KR100408108B1 - Agents having a differentiation effect for a neuroblastoma containing a leucocyanidin - Google Patents

Abstract

The present invention relates to neuroblastoma (NB) cell differentiators containing leucocyanidine as an active ingredient.

Description

AGENTS HAVING A DIFFERENTIATION EFFECT FOR A NEUROBLASTOMA CONTAINING A LEUCOCYANIDIN}

Cancer is one of the largest single signs of both sexes, killing more than 6 million people worldwide each year. Neuroblastoma (NB) is the most common cancer affecting 8-10% of children ages 0-14. NB can be cured limitedly by surgery, but if it has already spread through the bone marrow, it will have fatal consequences. See also Prognostic factor in neuroblastoma. Cancer , Jun 1; 59 (11): 1853-9, 1987, Evans AE, D'Angio GJ, Propert K, Anderson J, Hann HW.

A rudimentary but effective method of treating cancer is the use of DNA damaging agents to kill proliferating cells [Uncoupling of S phase and mitosis induced by anticancer agents in cells lacking p21. Nature. , Jun 20; 381 (6584): 713-6, 1996, Waldman T, Lengauer C, Kinzler KW, Vogelstein B. However, the problem with this method is whether the DNA damaging agent specifically acts on tumor cells. Thus, in recent years, attention has been focused on the use of substances that can influence cellular control pathways to induce differentiation in particular cell types. Differentiation of NB cells is a vitamin {Vitamin E increase the growth inhibitory and differentiating effects of tumor therapeutic agents on neuroblastoma a gliomma cells in culture. Proc Soc exp Biol Med. , 164: 158-163, 1980, Prasad KN, Edwards-Prasad J, Ramanuja S, Sakamoto A, In Saunders Cell differentiation and neoplasia, p. 111 (Ravan Press, New York, 1978), Prasad KN, Sinha PK, Retinoic acid induced growth inhibition and morphological differentiation of human neuroblastoma cells in vitro. J Natl Cancer Inst. , 68: 589-593, 1982, Sindell N] }, see, ball four ester (phorbolester) {literature [Vitamin E increase the growth inhibitory and differentiating effects of therapeutic agents on tumor neuroblastoma a glioma cells in culture. Proc Soc exp Biol Med. , 164: 158-163, 1980, Prasad KN, Edwards-Prasad J, Ramanuja S, Sakamoto A, In Saunders Cell differentiation and neoplasia., P. 111 (Ravan Press, New York, 1978), Prasad KN, Sinha PK, Retinoic acid induced growth inhibition and morphological differentiation of human neuroblastoma cells in vitro. J Natl Cancer Inst. , 68: 589-593, 1982, Sindell N, Differential effects of phorbol ester on the beta-adrenergic response of normal and ras-transformed NIH3T3 cells. Biochem Biophys Res Commun. , Dec 17; 133 (2): 702-8, 1985, Garte SJ, Differential effects of the tumor promoter phorbol-12-myristate-13-acetate on the morphological and biochemical differentiation of N-18 mouse neuroblastoma cells. J Cell Physiol. , Dec; 125 (3): 387-92, 1985, Liu AY, ChenKY, Effects of phorbol ester tumor promoters and nerve growth factor on neurite outgrowth in cultured human neuroblastoma cells. Cancer Res. , Dec; 42 (12): 5067-73, 1982, Spinelli W, Sonnenfeld KH, Ishii DN]}, hormones , cAMP {Adrenosine analogues stimulate cyclic AMP-accumulation in cultured neuroblastoma and glioma cells. Acta pharmac. , 55: 297-302, 1984, Elfman L, Lindgren E, Fredholm BB, Blockade of NIE-115 murine neuroblastoma muscarinic receptor function by agents that affect the metabolism of arachidonic acid. Biochem Pharmacol. , Jul 15; 35 (14): 2389-97, 1986, McKinney M, Richelson E], Role of cyclic AMP in differentiation of human neuroblastoma cells in culture. Cancer. , Oct; 36 (4): 1338-43, 1975, Prasad KN, Kumar S, Cyclic AMP-induced differentiated neuroblastoma cells: changes in total nucleic acid and protein contents. Biochem Biophys Res Commun. , Feb 20; 50 (4): 973-7, 1973, Prasad KN, Kumar S, Gilmer K, Vernadakis A, Morphological differentiation of a murine neuroblastoma clone in monolayer culture induced by dexamethasone. Exp Cell Res. , May; 113 (2): 375-81, 1978, Sandquist D, Williams TH, Sahu SK, Kataoka S]}, cell proliferation inhibitors , X-ray {X-ray-induced morphological differentiation of mouse neuroblastoma cells in vitro. Nature. , Dec 24; 234 (5330): 471-3, 1971, Prasad KN], and natural extracts . In particular, the interest in natural extracts has recently been greatly amplified. This is because natural extracts are grown all over the world and can be easily prepared as food. Resveratol, a type of phytoalexin contained in grapes and other foods, induces cell differentiation (pro- cessive activity) of promyelocytic leukemia, and polyphenol extract derived from barley bran is HL-60. It generates biochemical markers of cell differentiation. Polyphenols from Camellia Sinenis and proanthocyanidins from grape seeds have been reported to have anticancer activity. This is described in Cancer chemopreventive activity of resveratol, a natural product derived from grapes. Science. , Jan 10; 275 (5297): 218-20, 1997, Jang M, Cai L, Udeani GO, Slowing KV, Thomas CF, Beecher CW, Fong HH, Farnsworth NR, Kinghorn AD, Mehta RF, Moon RC, Pezzuto JM. . A common feature of these materials is that they have antioxidant activity that inhibits the production of free radicals that can promote cancer, namely superoxides, hydroperoxy radicals, singlet oxygen, hydroxyl radicals and hydrogen peroxide. will be. Thus, polyphenols, grape seed extracts, are also expected to act as anticancer agents and cell differentiation inducing agents. Flavan -3,4-diol-leucocyanidine (LC) represented by the following Chemical Formula 1, which is one of the extracts of grape seed ( Vitis vinifera ), exhibits vascular disorder preventing effect, strong free radical scavenging activity, and antimutagenic effect. Although reported, the efficacy as a cell differentiation inducing agent is not yet known.

