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PubMed2005

Glutathione S-transferase pi1 promotes tumorigenicity in HCT116 human colon cancer cells.

GSTP1 is a member of the glutathione S-transferase enzyme superfamily, which catalyzes the conjugation of electrophiles with glutathione in the process of detoxification. GSTP1 is widely overexpressed in colorectal cancer, from aberrant crypt foci to advanced carcinomas. Increased expression of GSTP1 is associated with multidrug resistance and a worse clinical prognosis. However, GSTP1-null mice have an increased risk of tumor formation. Thus, the biological function of GSTP1 in colorectal cancer biology remains speculative. In an effort to gain further insights into the role of GSTP1 in tumorigenesis, we disrupted the GSTP1 gene in HCT116 human colorectal cancer cells using targeted homologous recombination. We find that loss of GSTP1 resulted in impaired clonogenic survival and proliferation. Specifically, under growth-limiting conditions, (a) GSTP1 protected HCT116 cells from oxidative stress and associated apoptosis and (b) promoted mitogen-activated protein kinase-extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase-mediated G1-S cell cycle progression. In vivo, GSTP1 was critical for engraftment and growth of HCT116 tumor xenografts. These studies directly show that GSTP1 promotes clonogenic survival and proliferation in HCT116 human colon cancer cells.

Animals↗

PubMed2006

Activating transcription factor 3 and early growth response 1 are the novel targets of LY294002 in a phosphatidylinositol 3-kinase-independent pathway.

LY294002, a phosphatidylinositol 3-kinase (PI3K) inhibitor, has been widely used to study the function of PI3K in cellular responses. Based on its inhibitory effect on PI3K, LY294002 has been shown to exert antitumorigenic effect in vivo and in vitro. Here, we report that LY294002 alters early growth response 1 (EGR-1) phosphorylation and subsequently enhances activating transcription factor 3 (ATF3) expression independently of PI3K inhibition. This pathway may be, in part, responsible for the antitumorigenic effect of LY294002 in human colorectal cancer cells. ATF3 expression was increased by LY294002, followed by the induction of apoptosis in several colorectal cancer cell lines. This is consistent with results showing that the down-regulation of the ATF3 gene by small interfering RNA suppressed LY294002-induced apoptosis in HCT-116 cells. On the other hand, ATF3 expression was not affected by another PI3K inhibitor, wortmannin, as well as phosphatase and tensin homologue or dominant-negative Akt overexpression. We also found that LY294002 increases ATF3 promoter activity and the transactivation is partly mediated by a GC-rich sequence located in the promoter. EGR-1 binds to the ATF3 promoter as assessed by gel shift assay. Furthermore, phosphorylated EGR-1 was highly increased in LY294002-treated cells, indicating that EGR-1 phosphorylation induced by LY294002 may facilitate ATF3 transactivation. Our data suggest that EGR-1 acts as a mediator in LY294002-induced ATF3 expression via a PI3K-independent pathway. ATF3 and EGR-1 may provide a novel explanation for the antitumorigenic properties of LY294002 in human colorectal cancer cells.

Activating Transcription Factor 3↗

PubMed2005

Destabilization of Krüppel-like factor 4 protein in response to serum stimulation involves the ubiquitin-proteasome pathway.

