Abstract: Recurrence of colon cancer, which affects nearly 50% of patients treated by conventional therapeutics, is thought to be due to re-emergence of chemotherapy-resistant cancer stem/stem-like cells (CSCs). Therefore, development of therapeutic strategies for targeted elimination of CSCs would be a novel strategy. The current study examines whether difluorinated-curcumin (CDF), a novel analog of the dietary ingredient of curcumin, in combination with 5-fluorouracil and oxaliplatin (5-FU + Ox), the mainstay of colon cancer chemotherapeutic, would be effective in eliminating colon CSCs.
Abstract: Colorectal cancer is the third most common form of malignancy, behind prostate and lung cancers. Despite recent advances in medicine, mortality from colorectal cancer remains high, highlighting the need for improved therapies. Numerous studies have demonstrated increased activation of EGFR and its family members (EGFRs), IGF-1R as well as c-Src in colorectal cancer. The current study was undertaken to examine the effectiveness of combination therapy of dasatinib (BMS-354825; Bristol-Myers Squibb), a highly specific inhibitor of Src family kinases (SFK) and a nontoxic dietary agent; curcumin (diferuloylmethane), in colorectal cancer in in vitro and in vivo experimental models. For the latter, we utilized C57BL/6 APC(Min+/-) mice. Initial in vitro studies revealed synergistic interactions between the two agents. Additionally, we have observed that combination treatment causes a much greater inhibition of the following metastatic processes than either agent alone: (i) colony formation, (ii) invasion through extracellular matrix and (iii) tubule formation by endothelial cells. Dasatinib affects the cell adhesion phenotype of colon cancer HCT-116 cells whereas the combination therapy enhances this effect to a greater extent. Preclinical investigation revealed that the combination therapy to be highly effective causing an over 95% regression of intestinal adenomas in Apc(Min+/-) mice, which could be attributed to decreased proliferation and increased apoptosis. In conclusion, our data suggest that combination treatment of dasatinib and curcumin could be a potential therapeutic strategy for colorectal cancer.
Abstract: Recent evidence suggests that epithelial cancers, including colorectal cancer are driven by a small sub-population of self-renewing, multi-potent cells termed cancer stem cells (CSCs) which are thought to be responsible for recurrence of cancer. One of the characteristics of CSCs is their ability to form floating spheroids under anchorage-independent conditions in a serum-free defined media. The current investigation was undertaken to examine the role of Wnt/beta-catenin pathway in regulating the growth and maintenance of colonospheres. Human colon cancer cells HCT-116 (p53 wild type; K-ras mutant), HCT-116 (p53 null; K-ras mutant) and HT-29 (p53 mutant) were used.
Abstract: The intestinal epithelium is subjected to repetitive deformation during normal gut function by peristalsis and villous motility. In vitro, cyclic strain promotes intestinal epithelial proliferation and induces an absorptive phenotype characterized by increased dipeptidyl dipeptidase (DPPIV) expression. Schlafen 3 is a novel gene recently associated with cellular differentiation. We sought to evaluate whether Schlafen 3 mediates the effects of strain on the differentiation of intestinal epithelial cell (IEC)-6 in the absence or presence of cyclic strain. Strain increased Schlafen 3 mRNA and protein. In cells transfected with a control-nontargeting siRNA, strain increased DPPIV-specific activity. However, Schlafen 3 reduction by siRNA decreased basal DPPIV and prevented any stimulation of DPPIV activity by strain. Schlafen 3 reduction also prevented DPPIV induction by sodium butyrate (1 mM) or transforming growth factor (TGF)-beta (0.1 ng/ml), two unrelated differentiating stimuli. However, Schlafen-3 reduction by siRNA did not prevent the mitogenic effect of strain or that of EGF. Blocking Src and phosphatidyl inositol (PI3)-kinase prevented strain induction of Schlafen 3, but Schlafen 3 induction required activation of p38 but not ERK. These results suggest that cyclic strain induces an absorptive phenotype characterized by increased DPPIV activity via Src-, p38-, and PI3-kinase-dependent induction of Schlafen 3 in rat IEC-6 cells on collagen, whereas Schlafen 3 may also be a key factor in the induction of intestinal epithelial differentiation by other stimuli such as sodium butyrate or TGF-beta. The induction of Schlafen 3 or its human homologs may modulate intestinal epithelial differentiation and preserve the gut mucosa during normal gut function.
