Trichoderma harzianum m 99/51 micromycete strain used as producer for preparing biologically active anticancer preparations

FIELD: medicine.

SUBSTANCE: what is offered is the Trichoderma harzianum rifai M 99/51 strain deposited in the Russian National Collection of Industrial Microorganisms, No. F-1027. The strain is characterised by synthesis of active anticancer non-peptide compounds having no amide bonds. Besides, the strain is marked by producing the anticancer compounds. The strain has high cytotoxic activity with respect to the anticancer cell lines (human HaCaT-keratinocytes; THP-1 macrophage-like human cell line; A431 - human epidermoid carcinoma line; JurKat - human T-cells; K562 - chronic myeloid leukemia cell line; HEK293T - human embryonic kidney cells) and may be used for producing anticancer preparations.

EFFECT: using this strain as a producer for making the based anticancer preparations.

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The invention relates to the field of Microbiology, relates to a new strain M 99/51 Trichoderma harzianum Rifai (VKPM F-1027), and can be used for anticancer drugs.

It is known that the composition of the components of the higher basidiomycetes, such as Fomitopsis officinalis, Ganoderma lucidum, Coriolus pubescens, are drugs that have immunomodulatory effects [Bogaev A.G., 2007. / Study of the induction of immunomodulatory effects of oil extracts of fruiting bodies of basidiomycetes in mice inoculated with tumor carcinoma. / Aggai, Avellini, Magagazine. // Advances in medical Mycology. V.9, 2007. - P.148-150; Gortina Y.S. Biotechnological preparation of medicinal mushroom of coriole pubescent. // Eshurinu, MOV, Weglicki. / Modern Mycology Russia. Abstracts of the first Congress of mycologists Russia. - M.: national Academy of Mycology. - W. - S; Mahajna et al. Mushroom extracts having anticancer activity // Pub. No.: US 2006/0045887 A1. Pub. Date: Mar. 2, 2006. - p.21].

Of the polysaccharides of the fungus Trametes. versicolor was obtained and well studied clinically drug Japanese biotechnology firms "SanKyo" "the Christening" (PSK). The drug has proved moderately effective and, most importantly, low-toxic oncostatic with maintenance therapy of cancer, reducing hematological suppression caused by other anticancer drugs, is also as prospecive and paregoric [Larosz, W.A. Effect of saponin called from Midicado saliva extract on biological activity of Trametes versicolor // W.A.Larosz, E.Malarczyk, .Larzysta / J. New Horizon of Bioscience in Forest products Field, 2003. - P. 213-220].

Research in recent years shows that the phytopathogenic fungi of the genus Trichoderma also possess immunomodulatory and antitumor properties. A number of authors have established some groups of active substances, such as Trichoderma modified dipeptides, isolated from a marine strain of T. virens. Viridifolia isolated from T. viride, have a cytotoxic effect on tumor cells. Trihedron and cycloheptanone derivatives, isolated from the culture of T. harzianum, a parasite of Halichondria okadai, have a cytotoxic effect on the line of tumor cells [Reino, J.L. Secondary metabolites from species of the biocontrol agent Trichoderma / J.L.Reino, R.F.Guerriero, R.Hema'ndez-Gala', I.G.Collado // Phytochem Rev. - 2008. - V.7. - p.89-123].

Currently, the world microbiological industry not mastered the production of antitumor biological products on the basis of active strains of phytopathogenic fungi of the genus Trichoderma.

The technical result of the invention is a strain of M 99/51 (VKPM F-1027)with antitumor activity.

The objective is achieved by the use of strain M 99/51 (VKPM F-1027) to suppress the growth of tumor cells. Feature of the strain is unique biochemical composition. The strain at the surface and deep cultivation is assured secretes into the culture fluid metabolites, with antitumor properties against lines of tumor cells: Nasal - human keratinocytes; TNR-1 - macrophagecolony line of human cells; A431 - line epidermoid carcinoma of the person; Jurkt-T-cells; C - line cells of chronic myeloid leukemia; SOME T cells embryonic human kidney. Active principle active compounds is not a protein or peptide, refers to molecules that do not have amide bonds.

Offered the use of this strain as bioproducts for creation on its basis of anticancer drugs.

The strain obtained as monopoloy clone through breeding on the antitumor activity of wild strain M-99/5 Trichoderma harzianum (VKPM F-888), isolated from mulch Mininskom forestry Krasnoyarsk region.

Strain M-99/51 Trichoderma harzianum is characterized by the following features:

Micromorphologically signs:

Diagnostic environment (Chapek's medium at 25°C) - growth is very fast, the edge of the colony is fringed mycelium vatoobraznye; Radius colonies after 72 hours of growth is 72-75 mm at a temperature of 22°C. the Aerial mycelium is white, the Color of the colonies from grass-green to green malachite. The pigment in the environment does not evolve (figure 1).

