Sensing Small Changes in Protein Abundance: Stimulation of Caco-2 Cells by Human Whey Proteins.

Mass spectrometry (MS)-based proteomic approaches have largely facilitated our systemic understanding of cellular processes and biological functions. Cutoffs in protein expression fold changes (FCs) are often arbitrarily determined in MS-based quantification with no demonstrable determination of small magnitude changes in protein expression. Therefore, many biological insights may remain veiled due to high FC cutoffs. Herein, we employ the intestinal epithelial cell (IEC) line Caco-2 as a model system to demonstrate the dynamicity of tandem-mass-tag (TMT) labeling over a range of 5-40% changes in protein abundance, with the variance controls of ± 5% FC for around 95% of TMT ratios when sampling 9-12 biological replicates. We further applied this procedure to examine the temporal proteome of Caco-2 cells upon exposure to human whey proteins (WP). Pathway assessments predict subtle effects due to WP in moderating xenobiotic metabolism, promoting proliferation and various other cellular functions in differentiating enterocyte-like Caco-2 cells. This demonstration of a sensitive MS approach may open up new perspectives in the system-wide exploration of elusive or transient biological effects by facilitating scrutiny of narrow windows of proteome abundance changes. Furthermore, we anticipate this study will encourage more investigations of WP on infant gastrointestinal tract development.

The soybean-derived peptide lunasin inhibits non-small cell lung cancer cell proliferation by suppressing phosphorylation of the retinoblastoma protein.

Lunasin, a soybean bioactive peptide, has both chemopreventive and chemotherapeutic activities. The aim of this study was to determine the chemotherapeutic potential of lunasin against human lung cancer. Treatment of non-small cell lung cancer (NSCLC) cells with highly purified soybean-derived lunasin caused limited, cell-line specific anti-proliferative effects on anchorage-dependent growth whereas two normal bronchial epithelial cell lines were unaffected. Lunasin's antiproliferative effects were potentiated upon utilization of anchorage-independent conditions. Furthermore, NSCLC cell lines that were unaffected by lunasin in anchorage-dependent assays exhibited a dose-dependent inhibition in colony formation or colony size. Mouse xenograft studies revealed that 30 mg lunasin/kg body weight per day decreased NSCLC H1299 tumor volume by 63.0% at day 32. Mechanistic studies using cultured NSCLC H661 cells showed that lunasin inhibited cell cycle progression at the G1/S phase interface without inducing apoptosis. Immunoblot analyses of key cell-cycle proteins demonstrated that lunasin altered the expression of the G1 specific cyclin-dependent kinase complex components, increased levels of p27Kip1, reduced levels of phosphorylated Akt, and ultimately inhibited the sequential phosphorylation of the retinoblastoma protein (RB). These results establish for the first time that lunasin can inhibit NSCLC proliferation by suppressing cell-cycle dependent phosphorylation of RB.

Lunasin sensitivity in non-small cell lung cancer cells is linked to suppression of integrin signaling and changes in histone acetylation.

Lunasin is a plant derived bioactive peptide with both cancer chemopreventive and therapeutic activity. We recently showed lunasin inhibits non-small cell lung cancer (NSCLC) cell proliferation in a cell-line-specific manner. We now compared the effects of lunasin treatment of lunasin-sensitive (H661) and lunasin-insensitive (H1299) NSCLC cells with respect to lunasin uptake, histone acetylation and integrin signaling. Both cell lines exhibited changes in histone acetylation, with H661 cells showing a unique increase in H4K16 acetylation. Proximity ligation assays demonstrated lunasin interacted with integrins containing αv, α5, ß1 and ß3 subunits to a larger extent in the H661 compared to H1299 cells. Moreover, lunasin specifically disrupted the interaction of ß1 and ß3 subunits with the downstream signaling components phosphorylated Focal Adhesion Kinase (pFAK), Kindlin and Intergrin Linked Kinase in H661 cells. Immunoblot analyses demonstrated lunasin treatment of H661 resulted in reduced levels of pFAK, phosphorylated Akt and phosphorylated ERK1/2 whereas no changes were observed in H1299 cells. Silencing of αv expression in H661 cells confirmed signaling through integrins containing αv is essential for proliferation. Moreover, lunasin was unable to further inhibit proliferation in αv-silenced H661 cells. This indicates antagonism of integrin signaling via αv-containing integrins is an important component of lunasin's mechanism of action.