Abstract: Curcumin (diferuloylmethane), which has no discernible toxicity, inhibits initiation, promotion and progression of carcinogenesis. 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but produces an incomplete response resulting in survival of cells (chemo-surviving cells) that may lead to cancer recurrence. The present investigation was, therefore, undertaken to examine whether addition of curcumin to FOLFOX is a superior therapeutic strategy for chemo-surviving cells. Forty-eight-hour treatment of colon cancer HCT-116 and HT-29 cells with FOLFOX resulted in 60-70% survival, accompanied by a marked activation of insulin like growth factor-1 receptor (IGF-1R) and minor to moderate increase in epidermal growth factor receptor (EGFR), v-erb-b2 erythroblastic leukemia viral oncogene homolog 2 (HER-2) as well as v-akt murine thymoma viral oncogene homolog 1 (AKT), cyclooxygenase-2 (COX-2) and cyclin-D1. However, inclusion of curcumin to continued FOLFOX treatment for another 48 h greatly reduced the survival of these cells, accompanied by a concomitant reduction in activation of EGFR, HER-2, IGF-1R and AKT, as well as expression of COX-2 and cyclin-D1. More importantly, EGFR tyrosine kinase inhibitor gefitinib or attenuation of IGF-1R expression by the corresponding si-RNA caused a 30-60% growth inhibition of chemo-surviving HCT-116 cells. However, curcumin alone was found to be more effective than both gefitinib and IGF-1R si-RNA mediated growth inhibition of chemo-surviving HCT-116 cells and addition of FOLFOX to curcumin did not increase the growth inhibitory effect of curcumin. Our data suggest that inclusion of curcumin in conventional chemotherapeutic regimens could be an effective strategy to prevent the emergence of chemoresistant colon cancer cells.
Abstract: Many solid tumors, including breast cancer, show increased activation of several growth factor receptors, specifically epidermal growth factor receptor (EGFR) and its family members as well as c-Src, a nonreceptor tyrosine kinase that promotes proliferation, inhibits apoptosis, and induces metastasis. We hypothesize that inhibition of c-Src and EGFRs will be an effective therapeutic strategy for triple-negative breast cancer. To test our hypothesis, we used a c-Src-specific inhibitor dasatinib (BMS-354825; Bristol-Myers Squibb) and our newly developed ErbB-inhibitory protein (EBIP), a potential pan-ErbB inhibitor, in breast cancer cells. EBIP is composed of 1 to 448 amino acids of the ectodomain of human EGFR to which the 30-amino acid epitope (known as "U" region) of rat EGFR-related protein is fused at the COOH-terminal end. The combination of dasatinib and EBIP was found to be highly effective in inhibiting the growth of four different breast cancer cells (MDA-MB-468, SKBr-3, MDA-MB-453, and MDA-MB-231) that express different levels of EGFRs. In EGFR-overexpressing MDA-MB-468 cells, the combination, but not monotherapy, markedly stimulated apoptosis mediated by caspase-9 and caspase-8 and attenuated activation of EGFR and Src as well as tyrosine kinase activity. EBIP also inhibited heregulin-induced activation of HER-2 and HER-3 in MDA-MB-453 breast cancer cells. The combination therapy was highly effective in suppressing tumor growth ( approximately 90% inhibition) in MDA-MB-468-derived xenografts in severe combined immunodeficient mice. The latter could be attributed to induction of apoptosis. We conclude that combining dasatinib and EBIP could be an effective therapeutic strategy for breast cancer by targeting EGFRs and Src signaling.
Abstract: Schlafen-3 (Slfn-3), a novel gene, has been shown to be a negative regulator of proliferation. The current investigation was undertaken to determine whether Slfn-3 might play a role in regulating cellular differentiation. Butyric acid, a short chain fatty acid, which induced differentiation of intestinal cells as evidenced by increased alkaline phosphatase (ALP) activity in the rat small intestinal IEC-6 cells, also produced a marked increase in Slfn-3 expression. Furthermore, overexpression of Slfn-3 caused stimulation of ALP activity in IEC-6 cells, which was exacerbated by butyrate. On the other hand, downregulation of Slfn-3 by slfn-3-si-RNA greatly attenuated the butyrate-mediated induction of differentiation of IEC-6 cells. Additionally, we observed that increased expression of Slfn-3 in colon cancer HCT-116 cells stimulated TGF-beta expression and modulated expression of its downstream effectors as evidenced by increased expression of p27kip1 and downregulation of CDK-2. In addition, Slfn-3 increases E-cadherin expression but downregulates beta-catenin. In conclusion, our data show that Slfn-3 plays a critical role in regulating intestinal mucosal differentiation. Furthermore our data also show that TGF-beta signaling pathway plays an important role in mediating slfn-3 induced differentiation.
