Jasminka Pavelić

Natural zeolite clinoptilolite: new adjuvant in anticancer therapy

Natural silicate materials, including zeolite clinoptilolite, have been shown to exhibit diverse biological activities and have been used successfully as a vaccine adjuvant and for the treatment of diarrhea. We report a novel use of finely ground clinoptilolite as a potential adjuvant in anticancer therapy. Clinoptilolite treatment of mice and dogs suffering from a variety of tumor types led to improvement in the overall health status, prolongation of life-span, and decrease in tumors size. Local application of clinoptilolite to skin cancers of dogs effectively reduced tumor formation and growth. In addition, toxicology studies on mice and rats demonstrated that the treatment does not have negative effects. In vitro tissue culture studies showed that finely ground clinoptilolite inhibits protein kinase B (c-Akt), induces expression of p21WAF1/CIP1 and p27KIP1 tumor suppressor proteins, and blocks cell growth in several cancer cell lines. These data indicate that clinoptilolite treatment might affect cancer growth by attenuating survival signals and inducing tumor suppressor genes in treated cells.

Gastric cancer: the role of insulin-like growth factor 2 (IGF 2) and its receptors (IGF 1R and M6-P/IGF 2R).

Insulin‐like growth factor 2 (IGF 2) appears to be involved in the progression of many tumours. It binds to at least two different types of receptor: IGF type 1 (IGF 1R) and mannose 6‐phosphate/IGF type 2 (M6‐P/IGF 2R). Ligand binding to IGF 1R provokes mitogenic and anti‐apoptotic effects. M6‐P/IGF 2R has a tumour suppressor function—it mediates IGF 2 degradation. Mutation of M6‐P/IGF 2R causes both diminished growth suppression and augmented growth stimulation. The aim of this study was to investigate the role of IGF 2 and its receptors (IGF 1R and IGF 2R) in human gastric cancer. The expression of IGF 2 and its receptors was measured in order to analyse the possible correlation between the activity of these genes and cell proliferation in two different gastric tumour types: diffuse and intestinal. The effect of IGF 1 receptor blockage on cell proliferation and anchorage‐independent cell growth was also examined. Increased expression of IGF 2 and IGF 1R genes (at the mRNA and protein level) was found in gastric cancer when compared with non‐tumour tissue. Furthermore, there was a significant difference between IGF 2 expression in the more aggressive diffuse type and that in the intestinal type of gastric cancer. Moreover, the IGF 2 peptide level in the culture media obtained from the diffuse type of cancer cells was significantly higher when compared with the intestinal type. The level of IGF 2 peptide in the conditioned media strongly correlated with [3H]thymidine incorporation and cell proliferation. On the contrary, IGF 2R mRNA expression was much higher in the intestinal type of cancer than in the diffuse type. In addition, IGF 2R protein expression was substantially lower with progression of the diffuse cancer type to a higher stage. The αIR3 monoclonal antibody strongly inhibited [3H]thymidine incorporation and decreased the number of colonies in soft agar of cells overexpressing IGF 2. These findings suggest that members of the IGF family are involved in the pathogenesis of gastric cancer, probably by autocrine/paracrine stimulation of cell growth. Such tumours might be excellent candidates for therapeutic strategies aimed at interference with this pathway. Copyright

PCR amplification of DNA from stained cytological smears.

DNA from archival Papanicolaou stained smears was successfully amplified using the polymerase chain reaction (PCR) to see if it could be used for retrospective genome studies such as detection of the presence of human papilloma virus (HPV) and changes in p53 gene expression. DNA was isolated and purified by treatment with proteinase K, phenol/chloroform, and isoamyl alcohol. Segments of the human beta actin and TGF beta 1 gene were amplified by PCR. Of all stains used in the preparation of Papanicolaou smears, only eosin was detectable as a greenish band in ethidium bromide treated DNA gels under ultraviolet illumination.

Endogenous p21WAF1/CIP1 status predicts the response of human tumor cells to wild-type p53 and p21WAF1/CIP1 overexpression.