Although the zinc finger transcription factor Krüppel-like factor 4 (KLF4) has been shown to be a negative regulator of cell proliferation, the mechanisms underlying the posttranslational modification of KLF4, especially at the level of protein degradation, are poorly understood. Here, we show that KLF4 protein levels in quiescent cells were high, but decreased rapidly as cells entered the proliferating stage following serum stimulation. This decrease was partially reversed by pretreatment with MG132, a proteasome inhibitor. Moreover, KLF4 was an unstable protein that underwent rapid turnover, and exhibited a relatively short half-life (t1/2 approximately 120 minutes). To investigate the involvement of the ubiquitin-proteasome pathway in the regulation of the stability of KLF4, HCT116 cells were treated with proteasome inhibitors. Our results showed that, following lactacystin treatment, levels of endogenous KLF4 increased in a time- and dose-dependent manners. Using a cell-free system, in vitro-translated 35S-labeled KLF4 protein was degraded by protein extracts prepared from exponentially growing HCT116 cells in the presence of ATP. These effects were prevented by pretreatment with MG132 or replacement of ATP with ATP-gamma-S, a nonhydrolyzable analogue of ATP, suggesting that ATP is required for KLF4 degradation by the 26S proteasome. In addition, KLF4 was subject to ubiquitination when cells were treated with the proteasome inhibitor or transfected with exogenous ubiquitin. Collectively, these results indicate that destabilization of KLF4 following serum stimulation is mediated, at least in part, through a ubiquitin-proteasome pathway.

Adenosine Triphosphate↗

PubMed2006

Silencing of bidirectional promoters by DNA methylation in tumorigenesis.

CpG island methylation within promoters is known to silence individual genes in cancer. The involvement of this process in silencing gene pairs controlled by bidirectional promoters is unclear. In a screen for hypermethylated CpG islands in cancer, bidirectional promoters constituted 25.2% of all identified promoters, which matches with the genomic representation of bidirectional promoters. From the screen, we selected three bidirectional gene pairs for detailed analysis, WNT9A/CD558500, CTDSPL/BC040563, and KCNK15/BF195580. Levels of mRNA of all three pairs of genes were inversely correlated with the degree of promoter methylation in multiple cancer cell lines. Hypomethylation of these promoters induced by 5-aza-2'-deoxycytidine treatment reactivated or enhanced gene expression bidirectionally. The bidirectional nature of the WNT9A/CD558500 promoter was confirmed by luciferase assays, and hypermethylation down-regulated expression of both genes in the pair. Methylation of WNT9A/CD558500 and CTDSPL/BC040563 promoters occurs frequently in primary colon cancers and acute lymphoid leukemias (ALL), respectively, and methylation was correlated with decreased gene expression in ALL patient samples. Our study shows that hypermethylation of bidirectional promoter-associated CpG island silences two genes simultaneously, a property that should be taken into account when studying the functional consequences of hypermethylation in cancer.

Cell Line, Tumor↗

PubMed2006

Integrin alpha4beta1 promotes monocyte trafficking and angiogenesis in tumors.

Monocytes and macrophages extensively colonize solid tumors, where they are thought to promote tumor angiogenesis. Here, we show that integrin alpha4beta1 (VLA4) promotes the invasion of tumors by myeloid cells and subsequent neovascularization. Antagonists of integrin alpha4beta1, but not of other integrins, blocked the adhesion of monocytes to endothelium in vitro and in vivo as well as their extravasation into tumor tissue. These antagonists prevented monocyte stimulation of angiogenesis in vivo, macrophage colonization of tumors, and tumor angiogenesis. These studies indicate the usefulness of antagonists of integrin alpha4beta1 in suppressing macrophage colonization of tumors and subsequent tumor angiogenesis. These studies further indicate that suppression of myeloid cell homing to tumors could be a useful supplementary approach to suppress tumor angiogenesis and growth.

Animals↗

PubMed2006

Tumor-derived matrix metalloproteinase-1 targets endothelial proteinase-activated receptor 1 promoting endothelial cell activation.

