Cytotoxic effect of the biotechnologically-derived justicidin B on human lymphoma cells.

Purpose of work: The study was aimed to assess the antineoplastic activity of justicidin B in vitro and to search for its general toxicological profile in vivo. The anti-neoplastic activity of the arylnaphthalene lignin, justicidin B, was assessed in a panel of human lymphoma cell lines and compared with etoposide as a reference compound. A screening of the cytotoxicity after 24, 48 and 72 h exposure was performed by the MTT-dye reduction assay. Dose- and time-dependent cytotoxic effect was observed and the IC50 values ranged from 0.17 µM (RPMI-8226, 72 h) to 183 µM (U-266, 24 h) and more than 200 µM (HD-MY-Z, 24 and 48 h). Activation of caspase 3 and 8 was involved in the induction of programmed cell death in DOHH-2 cell line. NF-κB modulation occurred in DOHH-2 and HH cells. The general toxicity in mice after i.p. injection was also tested. The highest applied dose (50 mg/kg = 137.25 µM) did not show any toxicity. Justicidin B possesses definite and potent selective antineoplastic activity, related to its ability to induce programmed cell death in NHL-derived human cell lines at concentrations that can be reached in mice without toxicity.

Anticancer lignans--from discovery to biotechnology.

Malignant diseases are the second mortality cause within the human population. Despite the serious progress in establishing and introduction of novel specifically targeted drugs the therapy of these diseases remains severe medical and social problem. Some of the most effective cancer treatments to date are natural products or compounds derived from plant products. Isolation of anticancer pharmaceuticals from plants is difficult due to their extremely low concentrations. The industry currently lacks sufficient methods for producing all of the desired plant-derived pharmaceutical molecules. Some substances can only be isolated from extremely rare plants. Plant cell cultures are an attractive alternative source to whole plant for the production of high-value secondary metabolites. The biotechnological method offers a quick and efficient method for producing these high-value medical compounds in cultivated cells. Due to the pharmaceutical importance and the low content in the plants the present review focuses on discovery and alternative production systems for anticancer lignans--aryltetralin and arylnaphthalene lignans. The aim is to focus on recent progress of in vitro production of anticancer lignans, together with structure elucidation, the methods of increasing the levels of desired substances in plant cell and tissue cultures in general. Experience of different authors, working worldwide on plant biotechnology, has been discussed to show positive results in experiments.

Effect of justicidin B - a potent cytotoxic and pro-apoptotic arylnaphtalene lignan on human breast cancer-derived cell lines.

Justicidin B produced by genetically transformed cultures of Linum leonii was tested for cytotoxic activity and induction of apoptosis in MDA-MB-231 and MCF-7 breast cancer derived cell lines. The tested lignan evoked strong, concentration dependent cytotoxicity in both cell lines, whereby MCF-7 proved to be far more sensitive as compared to MDA-MB-231. The 24 h treatment of both cell lines increased the level of apoptotic DNA fragmentation; however the proapoptotic activity is completely inhibited if the cells are co-incubated with the non-selective pan-caspase inhibitor Boc-Asp(OMe)-fluoromethyl ketone (PCI), which implies that justicidin B, activates programmed cell death via caspase -dependent mechanisms. Exposure of MDA-MB-231 cells with justicidin B leads to concentration dependent decrease in the expression of NFkB; whereas the treatment of MCF-7, is consistent with strong increase in the expression of this transcription factor.

Production of podophyllotoxin in Linum linearifolium in vitro cultures.

For the first time, callus and suspension cultures of Linum linearifolium were initiated. Podophyllotoxin (PTOX), a strong antitumor precursor, was isolated from the calli and suspension, as a main lignan besides smaller amount of 6-methoxypodophyllotoxin (6MPTOX). L. linearifolium is now the third Linum species of section Syllinum, with PTOX as the main lignan. The amounts of lignans, especially PTOX, found in L. linearifolium cell cultures are quite high within the studied Linum species until now. The antiproliferative effects of extracts were tested in a panel of human tumor cell lines, using the MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide]-dye reduction assay. The lignan mixtures caused concentration-dependent inhibition of malignant cell proliferation and showed moderate cytotoxic activity. The results clearly demonstrate that the lignan mixture of L. linearifolium exerts inhibitory effects against malignant cells.