Scalable purification and characterization of the anticancer lunasin peptide from soybean.

Lunasin is a peptide derived from the soybean 2S albumin seed protein that has both anticancer and anti-inflammatory activities. Large-scale animal studies and human clinical trials to determine the efficacy of lunasin in vivo have been hampered by the cost of synthetic lunasin and the lack of a method for obtaining gram quantities of highly purified lunasin from plant sources. The goal of this study was to develop a large-scale method to generate highly purified lunasin from defatted soy flour. A scalable method was developed that utilizes the sequential application of anion-exchange chromatography, ultrafiltration, and reversed-phase chromatography. This method generates lunasin preparations of >99% purity with a yield of 442 mg/kg defatted soy flour. Mass spectrometry of the purified lunasin revealed that the peptide is 44 amino acids in length and represents the original published sequence of lunasin with an additional C-terminal asparagine residue. Histone-binding assays demonstrated that the biological activity of the purified lunasin was similar to that of synthetic lunasin. This study provides a robust method for purifying commercial-scale quantities of biologically-active lunasin and clearly identifies the predominant form of lunasin in soy flour. This method will greatly facilitate the development of lunasin as a potential nutraceutical or therapeutic anticancer agent.

TNF potentiates anticancer activity of bortezomib (Velcade) through reduced expression of proteasome subunits and dysregulation of unfolded protein response.

Bortezomib (Velcade) exploits proteasome inhibition as a unique mechanism of anticancer activity. The effectiveness of bortezomib is, however, limited, therefore, the search for therapeutic regimens combining bortezomib with other agents. In the present work we demonstrate enhanced anticancer activity of bortezomib by its combination with tumor necrosis factor (TNF) in the experimental model of C-26 colon carcinoma in mice. This interaction likely relies on the induction of a dysregulated response to ER stress, leading to apoptosis of cancer cells, evidenced by caspase-3 cleavage, p53 accumulation as well as increased SAPK/JNK phosphorylation. ER stress induced by the combination of TNF and bortezomib is corroborated by upregulation of BiP, PDI and calnexin as well as cleavage of caspase-12; however, in contrast to the classic pathway, it is also associated with decreased phosphorylation of eIF2 alpha and prevention of XBP-1 splicing. TNF prevented the upregulation of Hsp27 induced by bortezomib, which may contribute to enhanced ER stress. Moreover, TNF interfered with bortezomib-induced upregulation of distinct subunits of the 26S proteasome. Bortezomib concentration used in this study was not sufficient to prevent TNF from inducing nuclear translocation of p65/RelA; however, the combination of both agents reduced total p65/RelA levels. Combined treatment of tumor-bearing mice with bortezomib and TNF not only inhibited tumor growth but also significantly prolonged animal survival. Therefore, combination of bortezomib with TNF is an attractive option for further clinical studies.

Lymphoma creating colojejunal fistula: report of a case and review of the literature.

Malignant fistula of the colon to the small bowel is rare and is most often due to adenocarcinoma. Colonic lymphoma is unusual, representing only 0.5 percent of all colonic malignancies. We report a case of intestinal lymphoma presenting with diarrhea and malnutrition. A colojejunal fistula was discovered during colonoscopy by biopsy of small bowel through a fistula in the sigmoid colon. Celiotomy revealed a 12 cm mass in the sigmoid colon with a fistula to the jejunum. Pathology was consistent with T-cell lymphoma. This is a rare entity in a nonimmunocompromised host and has not been described in the English literature.

Depletion of activated Vbeta8+ T cells disrupts bispecific antibody directed antitumor immunity.

INTRODUCTION: Activation of Vbeta8+ T cells with superantigen staphylococcal enterotoxin B (SEB) and use of an antitumor, anti-CD3 bispecific antibody (BsAb) leads to tumor protective immunity. We hypothesize that Vbeta8+ T-cell activation in combination with BsAb is crucial for tumor protective immunity in this model. METHODS: Adolescent C3H/HeN mice were intravenously injected with syngeneic CL62 melanoma to establish pulmonary metastasis. Three days after establishing pulmonary metastasis, predominantly Vbeta8+ T cells are activated with 50 mug of intraperitoneal superantigen SEB. T cells were depleted at different time points in relation to SEB administration to assess the effect on protective immunity against a second tumor challenge. RESULTS: Protective immunity is significantly (P < 0.008) decreased when Vbeta8+ depletion occurs 6 h after SEB injection, as growth of rechallenged CL62 melanoma occurred in 43%. Protective immunity is present at all other time points when mice survive Vbeta8+ T-cell depletion. Survival of animals treated with SEB/BsAb (82%) is significantly better (P < 0.002) than with SEB alone (60%) or nontreated control (0%). Survival when Vbeta8+ T-cell depletion occurred at 6 h and 48 h post-SEB is 72% and 77%, respectfully, and is statistically indistinguishable (P < 0.232 and P < 0.602). If T-cell depletion was conducted before SEB administration, however, the combination of SEB and BsAb did not result in significant protective immunity. T-cell depletion before the use of SEB alone, without BsAb, failed to result in significant protective immunity. CONCLUSIONS: Depletion of Vbeta8+ T cells 6 h after activation disrupts the development of protective immunity.

