Catja Freiburghaus

Lactoferricin treatment decreases the rate of cell proliferation of a human colon cancer cell line

Food components modify the risk of cancer at a large number of sites but the mechanism of action is unknown. In the present investigation, we studied the effect of the peptide lactoferricin derived from bovine milk lactoferrin on human colon cancer CaCo-2 cells. The cells were either untreated or treated with 2.0, 0.2, or 0.02 microM lactoferricin. Cell cycle kinetics were investigated with a bromodeoxyuridine DNA flow cytometric method. The results show that lactoferricin treatment slightly but significantly prolonged the S phase of the cell cycle. Lactoferricin treatment lowered the level of cyclin E1, a protein involved in the regulation of genes required for G(1)/S transition and consequently for efficient S phase progression. The slight prolongation of the S phase resulted in a reduction of cell proliferation, which became more apparent after a long treatment time.

TP53 mutations identify younger mantle cell lymphoma patients who do not benefit from intensive chemoimmunotherapy

Despite recent advances in lymphoma treatment, mantle cell lymphoma (MCL) remains incurable, and we are still unable to identify patients who will not benefit from the current standard of care. Here, we explore the prognostic value of recurrent genetic aberrations in diagnostic bone marrow (BM) specimens from 183 younger patients with MCL from the Nordic MCL2 and MCL3 trials, which represent current standard-of-care regimens. In the univariate model, mutations of TP53 (11%) and NOTCH1 (4%), and deletions of TP53 (16%) and CDKN2A (20%), were significantly associated with inferior outcomes (together with MIPI, MIPI-c, blastoid morphology, and Ki67 > 30%); however, in multivariate analyses, only TP53 mutations (HR, 6.2; P < .0001) retained prognostic impact for overall survival (OS), whereas TP53 mutations (HR, 6.9; P < .0001) and MIPI-c high-risk (HR, 2.6; P = .003) had independent prognostic impact on time to relapse. TP53-mutated cases had a dismal outcome, with a median OS of 1.8 years, and 50% relapsed at 1.0 years, compared to a median OS of 12.7 years for TP53-unmutated cases (P < .0001). TP53 mutations were significantly associated with Ki67 > 30%, blastoid morphology, MIPI high-risk, and inferior responses to both induction- and high-dose chemotherapy. In conclusion, we show that TP53 mutations identify a phenotypically distinct and highly aggressive form of MCL with poor or no response to regimens including cytarabine, rituximab, and autologous stem-cell transplant (ASCT). We suggest patients with MCL should be stratified according to TP53 status, and that patients with TP53 mutations should be considered for experimental frontline trials exploring novel agents.

Reduction of ultraviolet light-induced DNA damage in human colon cancer cells treated with a lactoferrin-derived peptide

Treatment of Caco-2 cells with the peptide lactoferricin(4-14), results in reduction of the growth rate by prolongation of the S phase of the cell cycle. Lactoferricin(1-25) is formed in the gut by cleavage from lactoferrin and the bioactive amino acids are found within lactoferricin(4-14). Our hypothesis is that the reduction of the rate of S phase progression may result in increased DNA repair. To test this hypothesis, Caco-2 cells were subjected to UV light that caused DNA lesions and then the cells were grown in the absence or presence of 2.0 μM lactoferricin(4-14). Evaluation of DNA strand breaks using the comet assay showed that lactoferricin(4-14) treatment indeed resulted in a reduction of comets showing damaged DNA. In the search for a mechanism, we have investigated the levels of several proteins involved in cell cycle regulation, DNA replication, and apoptosis using Western blot. Lactoferricin(4-14) treatment resulted in an increased expression of flap endonuclease-1 pointing to increased DNA synthesis activity. Lactoferricin(4-14) treatment decreased the expression of the proapoptotic protein B-cell lymphoma 2-associated X protein (or Bax), indicating decreased cell death. As we have found previously, lactoferricin(4-14) treatment reduced the expression of cyclin E involved in the G(1)/S transition. Immunofluorescence microscopy showed that a lower γ-H2AX expression in lactoferricin(4-14)-treated cells, pointing to more efficient DNA repair. Thus, altogether our data show that lactoferricin(4-14) treatment has beneficial effects.

Identification of ubiquitin in bovine milk and its growth inhibitory effects on human cancer cell lines

Bovine milk is associated with improved health and reduced risk of several diseases, among them cancer. Milk is a complex mixture of known and unknown components. The components and the mechanisms that contribute to the cancer-preventive effects are largely unknown. We set out to find new peptides in milk and identified ubiquitin (Ub) using matrix-assisted laser desorption ionization-time of flight mass spectrometry and Western blot. Using quantitative Western blot, we estimated the Ub concentration to be about 0.003 micromol/L in milk. We then decided to investigate the effect of treating human colon cancer CaCo-2 cells with Ub, using higher concentrations than in milk. CaCo-2 cells treated with 0.02 to 2.0 micromol/L Ub showed significantly decreased proliferation compared with untreated control cells. A higher growth inhibitory effect than in CaCo-2 cells was found in the neuroblastoma cell line SH-SY5Y treated with 0.02 to 0.2 micromol/L Ub. A bromodeoxyuridine DNA flow cytometric method was used to study cell cycle kinetics in Ub-treated CaCo-2 cells. The data point toward a prolongation of the G(1) phase. The levels of several cell cycle regulatory proteins were affected. Our data point to Ub possibly being one of the components in milk reducing the risk of cancer.