Micromorphology:

Conidiophores unpainted smooth, straight or slightly sinuous 4,0-8,0 µm in diameter. Fieldy obrany in groups of 2-5, with the expanded basal part and a short narrow neck 5-7×3-5 µm, terminal of feality more elongated to 12 µm in length. Conidia ovoid, green, located on the hyphae of the terminal or intercalate size of 2.7 to 3.5×1,8-3,0 µm in diameter (figure 2). Forms chlamydospores.

Other media used for cultivation, - wort-agar, oatmeal agar.

Molecular genetic characteristics:

The strain has the following sequence:

The degree of similarity with collection of samples according to homology with other strains (NCBI): No. Z68189.1 Trichoderma harzianum strain CBS 819.68 is 97,0%.

According to the experts of the Krasnoyarsk regional veterinary laboratory, this strain of avirulent, non-toxic, netoxygen in relation to warm-blooded organisms and does not cause infectious diseases.

Figure 1-8 presents the following picture:

Figure 1. The morphology of colonies of strain M-99/51 Trichoderma harzianum on wort agar.

Figure 2. The micromorphology of the mycelium, conidiophores and conidia of strain M 99/51 Trichoderma harzianum:

1. The micromorphology of conidiophores and mycelium;

2. - Morphology of conidia (electron microscopy in scanning mode).

Figure 3. The index of inhibition of the culture fluid of strain M 99/51 Trichoderma harzianum lines of tumor cells.

Figure 4. The elution profile of the culture fluid of strain Trichodera harzianum M 99/51 on a column with reversed phase, wavelength -214 nm.

Fig 5. Assessment of apoptosis cell line A431 action faction F8 from the culture fluid of Trichoderma harzianum strain M99/51 (confocal microscopy cells, incubation for 20 h, the increase h).

6. The index of inhibition of cell TNR-1 and SOME T 72 h 15 fractions strain Trichoderma harzianum M 99/51.

Fig 7. The effect of fraction 8 cultural liquid of Trichoderma harzianum strain M 99/51 on the cell cycle C. The white area control, gray - adding fractions.

Fig. Antitumor activity of fraction 8 cultural liquid of Trichoderma harzianum strain M 99/51 in the cells C without treatment after treatment with trypsin.

Example 1. Deep cultivation of the strain M 99/51 Trichoderma harzianum

To study the activity of metabolites of the fungi Trichoderma against tumor cells receive the culture fluid depth and the mixed liquid-phase cultivation on a nutrient medium of čapek following composition: sucrose 30 g, sodium nitrite - 2 g of magnesium sulfate 0.5 g, potassium chloride 0.5 g, potassium phosphate disubstituted - 1,0,

Sowing produce culture of strain at the rate of 1.5-2×106conidia per 1 ml of culture medium. As inoculum using a culture of the strain grown on stubble wort-agar. From tubes do flush in flasks with sterile water and then determine the titer of the dispute in the resulting suspension of mikros epicheskim way the camera Goriaev and the method of conversion for the entire volume of the nutrient medium using the required amount of suspension to be planted. Sowing produced in the Erlenmeyer flask with a volume of 750 ml 200 ml of culture medium.

Cultivation of strain conduct in-depth way on the rocking chair at 24-26°C in flasks 750 ml for 5 and 10 days until the fungus does not reach the stationary phase of growth. The obtained culture liquid and centrifuged to remove the precipitate, sterilized by filtering through a filter with a pore diameter of 0.22 μm and test on the lines of tumor cells.

Example 2. Analysis of the cytotoxic activity of metabolites of the strain M 99/51 Trichoderma harzianum

For detection of antitumor activity analyze cytotoxic and cytostatic effects in vitro using the MTT test. MTT-test is used to evaluate the cytotoxicity of potentially anticancer compounds in the experiment, which is based on the ability of the dehydrogenases of living cells to restore unpainted form 3-4,5-dimethylthiazol-2-yl-2,5-diphenylthiazole (MTT reagent) to blue crystalline parmesana, soluble in dimethyl sulfoxide.

For this purpose, cells of various cell lines contribute in flat-bottomed 96-well plate, 50 thousand per well, in which pre-titrated culture fluid of strain M 99/51 Trichoderma harzianum. Tablets incubated in CO2-the incubator for 72 h and on the last 4 hours add 250 µg/ml MTT. After incubation the supernatant is removed and the wells add 100 ál of dimethyl sulfoxide (Reachim, Moscow) for dissolution of formazan. The tablets analyzed on a tablet spectrophotometer (Titertek, UK)at a wavelength of 540 nm. The results processed using the software package Excel (Microsoft). The effect of the drugs assessed by the index of inhibition (AI), calculated according to the formula: AI=[1-(ODop/OPpin)]*100%, where OPop- the optical density in the pilot hole, OPpin- the optical density in the control wells.