Abstract: It is becoming increasingly evident that cancer stem cells play a vital role in development and progression of cancers and relapse following chemotherapy. The present study examines the presence of cancer stem-like cells (CSC) in adenomatous polyps and in normal appearing colonic mucosa in humans during aging. The number of polyps was found to increase linearly with advancing age (r(2)=0.92, p<0.02). Immunohistochemical analysis revealed co-localization of CSC markers CD44 and CD166 in colonic polyps. Real-time RT-PCR analysis of normal appearing mucosa from subjects with adenomatous polyps showed an age-related rise in CSC as evidenced by the increased expression of CD44, CD166 and ESA. A similar phenomenon was also observed for EGFR. In addition, the expression each CSC marker was found to be about 2-fold higher in subjects with 3-4 polyps than those with 1-2 polyps. In conclusion, our results show that colon cancer stem-like cells are present in the premalignant adenomatous polyps as well in normal appearing colonic mucosa. Moreover, our observation of the age-related rise in CSC in macroscopically normal colonic mucosa suggests a predisposition of the organ to developing colorectal cancer.
Abstract: Although aging is associated with increased proliferation and decreased apoptosis in the colonic mucosa of Fischer 344 rats, the regulatory mechanisms are poorly understood. Gene expression profiling (Illumina platform) was carried out in freshly isolated colonic mucosal cells from young (4-6 mo old) and aged (22-24 mo old) Fischer 344 rats. Sixty-six genes were differentially expressed in the colonic mucosa between young and old animals (P<0.05). In particular, the expression of schlafen 3, a negative regulator of proliferation, was decreased by 8- to 10-fold in the colonic mucosa of aged rats. Administration of wortmannin, which inhibited colonic mucosal proliferation in the colonic mucosa of aged rats, stimulated the expression of schlafen 3, indicating a growth regulatory role of this gene. To further determine the growth regulatory properties of schlafen 3 gene, schlafen 3 cDNA was transfected in colon cancer HCT-116 cells. This resulted in a 30-40% inhibition of cellular growth, accompanied by decreased expression of PCNA and cyclin D1 and reduced phosphorylation of retinoblastoma protein. In conclusion, our present study demonstrates that several genes involved in proliferation and apoptosis are differentially expressed in the colonic mucosa of young and aged rats. Schlafen 3, a novel negative regulator of growth, which is markedly downregulated in the colonic mucosa of the aged, may play a role in regulating colonic mucosal growth during aging.
Abstract: 5-Fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics but with limited success. This could partly be due to the enrichment of cancer stem cells (CSCs) that are resistant to conventional chemotherapy. Therefore, validation of a nontoxic agent that can either cause reversal of chemoresistance or promote the killing of CSCs would be highly desirable. The current study examines whether curcumin, the major active ingredient of turmeric, either alone or together with FOLFOX, would be an effective strategy to eliminate colon CSCs. Exposure of colon cancer HCT-116 or HT-29 cells to FOLFOX that inhibited their growth led to the enrichment of CSC phenotype as evidenced by increased proportion of CD133-, CD44-, and/or CD166-positive cells and epidermal growth factor receptor (EGFR) levels. Treatment of FOLFOX-surviving colon cancer cells with either curcumin alone or together with FOLFOX resulted in a marked reduction in CSCs, as evidenced by the decreased expression of CD44 and CD166 as well as EGFR and by their ability to form anchorage-dependent colonies. They also caused disintegration of colonospheres. Increased expression of EGFR in FOLFOX-surviving cells could be attributed to hypomethylation of the EGFR promoter, whereas an opposite phenomenon was observed when the FOLFOX-surviving cells were treated with curcumin and/or FOLFOX. These changes were accompanied by parallel alterations in the levels of DNA methyltransferase 1. In conclusion, our data suggest that curcumin by itself or together with the conventional chemotherapeutic could be an effective treatment strategy for preventing the emergence of chemoresistant colon cancer cells by reducing/eliminating CSCs.