In the vascular system, circulating tumor cells interact with endothelial cells. Tumor-endothelial cross-talk transforms the intravascular milieu to a prothrombotic, proinflammatory, and cell-adhesive state called endothelial cell activation (ECA). In the present study, we analyze the potential of metastatic tumor-derived soluble factors to transform the vascular endothelium into a prothrombotic and proinflammatory activated state. Supernatant from cultured melanoma and colon cancer cells (A375, WM9, A7, and HT-29) induced an acute activation of macrovascular and microvascular endothelial cells (human umbilical vein endothelial cells and human dermal microvascular endothelial cells) as shown by intracellular calcium flux and secretion of von Willebrand factor and interleukin-8, all markers of acute ECA. This process was inhibited using specific proteinase-activated receptor 1 (PAR1) inhibitors (RWJ-58259 and SCH-79797), indicating a mediating role for endothelial thrombin receptors. Immunofluorescence, Western blot analysis, and collagenase activity assay of tumor cells and culture supernatant revealed the presence of matrix metalloproteinase-1 (MMP-1), a recently described activator of PAR1. Inhibition of MMP-1 in supernatant from cultured tumor cells significantly attenuated ECA. Additional studies using isolated human MMP-1 (5 nmol/L) proved the presence of a functional MMP-1/PAR1 axis in tumor-endothelial communication. These findings show a new pathway of tumor-endothelial cross-talk via an intravascular MMP1/PAR1 axis in microvascular and macrovascular endothelium. Inhibition of this cross-talk may be a powerful means to prevent tumor-induced ECA and thus thrombotic and inflammatory cell adhesion.

Animals↗

PubMed2006

KRAS(D13) Promotes apoptosis of human colorectal tumor cells by ReovirusT3D and oxaliplatin but not by tumor necrosis factor-related apoptosis-inducing ligand.

Colorectal tumors frequently contain activating mutations in KRAS. ReovirusT3D is an oncolytic virus that preferentially kills tumor cells with an activated Ras pathway. Here we have assessed the contribution of endogenous mutant KRAS in human colorectal cancer cell lines to ReovirusT3D replication and to tumor cell oncolysis. In addition, treatment combinations involving ReovirusT3D, oxaliplatin, and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) were tested for their efficacy in tumor cell killing. The mutation status of KRAS did not predict the sensitivity of a panel of human colorectal cancer cell lines to ReovirusT3D. Virus replication was observed in all cell lines tested regardless of KRAS status and was not affected by deletion of endogenous mutant KRAS(D13). However, deletion of KRAS(D13) or p53 did reduce apoptosis induction by ReovirusT3D whereas deletion of beta-catenin(DeltaS45) had no effect. Likewise, KRAS(D13)- or p53-deficient cells display reduced sensitivity to oxaliplatin but not to death receptor activation by TRAIL. Finally, the treatment of colorectal cancer cells with ReovirusT3D combined with either oxaliplatin or TRAIL resulted in a nonsynergistic increase in tumor cell killing. We conclude that oncolysis of human tumor cells by ReovirusT3D is not determined by the extent of virus replication but by their sensitivity to apoptosis induction. Oncogenic KRAS(D13) increases tumor cell sensitivity to activation of the cell-intrinsic apoptosis pathway without affecting ReovirusT3D replication.

Apoptosis↗

PubMed2006

Direct transcriptional up-regulation of cyclooxygenase-2 by hypoxia-inducible factor (HIF)-1 promotes colorectal tumor cell survival and enhances HIF-1 transcriptional activity during hypoxia.

