Sarah Kraus

Cell desensitization by sublytic C5b-9 complexes and calcium ionophores depends on activation of protein kinase C

Basal cell resistance to lysis by complement C5b‐9 complexes depends on extracellular and intracellular protection. Cell membrane regulatory proteins and enzymes interfere with complement activation and intracellular processes of protein phosphorylation and synthesis support cell resistance and damage repair. K562 human erythroleukemic cells treated with sublytic complement doses become protected from lytic doses of complement within 50u2004min. The early signaling processes leading to cell desensitization to complement‐mediated lysis were studied. Treatment with calcium ionophores or phorbol 12‐myristate 13‐acetate rapidly induced in K562 cells protection from complement as well as synthesis of a large protein complex similar to the large complement‐induced protein complex L‐CIP induced by sublytic complement. Both ionophore‐ and complement‐induced protection were blocked by treatment with protein kinase C (PKC) inhibitors. Calphostin C, sphingosine and GF109203X abrogated complement‐induced protection almost completely, whereas Go6976 inhibited it only partially. Since Go6976 is a selective inhibitor of the conventional PKC type, it is proposed that sublytic complement doses activate both conventional and non‐conventional PKC types. Immunofluorescence analysis of K562 cells demonstrated sublytic complement‐induced translocation of the conventional PKCα and PKCβII from the cytoplasm to the plasma membrane. These results indicate that PKC activation is an early obligatory signal in cell desensitization by sublytic C5b‐9 or calcium ionophore.

Recent advances in personalized colorectal cancer research.

Colorectal cancer is one of the most prevalent cancers and a leading cause ofcancer-related death. It is also curable if detected early. The prognosis for metastatic colorectal cancer remains poor and resistance to chemotherapy is still a major obstacle in effective treatment. While many patients do not clinically benefit from chemotherapy, others experience adverse reactions resulting in dose modifications or treatment withdrawal, thereby reducing treatment efficacy. Researchefforts attempt to identify reliable biomarkers which will guide clinicians in decision making, while matching suitable therapeutic regimens. We here review currently known molecular biomarkers used for the personalized treatment of colorectal cancer.

COX-2 Active Agents in the Chemoprevention of Colorectal Cancer

Chemopreventive strategies for colorectal cancer (CRC) have been extensively studied to prevent the recurrence of adenomas and/or delay their development in the gastrointestinal tract. The non-steroidal anti-inflammatory drugs (NSAIDs) and selective cyclooxygenase (COX)-2 inhibitors have been proven as promising and the most attractive candidates for CRC clinical chemoprevention. The preventive efficacy of these agents is supported by a large number of animal and epidemiological studies which have clearly demonstrated that NSAID consumption prevents adenoma formation and decreases the incidence of, and mortality from CRC. On the basis of these studies, aspirin chemoprevention may be effective in preventing CRC within the general population, while aspirin and celecoxib may be effective in preventing adenomas in patients after polypectomy. Nevertheless, the consumption of NSAID and COX-2 inhibitors is not toxic free. Well-known serious adverse events to the gastrointestinal, renal and cardiovascular systems have been reported. These reports have led to some promising studies related to the use of lower doses and in combination with other chemopreventive agents and shown efficacy. In the intriguing jigsaw puzzle of cancer prevention, we now have a definite positive answer for the basic question if, but several other parts of the equation-proper patient selection, the ultimate drug, optimal dosage and duration are still missing.

An Immunoconjugate of Anti-CD24 and Pseudomonas Exotoxin Selectively Kills Human Colorectal Tumors in Mice

BACKGROUND & AIMSnEffective and selective treatment options are needed for patients with colorectal cancer (CRC). The CD24 mucin-like glycoprotein is overexpressed in CRCs; monoclonal antibodies (mAbs) against CD24 inhibit tumor cell growth in vitro and in vivo. Based on the tumor-specific expression of CD24, we investigated the potential of anti-CD24 SWA11 mAb, to deliver a cytotoxic agent into CRC cells.nnnMETHODSnWe conjugated SWA11 to a Pseudomonas exotoxin derivative (PE38) via an Fc-binding ZZ domain from Staphylococcal protein A (which binds the Fc domain of mouse IgG2a immunoglobulins) to generate the immunotoxin SWA11-ZZ-PE38; IgG-ZZ-PE38 was used as control. Human HT-29 and COLO320 (CD24-positive) and HCT116 (CD24-negative) CRC cell lines were assayed for immunotoxin binding, cytotoxicity, viability, and apoptosis. Toxicity and antitumor efficacy were tested in mice.nnnRESULTSnThe immunotoxin preserved the affinity and specificity of SWA11, bound and selectively killed CD24-expressing CRC cells via apoptosis. IC(50) values ranged from 20 to 50 ng/mL-several orders of magnitude lower than that of the mAb alone. The immunotoxins were not toxic to mice at the maximum dose of 0.75 mg/kg. Growth of HT-29 xenograft tumors was significantly reduced in mice given SWA11-ZZ-PE38 (by 78%) compared to untreated mice.nnnCONCLUSIONSnAnti-CD24 SWA11 mAb can deliver a PE exotoxin derivative to CRC cells and cause them to undergo apoptosis, without toxicity to normal tissues. This immunotoxin might be developed as a therapeutic treatment for patients with CRC.

