and under conditions of increased extracellular pressure. Using SCC25 tongue squamous cancer cells stably transfected with either an empty selection vector or paxillin expression and selection vectors, we studied adhesion to collagen, paxillin, FAK, and Src expression and phosphorylation in cells maintained for 30 min under ambient or 15 mmHg increased pressure conditions. Paxillin-overexpressing cells exhibited adhesion 121 +/- 2.9% of that observed in vector-only cells (n = 6, P < 0.001) under ambient pressure. Paxillin-overexpression reduced FAK phosphorylation. Pressure stimulated adhesion to 118 +/- 2.3% (n = 6, P < 0.001) of baseline in vector-only cells, similar to its effect in the parental line, and induced paxillin, FAK, and Src phosphorylation. However, increased pressure did not stimulate adhesion or phosphorylate paxillin, FAK, or Src further in paxillin-overexpressing cells. Metastasizing squamous cancer cell adhesiveness may be increased by paxillin-overexpression or by paxillin activation by extracellular pressure during surgical manipulation or growth within a constraining compartment. Targeting paxillin in patients with malignancy and minimal tumor manipulation during surgical resection may be important therapeutic adjuncts.

Cell Adhesion↗

PubMed2006

Pressure stimulates breast cancer cell adhesion independently of cell cycle and apoptosis regulatory protein (CARP)-1 regulation of focal adhesion kinase.

BACKGROUND: Pressure stimulates colon cancer adhesion via focal adhesion kinase (FAK). Extracellular pressures reaching 29 mm Hg have been reported in rapidly growing breast cancers, and tumors experience pressure during surgical manipulation. We hypothesized that pressure stimulates breast cancer adhesion and that CARP-1, which influences cancer biology, inhibits FAK, and modulates pressure effects. METHODS: We compared MDA-MB-468 breast cancer cells under ambient or 15-mm Hg increased pressure. We studied FAK-397 autophosphorylation, which parallels activation, after CARP-1 overexpression, and investigated whether CARP-1 stable overexpression or reduction alters pressure-stimulated adhesion. RESULTS: Pressure increased MDA-MB-468 adhesion 25% (n = 30, P < .05). CARP-1 overexpression inhibited FAK-397 phosphorylation. However, pressure stimulated adhesion equivalently in CARP-1-overexpressing and CARP-1-reduced lines (n = 6, P < .05). CONCLUSIONS: Pressure within proliferative tumors or during manipulation may activate breast cancer cells. Thus, inhibiting pressure signaling in rapidly growing breast tumors may be beneficial. CARP-1 does regulate FAK, but CARP-1 modulation does not alter pressure-stimulated adhesion. Targeting CARP-1 is unlikely to manipulate this pathway.

Apoptosis Regulatory Proteins↗

PubMed2006

Focal adhesion kinase protein levels in gut epithelial motility.

Mucosal healing requires migration and proliferation. Most studies of focal adhesion kinase (FAK), a protein that regulates motility, proliferation, and apoptosis, have focused on rapid phosphorylation. We reported lower FAK protein levels in motile Caco-2 colon cancer cells and postulated that this reduction in FAK available for activation might impact cell migration and mucosal healing. Therefore, total and active FAK (FAK(397)) immunoreactivity was assessed at the migrating fronts of human Caco-2 and rat IEC-6 intestinal epithelial cells. Caco-2 and IEC-6 motility, quantitated as migration into linear or circular wounds, was examined following FAK protein inhibition by small interfering RNA (siRNA). FAK protein stability and mRNA expression were ascertained by cycloheximide decay, RT-PCR, and in situ hybridization in static and migrating Caco-2 cells. Cells at the migrating front of Caco-2 and IEC-6 monolayers exhibited lower immunostaining for both total and activated FAK than cells immediately behind the front. Western blot analysis also demonstrated diminished FAK protein levels in motile cells by >/=30% in both the differential density seeding and multiple scrape models. siRNA FAK protein inhibition enhanced motility in both the linear scrape (20% in Caco-2) and circular wound (16% in Caco-2 and 19% in IEC-6 cells) models. FAK protein degradation did not differ in motile and static Caco-2 cells and was unaffected by FAK(397) phosphorylation, but FAK mRNA was lower in migrating Caco-2 cells. Thus FAK protein abundance appears regulated at the mRNA level during gut epithelial cell motility and may influence epithelial cell migration coordinately with signals that modify FAK phosphorylation.

