Growth Of Human Colon Cancer Cells Research Articles

2-Hydroxycinnamaldehyde Inhibits SW620 Colon Cancer Cell Growth Through AP-1 Inactivation

Cinnamaldehyde derivatives isolated from Cinnamomum cassia have been widely used for treating dyspepsia, gastritis, and inflammatory disease as well as cancer. To investigate the anti-tumor activities of several cinnamaldehyde derivatives, we compared the inhibitory effect of cinnamaldehyde derivatives on cell growth and AP-1 transcriptional activity in SW620 human colon cancer cells since AP-1 is a transcriptional factor implicated to control cancer cell growth. Among the derivatives, 2’-hydroxycinnamaldehyde (HCA) most significantly inhibited cancer cell growth and AP-1 transcriptional activity in a dose-dependent manner with an IC50 value of 12.5 and 9 µg/ml, respectively. In further studies on the mechanism, we found that consistent with the inhibitory effect on cell growth, HCA dose-dependently (0 – 20 µg/ml) inhibited DNA binding activity of AP-1 accompanied with down regulation of c-Jun and c-Fos expressions. HCA also induced apoptotic cell death as well as expression of the apoptosis-regulating gene caspase-3, but inhibited the anti-apoptosis regulating gene bcl-2 in a dose-dependent manner. These results suggested that HCA has the most potent inhibitory effect against human colon cancer cell growth, and AP-1 may be an important target of HCA.

Sequence-dependent inhibition of human colon cancer cell growth and of prosurvival pathways by oxaliplatin in combination with ZD6474 (Zactima), an inhibitor of VEGFR and EGFR tyrosine kinases

To date, clinical studies combining the new generation of targeted therapies and chemotherapy have had mixed results. Preclinical studies can be used to identify potential antagonism/synergy between certain agents, with the potential to predict the most efficacious combinations for further investigation in the clinical setting. In this study, we investigated the sequence-dependent interactions of ZD6474 with oxaliplatin in two human colon cell lines in vitro. We evaluated the in vitro antitumor activity of ZD6474, an inhibitor of vascular endothelial growth factor receptor (VEGFR), epidermal growth factor receptor (EGFR) and RET tyrosine kinase activity, and oxaliplatin using three combination schedules: ZD6474 before oxaliplatin, oxaliplatin before ZD6474, and concurrent exposure. Cell proliferation studies showed that treatment with oxaliplatin followed by ZD6474 was highly synergistic, whereas the reverse sequence was clearly antagonistic as was concurrent exposure. Oxaliplatin induced a G(2)-M arrest, which was antagonized if the cells were previously or concurrently treated with ZD6474. ZD6474 enhanced oxaliplatin-induced apoptosis but only when added after oxaliplatin. The sequence-dependent antitumor effects appeared, in part, to be based on modulation of compensatory prosurvival pathways. Thus, expression of total and active phosphorylated EGFR, as well as AKT and extracellular signal-regulated kinase, was markedly increased by oxaliplatin. This increase was blocked by subsequent treatment with ZD6474. Furthermore, the synergistic sequence resulted in reduced expression of insulin-like growth factor-I receptor and a marked reduction in secretion of vascular endothelial growth factor protein. ZD6474 in combination with oxaliplatin has synergistic antiproliferative properties in human colorectal cancer cell lines in vitro when oxaliplatin is administered before ZD6474.

3,3′‐Diindolylmethane (DIM) induces cell cycle arrest in HT‐29 human colon cancer cells