Population-based incidence of complicated diverticular disease of the sigmoid colon based on gender and age.

PURPOSE: The purpose of this study was to characterize the gender and age differences in patients with clinically symptomatic sigmoid diverticular disease requiring surgery. METHODS: All surgical patients hospitalized with proven diverticular disease requiring sigmoid resection from January 1988 to January 1998 were reviewed. RESULTS: A total of 934 patients requiring surgical resection for diverticular disease were admitted. There were 443 men and 491 women with an average age of 64. Forty-nine patients presented with massive rectal bleeding (males, 3.6 percent; females, 1.6 percent), 329 with chronic diverticulitis (males, 15.8 percent; females, 19.3 percent), 61 with obstructive symptoms (males, 2.7 percent; females, 3.9 percent), 148 with fistulas (males, 8.0 percent; females, 7.8 percent), 170 with perforation (male, 8.7 percent; female, 9.4 percent), 79 with abscess (males, 4.0 percent; females, 4.5 percent), 59 with stricture (males, 2.2 percent; females, 4.0 percent), and 39 with acute diverticulitis (males, 2.2 percent; females, 1.9 percent). Overall, patients younger than 50 presented more often with chronic or recurrent diverticulitis. CONCLUSIONS: Female patients present, on average, five years later than male with complications requiring surgery. Overall, men have a higher incidence of bleeding (P = 0.015), whereas women present more often with stricture and obstruction (P = 0.02). Young males present more with fistula (P = 0.03), whereas older males present with bleeding (P = 0.001). Young females present with perforation (P = 0.002), and older females present with chronic diverticulitis (P = 0.04) and stricture (P = 0.04).

Melanoma metastatic to the colon: case series and review of the literature with outcome analysis.

PURPOSE: Symptomatic melanoma of the colon is rare. The aim of this study was to determine the incidence, presenting signs and symptoms, and survival correlation. METHODS: A retrospective review was performed of all patients treated in Mayo Clinic facilities from 1960 to 2000 for primary and metastatic melanoma. We identified 24 patients with metastatic melanoma to the colon. RESULTS: There were 24 patients (14 males) with an average age of 60.4 years at the time of metastatic involvement. The interval time between diagnosis of the primary and metastatic disease to the colon was 7.47 years. The most common presentation was bleeding. Colonoscopy was used in 11 patients and diagnostic in 9. Eighteen patients underwent resection, and seven patients had positive nodes. The average time until death after operation was 27.5 months (range, 30 days to 65 months). Nonoperative candidates died within 7.8 months after diagnosis. One-year and five-year survival for resected patients were 37 and 21 percent, respectively. Patients with negative nodes had an average survival time of 34.7 months compared with 20.4 months in patients with positive nodes. Perforation and bowel obstruction directly correlated with poor survival, with an average life expectancy of ten months (P = 0.03). CONCLUSIONS: Metastatic melanoma of the colon is rare. Segmental resection is justified and can be successfully completed in 95 percent of patients who undergo attempted resection. Survival of less than ten months is most accurately predicted by signs and symptoms of obstruction or perforation at presentation (P = 0.03).

Dendritic cell-tumor cell fusion and staphylococcal enterotoxin B treatment in a pancreatic tumor model.

