Effects of two disiloxanes ALIS-409 and ALIS-421 on chemoprevention in model experiments.

Morpholino-disiloxane (ALIS-409) and piperazino-disiloxane (ALIS-421) compounds were developed as inhibitors of multidrug resistance of various types of cancer cells. In the present study, the effects of ALIS-409 and ALIS-421 compounds were investigated on cancer promotion and on co-existence of tumor and normal cells. The two compounds were evaluated for their inhibitory effects on Epstein-Barr virus immediate-early antigen (EBV-EA) expression induced by tetradecanoyl-phorbol-acetate (TPA) in Raji cell cultures. The method is known as a primary screening test for antitumor effect, below the (IC50) concentration. ALIS-409 was more effective in inhibiting EBV-EA (100 µg/ml) and tumor promotion, than ALIS-421, in the concentration range up to 1000 µg/ml. However, neither of the compounds were able to reduce tumor promotion significantly, expressed as inhibition of TPA-induced tumor antigen activation. Based on the in vitro results, the two disiloxanes were investigated in vivo for their effects on mouse skin tumors in a two-stage mouse skin carcinogenesis study. The application of dimethyl-benzanthracene (DMBA; 390 nmol) as a tumor initiator was followed by exposure to TPA (1.7 nmol/l) as a tumor promoter. The experiments showed that ALIS-409 at a concentration of 85 nmol/l had a weak EBV-EA inhibitory effect in vitro and a moderate antitumor activity, compared to the positive control of DMBA plus TPA-treated mice. Flow cytometry by differential staining demonstrated interactions in co-cultures of MCF7 breast cancer and MRC5 human lung fibroblasts. The growth rate of tumor cells in mixed populations of MCF7 breast cancer and MRC5 normal fibroblast cells was reduced in the presence of ALIS-409, as compared to the control non-treated cell populations. The two disiloxanes were moderately-effective in chemoprevention in DMBA-induced and TPA-promoted in vivo tumor formation. Authors suggest that the inhibition of tumor cell and fibroblast interaction by ALIS409 might have some perspective in the development of anti-stromal therapy.

In vitro antiproliferation in prostate cancer cell lines with cytostatics and combinations with resistance modifiers.

The treatment of prostate cancer in an advanced state is still unsatisfactory. In the event of the ineffectiveness of total androgen blockade (TAB) therapy, cytostatic administration may be attempted. In this study, we modelled the drugs used in practice on human prostate cancer cell lines. Studies aimed at decreasing multidrug resistance were performed on PC-3 cells. With the use of various cytostatics, the cell proliferation-inhibiting effects were measured under in vitro conditions on human prostate cancer cell lines LNCaP-FGC and PC-3. Under the given experimental conditions, the examined cytostatics exhibited antiproliferative effects on each of the investigated cell lines. Our results indicate that it is not necessary to wait until the development of a hormone-resistant state. In the studies on the PC-3 cell line, we did not find a multidrug-resistant efflux activity responsible for the resistance of the tumour.

Modulation of multidrug resistance and apoptosis of cancer cells by selected carotenoids.

The multidrug resistance (MDR) proteins that belong to the ATP-binding casette superfamily are present in a majority of human tumors and are an important final cause of therapeutic failure. Therefore, compounds which inhibit the function of the MDR-efflux proteins may improve the cytotoxic action of anticancer chemotherapy. The effects of carotenoids were studied on the activity of the MDR-1 gene-encoded efflux pump system. The carotenoids, isolated from paprika and other vegetables, were tested on the rhodamine 123 accumulation of human MDR-1 gene-transfected L1210 mouse lymphoma cells and human breast cancer cells MDA-MB-231 (HTB-26). Capsanthin and capsorubin enhanced the rhodamine 123 accumulation 30-fold relative to nontreated lymphoma cells. Lycopene, lutein, antheraxanthin and violaxanthin had moderate effects, while alfa- and beta-carotene had no effect on the reversal of MDR in the tumor cells. Apoptosis was induced in human MDR1 transfected mouse lymphoma cells and human breast cancer MDA-MB-231 (HTB-26) cell lines in the presence of lycopene, zeaxanthin and capsanthin. The data suggest the potential of carotenoids as possible resistance modifiers in cancer chemotherapy.

Chemosensitizing effect of vitamin C in combination with 5-fluorouracil in vitro.

The antiproliferative effect and apoptosis-inducing action of 5-fluorouracil (5-FU) in combination with vitamin C were tested in vitro against the chemosensitive mouse lymphoma, the chemoresistant HEp-2 and a human lung fibroblast cell line. Vitamin C itself had no antiproliferative effect on the fibroblasts, but increased the anticancer effect of 5-FU dose-dependently. In the case of the chemoresistant cell line, only a high concentration of vitamin C increased the cytotoxicity of 5-FU. A combination of 5-FU and vitamin C exerted a significantly enhanced apoptotic effect on the mouse lymphoma cell line, whereas for the HEp-2 cell line this effect was less marked and was achieved only at a high concentration of vitamin C. These findings suggest that the administration of a high dose of vitamin C in combination with 5-FU chemotherapy enhances the chemoresponsiveness of cancer cells and serves as a potential sensitizer, especially in chemo-resistant cell lines. One of the mechanisms by which vitamin C potentiates cytostatics could be apoptosis induction.

Reversal of multidrug resistance in mouse lymphoma cells by phenothiazines.