3,3′-Diindolylmethane (DIM), an indole derivative produced on consumption of broccoli and other cruciferous vegetables, has been shown to have multiple anti-cancer effects in both in vivo and in vitro models. To examine the effects of DIM on of HT-29 human colon cancer cell growth, cells were cultured with various concentrations (0–30 μmol/L) of DIM. Treatment of cells with increasing concentration of DIM led to a dose-dependent decrease in viable cell numbers in HT-29 cells. The decrease in cell growth was due to an increase in apoptosis and a decrease in DNA synthesis. Results from DNA flow cytometric analysis revealed that DIM induced G1 and G2/M phase arrests at 12 h and G2/M phase arrest at 24 h of culture. Western blot analyses of total cell lysates revealed that DIM decreased phosphorylated retinoblastoma proteins (Rb) and E2F-1, and increased hypophosphorylated Rb levels. DIM decreased cyclin A and D1 and increased cyclin-dependent kinase (CDK) 4 levels in a time-related manner. DIM also increased the protein levels of the CDK inhibitors p21 and p27. In addition, DIM decreased the levels of cell division cycle 2 and cyclin B1. These results suggest that DIM inhibits G1 or G2/M phase cell cycle progression by inhibiting CDK activities.

Curcumin inhibits human colon cancer cell growth by suppressing gene expression of epidermal growth factor receptor through reducing the activity of the transcription factor Egr-1

High expression of epidermal growth factor receptor (EGFR) is found in a variety of solid tumors, including colorectal cancer. EGFR has been identified as a rational target for anticancer therapy. Curcumin, the yellow pigment of turmeric in curry, has received attention as a promising dietary supplement for cancer prevention and treatment. We recently reported that curcumin inhibited the growth of human colon cancer-derived Moser cells by suppressing gene expression of cyclinD1 and EGFR. The aim of the present study was to explore the molecular mechanisms underlying curcumin inhibition of gene expression of EGFR in colon cancer cells. The generality of the inhibitory effect of curcumin on gene expression of EGFR was verified in other human colon cancer-derived cell lines, including Caco-2 and HT-29 cells. Promoter deletion assays and site-directed mutageneses identified a binding site for the transcription factor early growth response-1 (Egr-1) in egfr promoter as a putative curcumin response element in regulating the promoter activity of the gene in Moser cells. Electrophoretic mobility shift assays demonstrated that curcumin significantly reduced the DNA-binding activity of the transcription factor Egr-1 to the curcumin response element. In addition, curcumin reduced the trans-activation activity of Egr-1 by suppressing egr-1 gene expression, which required interruption of the ERK signal pathway and reduction of the level of phosphorylation of Elk-1 and its activity. Taken together, our results demonstrated that curcumin inhibited human colon cancer cell growth by suppressing gene expression of EGFR through reducing the trans-activation activity of Egr-1. These results provided novel insights into the mechanisms of curcumin inhibition of colon cancer cell growth and potential therapeutic strategies for treatment of colon cancer.

Selective CCK‐A but not CCK‐B receptor antagonists inhibit HT‐29 cell proliferation: synergism with pharmacological levels of melatonin

Some data suggest that cholecystokinin (CCK) receptor agonists stimulate the growth of colon cancer. Melatonin, an endogenous indoleamine with strong antioxidant properties, displays antiproliferative and proapoptotic properties both in vivo or in vitro in several types of tumors. We used HT-29 human colon cancer cells, expressing CCK receptors, to test the antiproliferative effects of several antagonists of CCK-A and/or CCK-B and their possible synergism with melatonin. HT-29 cells were cultured in RPMI 1640 medium supplemented with fetal bovine serum at 37 degrees C. Cell proliferation was assessed by the incorporation of [3H]-thymidine into DNA. Annexin V-FITC plus propidium iodine were used for flow cytometry apoptosis/necrosis evaluation. The following drugs were tested: gastrin (CCK-B agonist); CCK-8s (CCK-A agonist); proglumide (CCK-A plus CCK-B antagonist); lorglumide (CCK-A antagonist); PD 135,158 (CCK-B antagonist and weak CCK-A agonist); devazepide or L 364,718 (CCK-A antagonist); L 365,260 (CCK-B antagonist), and melatonin. The results shown a lack of effects of gastrin on HT-29 cell proliferation, whereas CCK-8s induced proliferation at high doses. The order of the antiproliferative effect of the other drugs was devazepide > lorglumide > proglumide. These drugs produce cell death mainly inducing apoptosis. Melatonin showed strong antiproliferative effect at millimolar concentrations, and it induced apoptotic cell death. Melatonin generally enhanced the antiproliferative effects of devazepide, lorglumide and proglumide and increased the proglumide-induced apoptosis. These results suggest that melatonin and CCK-A antagonists are useful for controlling human colon cancer cell growth in culture and in combined therapy significantly increases their efficiency.