Expression of exogenous wild-type (wt) p53 protein can suppress the growth and/or induce apoptosis in different tumor cells. The effect of exogenous p21WAF1/CIP1 expression is more controversial: while it can induce apoptosis in some cells, it can protect against p53-mediated apoptosis in others. We used adenoviral vectors to introduce p53 and p21WAF1/CIP1 genes into human tumor cell lines with different p53 and/or p21WAF1/CIP1 status. The cell growth inhibition and the induction of apoptosis were measured. Overexpression of wt p53 induced more efficient growth inhibition and apoptosis in SW 620 (mutant p53) and HeLa (inactivated p53 protein) than in MCF-7 (wt p53) and CaCo-2 cell line, which was the most resistant to p53 overexpression despite the p53 mutation. Unlike HeLa and SW 620 cells, the basal p21 protein level was readily detected in CaCo-2 and MCF-7 cells. Overexpression of p21WAF1/CIP1 gene induced somewhat less pronounced growth inhibition of all cell lines tested, but it also induced apoptosis in HeLa and SW 620 cells. These results suggest that the basal, but not the inducible, levels of p21WAF1/CIP1 protein in tumor cells could protect from p53-mediated apoptosis. On the other hand, overexpression of p21WAF1/CIP1 gene itself can induce apoptosis in cells with no basal p21WAF1/CIP1 protein level. Possible mechanisms of the differential response to these genes are discussed.

p53 Immunoreactivity in oligodendrogliomas

SummaryData are presented onp53 protein presence in human oligodendrogliomas whose progress from low grade to anaplastic oligodendroglioma can be followed. Expression was evaluated by formalin-fixed paraffin-embedded section immunohistochemistry, using monoclonal PAb 1801 antibody. The frequency ofp53 protein accumulation is related to the stage of tumor malignancy. All the samples (100%) of malignant oligodendrogliomas were positive forp53 protein. Of 14 type II oligodendroglioma samples, 9 were positive (64%) while among type I oligodendroglioma the positivity was 28%. The mean proportion of reactive cells was also higher in malignant oligodendrogliomas. However, mean intensity staining did not differ among various grades of tumors. Our results point to the direct link betweenp53 protein accumulation and the malignant stage of human oligodendrogliomas. However, the value ofp53 protein accumulation in predicting malignant behavior of oligodendrogliomas requires further confirmation.

Molecular pathology of hemangiopericytomas accompanied by severe hypoglycemia: oncogenes, tumor-suppressor genes and the insulin-like growth factor family

Abstract Relatively little is known about molecular genetic events that participate in the genesis and progression of hemangiopericytoma. In this study, we describe two cases of hemangiopericytoma accompanied by severe hypoglycemia. Tumor cells from patient 1 exhibited insulin-growth factor I (IGF I) and insulin-like growth factor I receptor (IGF IR) mRNA transcripts. Tumor cells from patient 2 exhibited IGF II, IGF IR and IGF binding proteins 1–3 mRNA. Serum from patient 2 contained IGF II, mostly in a large molecular form (“big” IGF II); the IGF II level did not change after the tumor removal. The presence of IGF IR in tumor cells was confirmed by immunoprecipitation with antibodies that recognize human IGF IR subunit (visualized as a 460-kDa band). The hemangiopericytoma cells derived from patient 1 expressed 210 000 IGF I receptors/cell. Specific binding of IGF I to the tumor cell membrane fraction was higher in tissue from patient 1, while the tissue of patient 2 showed relatively low IGF I binding. In contrast, IGF II binding was much higher in tissue from patient 2. Both tumor tissues showed positive immunostaining for c-Jun; one tumor showed strong immunostaining for c-Myc, H-Ras and p53, while the other exhibited strong reaction with H-Ras antibodies only. No loss of the heterozygosity at the genes APC, NFI and nm23-H1 loci in tumor tissue obtained from patient 1 was found. In effect, our results suggest multiple molecular genetic changes in hemangiopericytoma – activation of some oncogenes and the IGF growth factor family. IGF ligands together with IGF IR could be responsible for hypoglycemia and perhaps the transformed phenotype.

Loss of heterozygosity and protein expression of APC gene in renal cell carcinomas

This study evaluated the potential contribution of the APC gene to malignant transformation in patients with renal cell carcinoma. We tested 36 human renal cell carcinoma samples and 18 adjacent normal kidney tissues for the expression of APC protein, both wild and truncated types, by western blot using antibodies that recognize either the carboxy or the amino epitope of the APC protein. The same tumor samples together with autologous peripheral blood were also analyzed at the DNA level. Using specific oligonucleotide primers for exons 11 and 15, gene instability was followed by polymerase chain reaction/loss of heterozygosity (LOH) (on the basis of restriction fragment length polymorphism). Molecular data were also compared to pathohistological diagnosis, TNM stage, and patient’s age using multivariate statistical methods. All normal renal tissues revealed expression of the wild-type APC protein. Neither wild nor mutant type proteins were found in 36% (13/36) of tumor samples; the rest of tumor tissues expressed the wild-type protein (312 kDa). Mutated APC protein, with a molecular weight of 117 kDa, was found in only one tumor sample. From 36 tumor samples 16 (44.4%) were informative for RsaI exon 11 polymorphic site, while only half of these (8/16) demonstrated LOH. From 13 tumor samples that had no detectable protein product by western blot analysis eight were homozygous for the exon 11 polymorphism and were tested for another polymorphic site, MspI/exon 15. The overall proportion of LOH cases for both polymorphisms tested was 52.9% (9/17). Pathohistological diagnosis and molecular data showed no correlation. However, multivariate analysis determined a stage strong positive correlation of age and TNM with the presence of LOH and the absence of the wild-type APC protein. Out results suggest that the APC tumor suppressor gene plays a role in renal carcinogenesis. Alterations in this gene are responsible for tumor evolution and progression, but cannot be considered as a first event in tumor initiation.