Effects of cycloartane saponins from hairy roots of Astragalus membranaceus Bge., on human tumor cell targets.

For the first time three different natural compounds, isolated from hairy roots of Astragalus membranaceus, cultivated in airlift bioreactor were tested for their cytotoxic potential and apoptosis induction in a panel of human tumor cell lines. Root cultures, cultivated in bioreactor gave 18.5 g l(-1) dry wt roots with the highest astragaloside production in vitro up to now - 1.64% (astragaloside I), 1.12% (astragaloside II) and 1.08% (astragaloside III). In this manner the production in airlift bioreactor can be used as means of reliable supply of cycloartane saponins to extend the research to human clinical studies.

Lignans from Linum species of sections Syllinum and Linum.

The aryltetralin lignans 6-methoxypodophyllotoxin, 5'-demethoxy-6-methoxypodophyllotoxin as well as the corresponding 8'-epimers 6-methoxypicropodophyllin, and 5'-demethoxy-6-methoxypicropodophyllin were isolated from suspension cultures of Linum cariense, and 4'-demethyl-6-methoxypodophyllotoxin together with 6-methoxypodophyllotoxin from plants of L. tauricum, which both belong to section Syllinum of the genus Linum. Cell cultures of L. altaicum, L. austriacum ssp. euxinum and L. lewisii belonging to section Linum accumulate the naphthalene lignans justicidin B and isojusticidin B. The different lignans were identified by HPLC and spectroscopic methods.

Cytotoxic activity of a podophyllotoxin-like lignan from Linum tauricum Willd.

The present study describes the preliminary evaluation of the cytotoxic activity of a podophyllotoxin-like compound 4'-demethyl-6-methoxypodophyllotoxin (4'-DM-6-Mptox), isolated as one of the main lignans of Linum tauricum Willd. ssp. tauricum. The cytotoxic effects 4'-DM-6-Mptox were assessed by the MTT-dye reduction assay against the human leukemic cell lines HL-60, BV-173 and LAMA-84. DNA-fragmentation analysis and NF-kB inhibition assay were performed in order to elucidate some of the mechanistic aspects of the cytotoxic action of the investigated compound. 4'-DM-6-Mptox was found to exert prominent cytotoxicity, with IC50 values being several-fold lower than those of the referent antineoplastic agent etoposide. The DNA-fragmentation analysis revealed that 4'-DM-6-Mptox treatment triggered apoptosis in BV-173 and HL-60 cells. In our hands 4'-DM-6-Mptox was found to induce concentration-dependent NF-kB inhibition in HeLa cells as assessed by the IL-6 luciferase gene reporter assay, which though not quite prominent, at least partly contributes to the cytotoxic potential of the tested lignan. On the basis of the results obtained it could be concluded that 4'-DM-6-Mptox necessitates further pharmacological and toxicological evaluation as a possible chemotherapeutic agent. Furthermore due to its relatively high concentrations in the described plant source the possibility for its use as a precursor for the semisynthetic production of lignan-based drugs, could be considered.

Cytotoxic activity of extracts from Linum cell cultures.

Callus and suspension cultures of Linum narbonense and Linum leonii were developed to study the production of lignans and their cytotoxic activity. Justicidin B was determined to be the main lignan. The maximal yield of justicidin B up to 2.22 mg/g of the cell dry weight was detected in the callus cultures of L. leonii, followed by the callus cultures of L. narbonense (1.57 mg/g dwt). The cytotoxicity of the obtained extracts was measured using the MTT-dye assay. L. narbonense and L. leonii both showed cytotoxic activity.

HPTLC densitomeric determination of justicidin B in Linum in vitro cultures.

A convenient, sensitive and accurate method for the separation and determination of justicidin B was created. The values were calculated by linear calibration under standard conditions. The method can be applied for the quantification of justicidin B in plants or in vitro cultures.