Up-regulation of the enzymes involved in prostacyclin synthesis via Ras induces vascular endothelial growth factor

The constitutive activation of Ras is an important step in the development and progression of several different cancers and is known to increase the level of cyclooxygenase 2 (COX-2). Prostaglandins are the downstream bioactive lipid mediators produced by the COX-2 enzyme. We sought to determine the role of Ras-induced up-regulation of the enzymes involved in prostacyclin biosynthesis in nontransformed rat intestinal epithelial cells (IECs). Messenger RNA (mRNA) and protein expression were analyzed by Northern and Western analysis, respectively, to determine the level of enzymes induced by Ras. In vitro assays were used to determine the production of vascular endothelial growth factor (VEGF) and prostaglandins as well as the promoter and enzymatic activation of the rate-limiting enzyme in prostaglandin production (phospholipase A(2) [cPLA(2)]). The inducible expression of Ha-Ras(V12) increased the production of prostaglandin (PG)F(2alpha) and prostacyclin by 2- and 13-fold, respectively. The induction of Ha-Ras(V12) also up-regulated the mRNA and protein levels of cPLA(2), COX-2, and prostacyclin synthase, as well as the promoter and enzyme activity of cPLA(2). Furthermore, oncogenic Ras increased the production of the pro-angiogenic factor VEGF. The increase of VEGF was abolished after treatment with celecoxib, a selective COX-2 inhibitor. The addition of PGI 2 alone also induced the expression of VEGF. Inducible Ha-Ras(V12) increases the production of PGI(2) through the coordinate up-regulation of cPLA(2), COX-2, and prostacyclin synthase (PGIS). The production of PGI(2) leads to an increase in the level of the pro-angiogenic factor VEGF, which is known to play a crucial role in the regulation of tumor-associated angiogenesis.

Effects of a hyaluronan-based membrane (Seprafilm) on intraperitoneally disseminated human colon cancer cell growth in a nude mouse model.

The purpose of this study was to examine whether a hyaluronan-based membrane (Seprafilm Adhesion Barrier) could affect growth and metastasis of colon cancer in a human xenograft/nude mouse model. Male athymic (nude) mice underwent a midline abdominal incision followed by an intraperitoneal inoculation of KM12-L4 human colon cancer cells. Seprafilm membrane was placed under the incision or on the right lateral abdominal wall; control groups received no Seprafilm membrane. In another group, Vicryl mesh was placed on the right lateral abdominal wall and removed after 1 minute to control for surgical trauma associated with biomaterial placement. Intraperitoneal Seprafilm did not affect human colon cancer tumor metastasis, including the liver, spleen, and mesenteric lymph nodes. The application of a biomaterial such as Seprafilm or Vicryl mesh to the peritoneal sidewall away from the midline wound was associated with an increased rate of local tumor growth. This was likely because of the local trauma of biomaterial placement in the nude mouse model and not because of the presence of a foreign material. Our study suggests that Seprafilm does not affect tumor metastasis. Additionally, placement of biomaterials may cause local trauma that stimulates the formation of localized sidewall tumors in the nude mouse model. Further studies in other animal models and ultimately, in humans are required to unambiguously understand the safety of Seprafilm and other biomaterials in cancer patients.

Inhibition of human colon cancer cell growth by antisense oligodeoxynucleotides targeted at basic fibroblast growth factor.