Cyclooxygenase (COX)-2, the inducible key enzyme for prostanoid biosynthesis, is overexpressed in most colorectal carcinomas and a subset of colorectal adenomas. Genetic, biochemical, and clinical evidence indicates an important role for COX-2 in colorectal tumorigenesis. Although COX-2 can be induced by aberrant growth factor signaling and oncogene activation during colorectal tumorigenesis, the role of microenvironmental factors such as hypoxia in COX-2 regulation remains to be elucidated. For the first time, we report that under hypoxic conditions COX-2 protein levels increase in colorectal adenoma and carcinoma cells. Rigorous analyses reveal that COX-2 up-regulation is transcriptional and is associated with hypoxia-inducible factor (HIF)-1alpha induction. Oligonucleotide pull-down and chromatin immunoprecipitation assays reveal that HIF-1alpha binds a hypoxia-responsive element on the COX-2 promoter. COX-2 up-regulation during hypoxia is accompanied by increased levels of prostaglandin E(2) (PGE(2)), which promote tumor cell survival under hypoxic conditions. In addition, elevated levels of PGE(2) in hypoxic colorectal tumor cells enhance vascular endothelial growth factor expression and HIF-1 transcriptional activity by activating the mitogen-activated protein kinase pathway, showing a potential positive feedback loop that contributes to COX-2 up-regulation during hypoxia. This study identifies COX-2 as a direct target for HIF-1 in colorectal tumor cells. In addition, COX-2 up-regulation represents a pivotal cellular adaptive response to hypoxia with implication for colorectal tumor cell survival and angiogenesis. We propose that using modified COX-2-selective inhibitors, which are only activated under hypoxic conditions, could potentially be a novel more selective strategy for colorectal cancer prevention and treatment.

Base Sequence↗

PubMed2006

Repression of prostaglandin dehydrogenase by epidermal growth factor and snail increases prostaglandin E2 and promotes cancer progression.

Prostaglandin E(2) (PGE(2)), a proinflammatory bioactive lipid, promotes cancer progression by modulating proliferation, apoptosis, and angiogenesis. PGE(2) is a downstream product of cyclooxygenase (COX) and is biochemically inactivated by prostaglandin dehydrogenase (PGDH). In the present study, we investigated the mechanisms by which PGDH is down-regulated in cancer. We show that epidermal growth factor (EGF) represses PGDH expression in colorectal cancer cells. EGF receptor (EGFR) signaling induces Snail, which binds conserved E-box elements in the PGDH promoter to repress transcription. Induction of PGE(2) catabolism through inhibition of EGFR signaling blocks cancer growth in vivo. In human colon cancers, elevated Snail expression correlates well with down-regulation of PGDH. These data indicate that PGDH may serve a tumor suppressor function in colorectal cancer and provide a possible COX-2-independent way to target PGE(2) to inhibit cancer progression.

Animals↗

PubMed2005

Blockade of paracrine supply of insulin-like growth factors using neutralizing antibodies suppresses the liver metastasis of human colorectal cancers.

Environmental stimuli, such as organ-specific growth factors, can influence the metastatic potential of a tumor. The liver is the main source of insulin-like growth factors (IGFs). The importance of IGF signal in hepatic metastasis has been clarified mainly by IGF-I receptor targeting strategies. This study aims to confirm these precedent reports by novel tool, neutralizing antibodies against IGFs and to show that IGFs are promising therapeutic targets for hepatic metastasis in vivo. Hepatic metastasis was induced by intrasplenic injection of human colorectal cancer cell line, HT29. The antimetastatic effects of three antibodies (anti-mouse IGF-I, anti-mouse IGF-II, and anti-human/mouse IGF-II designated KM1468) were tested singly or in combination in the early phase of metastasis. The dose escalation effect of KM1468 and its survival benefit were examined in the early and late phases of metastasis. The mechanism of IGF neutralization was investigated with immunohistochemistry. Dual neutralization of paracrine IGF-I and IGF-II showed modest additive antimetastatic effects than single neutralization of IGF-I or IGF-II. In any phase of metastasis, neutralization led to significant tumor growth inhibition and longer survival. Dose escalation of KM1468 influenced survival only in the late phase of metastasis. Apoptosis increased significantly in the antibody-treated group compared with the control group (P = 0.0025) In conclusion, IGFs are promising therapeutic targets for hepatic metastases of colorectal cancers. However, the IGF dependency is probably variable in the metastatic process.

Animals↗

PubMed2005

The stress-responsive gene GADD45G is a functional tumor suppressor, with its response to environmental stresses frequently disrupted epigenetically in multiple tumors.