Abstract: Development and progression of many malignancies, including colorectal cancer, are associated with activation of multiple signaling pathways. Therefore, inhibition of these signaling pathways with noncytotoxic natural products represents a logical preventive and/or therapeutic approach for colon cancer. Curcumin and resveratrol, both of which inhibit the growth of transformed cells and colon carcinogenesis, were selected to examine whether combining them would be an effective preventive and/or therapeutic strategy for colon cancer. Indeed, the combination of curcumin and resveratrol was found to be more effective in inhibiting growth of p53-positive (wt) and p53-negative colon cancer HCT-116 cells in vitro and in vivo in SCID xenografts of colon cancer HCT-116 (wt) cells than either agent alone. Analysis by Calcusyn software showed synergism between curcumin and resveratrol. The inhibition of tumors in response to curcumin and/or resveratrol was associated with the reduction in proliferation and stimulation of apoptosis accompanied by attenuation of NF-kappaB activity. In vitro studies have further demonstrated that the combinatorial treatment caused a greater inhibition of constitutive activation of EGFR and its family members as well as IGF-1R. Our current data suggest that the combination of curcumin and resveratrol could be an effective preventive/therapeutic strategy for colon cancer.
Abstract: Because the colonic mucosa is in direct contact with digesta, luminal exposure to potentially carcinogenic or chemopreventive agents may be important in colorectal carcinogenesis, independently of the effects of systemic exposure through the circulation. To date, few biomarkers for luminal exposure have been identified, and isolation of reasonably good quality fecal human RNA remains difficult. In this study, we assessed the yield and quality of RNA extracted from 10 human stool samples after storage with several commercially available preservatives compared with stool samples immediately frozen in liquid nitrogen. This study shows that careful design of primer pairs which amplify a short length of DNA is key to obtaining interpretable and reproducible results. Moreover, the use of commercially available RNA preservation kits enables investigators to collect usable fecal samples from large populations. Of all the preservative methods tested, RNAlater had the best performance in terms of overall quality, quantity, and level of genomic DNA contamination, and thus deserves further investigation.
Abstract: Curcumin (diferuloylmethane), which has been shown to inhibit growth of transformed cells, has no discernible toxicity and achieves high levels in colonic mucosa. 5-fluorouracil (5-FU) or 5-FU plus oxaliplatin (FOLFOX) remains the backbone of colorectal cancer chemotherapeutics, but with limited success. The present investigation was, therefore, undertaken to examine whether curcumin in combination with conventional chemotherapeutic agent(s)/regimen will be a superior therapeutic strategy for colorectal cancer. Indeed, results of our in vitro studies demonstrated that curcumin together with FOLFOX produced a significantly greater inhibition (p < 0.01) of growth and stimulated apoptosis (p < 0.001) of colon cancer HCT-116 and HT-29 cells than that caused by curcumin, 5-FU, curcumin + 5-FU or FOLFOX. These changes were associated with decreased expression and activation (tyrosine phosphorylation) of EGFR, HER-2, HER-3 (72-100%) and IGF-1R (67%) as well as their downstream effectors such as Akt and cycloxygenase-2 (51-97%). Furthermore, while these agents produced a 2-3-fold increase in the expression of IGF-binding protein-3 (IGFBP-3), curcumin together with FOLFOX caused a 5-fold increase in the same, when compared to controls. This in turn led to increased sequestration of IGF by IGFBP-3 rendering IGF-1 unavailable for binding to and activation of IGF-1R. We conclude that the superior effects of the combination therapy of curcumin and FOLFOX are due to attenuation of EGFRs and IGF-1R signaling pathways. We also suggest that inclusion of curcumin to the conventional chemotherapeutic agent(s)/regimen could be an effective therapeutic strategy for colorectal cancer.