Basic fibroblast growth factor has been shown to be mitogenic in colon cancer cell lines. In human malignant melanoma cells, antisense oligodeoxynucleotides targeted against basic fibroblast growth factor messenger RNA significantly inhibit cell growth. However, the efficacy of such an antisense oligodeoxynucleotide strategy has not been evaluated for colon cancer cells. To investigate whether basic fibroblast growth factor can stimulate the growth of HT-29 human colon cancer cells and whether antisense oligodeoxynucleotides can inhibit growth of these cells at baseline. Western blotting analyses were used to confirm the presence of basic fibroblast growth factor protein in this cell line. Cell growth was assessed after 2, 4 and 6 days of treatment by cell counting using the trypan blue exclusion method. Phosphorothioate-modified oligodeoxynucleotides (10 microM) were used, complementary to codon 60 of the basic fibroblast growth factor messenger RNA. Cationic liposomes (DOTAP) were used to enhance the cellular uptake of the oligodeoxynucleotides. Western blotting demonstrated the presence of basic fibroblast growth factor protein in this cell line. Basic fibroblast growth factor (1-40 ng/mL) dose-dependently stimulated cell growth and peak values were obtained at a dose of 20 ng/mL. By contrast, antisense oligodeoxynucleotide treatment significantly inhibited cell growth compared with the sense oligodeoxynucleotide-treated cells (P=0.007). This inhibition was reversed by the addition of basic fibroblast growth factor, 20 ng/mL. Treatment targeted against basic fibroblast growth factor messenger RNA inhibits growth of HT-29 human colon cancer cells. This finding may provide a rationale for the therapeutic use of antisense oligodeoxynucleotides targeted at basic fibroblast growth factor for the treatment of colon cancer.

Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines.

We have constructed a series of 22 phosphorothioate 20-mer antisense oligonucleotides directed against different regions of the human (EGFR) mRNA. Treatment with EGFR antisense oligonucleotides showed a dose-dependent inhibition of human GEO colon cancer cell growth in soft agar. Western blot analysis demonstrated a significant reduction in EGFR expression after treatment with each EGFR antisense oligonucleotide. The ability to inhibit GEO anchorage-independent growth, however, varied among the EGFR antisense sequences with an IC(50) ranging between 0.5 and 3.5 microM. Two of these antisense oligonucleotides targeting the regions between 2457-2476 and 614-4633 bases of the human EGFR mRNA have been modified as hybrid DNA/RNA mixed backbone oligonucleotides (MBO) to examine their anticancer properties in vivo. The 2 EGFR antisense MBOs retained the same biological properties of the fully phosphorothioate EGFR antisense oligonucleotides targeting the same EGFR mRNA sequences, such as blocking EGFR synthesis, inhibiting cell growth and enhancing programmed cell death in human cancer cell lines that express functional EGFRs. Furthermore, a potentiation in the growth inhibitory effect on GEO cancer cells was observed after treatment with these EGFR antisense MBOs in combination with cytotoxic drugs, including cisplatin, doxorubicin, paclitaxel, or topotecan. These results show the antiproliferative activity of specific EGFR antisense oligonucleotides and allow to identify novel EGFR antisense MBOs that deserve further evaluation as potential selective anticancer agents alone or in combination with cytotoxic drugs in human carcinomas that express functional EGFRs.

A specific function for phosphatidylinositol 3-kinase alpha (p85alpha-p110alpha) in cell survival and for phosphatidylinositol 3-kinase beta (p85alpha-p110beta) in de novo DNA synthesis of human colon carcinoma cells.