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2'-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkin's lymphoma, 3 of 6 (50%) Hodgkin's lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2'-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

Antigens, Differentiation↗

PubMed2006

Irinotecan inactivation is modulated by epigenetic silencing of UGT1A1 in colon cancer.

PURPOSE: Irinotecan is used in the first-line treatment of metastatic colorectal cancer. The UGT1A1-metabolizing enzyme, expressed in liver and colon, is primarily involved in the inactivation of its active metabolite 7-ethyl-10-hydroxycamptothecin (SN-38). Herein, we explored the role of DNA methylation in the silencing of UGT1A1 gene expression in colon cancer and its influence on cellular SN-38 detoxification. EXPERIMENTAL DESIGN AND RESULTS: UGT1A1 mRNA was repressed in most primary tumors (41 of 50; 82%) and in three colon cancer cell lines (HCT-116, HCT-15, and COLO-320DM). Bisulfite sequencing of the UGT1A1 gene revealed the aberrant methylation of specific CpG islands in UGT1A1-negative cells. Conversely, hypomethylation was observed in HT-29, HT-115, and LOVO cells that overexpress UGT1A1. Direct methylation of the UGT1A1 promoter resulted in the complete repression of transcriptional activity. Treatment with demethylating and histone deacetylase inhibitor agents had the capacity to reverse aberrant hypermethylation and to restore UGT1A1 expression in hypermethylated UGT1A1-negative cells but not in hypomethylated cells. Loss of UGT1A1 methylation was further associated with an increase in UGT1A1 protein content and with an enhanced inactivation of SN-38 by 300% in HCT-116 cells. CONCLUSIONS: We conclude that DNA methylation represses UGT1A1 expression in colon cancer and that this process may contribute to the level of tumoral inactivation of the anticancer agent SN-38 and potentially influence clinical response.

Azacitidine↗

PubMed2006

Beta-escin inhibits colonic aberrant crypt foci formation in rats and regulates the cell cycle growth by inducing p21(waf1/cip1) in colon cancer cells.

Extracts of Aesculus hippocastanum (horse chestnut) seed have been used in the treatment of chronic venous insufficiency, edema, and hemorrhoids. Most of the beneficial effects of horse chestnut are attributed to its principal component beta-escin or aescin. Recent studies suggest that beta-escin may possess anti-inflammatory, anti-hyaluronidase, and anti-histamine properties. We have evaluated the chemopreventive efficacy of dietary beta-escin on azoxymethane-induced colonic aberrant crypt foci (ACF). In addition, we analyzed the cell growth inhibitory effects and the induction of apoptosis in HT-29 human colon cancer cell line. To evaluate the inhibitory properties of beta-escin on colonic ACF, 7-week-old male F344 rats were fed experimental diets containing 0%, 0.025%, or 0.05% beta-escin. After 1 week, the rats received s.c. injections of azoxymethane (15 mg/kg body weight, once weekly for 2 weeks) or an equal volume of normal saline (vehicle). Rats were continued on respective experimental diets and sacrificed 8 weeks after the azoxymethane treatment. Colons were evaluated histopathologically for ACF. Administration of dietary 0.025% and 0.05% beta-escin significantly suppressed total colonic ACF formation up to approximately 40% (P < 0.001) and approximately 50% (P < 0.0001), respectively, when compared with control diet group. Importantly, rats fed beta-escin showed dose-dependent inhibition (approximately 49% to 65%, P < 0.0001) of foci containing four or more aberrant crypts. To understand the growth inhibitory effects, HT-29 human colon carcinoma cell lines were treated with various concentrations of beta-escin and analyzed by flow cytometry for apoptosis and cell cycle progression. Beta-escin treatment in HT-29 cells induced growth arrest at the G1-S phase, which was associated with the induction of the cyclin-dependent kinase inhibitor p21(WAF1/CIP1), and this correlated with reduced phosphorylation of retinoblastoma protein. Results also indicate that beta-escin inhibited growth of colon cancer cells with either wild-type or mutant p53. This novel feature of beta-escin, a triterpene saponin, may be a useful candidate agent for colon cancer chemoprevention and treatment.