Loss of heterozygosity of thenm23-H1 gene in human renal cell carcinomas

This study evaluates the potential contribution of thenm23-H1 gene to malignant transformation in patients with renal cell carcinoma. Using specific oligonucleotide primers for thenm23-H1 microsatellite repetitive sequence, gene instability was followed by polymerase chain reaction/loss of heterozygosity assay on 54 tumor specimens and the corresponding normal tissue samples. We also determined, immunohistochemically, the relative concentration and localization of thenm23-H1 protein product. From 77.7% informative cases, DNA from 6 tumors exhibited loss of heterozygosity, regardless of the tumor stage (TNM). Out of 39 samples analyzed, 30 were negative for Nm23-H1 protein, while the others were only slightly positive. No correlation with tumor stage was found. Normal renal tissue was also negative for this protein. Our results provide the evidence for loss of heterozygosity, followed by means of microsatellite tandem-repeat polymorphism, at thenm23-H1 locus in renal cell carcinoma. However, since no correlation was found between the tumor stage or metastatic potential on the one hand, and allelic loss and specific protein expression on the other, it seems thatnm23-H1 does not play a key role in the invasiveness of this tumor type.

The exploitation of Toll-like receptor 3 signaling in cancer therapy.

Toll-like receptors (TLRs) are a group of transmembrane receptors that recognize molecular motifs of pathogen origin and activate immune response. Although TLRs were first identified in immune system cells, recent studies show they can also be expressed in tumor cells. TLR3 recognizes dsRNA or its synthetic ligand poly (I: C) and is responsible primarily for the defense against viral infections. Recent studies showed that TLR3 can trigger apoptosis in cancer cell. Furthermore, other dsRNA binding receptors (MDA5 and RIG-I), localized in cytoplasm, can also bind poly (I: C) and therefore contribute to this effect. With TLR3s capacity to induce apoptosis and activate the immune system at the same time, TLR3 ligands are an attractive therapeutic option for treatment of cancer. Novel therapies include combining poly (I: C) with other components such as chemotherapeutics, apoptosis enhancers, other TLR ligands and peptides activating the immune system. Slightly modified TLR3 agonists (Ampligen®, Hiltonol®, poly IC-LC) are already being used in clinical studies for cancer therapy as single agents or in combination with other drugs. On the other hand, latest studies forewarn that TLR3 activation can also have tumor promoting role so it is crucial to identify the terms by which TLR3 has pro-tumor/anti-tumor effect in order to safely implement TLR3 ligand based therapy into clinical trials.

Nm23-H1 promotes adhesion of CAL 27 cells in vitro

nm23‐H1 was found to diminish metastatic potential of carcinoma cell lines and therefore was placed in the group of metastatic suppressor genes. Its protein product has a function of a nucleoside diphosphate kinase (NDPK) as well as protein kinase and nuclease. Though it was found that Nm23‐H1 is involved in many cellular processes, it is still not known how it promotes metastatic suppressor activity. Since the process of metastasis is dependent on adhesion properties of cells, the goal of our work was to describe the adhesion properties of CAL 27 cells (oral squamous cell carcinoma of the tongue) overexpressing FLAG/nm23‐H1. In our experiments, cells overexpressing nm23‐H1 show reduced migratory and invasive potential. Additionally, cells overexpressing nm23‐H1 adhere stronger on substrates (collagen IV and fibronectin) and show more spread morphology than the control cells. Results obtained by EGF induction of migration revealed that the adhesion strength predetermined cell response to chemoattractant and that Nm23‐H1, in this cell type, does not interfere with, EGF induced, Ras signaling pathway. These data contribute to the overall knowledge about nm23‐H1 and its role in cell adhesion, migration, and invasion, especially in oral squamous cell carcinoma.