We have previously shown an important function of phosphatidylinositol 3-kinase (PI3K)alpha(p85alpha-p110alpha) and PI3Kbeta (p85-alpha-p110beta) for DNA synthesis induced by various mitogens in non transformed fibroblasts and we now report a specific role of these enzymes in human colon cancer cell growth. Using antibodies specific to p110alpha and to p110beta catalytic subunits, increase in PI3Kalpha and PI3Kbeta activities was detected in 15/19 human tumour biopsies relative to adjacent normal mucosa of human colon and bladder. Increase in such activities was also observed in adenocarcinoma cell lines CaCo2, CO115, HCT 116, LS 174T and WiDr relative to non-transformed fibroblasts. Maximal PI3Kalpha activity was observed for LS 174T and PI3Kbeta activity for WiDr cells. This was partly correlated with an increase in p110alpha and p110beta protein levels both in some primary tumours and established cell lines, suggesting that PI3K overexpression is involved in enzymatic deregulation. Functional consequence of such activation was assessed by a microinjection approach. An injection of neutralizing antibody specific to p110beta in WiDr, HCT116 and CO 115 cells inhibited de novo DNA synthesis, whereas antibodies specific to p110gamma had no effect. Neutralizing antibodies specific to p110alpha induced apoptosis, a response that was reverted by treating cells with the caspase inhibitor z-VAD-fmk. However anti-p110beta and anti-p110gamma antibodies did not affect cell survival. We concluded that PI3Kalpha and PI3Kbeta play important roles in human colon cancer cell growth with a specific function for PI3Kbeta in de novo DNA synthesis and an involvement of PI3Kalpha in cell survival.

Human colon cancer cell growth is inhibited by adenoviral delivery of chimeric P27/P16

Human colon cancer cell growth is inhibited by adenoviral delivery of chimeric P27/P16

INHIBITION OF CYCLOOXYGENASE-2 SUPPRESSES ANGIOGENESIS AND THE GROWTH OF PROSTATE CANCER IN VIVO

INHIBITION OF CYCLOOXYGENASE-2 SUPPRESSES ANGIOGENESIS AND THE GROWTH OF PROSTATE CANCER IN VIVO

Neurotensin and a non-peptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice.

The intestine is a large endocrine organ, but the dependence of colon cancer on hormones remains unknown. We show here that neurotensin, a paracrine/endocrine peptide in the gut, and the neurotensin receptor antagonist SR 48692 control colon cancer cell growth in vitro and in vivo by interacting with receptors that are ectopically expressed in colon cancers. In cell culture, neurotensin stimulates the growth of human colon cancer cell lines (SW480, SW620, HT29, HCT116 and Cl.19A) expressing the neurotensin receptor NTR1 but does not change the growth of Caco2 cells, which do not express NTR1. In SW480 cells, neurotensin is active in the 10(-10) to 10(-6) M concentration range (ED50 = 0.47 nM) while the neurotensin fragment (I-II) is inactive. Neurotensin also enhances the cellular cloning efficiency of SW480 cells in soft agar by inducing a 50% increase of colony formation. This effect is blocked by SR 48692, which alone does not alter colony formation. Subcutaneous delivery of neurotensin (0.54 micromol/kg every 24 hr) by osmotic pumps to nude mice that have been xenografted with SW480 cells results in a significant increase of tumor volume, i.e., up to 255% of control at day 20 of treatment. SR 48692 administered alone (1.7 micromol/kg every 24 hr) by daily i.p. injections reduces the development of tumors formed by xenografting SW480 cells in nude mice. A significant mean reduction of tumor volume of 38% is observed during the 22-day period of treatment. SR 48692 alone is also active at reducing tumor volume after xenografting HCT116 cells in nude mice. Our results support the notion that colon cancer growth may be dependent on blood-borne neurotensin and suggest that non-peptide neurotensin antagonists, such as SR 48692, may be useful for the development of novel therapeutic strategies of colon cancer.

Neurotensin and nonpeptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice

Neurotensin and nonpeptide neurotensin receptor antagonist control human colon cancer cell growth in cell culture and in cells xenografted into nude mice

Inhibition of human colon cancer cell growth by selective inhibition of cyclooxygenase-2.