Aesculus↗

PubMed2006

Rapamycin synergizes with the epidermal growth factor receptor inhibitor erlotinib in non-small-cell lung, pancreatic, colon, and breast tumors.

The receptor for epidermal growth factor (EGFR) is overexpressed in many cancers. One important signaling pathway regulated by EGFR is the phosphatidylinositol 3'-kinase (PI3K)-phosphoinositide-dependent kinase 1-Akt pathway. Activation of Akt leads to the stimulation of antiapoptotic pathways, promoting cell survival. Akt also regulates the mammalian target of rapamycin (mTOR)-S6K-S6 pathway to control cell growth in response to growth factors and nutrients. Recent reports have shown that the sensitivity of non-small-cell lung cancer cell lines to EGFR inhibitors such as erlotinib (Tarceva, OSI Pharmaceuticals) is dependent on inhibition of the phosphatidylinositol 3'-kinase-phosphoinositide-dependent kinase 1-Akt-mTOR pathway. There can be multiple inputs to this pathway as activity can be regulated by other receptors or upstream mutations. Therefore, inhibiting EGFR alone may not be sufficient for substantial inhibition of all tumor cells, highlighting the need for multipoint intervention. Herein, we sought to determine if rapamycin, an inhibitor of mTOR, could enhance erlotinib sensitivity for cell lines derived from a variety of tissue types (non-small-cell lung, pancreatic, colon, and breast). Erlotinib could inhibit extracellular signal-regulated kinase, Akt, and S6 only in cell lines that were the most sensitive. Rapamycin could fully inhibit S6 in all cell lines, but this was accompanied by activation of Akt phosphorylation. However, combination with erlotinib could down-modulate rapamycin-stimulated Akt activity. Therefore, in select cell lines, inhibition of both S6 and Akt was achieved only with the combination of erlotinib and rapamycin. This produced a synergistic effect on cell growth inhibition, observations that extended in vivo using xenograft models. These results suggest that combining rapamycin with erlotinib might be clinically useful to enhance response to erlotinib.

Animals↗

PubMed2006

Tautomycetin inhibits growth of colorectal cancer cells through p21cip/WAF1 induction via the extracellular signal-regulated kinase pathway.

Tautomycetin is an antifungal antibiotic retaining potent immunosuppressive function. We have identified the roles of tautomycetin on cellular proliferation and transformation of colorectal cancer cells. The proliferation and anchorage-independent growth of HCT-15, HT-29, and DLD-1 colorectal cancer cells were efficiently inhibited without induction of apoptosis by 150 nmol tautomycetin. These growth inhibitory effects were dependent on p21Cip/WAF induction via the extracellular signal-regulated kinase pathway, and the tautomycetin effects were abolished in HCT-116 colon cells and eight other types of cells that did not induce p21Cip/WAF by 150 nmol tautomycetin. The crucial role of p21Cip/WAF1 in the extracellular signal-regulated kinase pathway-dependent antiproliferative responses by tautomycetin was confirmed by using p21Cip/WAF1 gene-deleted HCT-116 cells. The growth inhibitory effect of tautomycetin was acquired by regulation of Raf-1 activity through inhibition of protein phosphatase type 1 and protein phosphatase type 2A with high preference toward protein phosphatase type 1. Tautomycetin could be a potential drug for colorectal cancer.

Animals↗

PubMed2006

Comparative effects of RRR-alpha- and RRR-gamma-tocopherol on proliferation and apoptosis in human colon cancer cell lines.