Abstract: Deregulated signaling by the epidermal growth factor receptor family of proteins is encountered in human malignancies including breast cancer. Cell cycle and apoptosis-regulatory protein-1 (CARP-1), a novel, perinuclear phosphoprotein, is a regulator of apoptosis signaling by epidermal growth factor receptors. CARP-1 expression is diminished in human breast cancers, and correlates inversely with human breast cancer grades which could be attributed to increased methylation. The expression of CARP-1, on the other hand, interferes with the ability of human breast cancer cells to invade through the matrigel-coated membranes, to form colonies in the soft agar, and to grow as s.c. tumors in severe combined immunodeficiency (SCID) mice. To test whether CARP-1 is a suppressor of human breast cancer growth, we generated transactivator of transcription (TAT)-tagged CARP-1 peptides. Treatment of human breast cancer cells with affinity purified, TAT-CARP-1 1-198, 197-454, and 896-1150 peptides caused inhibition of human breast cancer cell proliferation and elevated apoptosis. In contrast, TAT-tagged enhanced green fluorescent protein or CARP-1 (1-198(Y192/F)) peptide failed to inhibit cell proliferation or induce apoptosis. Apoptosis by CARP-1 peptides, with the exception of CARP-1 (1-198(Y192/F)), involves the activation of p38 stress-activated protein kinase and caspase-9. Moreover, administration of TAT-CARP-1 (1-198), but not TAT-tagged enhanced green fluorescent protein or TAT-CARP-1 (1-198(Y192/F)), inhibits growth of human breast cancer cell-derived tumor xenografts in SCID mice. We conclude that CARP-1 is a suppressor of human breast cancer growth, and its expression is diminished in tumors, in part, by methylation-dependent silencing.
Abstract: Although the regulatory mechanisms for the age-related rise in proliferation and reduction in apoptosis in the colonic mucosa are yet to be fully delineated, we have demonstrated that these events are associated with increased expression and activation of epithelial growth factor receptor (EGFR)/ErbB-1 and some of its receptor family members (EGFRs), indicating their involvement in these processes. However, the downstream signaling events of EGFR and/or its family members regulating age-related changes in mucosal proliferation and apoptosis remain to be delineated. Cell cycle and apoptosis regulatory protein-1 (CARP-1), a novel growth signaling regulator that we isolated, participates in EGFR-dependent signaling. In the current investigation, we examined the involvement of CARP-1 in colonic mucosal growth-related processes during aging. We report that the age-related reduction in apoptosis in the colonic mucosa is associated with increased expression and tyrosine phosphorylation of not only EGFR but also ErbB-2 and ErbB-3. In contrast, protein and mRNA levels of CARP-1 as well as tyrosine phosphorylation of CARP-1 are decreased. Additionally, we have observed that administration of wortmannin, an inhibitor of phosphatidylinositol 3-kinase activity that accelerates apoptosis in the colonic mucosa of aged rats, causes a marked increase in expression and tyrosine phosphorylation of CARP-1. The age-related decline in CARP-1 expression could partly be attributed to increased methylation of the CARP-1 promoter. Taken together, our data suggest that not only EGFR but also its other members are involved in regulating colonic mucosal growth during aging and that CARP-1 may play a crucial role in transducing EGFRs signals.
Abstract: Activation of the epidermal growth factor receptor (EGFR) and/or its family member(s) stimulates many processes of carcinogenesis, including cell invasion and the formation of new blood vessels, events that are critically involved in angiogenesis. Interference with the activation of EGFRs, therefore, represents a promising strategy for the development of novel and selective anticancer therapies. Previously, we reported that EGFR-related protein (ERRP), which we have isolated and characterized as a pan-erbB inhibitor, is a potential therapeutic agent for colorectal and other epithelial cancers. The present investigation was undertaken to determine whether ERRP would affect the invasion of colon cancer cells and formation of tubules, and the regulation of these processes. ERRP inhibited tubule formation by aortic endothelial cells and invasion of HCT-116 colon cancer cells through matrigel. These changes were associated with marked reductions in the synthesis and secretion of bFGF, VEGF and TGF-alpha by HCT-116 cells. Secretion of bFGF and VEGF by aortic endothelial cells was also inhibited by ERRP. Microarray analysis of ERRP-treated HCT-116 cells showed reduced levels of several growth regulatory proteins such as p21Rac1, Stratifin (14-3-3 Sigma), focal adhesion kinase (FAK) and mediators of the Ras-Raf-ERK pathway. ERRP treatments resulted in reduced expression of p21Rac1 and inhibited the constitutive activation of FAK and MEK2 in HCT-116 cells. Transfection of constitutively activate p21Rac1 or MEK2 into HCT-116 cells abrogated ERRP-induced inhibition of growth. In summary, it was demonstrated that ERRP not only inhibits cell growth, but also the processes of cell invasion and blood vessel formation that are critical for the development and progression of carcinogenesis.