A considerable amount of evidence collected from several different experimental systems indicates that cyclooxygenase-2 (COX-2) may play a role in colorectal tumorigenesis. Large epidemiologic studies have shown a 40-50% reduction in mortality from colorectal cancer in persons taking aspirin or other nonsteroidal antiinflammatory drugs on a regular basis. One property shared by all of these drugs is their ability to inhibit COX, a key enzyme in the conversion of arachidonic acid to prostaglandins. Two isoforms of COX have been characterized, COX-1 and COX-2. COX-2 is expressed at high levels in intestinal tumors in humans and rodents. In this study, we selected two transformed human colon cancer cell lines for studies on the role of COX-2 in intestinal tumorigenesis. We evaluated HCA-7 cells which express high levels of COX-2 protein constitutively and HCT-116 cells which lack COX-2 protein. Treatment of nude mice implanted with HCA-7 cells with a selective COX-2 inhibitor (SC-58125), reduced tumor formation by 85-90%. SC-58125 also inhibited colony formation of cultured HCA-7 cells. Conversely, SC-58125 had no effect on HCT-116 implants in nude mice or colony formation in culture. Here we provide evidence that there may be a direct link between inhibition of intestinal cancer growth and selective inhibition of the COX-2 pathway.

Insulin like growth factor I is an autocrine regulator of human colon cancer cell differentiation and growth

The polyanionic compound suramin triggers enterocyte-like differentiation of the human colic adenocarcinoma cell clone HT29-D4. We now demonstrate that suramin interferes with the binding of IGF-I to its receptor at the surface of HT29-D4 cells. Half-maximum inhibition of 125I-IGF-I binding was obtained in the presence of 25 μg/ml suramin. Moreover, the drug was able to dissociate 125I-IGF previously bound to its cell surface receptor. Affinity labeling experiments demonstrated that 125I-IGF-I bound to a polypeptide of 140 kDa. When HT29-D4 cells were cultured in the presence of 10 μg/ml of α-IR3, a monoclonal antibody directed against the binding site of IGF-I, an inhibition of cell proliferation and a stimulation of cell differentiation was observed. After 10 days of treatment with α-IR3, HT29-D4 cells formed a regular monolayer of enterocyte-like cells exhibiting an apical brush border and tight junctions delimiting two domains of the plasma membrane (apical and basolateral). Furthermore, we show that IGF-I significantly increased the initial rate of glucose uptake by HT29-D4 cells, while we have previously shown that suramin decreased glucose consumption. From these data we conclude that IGF-I secreted by the cells themselves, stimulates proliferation of HT29-D4 cells via an autocrine mechanism. Blockade of this stimulation by suramin or by a specific monoclonal antibody inhibits cell growth, glucose uptake and triggers the process of enterocytic differentiation.

Selective modulation of protein kinase isozymes by the site-selective analog 8-chloroadenosine 3',5'-cyclic monophosphate provides a biological means for control of human colon cancer cell growth.

Differential expression of type I and type II cAMP-dependent protein kinase isozymes has been linked to growth regulation and differentiation. We examined the expression of protein kinase isozymes in the LS 174T human colon cancer cell line during 8-chloroadenosine 3',5'-cyclic monophosphate (8-Cl-cAMP)-induced growth inhibition. Two species of RII (the regulatory subunit of protein kinase type II) with apparent Mr 52,000 (RII52) and Mr 56,000 (RII56) and a single species of RI (the regulatory subunit of protein kinase type I) with Mr 48,000 were identified in the cancer cells. RI and both forms of RII were covalently labeled with 8-azidoadenosine 3',5'-cyclic [32P]monophosphate, and two anti-RII antibodies that exclusively recognize either RII52 or RII56 resolved two forms of the RII receptors. 8-Cl-cAMP treatment induced a decrease of RI and an increase of both RII52 and RII56 in the cytosols of cancer cells and rapid translocation (within 10 min) of RII52 from the cytosol to nucleus. 8-Cl-cAMP caused transcriptional activation of the RII52 receptor gene and inactivation of the RI receptor gene. It also exhibited high-affinity site-1-selective binding to the purified preparations of both RII receptor proteins. Thus, differential regulation of various forms of cAMP receptor proteins is involved in 8-Cl-cAMP-induced regulation of cancer cell growth, and nuclear translocation of RII52 receptor protein appears to be an early event in such differential regulation.