Synergistic effects of agonistic costimulatory antibodies adsorbed to amphiphilic poly(γ-glutamic acid) nanoparticles

Agonistic antibodies targeting costimulatory pathways for the immune system hold great potential for cancer immunotherapy. However, systemic administration of immunoactivating antibodies can be associated with side effects such as cytokine release syndrome and liver toxicity. Polymeric nanoparticles (NPs) represent an exciting approach to control the release of therapeutic antibodies and to optimize the desired immune response via selective targeting. With increased potency as objective, agonistic antibodies targeting TNF receptors were adsorbed to immune stimulating biodegradable and self-assembled polymeric nanoparticles composed of g-glutamic acid (g-PGA NPs). The effects of antibodies targeting CD40, CD137 and OX40 were evaluated based on T and B cell proliferation as well as on cytokine release and phenotypic maturation of antigen-presenting cells. Our results demonstrated a strong synergistic effect on human B cell proliferation of CD40 monoclonal antibodies (mAbs) carrying NPs in vitro. In addition adsorption of anti-CD40 mAb to g-PGA NPs significantly reduced the systemic release of TNF-a ,I L-6, IL-10 and IL-12, compared to treatment with the soluble mAb. Preliminary results indicate increased T cell proliferation and activation by CD137 and OX40 agonistic antibodies in combination with g-PGA NPs. Combining NPs with agonistic antibodies for cancer immunotherapy offers intriguing opportunities for increased therapeutic efficacy and safety.

Bortezomib prevents cytarabine resistance in MCL, which is characterized by down-regulation of dCK and up-regulation of SPIB resulting in high NF-κB activity

BackgroundThe addition of high-dose cytarabine to the treatment of mantle cell lymphoma (MCL) has significantly prolonged survival of patients, but relapses are common and are normally associated with increased resistance. To elucidate the mechanisms responsible for cytarabine resistance, and to create a tool for drug discovery investigations, we established a unique and molecularly reproducible cytarabine resistant model from the Z138 MCL cell line.MethodsEffects of different substances on cytarabine-sensitive and resistant cells were evaluated by assessment of cell proliferation using [methyl-14C]-thymidine incorporation and molecular changes were investigated by protein and gene expression analyses.ResultsGene expression profiling revealed that major transcriptional changes occur during the initial phase of adaptation to cellular growth in cytarabine containing media, and only few key genes, including SPIB, are deregulated upon the later development of resistance. Resistance was shown to be mediated by down-regulation of the deoxycytidine kinase (dCK) protein, responsible for activation of nucleoside analogue prodrugs. This key event, emphasized by cross-resistance to other nucleoside analogues, did not only effect resistance but also levels of SPIB and NF-κB, as assessed through forced overexpression in resistant cells. Thus, for the first time we show that regulation of drug resistance through prevention of conversion of pro-drug into active drug are closely linked to increased proliferation and resistance to apoptosis in MCL. Using drug libraries, we identify several substances with growth reducing effect on cytarabine resistant cells. We further hypothesized that co-treatment with bortezomib could prevent resistance development. This was confirmed and show that the dCK levels are retained upon co-treatment, indicating a clinical use for bortezomib treatment in combination with cytarabine to avoid development of resistance. The possibility to predict cytarabine resistance in diagnostic samples was assessed, but analysis show that a majority of patients have moderate to high expression of dCK at diagnosis, corresponding well to the initial clinical response to cytarabine treatment.ConclusionWe show that cytarabine resistance potentially can be avoided or at least delayed through co-treatment with bortezomib, and that down-regulation of dCK and up-regulation of SPIB and NF-κB are the main molecular events driving cytarabine resistance development.

Frequency and clinical implications of SOX11 expression in Burkitt lymphoma

Burkitt lymphoma (BL) is a rare, aggressive neoplasm that constitutes 1–2% of adult lymphomas. It derives from mature B-cells in the germinal center and is characterized by a highly proliferative s...

BORTEZOMIB PREVENTS DEVELOPMENT OF CYTARABINE RESISTANCE IN A MANTLE CELL LYMPHOMA IN VITRO MODEL

The NFκB pathway plays a crucial role in cancer cell survival, proliferation and acquisition of resistance to therapy. Mucosa‐associated lymphoid tissue lymphoma translocation protein 1 (MALT1) is an intermediary protein paracaspase important in the classical activation of NFκB. Previous studies have postulated an important role for NFκB in facilitating BCR signaling in CLL cells. Mi‐2 is a novel irreversible small molecule inhibitor of MALT1.Herein, we studied the effect of MALT‐1 inhibition, using Mi‐2, on the survival of CLL cells. Mi‐2 rapidly reduced CLL cell viability in a dose‐dependent manner. This effect was associated with the intrinsic apoptotic pathway activation, a robust increase in cleaved PARP and accompanied by a prominent decrease in Mcl‐1 and BCL2 levels. The pro‐apoptotic effect of Mi‐2 was observed in CLL cells of both low and high‐risk prognostic features. Accordingly pre‐treatment with Mi‐2 resulted in inhibition of IκB phosphorylation after BCR activation or even reduction below its basal level.Taken together, our results provide proof of the concept that MALT1 inhibition by the small molecule Mi‐2 can efficiently induce apoptosis in CLL cells. In light of the importance of the BCR and NFκB pathways in the pathogenesis of CLL, MALT1 is a potential therapeutic target in this disease. This is particularly important in the era of novel agents used to treat CLL, when resistance to these drugs emerges over time.