Animals↗

PubMed2005

The role of the cytoskeleton in differentially regulating pressure-mediated effects on malignant colonocyte focal adhesion signaling and cell adhesion.

Increased extracellular pressure stimulates colon cancer cell adhesion by activating focal adhesion kinase (FAK) and Src. We investigated the role of the cytoskeleton in pressure-induced inside-out FAK and Src phosphorylation and pressure-stimulated adhesion. We perturbed actin polymerization with phalloidin, cytochalasin D and latrunculin B, and microtubule organization with colchicine and paclitaxol. We compared the effects of these agents on pressure-induced SW620 and human primary colon cancer cell adhesion and inside-out FAK/Src activation with outside-in adhesion-dependent FAK/Src activation. Cells pretreated with cytoskeletal inhibitors were subjected to 15 mmHg increased pressure and allowed to adhere to collagen I coated plates or prevented from adhesion to pacificated plates for 30 min. Phalloidin, cytochalasin D, latrunculin B and colchicine pretreatment completely prevented pressure-stimulated and significantly inhibited basal SW620 cell adhesion. Taxol did not inhibit pressure-induced colon cancer cell adhesion, but significantly lowered basal adhesion. Cytochalasin D and colchicine had similar effects in pressure-stimulated primary human malignant colonocytes. Phalloidin, cytochalasin D, latrunculin B and colchicine prevented pressure-induced SW620 FAK phosphorylation but not Src phosphorylation. FAK phosphorylation in response to collagen I adhesion was significantly attenuated but not completely prevented by these inhibitors. Although Src phosphorylation was not increased on adhesion, the cytoskeleton disrupting agents significantly lowered basal Src phosphorylation in adherent cells. These results suggest that both cytoskeleton-dependent FAK activation and cytoskeleton-independent Src activation may be required for extracellular pressure to stimulate colon cancer cell adhesion. Furthermore, the cytoskeleton plays a different role in pressure-activated FAK and Src signaling than in FAK and Src activation in adherent cells. We, therefore, hypothesize that cytoskeletal interactions with focal adhesion signals mediate the effects of extracellular pressure on colon cancer cell adhesion.

Cell Adhesion↗

PubMed2005

Divalent cations influence colon cancer cell adhesion in a murine transplantable tumor model.

BACKGROUND: Cancer cells adhere principally by integrins, matrix receptors that may be influenced by divalent cations. Surgical wound fluid is high in Mg2+ and low in Ca2+. We hypothesized that Mg+ and Mn2+ promote perioperative adhesion of shed cancer cells to surgical sites and that washing surgical wounds with Ca2+ inhibits implantation. METHODS: We tested our hypothesis in a murine colon 26 adenocarcinoma model. We added 10 mmol/L CaCl2, 0.25 mmol/L MgCl2, or 0.5 mmol/L MnCl2 to suspended murine colon 26 cancer cells and placed these suspensions into wounds in anesthetized mice. After 30 minutes, we washed away nonadherent cells. In some studies, we 51Cr-labeled the cells and assayed tumor adhesion by wound radioactivity. In parallel studies, we closed the wounds and observed the mice for 90 days. RESULTS: Mg2+ increased adhesion to 188% +/- 15% of control (n = 10, P < .001) and Mn2+ to 130% +/- 6% (n = 7, P < .001). However, Ca2+ inhibited adhesion to 61% +/- 12% (n = 7, P = .006) of control. Seventy-two percent of survival controls developed tumors during follow-up. Mg2+ and Mn2+ stimulated tumor formation to 96% and 92%, respectively, but adding Ca2+ to the wounds reduced subsequent tumor formation to 56% without altering serum Ca2+. The survival curves each differed significantly by log-rank test (P < .01 each). All pair-wise multiple comparisons were significant (Holm-Sidak, P < .05 each). CONCLUSION: Thus, the high Mg2+ in endogenous wound fluid may potentiate tumor cell adhesion. However, 10 micromol/L Ca2+ inhibits cancer cell adhesion to murine wounds and subsequent tumor development. Irrigating with dilute CaCl2 could decrease local tumor recurrence by inhibiting the adhesion of shed tumor cells.