BACKGROUND: Mediterranean societies, with diets rich in vitamin E isoforms, have a lower risk for colon cancer than those of northern Europe and the Americas. Vitamin E rich diets may neutralize free radicals generated by fecal bacteria in the gut and prevent DNA damage, but signal transduction activities can occur independent of the antioxidant function. The term vitamin E represents eight structurally related compounds, each differing in their potency and mechanisms of chemoprevention. The RRR-gamma-tocopherol isoform is found primarily in the US diet, while RRR-alpha-tocopherol is highest in the plasma. METHODS: The effectiveness of RRR-alpha- and RRR-gamma-tocopherol at inhibiting cell growth and inducing apoptosis in colon cancer cell lines with varying molecular characteristics (SW480, HCT-15, HCT-116 and HT-29) and primary colon cells (CCD-112CoN, nontransformed normal phenotype) was studied. Colon cells were treated with and without RRR-alpha- or RRR-gamma-tocopherol using varying tocopherol concentrations and time intervals. Cell proliferation and apoptosis were measured using the trypan blue assay, annexin V staining, DNA laddering and caspase activation. RESULTS: Treatment with RRR-gamma-tocopherol resulted in significant cell death for all cancer cell lines tested, while RRR-alpha-tocopherol did not. Further, RRR-gamma-tocopherol treatment showed no cytotoxicity to normal colon cells CCD-112CoN at the highest concentration and time point tested. RRR-gamma-tocopherol treatment resulted in cleavage of PARP, caspase 3, 7, and 8, but not caspase 9. Differences in the percentage cell death and apoptosis were observed in different cell lines suggesting that molecular differences in these cell lines may influence the ability of RRR-gamma-tocopherol to induce cell death. CONCLUSION: This is the first study to demonstrate that multiple colon cancer cell lines containing varying genetic alterations will under go growth reduction and apoptosis in the presence of RRR-gamma-tocopherol without damage to normal colon cells. The amount growth reduction was dependent upon the molecular signatures of the cell lines. Since RRR-gamma-tocopherol is effective at inhibition of cell proliferation at both physiological and pharmacological concentrations dietary RRR-gamma-tocopherol may be chemopreventive, while pharmacological concentrations of RRR-gamma-tocopherol may aid chemotherapy without toxic effects to normal cells demonstrated by most chemotherapeutic agents.

Antioxidants↗

PubMed2006

Localization of StarD5 cholesterol binding protein.

Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.

Adaptor Proteins, Vesicular Transport↗

PubMed2006

Mechanisms of curcumin- and EGF-receptor related protein (ERRP)-dependent growth inhibition of colon cancer cells.

Numerous dietary and pharmacological agents have been proposed as alternative strategies for treatment and prevention of colorectal cancer. Curcumin, an active ingredient of turmeric, that inhibits growth of malignant neoplasms, has a promising role in the prevention and treatment of colorectal cancer. EGF-R related protein (ERRP), a recently identified pan-erbB inhibitor, is a potential therapeutic agent for colorectal cancer. Here we examine whether curcumin together with ERRP will cause a greater inhibition of growth of colon cancer cells than either agent alone and the mechanisms of this inhibition. Human colon cancer HCT-116 or HT-29 cells were incubated with increasing doses of curcumin (up to 10 microM) or ERRP (up to 5 microg/ml), or a combination of both for 48 h. We observed that the cell growth inhibition and stimulation of apoptosis in response to the combinatorial treatment was significantly greater than that caused by either agent alone. These changes were associated with decreased activation (tyrosine phosphorylation) of EGFR, ErbB-2, ErbB-3, and/or IGF-1R. Whereas curcumin inhibited constitutive activation of both EGFR and IGF-1R, ERRP decreased activation of EGFR, ErbB-2, and ErbB-3 but had no effect on IGF-1R. Further, the combination therapy caused a greater attenuation of downstream effectors such as NF-kappaB, Akt and BAD activation, and down-regulation of procaspase-3 than that noted with either agent alone. The superior effects of the combinatorial treatment could partly be attributed to inhibition of constitutive activation of EGFRs and IGF-1R signaling pathways.

Antineoplastic Agents↗