Impact of genetic polymorphisms on adenoma recurrence and toxicity in a COX2 inhibitor (celecoxib) trial: results from a pilot study.

Objective Chemoprevention trials have shown that celecoxib reduces adenoma recurrence but can cause cardiovascular toxicity. In this pilot study, we evaluated associations between genetic variation in several candidate pathways (e.g. prostaglandin synthesis) and adenoma recurrence and cardiovascular and gastrointestinal toxicities. Methods Genotyping analysis was carried out on 117 Israeli colorectal adenoma patients who participated in the Prevention of Colorectal Sporadic Adenomatous Polyps trial. Reassessment followed after 3 years on celecoxib and after 2 years from termination of treatment with celecoxib. Efficacy (absence of colorectal adenomas) was measured by colonoscopy at years 1, 3, and 5. Toxicities were assessed by investigators during celecoxib treatment and by self-report post-treatment. A linkage disequilibrium-based selection algorithm (r2≥0.90, MAF≥4%) identified 255 tagSNPs in 25 analyzed candidate genes. Genotyping was performed by using Illumina GoldenGate technology. Results Multiple genetic variants were associated with adenoma recurrence and toxicity. Genetic variability in COX1, COX2, and ALOX12/15 genes played a role in adenoma recurrence, particularly among patients on placebo. More gene variants (especially variants in PGES, CRP, SRC, and GPX3) were associated with increased risk for cardiovascular toxicity and symptoms, compared with gastrointestinal toxicity and symptoms. The increased risk for cardiovascular toxicity/symptoms associated with the SRC gene variants (rs6017996, rs6018256, rs6018257) ranged from 6.61 (95% confidence interval 1.66–26.36, P<0.01) to 10.71 (95% confidence interval 1.96–58.60, P<0.01). Conclusion Genetic polymorphisms in multiple inflammation-related genes appear to interact with celecoxib on adenoma recurrence and its attendant toxicity, particularly cardiovascular toxicity/symptoms. Larger studies validating these pharmacogenetic relationships are needed.

Role of CD24 polymorphisms in the susceptibility to inflammatory bowel disease

Background Inflammatory bowel disease (IBD) results from an inappropriate inflammatory response in which genetic, immune, and environmental factors all play important roles. Recently, single nucleotide polymorphisms (SNPs) in the CD24 gene have been associated with the development of several autoimmune diseases. Aim To evaluate whether CD24 SNPs, are associated with risk of ulcerative colitis (UC) and Crohns disease (CD). Methods The CD24 polymorphisms C170T (rs8734), TG1527del (rs3838646), A1626G (rs1058881), and A1056G (rs1058818) were assessed in a case-control study of an Israeli cohort comprising 117 IBD patients and 105 age and gender-matched healthy controls. Restriction fragment length polymorphism (RFLP) analysis was performed using BstX1, Bsr1, Mfe1, and BstU1 restriction enzymes. Odds ratios (OR) and 95% confidence intervals (CI) were estimated by logistic regression models. Results Carriers of the C170T SNP were at increased risk of IBD (OR=3.022, 95% CI: 1.748–5.223, p=0.001), UC (OR=3.002, 95% CI: 1.661–5.427, p=0.001) and CD (OR=3.077, 95% CI: 1.334–7.095, p=0.008). Carrying the A1626G and A1056G SNPs was found to be a risk factor for IBD (OR=2.460, 95% CI: 1.420–4.259, p=0.001 and OR=1.856, 95% CI: 1.011–3.405, p=0.01), UC (OR=2.218, 95% CI: 1.207–4.075, p=0.01 and OR=1.944, 95% CI: 0.995–3.798, p=0.01) but not for CD (p=0.086 and p=0.299). The A1626G and TG1527del were found to be associated with younger age of IBD onset (p=0.022 and p=0.027, respectively). Conclusions The CD24 C170T polymorphism is associated with IBD risk. The A1626G and A1056G SNPs might be associated only with UC risk. These findings suggest CD24 as a new genetic susceptibility factor, with clinical implications in the prediction of IBD prognosis and therapy.

Curcumin potentiates the pro-apoptotic effects of sulindac sulfone in colorectal cancer

Objective: The use of sulindac sulfone (SFN) for colorectal cancer (CRC) therapy is limited due to its toxicity. The present study was carried out to examine whether curcumin, a novel chemopreventive agent, can potentiate the effects of low dosages of SFN in CRC treatment. Methods: HT-29 CRC cells were exposed to SFN (200 – 400 microM), curcumin (5 – 10 microM) or their combination. The cytotoxic effects of the drugs were evaluated using growth inhibition assays. Annexin V/PI and cell cycle analysis were employed to study the mechanism of action of the drugs. The therapeutic efficacy of the drugs in vivo was examined using the aberrant crypt foci (ACF) model. The treatment groups included eight rats/group. Results: Treatment of cells with curcumin and SFN resulted in a synergistic inhibitory effect of 50 – 90% (p <u20090.005) on cell growth. Growth inhibition was associated with inhibition of proliferation, G2/M arrest and induction of apoptosis. Administration of curcumin (0.6%) and SFN (0.06%) to 1, 2-dimethylhydrazine treated rats significantly reduced (by 75%, p <u20090.01) the number of ACF. Conclusions: Curcumin augments the therapeutic effects of SFN. This may be clinically important since the addition of curcumin to low dosages of SFN may encourage a safer and potent combinatorial treatment regimen for CRC.