Adenocarcinoma↗

PubMed2004

Colon cancer cell adhesion in response to Src kinase activation and actin-cytoskeleton by non-laminar shear stress.

Malignant cells shed from tumors during surgical resection or spontaneous metastasis experience physical forces such as shear stress and turbulence within the peritoneal cavity during irrigation, laparoscopic air insufflation, or surgical manipulation, and within the venous or lymphatic system. Since physical forces can activate intracellular signals that modulate the biology of various cell types in vitro, we hypothesized that shear stress and turbulence might increase colon cancer cell adhesion to extracellular matrix, potentiating metastatic implantation. Primary human malignant colon cancer cells isolated from resected tumors and SW620 were subjected to shear stress and turbulence by stirring cells in suspension at 600 rpm for 10 min. Shear stress for 10 min increased subsequent SW620 colon cancer cell adhesion by 40.0 +/- 3.0% (n = 3; P < 0.001) and primary cancer cells by 41.0 +/- 3.0% to collagen I when compared to control cells. In vitro kinase assay (1.5 +/- 0.13 fold) and Western analysis (1.34 +/- 0.04 fold) demonstrated a significant increase in Src kinase activity in cells exposed shear stress. Src kinase inhibitors PP1 (0.1 microM), PP2 (20 microM), and actin-cytoskeleton stabilizer phalloidin (10 microM) prevented the shear stress stimulated cell adhesion to collagen I. Furthermore, PP2 inhibited basal (50.0 +/- 2.8%) and prevented shear stress induced src activation but phalloidin pretreatment did not. These results raise the possibility that shear stress and turbulence may stimulate the adhesion of malignant cells shed from colon cancers by a mechanism that requires both actin-cytoskeletal reorganization an independent physical force activation of Src kinase. Blocking this pathway might reduce tumor metastasis during surgical resection.

Actins↗

PubMed2004

Extracellular pressure stimulates colon cancer cell adhesion in vitro and to surgical wounds by Src (sarcoma protein) activation.

BACKGROUND: We hypothesized that pressure stimulates colon cancer cell adhesion to surgical wounds. METHODS: We quantitated adhesion of murine 26/51 transplantable colon cancer cells by cell counting or chromium 51-labeling. Tumor cells were added to murine surgical wounds after 30 minutes preincubation under ambient or 15 mm Hg increased pressure. Src activation was assayed by immunoblotting for phosphorylated Src and inhibited by 4-amino-5-(4chlorophenyl)-7-(t-butyl)pyrazolo-[3-4-d]pyrimidine (PP2). RESULTS: Pressure stimulated colon 26/51 cell adhesion to murine wounds by 43% to 52% (n = 9, P <0.05 each). Pressure stimulated Src phosphorylation by 39% +/- 4% (n = 5, P = 0.004) in colon 26 cells. The Src inhibitor PP2 (20 mumol/L) did not inhibit Src phosphorylation at ambient pressure but prevented pressure stimulation of Src phosphorylation. Src blockade by PP2 did not affect basal adhesion of either tumor to murine wounds but completely blocked pressure stimulation of adhesion (n = 4, P <0.001 each). CONCLUSIONS: Increased pressure may activate cancer adhesion to surgical wounds via Src. Src antagonists might inhibit this process.

Animals↗

PubMed2004

Pressure activates colon cancer cell adhesion by inside-out focal adhesion complex and actin cytoskeletal signaling.