BACKGROUND: Surgical resection of pancreatic tumors removes gross disease but not metastases. Adjuvant therapy such as chemotherapy and radiation treatment is of little value in metastatic pancreatic cancer. The hypothesis of this investigation is that specific and effective immunotherapeutic vaccine (dendritic/tumor cell fusion) will activate cytotoxic T lymphocytes (CTLs), leading to the eradication of spontaneous pancreatic cancer. METHODS: We have developed a double transgenic mouse model (MET) that forms spontaneous pancreatic tumors and expresses the human MUC1 antigen. Seven-week-old MET mice (n = 8) were treated every 3 weeks with the vaccine. In addition, these mice received 50 microg of superantigen staphylococcal enterotoxin B (SEB), a known T cell stimulant, prior to the first vaccination. A second treatment group received SEB alone (n = 8) and controls received no treatment (n = 9). MUC1-specific CTLs were measured by chromium release assay. At 10 weeks of age and at necropsy, MUC1 serum levels were measured using a MUC1-specific ELISA. RESULTS: Mice were known to harbor microscopic foci of cancer at birth. Survival was enhanced in vaccine as well as SEB-treated mice (75% CI +/- 0.42) compared to controls (11% CI +/- 0.28) and both groups of treated mice exhibited mature CTLs without in vitro stimulation. MUC1 serum levels of the vaccine group were 50% less than that of control (P < 0.04) at 10 weeks. MUC1 serum levels directly correlated with tumor weight at necropsy (r = 0.86). CONCLUSIONS: This is the first evidence that MUC1-specific CTLs can be stimulated to enhance survival in a spontaneous tumor model.

ErbB-beta-catenin complexes are associated with human infiltrating ductal breast and murine mammary tumor virus (MMTV)-Wnt-1 and MMTV-c-Neu transgenic carcinomas.

Simultaneous deregulation of both Wnt and ErbB growth factors has previously been shown to result in the cooperative induction of mammary gland tumors. Using the murine mammary tumor virus (MMTV)-Wnt-1 transgenic model of mammary carcinoma, we have identified an unvarying association between beta-catenin and epidermal growth factor receptor/c-Neu (ErbB1/ErbB2) heterodimers in mammary gland tumors, indicating a requirement for ErbB signaling in Wnt-mediated tumorigenesis. Expansion of these observations to a second transgenic model, MMTV-c-Neu, demonstrated similar tumor-specific interactions, including an ErbB1 ligand-inducible phosphorylation of both beta-catenin and c-Neu. Direct relevance of these findings to human breast cancer was established upon examination of a set of human infiltrating ductal breast adenocarcinoma and lymph node metastasis tissues taken at surgery. These data revealed increased levels of beta-catenin in tumors and metastases versus normal breast as well as an association between beta-catenin and c-Neu that measurably occurs only in neoplasia, most strongly in metastatic lesions. These studies have identified a seemingly indispensable interaction between beta-catenin and epidermal growth factor receptor/c-Neu heterodimers in Wnt-1-mediated breast tumorigenesis that may indicate a fundamental signaling event in human metastatic progression.

Intracellular Ca2+ homeostatic regulation and 4-hydroxynonenal-induced aortic endothelial dysfunction.

The aldehydic lipid peroxidation product 4-hydroxynonenal (HNE) is known to compromise erythrocyte passive Ca2+ permeability and to irreversibly inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase and Ca2+-transport. To measure the effects of HNE on passive and active Ca2+ transport in endothelial cells, we first characterized 45Ca2+ uptake and efflux in cultured porcine aortic endothelial cells (PAEC). PAEC exchanged 45Ca2+ to a cumulative near-isotopic equilibrium of about 4.5 pmole 45Ca2+/10(6) cells in 120 min at 37 degrees C. This Ca2+ pool was diminished by thapsigargin, cyclopiazonic acid, oligomycin B, and sodium azide. In contrast, ouabain enhanced Ca2+ uptake capacity from 5.17 to 5.77 pmole/10(6) cells. Accumulated 45Ca2+ was extruded at rate of 8.7 fmole 45Ca2+/10(6) cells/min or shunted rapidly by the ionophore A23187. HNE increased total 45Ca2+ accumulation in a time- and concentration-dependent manner by as much as 562% with an EC50 of 64.0 wM. Concomitant morphological analysis of PAEC revealed vacuolization, nuclear swelling, cell shrinking, and cell detachment. Initial structural changes, such as vacuolization, began well before any changes in Ca2+ accumulation were observed. These functional and morphological changes indicate that HNE significantly increases intracellular Ca2+ accumulation in vascular endothelium, which may explain the cytotoxic effects associated with HNE exposure and provide further evidence that atherogenic effects of HNE may, in part, be caused by disturbances in Ca2+ homeostasis.