Novel approach to abuse the hyperactive K-Ras pathway for adenoviral gene therapy of colorectal cancer.

BACKGROUNDnFunctional activation of oncogenic K-Ras signaling pathway plays an important role in the early events of colorectal carcinogenesis (CRC). K-Ras proto-oncogene is involved in 35-40% of CRC cases. Mutations in the Ras gene trigger the transduction of proliferative and anti-apoptotic signals, even in the absence of extra cellular stimuli. The objective of the current study was to use a gene-targeting approach to kill human CRC cells selectively harboring mutated K-Ras.nnnRESULTSnA recombinant adenovirus that carries a lethal gene, PUMA, under the control of a Ras responsive promoter (Ad-Py4-SV40-PUMA) was used selectively to target CRC cells (HCT116, SW480, DLD1 and RIE-Ras) that possess a hyperactive Ras pathway while using HT29 and RIE cells as a control that harbors wild type Ras and exhibit very low Ras activity. Control vector, without the Ras responsive promoter elements was used to assess the specificity of our gene therapy approach. Both adenoviral vectors were assed in vitro and in xenograft model in vivo. Ad-Py4-SV40-PUMA showed high potency to induce ~50% apoptosis in vitro, to abolish completely tumor formation by infecting cells with the Ad-Py4-SV40-PUMA prior xenografting them in nude mice and high ability to suppress by ~35% tumor progression in vivo in already established tumors.nnnCONCLUSIONSnSelective targeting of CRC cells with the activated Ras pathway may be a novel and effective therapy in CRC. The high potency of this adenoviral vector may help to overcome an undetectable micro metastasis that is the major hurdle in challenging with CRC.

Delayed Wound Healing in Heat Stable Antigen (HSA/CD24)-Deficient Mice

Background Healthy individuals rarely have problems with wound healing. Most skin lesions heal rapidly and efficiently within one to two weeks. However, many medical and surgical complications can be attributed to deficiencies in wound repair. Open wounds have lost the barrier that protects tissues from bacterial invasion and allows the escape of vital fluids. Without expeditious healing, infections become more frequent. The CD24 gene encodes a heavily-glycosylated cell surface protein anchored to the membrane by phosphatidylinositol. CD24 plays an important role in the adaptive immune response and controls an important genetic checkpoint for homeostasis and autoimmune diseases in both mice and humans. We have previously shown that overexpression of CD24 results in increased proliferation and migration rates. Aim To examine the role of CD24 in the wound healing process. Methods An excisional model of wound healing was used and delayed wound healing was studied in genetically modified heat stable antigen (HSA/CD24)-deficient mice (HSA -/-) compared to wild-type (WT) mice. Results Large full-thickness skin wounds, excised on the back of mice, exhibited a significant delay in the formation of granulation tissue, and in wound closure when compared to their WTHSA +/+ littermates. Wounds were histologically analyzed and scored, based on the degree of cellular invasion, granulation tissue formation, vascularity, and re-epithelialization. Additionally, in stitched wounds, the HSA -/- mice failed to maintain their stitches; they did not hold and fell already 24 hours, revealing erythematous wound fields. Re-expression of HSA, delivered by lentivirus, restored the normal healing phenotype, within 24 hours post-injury, and even improved the healing in WT, and in BalbC mice. Conclusions Delayed wound-healing in the absence of HSA/CD24 suggests that CD24 plays an important role in this process. Increased expression of CD24, even in the normal state, may be used to enhance wound repair.

Colorectal cancer chemoprevention: the potential of a selective approach

Evaluation of: Zhang L, Ren X, Alt E et al. Chemoprevention of colorectal cancer by targeting APC-deficient cells for apoptosis. Nature 464(7291), 1058–1061 (2010). Colorectal cancer (CRC) is a leading cause of cancer death, and therefore demands special attention. Novel recent approaches for the chemoprevention of CRC focus on selective targeting of key pathways. We review the study by Zhang and colleagues, evaluating a selective approach targeting APC-deficient premalignant cells using retinoid-based therapy and TNF-related apoptosis-inducing ligand (TRAIL). This study demonstrates that induction of TRAIL-mediated death signaling contributes to the chemopreventive value of all-trans-retinyl acetate (RAc) by sensitizing premalignant adenoma cells for apoptosis without affecting normal cells. We discuss these important findings, raise few points that deserve consideration, and may further contribute to the development of RAc-based combination therapies with improved efficacy. The authors clearly demonstrate a synergistic interaction between TRAIL, RAc and APC, which leads to the specific cell death of premalignant target cells. The study adds to the growing body of literature related to CRC chemoprevention, and provides solid data supporting a potentially selective approach for preventing CRC using RAc and TRAIL.