BACKGROUND AND AIMS: Few circulating tumor cells implant or cause metastasis. We hypothesized that venous or lymphatic pressure or iatrogenic pressure during resection activates signals governing malignant colonocyte adhesion. METHODS: We studied the effect of 15 mm Hg increased pressure for 30 minutes on adhesion of primary human colon cancer cells and SW620 colonocytes to collagen and endothelial cells. We modulated integrin affinity with extracellular cations. We assessed binding affinity by detachment assay; integrin surface expression by flow cytometry; and focal adhesion kinase (FAK), Src, and extracellular signal-regulated kinase (ERK) activation by Western analysis and Src in vitro kinase assay. We inhibited Src (PP2), FAK (small RNA interference, SiRNA, or FRNK transfection), MEK (PD98059), PKC (calphostin C), and actin destabilization (phalloidin). RESULTS: Pressure and manganese stimulated primary and SW620 colonocyte adhesion to collagen. Pressure also stimulated SW620 adhesion to endothelial monolayers. Pressure strengthened SW620 binding force to matrix without changing integrin surface expression. Pressure activated SW620 FAK and Src, but not ERK. Manganese did not. Calcium-inhibited adhesion but stimulated FAK (but not Src). PP2 prevented pressure activation of Src, Src phosphorylation of FAK576, and pressure-stimulated adhesion but not FAK397 autophosphorylation. FRNK transfection or FAK SiRNA also prevented pressure-stimulated adhesion. FAK SiRNA ablated pressure-activated FAK397, Src, and FAK576 phosphorylation. Neither Src nor FAK inhibition blocked cation effects. Phalloidin prevented pressure-stimulated adhesion. PD98059 or calphostin C did not. CONCLUSIONS: In contrast to divalent cations, extracellular pressure may increase integrin affinity and promote colon cancer adhesion via actin dependent inside-out FAK and Src signals. This mechanotransduced pathway may regulate metastasizing tumor cell adhesion.

Actins↗

PubMed2003

Divalent cations modulate human colon cancer cell adhesion.

BACKGROUND: Iatrogenic tumor implantation within surgical sites can compromise curative cancer surgery. Cancer cell adhesion to extracellular matrix proteins is mediated by diverse matrix receptors, most notably integrins. Divalent cations may modulate integrin-ligand interactions in some cells. MATERIALS AND METHODS: We studied adhesion of SW620 and Caco-2 human colon cancer cells to collagen I, the dominant collagen of the interstitial matrix, and confirmed our results in primary human colon cancer cells from surgical specimens. Single cell suspensions in either HEPES/NaCl buffer or media supplemented with 0-1 mM Mn2+ or Mg2+, and 0-10 mM Zn2+ or Ca2+ were plated onto collagen-I-precoated dishes for 30 min. RESULTS: Supplementation of the HEPES/NaCl/BSA buffer with 1 mM Mn2+, Mg2+, Zn2+, or Ca2+ affected adhesion differently. Mn2+ (1 mM) markedly promoted SW620 adhesion vs control (21.17 +/- 0.08-fold). Mg2+ (1 mM) had a similar but lesser effect (14.71 +/- 0.02-fold). However, 1-10 mM Ca2+ inhibited basal cell adhesion by 22.0 +/- 3.1 to 88.0 +/- 7.3 % inhibition. Ca2+ (2.5-10 mM) also inhibited Mn2+-induced adhesion. Zn2+ stimulated basal adhesion slightly at lower concentrations but inhibited Mn2+-stimulated adhesion similarly to Ca2+ at higher concentrations. Results were duplicated in conventional serum containing culture medium supplemented with these cations. Caco-2 cells and primary cancer cells yielded similar results. All results are significant to P < 0.01. DISCUSSION: Integrin-mediated colon cancer cell adhesion is affected by extracellular divalent cation concentrations. Washing the surgical site with dilute calcium or zinc solutions might diminish perioperative tumor implantation.

Adenocarcinoma↗

PubMed2003

Absence of adhesion triggers differential FAK and SAPKp38 signals in SW620 human colon cancer cells that may inhibit adhesiveness and lead to cell death.