Abstract: CARP-1, a novel apoptosis inducer, regulates apoptosis signaling by diverse agents, including adriamycin and growth factors. Epidermal growth factor receptor (EGFR)-related protein (ERRP), a pan-ErbB inhibitor, inhibits EGFR and stimulates apoptosis. Treatments of cells with ERRP or Iressa (an EGFR tyrosine kinase inhibitor) results in elevated CARP-1 levels, whereas antisense-dependent depletion of CARP-1 causes inhibition of apoptosis by ERRP. CARP-1 is a tyrosine-phosphorylated protein, and ERRP treatments cause elevated tyrosine phosphorylation of CARP-1. CARP-1 contains multiple, nonoverlapping apoptosis-inducing subdomains; one such subdomain is present within amino acids 1-198. Wild-type or CARP-1-(1-198) proteins that have substitution of tyrosine 192 to phenylalanine abrogate apoptosis by ERRP. In addition, apoptosis mediated by wild type or CARP-1-(1-198), and not CARP-1-(1-198(Y192F)), results in activation of caspase-9 and increased phosphorylation of p38 MAPK. However, the expression of dominant-negative forms of p38 MAPK activators MKK3 or MKK6 proteins inhibits apoptosis induced by both the full-length and truncated (amino acids 1-198) proteins. Together, data demonstrate that tyrosine 192 of CARP-1 is a target of apoptosis signaling, and CARP-1, in turn, promotes apoptosis by activating p38 MAPK and caspase-9.
Abstract: Inactivation of epidermal growth factor receptor (EGFR) family members represents a promising strategy for the development of selective therapies against epithelial cancers. Current anti-EGFR therapies, such as cetuximab (Erbitux), gefitinib (Iressa), or trastuzumab (Herceptin), target EGFR or HER-2 but not both. Because solid tumors express different EGFRs, identification of inhibitor(s), targeting multiple EGFR family members may provide a therapeutic benefit to a broader patient population. We have identified a natural inhibitor of EGFRs called EGFR-related protein (ERRP), a 53 to 55 kDa protein that is present in most, if not all, normal human epithelial cells. The growth of colon (HCT-116, Caco2, and HT-29) and breast (MDA-MB-468 and SKBR-3) cancer cells expressing varying levels of EGFR, HER-2, and/or HER-4 was inhibited by recombinant ERRP in a dose-dependent manner. In contrast, ERRP caused no inhibition of growth of normal mouse fibroblast cell lines (NIH-3T3, NIH-3T3/P67), and the growth of nontransformed rat small intestinal IEC-6 cells expressing relatively low levels of EGFRs was inhibited only at high doses of ERRP. Transforming growth factor-alpha or heparin-binding epidermal growth factor-induced activation of EGFR and HER-2 was inhibited by ERRP in colon and breast cancer cells expressing high levels of EGFR or HER-2. In contrast, cetuximab inhibited the growth- and ligand-induced activation of EGFR in cell lines expressing high levels of EGFR, whereas trastuzumab was effective only in HER-2-overexpressing cells. ERRP and trastuzumab, but not cetuximab, attenuated heregulin-alpha-induced activation of colon and breast cancer cells that expressed high levels of HER-2. Furthermore, ERRP, but not cetuximab or trastuzumab, significantly induced apoptosis of colon and breast cancer cells. None of these agents induced apoptosis of either NIH-3T3 mouse fibroblast or normal rat small intestinal IEC cells. Our results suggest that ERRP is an effective pan-erbB inhibitor and, thus, may be a potential therapeutic agent for a wide variety of epithelial cancers expressing different levels and subclasses of EGFRs.
Abstract: Epidermal growth factor receptor is frequently implicated in epithelial cancers and is, therefore, being considered as a potential target for therapy. Recently, we reported the isolation and characterization of epidermal growth factor receptor-related protein, a negative regulator of epidermal growth factor receptor. To discern whether epidermal growth factor receptor-related protein could be an effective therapeutic agent for colorectal cancer, we generated epidermal growth factor receptor-related protein fusion protein and studied its effect on the growth of colon cancer cells in vivo and in vitro. We also studied whether epidermal growth factor receptor-related protein expression is altered in colorectal cancer.