Various compounds were tested with regard to their reversal of multidrug resistance (MDR) in mouse tumor cells transfected with the human MDR1 gene. Phenothiazines containing aromatic moieties were bound through stacking interaction involving the polarization of the aromatic aminoacid substituents at the target site of p-glycoprotein (Pgp) 170, as a consequence of their large dipoles (as in the binding of phenothiazine to calmodulin-like structures). Acting as a calcium channel blocker, verapamil may induce conformational changes in the calcium channel-like structures of the transmembrane regions of Pgp. Most probably the tyrosine moieties of Pgp are involved in the action of verapamil and phenothiazines. Tomato lectin specifically binds to the polylactosamine moiety of Pgp170 at the first loop of Pgp. Other targets in the membrane may exist in close proximity to Pgp170, such as conA-reactive glycoproteins with terminal mannosyl residues. WGA-reactive N-acetyl glucosamine residues can also be modified resulting in conformational changes in trans-membrane regions of the ABC transporter. Our results demonstrate that MDR can be reversed by interaction of various compounds with Pgp or by modification of the membrane structure around the Pgp.

Cancer prevention and therapy with kiwifruit in Chinese folklore medicine: a study of kiwifruit extracts.

Kiwi gold fruits were extracted successively with hexane, acetone, methanol and 70% methanol, and further fractionated by silica gel and ODS column chromatographies for the assays of various biological activities. Five fractions H1, H2 (hexane extract), Al, A2 (acetone extract) and M2 (methanol extract) showed selective cytotoxic activity against human oral tumor cell lines, which was more sensitive than human gingival fibroblasts. More hydrophilic fractions [70M3, 70M4, 70M5] of 70% methanol extract displayed higher anti-HIV activity, radical generation and O2- scavenging activity. The antibacterial activity of 70% methanol extracts [70M0, 70M1, 70M2, 70M3, 70M4] was generally lower than that of more lipophilic fractions (hexane, acetone, methanol extracts), although each fraction did not show any specific antimicrobial action. All fractions were inactive against Helicobacter pylori. These results demonstrate that gold kiwifruit extracts contain valuable, various bioactive materials, which can be separated with each other.

Combined effect of cisplatin and 5-fluorouracil with irradiation on tumor cells in vitro.

The concomitant administration of chemotherapy and radiation is an alternative tool in cancer therapy. The antiproliferative and the radiosensitizing effect of Cisplatin and 5-Fluorouracil (5-FU) were studied on mouse lymphoma cells transfected with human MDR1 gene (mdr) and its parent cell line (par) combined with and without radiation. HEp2 radioresistant cell culture was used in our experiments as a model of radioresistance. The growth rate and antiproliferative effect was measured by the MTT method. Significant inhibition of tumor cell growth was observed at a low concentration of Cisplatin and 5-FU combined with radiation on the mouse lymphoma cell lines. However an extremely high dose of Cisplatin and 5-FU resulted in moderate growth inhibition in the case of the HEp2 cell line. We assume that the radiosensitizing effect of 5-FU and Cisplatin can be considered as a synergistic antitumor effect at low doses of chemotherapy and radiation in a radiosensitive cell line. In the case of a radio-and chemoresistant cell line, high doses of radiation and chemotherapeutic agent achieved moderate tumour growth inhibition without significant synergistic effect. In addition the simultaneous application of both treatments can result in remarkable toxicity.

Peptide based vaccine design: synthesis and immunological characterization of branched polypeptide conjugates comprising the 276-284 immunodominant epitope of HSV-1 glycoprotein D.

The importance of the length and conjugation site of a protective epitope peptide (276SALLEDPVG284) from glycoprotein D of herpes simplex virus in branched polypeptide conjugates has been investigated. A new set of peptides, with a single attachment site and truncated sequences, was prepared. The immunogenicity of conjugates and the specificity of antibody responses elicited were investigated in BALB/c, C57/B1/6 and CBA mice. It was found that the covalent coupling of the peptide comprising the 276-284 sequence of gD through its Asp residue at position 281 did not influence the immunogenic properties of the epitope, while involvement of the side chain of Glu at position 280 almost completely abolished immunogenicity. These results clearly indicated that the conjugation site of the epitope peptide influenced the intensity and specificity of antibody responses. Comparison of the immunological properties of conjugates containing truncated gD peptides revealed the presence of two epitopes within the 276-284 region. One of the proposed epitopes is situated at the N-terminal (276-281) region, while the other is located at the C-terminal end of the sequence (279-284). Binding data demonstrated that some of the peptides comprising these epitopes induced gD-specific responses in their conjugated form and also elicited an immune response that conferred protection against lethal HSV-1 infection. The correlation of peptide- and gD-specific antibody responses with the protective effect of the immune response is discussed.

Interaction between various resistance modifiers and apoptosis inducer 12H-benzo[alpha]phenothiazine.

The effect of some resistance modifiers on apoptosis induction by a benzo[alpha]phenothiazine derivative was studied on the L5178Y mouse lymphoma cells (parent) and its multidrug resistant (MDR) subline. For evaluation of apoptosis the cells were stained with FITC-labelled annexin V and propidium iodide and the results were analysed by flow cytometry. 12H-benzo[alpha]phenothiazine [M627] induced apoptosis both in the parent cells and in the MDR cells. The apoptosis induction by [M627] was not affected significantly by post- or pre-treatment with resistance modifiers, while in the cells treated by (+/-)-verapamil before and after apoptosis induction with [M627], the apoptosis was somewhat higher. The resistance modifier compounds alone also induced apoptosis and it was slightly higher in the parent cells than its MDR1/A gene-transformed subline.