BACKGROUND: Upon adhesion, anchorage-dependent cells transmit survival signals from the matrix into the cell. Loss of anchorage leads to anoikis. Resistance to anoikis may influence tumor progression and metastasis. To better understand the pathways that regulate the choice between adhesion and cell death, we examined FAK, c-Src andMAPKinase activities in SW620 human colon cancer cells. METHODS: SW620 cell suspensions were first allowed to adhere to collagen I for 30 minutes and adherent cells were subsequently counted. FAK, p38, c-Src and ERK1/2 phosphorylation were assessed by Western blot in adherent cells and in cells prevented from adhesion by plating unto BSA-pacificated dishes. p38 and FAK were inhibited by SB203580 (20 microM) or by specific FAK antisense nucleotides or FAK siRNA, respectively, and adhesion quantitated. Apoptosis (anoikis) after lack of adhesion was measured colorimetrically in control cells and in cells treated with SB203580. RESULTS: Adhesion to collagen I nearly doubled FAK phosphorylation at Y397, the autophosphorylation site, and decreased p38 activation by 60% (p<0.001) but did not affect FAK phosphorylation at Y576, the c-Src dependent site. Lowering FAK expression with FAK antisense decreased adhesion to collagen I; the larger decrease in FAK expression obtained with the siRNA (43 +/- 2%) resulted in significantly greater inhibition of adhesion not only to collagen I but also to collagen IV and fibronectin. The p38 inhibitor diminished anoikis and enhanced adhesion. Interestingly, the SB compound also significantly inhibited FAK phosphorylation at Y397 (23 +/- 3% in adherent, 30 +/- 4% in non-adherent cells at 30 minutes and 35 +/- 4% in adherent, 46 +/- 14% in non-adherent cells after 6 hours, p<0.05 for each) and greatly enhanced phosphorylation of ERK1/2, a putative anti-apoptotic component of the MAPK cascade. CONCLUSIONS: In the absence of adhesion, SW620 cells exhibit increased p38 but decreased FAK activation, signals that may promote cell death. Our observations with the p38 inhibitor SB203580 indicate that inside-out signals, from p38 to FAK, may regulate both adhesion and anoikis in SW620 cells. In addition, the data suggest the presence of cross-talk between the pro-apoptotic p38 and anti-apoptotic ERK1/2 pathways.

Apoptosis↗

PubMed2002

Sphingosine-1-phosphate stimulates human Caco-2 intestinal epithelial proliferation via p38 activation and activates ERK by an independent mechanism.

Sphingosine-1-phosphate (S-1-P) has been identified as an extracellular mediator and an intracellular second messenger that may modulate cell motility, adhesion, proliferation, and differentiation and cancer cell invasion. Widely distributed, S-1-P is most abundant in the intestine. Although S-1-P is likely to modulate various intracellular pathways, activation of the mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase 1 (ERK1), ERK2, and p38 is among the best-characterized S-1-P effects. Because the MAPKs regulate proliferation, we hypothesized that S-1-P might stimulate intestinal epithelial cell proliferation by MAPK activation. Human Caco-2 intestinal epithelial cells were cultured on a fibronectin matrix because fibronectin is an important constituent of the gut mucosal basement membrane. We assessed ERK1, ERK2, and p38 activation by Western blotting with antibodies specific for their active forms and proliferation by Coulter counting at 24 h. Specific MAP kinase kinase (MEK) and p38 inhibitors PD98059 (20 microM) and SB202190 and SB203580 (10 and 20 microM) were used to probe the role of ERK and p38 in S-1-P-mediated proliferation. Three or more similar studies were pooled for the analysis. S-1-P stimulated Caco-2 proliferation and dose-responsively activated ERK1, ERK2, and p38. Proliferation peaked at 5 microM, yielding a cell number 166.3 +/- 2.7% of the vehicle control (n = 6, P < 0.05). S-1-P also maximally stimulated ERK1, ERK2, and p38 at 5 microM, to 164.4 +/- 19.9%, 232.2 +/- 38.5%, and 169.2 +/- 20.5% of the control, respectively. Although MEK inhibition prevented S-1-P activation of ERK1 and ERK2 and slightly but significantly inhibited basal Caco-2 proliferation, MEK inhibition did not block the S-1-P mitogenic effect. However, pretreatment with 10 microM SB202190 or SB203580 (putative p38 inhibitors) attenuated the stimulation of proliferation by S-1-P. Twenty micromolars of SB202190 or SB203580 completely blocked the mitogenic effect of S-1-P. Ten to twenty micromolars of SB202190 and SB203580 also dose-dependently ablated the effects of 5 microM S-1-P on heat shock protein 27 accumulation, a downstream consequence of p38 MAPK activation. Consistent with the reports in some other cell types, S-1-P appears to activate ERK1, ERK2, and p38 and to stimulate proliferation. However, in contrast to the mediation of the S-1-P effects in some other cell types, S-1-P appears to stimulate human intestinal epithelial proliferation by activating p38. ERK activation by S-1-P is not required for its mitogenic effect.

Caco-2 Cells↗