Abstract: CD437, a novel retinoid, causes cell cycle arrest and apoptosis in a number of cancer cells including human breast carcinoma (HBC) by utilizing an undefined retinoic acid receptor/retinoid X receptor-independent mechanism. To delineate mediators of CD437 signaling, we utilized a random antisense-dependent functional knockout genetic approach. We identified a cDNA that encodes approximately 130-kDa HBC cell perinuclear protein (termed CARP-1). Treatments with CD437 or chemotherapeutic agent adriamycin, as well as serum deprivation of HBC cells, stimulate CARP-1 expression. Reduced levels of CARP-1 result in inhibition of apoptosis by CD437 or adriamycin, whereas increased expression of CARP-1 causes elevated levels of cyclin-dependent kinase inhibitor p21WAF1/CIP1 and apoptosis. CARP-1 interacts with 14-3-3 protein as well as causes reduced expression of cell cycle regulatory genes including c-Myc and cyclin B1. Loss of c-Myc sensitizes cells to apoptosis by CARP-1, whereas expression of c-Myc or 14-3-3 inhibits CARP-1-dependent apoptosis. Thus, apoptosis induction by CARP-1 involves sequestration of 14-3-3 and CARP-1-mediated altered expression of multiple cell cycle regulatory genes. Identification of CARP-1 as a key mediator of signaling by CD437 or adriamycin allows for delineation of pathways that, in turn, may prove beneficial for design and targeting of novel antitumor agents.
Abstract: Although epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, differentiation, and transformation in many tissues, little is known about the factor(s) that may modulate its function. We have isolated a cDNA clone from the rat gastroduodenal mucosa whose full length revealed 1,958 bp that contained 227 bp of 5'-untranslated region (UTR) and an open-reading frame encoding 479 amino acids, followed by 290 bp of 3'-UTR. It showed ~85% nucleotide homology to the external domain of the rat EGFR. We refer to the product of the newly isolated cDNA as EGFR-related protein (ERRP). In Northern blot analysis with poly(A)(+) RNA from different rat tissues, ERRP cDNA hybridized to several mRNA transcripts with the strongest reaction noted with a transcript of approximately 2 kb. Maximal expression of the 2-kb mRNA transcript was observed in the small intestine, followed by colon, liver, gastric mucosa, and other tissues. Transfection of ERRP cDNA into a colon cancer cell line, HCT116, resulted in a marked reduction in proliferation in monolayer and colony formation in soft agar compared with the vector-transfected controls. In another colon cancer cell line, Caco-2, with a tetracycline-regulated promoter system, induction of ERRP expression in the absence of doxycycline was associated with a marked reduction in EGFR activation and proliferation. We conclude that the ERRP cDNA may represent a new member of the EGFR gene family and that ERRP plays a role in regulating cell proliferation by modulating the function of EGFR.
Abstract: Although in Fischer 344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about specific changes in the regulatory mechanisms of this process. To determine whether changes in cell cycling events could partly contribute to the age-related rise in gastric mucosal proliferative activity, the present investigation examines changes in cyclin-dependent kinase (Cdk2) activity and the regulation of this process in the gastric mucosa of Fischer 344 rats aged 4 (young), 13 (middle aged), and 24 (old) mo. We observed that aging is associated with a progressive rise in activity and protein levels of Cdk2 in the gastric mucosa. This is also found to be accompanied by a concomitant increase in cyclin E but not cyclin D1 levels. On the other hand, the levels of p21(Waf1/Cip1) (total as well as the fraction associated with Cdk2), a nuclear protein that is known to inhibit different cyclin-Cdk complexes, are found to decline in the gastric mucosa with advancing age. In contrast, with aging, there was a steady rise in p53 levels in the gastric mucosa. We have also observed that the levels of phosphorylated retinoblastoma protein, a form that participates in regulating progression through the S phase, are markedly elevated in the gastric mucosa of aged rats. In conclusion, our data suggest that, in the gastric mucosa, aging enhances transition of G(1) to S phase as well as progression through the S phase of the cell cycle. However, the age-related decline in p21(Waf1/Cip1) in the gastric mucosa appears to be independent of p53 status.