The culture fluid obtained in submerged cultivation of strain M 99/51 within 10 days on the environment of čapek, causing 100% mortality of cell lines K, Jukat and SOME T (figure 3).

Example 3. Analysis of the proapoptotic activity of metabolites in the culture fluid of strain M 99/51 Trichoderma harzianum

Assessment of the proapoptotic activity of the culture liquid strain is carried out with the help of experienced staining of the cell 10-N-nanosil-acridine orange (NAO), which binds to cardiolipin in mitochondria. Cardiolipin is oxidized under the action of apoptotic stimuli [Lutsenko GV Cytometrics method of registration of apoptosis using fluorophore 10-N-nonyl-acridine orange, Biological membranes, 2010, vol 27, No. 5, S-439].

To assess apoptotic actions the culture fluid of cells A431 werasit on sterile slides to a confluent monolayer. The monolayer add to liturally liquid strain concentration LD 50(50% lethal dose at which there is 50% inhibition of cell growth) and incubated for 20 hours Assessment apoptosis spend staining cells within 45 minutes of mitochondrial dyes Nonyl Acridine Orange (NAO), in accordance with the Protocol. Visualization of the cells is performed using staining nuclei dye Hoechst33342 (Sigma). After staining, the cells fixed with 2%solution of paraformaldehyde for 1 h, then washed three times FB and polimerizuet on slides using resin Mowiol 4.88 (Calbiochem, Germany). Detection is performed using confocal microscopy the microscope Eclipse A company Nikon (Japan).

As a control in the evaluation of apoptotic activity in the culture fluid used anticancer drug Actinomycin D.

As a result of experiments on the analysis of cytotoxicity and proapoptotic activity found that a strain exhibits significant activity against three cell lines: A431 (figure 5). One of the mechanisms of action is the induction of apoptosis of late-stage (20 h).

Example 4. Fractionation of metabolites of strain M 99/51 Trichoderma harzianum by high performance liquid chromatography (HPLC).

For a more detailed analysis of the current start spending fractionating the supernatant by the method of VAS is. Figure 4 shows the elution profile of the fractions in the gradient of acetonitrile. The fractions obtained lyophilizer, dissolved in water. Then analyze activity MTT-test. Fractions obtained from the culture filtrate of strain M 99/51, tested on two lines of tumor cells TNR-1 and SOME T. The results of the experiment are presented as histograms in Fig 6. After analyzing the results of the experiment, note that the fraction F8 has a toxic effect on the two lines of cells TNR-1 and SOME T and is of interest for the selection of anticancer compounds.

Example 5. The cell cycle analysis of active fractions of strain M 99/51 Trichoderma harzianum

The impact analysis of the fractions obtained from the culture fluid of strain M 99/51 under cultivation of strain within 10 days, the cell cycle is performed on the suspension cell lines Jurkat and C. Fraction added to the cells at 24 h after incubation, the cells are transferred to phosphate buffer (FB), centrifuged, and the sediment is fixed with cold 70%ethanol for 12 hours at minus 2°C. after fixation, the cells washed FB and stained with a solution of propidium iodide (50 µg/ml) in the presence of 10 μg/ml RNase (Evrogen, Moscow, Russia) for 1 hour. Followed by cell cycle analysis on FACScan instrument (BD). When analysing put the region on the area and peak width to select the singlet what's cells (Fig.7). As a result of comparison of the two histograms revealed no action nadeshiko on the cell cycle, which means that no activity inhibitor of the cell cycle.

Example 6. Antitumor effects of culture medium and the active fractions of strain M 99/51 Trichoderma harzianum substances peptide

For analysis of the role of peptides and proteins in the antitumor action use the fraction 8 culture fluid of strain M 99/51 Trichoderma harzianum. Carry out the hydrolysis using 0.05% trypsin for 16 h, the Cytotoxicity of the resulting hydrolysate trypsin to assess cell lines Nasat b THP-1 using the MTT-test. Fraction 8 culture liquid after the hydrolysis contribute to the culture of cells in different quantities. As a control, use the original fraction, which stand for 16 hours without the addition of trypsin. Analysis of cytotoxicity at different concentrations of fraction 8 cultural liquid of the fungus showed no changes in the activity of the culture fluid in the components hydrolysis by trypsin, which means the absence of the peptides in the composition of the current start of study drug (Fig).

Strain Trichoderma harzianum Rifai M 99/51 (VKPM F-1027), used for receiving anticancer drugs.



 

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