Abstract: To study the role of EGF-R in small intestinal adaptation to hemorrhage and I/R, anesthetized rabbits were implanted aseptically with arterial and venous catheters and bilateral renal artery Doppler flow probes and silastic occluders and allowed to recover. Rabbits were then randomly assigned to one of six groups: time control; hemorrhage (22.5 mL/kg) and 2.5 hours of renal occlusion (hemorrhage plus I/R); hemorrhage plus I/R and 2:1 LRS resuscitation; hemorrhage plus I/R and 3:1 LRS resuscitation; hemorrhage alone; or I/R alone. Rabbits were killed 48 hours after hemorrhage, and a section of duodenum was collected for analysis. Hemorrhage plus I/R induced a 2.5-fold increase in EGF-R tyrosine kinase activity compared with that found in the control group (P < .05), and this effect was not modified by either LRS resuscitation regimen. This increased activity was associated with similar Increases in EGF-R protein concentrations and approximately a 50% increase in EGF-R messenger (m)RNA levels compared with levels found in the control group. Further analysis of possible regulatory mechanisms for the increased EGF-R expression after hemorrhage plus I/R detected higher levels of EGF-R phosphorylation compared with those found in the control group but no significant increases in transforming growth factor-alpha mRNA levels. These data, coupled with a significant increase in duodenal thlobarbituric acid-reactive substance concentrations from rabbits in the hemorrhage plus I/R group, support the hypothesis that tyrosine kinase signal transduction pathways involving the EGF-R are activated in the small intestine after hemorrhage, renal I/R, or both, and this process may be mediated, at least in part, by oxidant stress.
Abstract: Aging is associated with decreased reparative ability of the gastric mucosa. Our recent data suggest a role for epidermal growth factor receptor (EGFR) in the mucosal reparative processes. Thus we examined changes in EGFR tyrosine kinase activity as well as expression and subcellular localization of EGFR and its ligand transforming growth factor-alpha (TGF-alpha) in the gastric mucosa of young (4-mo-old) and aged (24-mo-old) Fischer 344 male rats during the early reparative phase after acute injury induced by 2 M NaCl. Within 240 min of injury, significant epithelial restitution was observed in the gastric mucosa of young but not of aged rats. Expansion of the neck region and initiation of foveolar cell migration could be seen within 45 min of injury in young rats but not until 90 min in aged rats. In young rats mucosal EGFR tyrosine kinase activity increased at 45 min after injury and subsequently fell to basal levels. Mucosal EGFR mRNA increased throughout the reparative phase as did content of the EGFR ligand TGF-alpha. In contrast, although the basal tyrosine kinase activity and levels of EGFR mRNA and TGF-alpha were elevated in the gastric mucosa of aged rats, injury did not cause increases in these parameters. Immunofluorescent localization suggests that internalization and/or degradation of EGFR may be higher in aged than in young rats. We suggest that diminished induction of EGFR tyrosine kinase activity and increased EGFR internalization after injury may in part be responsible for the age-related decrease in the reparative capacity of the gastric mucosa.
Abstract: Although in Fischer-344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about the intracellular events that regulate this process. The present investigation examines the age-related changes in gastric mucosal tyrosine kinase activity and expression of epidermal growth factor receptor(EGFR) and its structural and functional analog p185c-erbB-2, the protein product of c-erbB-2/c-neu protooncogene. We observed a significantly higher intrinsic tyrosine kinase activity and tyrosine phosphorylation of EGFR and p185c-erbB-2 in the gastric mucosa of 24-mo-old (aged) rats than in that of their 4- or 12-mo-old counterparts. This was associated with increased levels of EGFR protein and steady-state mRNA levels of EGFR and p185c-erbB-2. In addition, we also observed threefold higher steady-state mRNA levels of transforming growth factor-alpha (TGF-alpha; one of the primary ligands of EGFR) in the gastric mucosa of aged rats than in that of 4-mo-old (young) animals. This was accompanied by a fivefold increase in the relative concentration of the 18-kDa precursor form of TGF-alpha in gastric mucosal membranes but not in the cytosol. In conclusion, our data demonstrate that aging is associated with increased tyrosine kinase activity of EGFR and p185c-erbB-2 in the gastric mucosa. Moreover, the observation that aging results in increased accumulation of TGF-alpha in gastric mucosal membranes raises the possibility that the membrane-bound TGF-alpha could partly be responsible for the constitutively active EGFR-induced signaling pathway in the gastric mucosa of aged rats and, in turn, for stimulation of mucosal proliferative activity.