Nitric oxide (NO) is a free radical known as an activator synthesized in many mammalian cells in recent decades. The action of NO is associated with biological mechanisms such as neurotransmission, death of tumor cells, immunity, and inflammation. NO has also been reported to provide cytoprotective effects.

The production of NO is catalyzed by NADPH dependent NO synthetase (NOS), which produces L-arginine in a 1: 1 stoichiometric ratio of L-citrulline and NO, while competitively by N ^G substituted L-arginine analogs. Suppressed. There are three or more different isoenzymes for NOS enzymes that catalyze the production of NO. One of the two NOS gene products, eNOS, is structurally expressed in endothelial cells and another, nNOS, is structurally expressed in nerve cells, stomach and uterus. INOS, the third species of NOS, is expressed by immune stimulation. For the production of NO, see Scheme 1 below.

Studies in many cell models have shown that NO has the effect of inhibiting cell proliferation and induces cell death by cytokines in murine activated macrophages. Inhibition of such cell proliferation is often made during cell differentiation. NO induces cell differentiation of promyelocytic leukemia cells like retinoic acid (RA). In addition, NO provided by the NO donor promotes differentiation by RA of monocyte cell line U-937. In this sense, NO is associated with several differentiation mechanisms.

The present inventors induce differentiation of human NB cell lines upon administration of LC, and in this differentiated cell, NOS activity is increased to increase the amount of NO produced, and the generated NO further promotes the differentiation effect of LC. It has been found that differentiation effects of NB cells are provided.

1 is a graph showing the inhibition of proliferation of cells measured after treatment of SH-SY5Y cells with LC.

2A and 2B are photographs of the morphology of differentiated SH-SY5Y cells and control cells.

3 is a graph showing the number of time slots of cells having neurites.

Figure 4 graphically shows the total neurites length of cells over time after each treatment with fresh medium and LC.

5 is a graph showing NO ₂ − production in SH-SY5Y cells in the presence of LC.

6 is a graph showing the total NO production in the supernatant of control and LC treated cells.

7 is a graph showing NOS activity (L-citrulline concentration) over time in SH-SY5Y cells.

8 shows the AChE activity of differentiating SH-SY5Y cells.

The present invention relates to novel uses of known compounds, ie LCs. More specifically, the present invention relates to a human NB cell differentiation agent containing the LC of Formula 1 as an active ingredient.

The present inventors have shown through the clinical experiments presented in the Examples below that i) whether LC causes differentiation of human NB cell lines, ii) whether NO production occurs in such differentiated NB cell lines, and iii) the amount of NO produced. Learned.

In this experiment, SH-SY5Y cells were used as human NB cell lines. These SH-SY5Y cells were cloned from NB SK-N-SH and are cell lines used in many differentiation induction experiments. SH-SY5Y cells, like other NB cells, are retinoic acid, Fourbol 12-myristate 13-acetate (PMA), 12-0-tetradecanoylpobol-13-acetate (TPA), adenosine 3 ', 5'-cyclic It is differentiated by monophosphate (cAMP), neurotrophic factor and staurosporine and the like. Changes in differentiation include neurite outgrowth formation, decreased proliferation rate, increased synthesis of neurotransmitters and receptors, upregulation of RET proteomic gene expression, and downregulation of metallopeptidase activity.

An effective amount of leucocyanidine (LC) is at least in part an amount that can achieve the desired human neuronal cell differentiation effect, which may vary depending on the condition of the subject to be administered, but is generally 0.1 to 1500 mg per kg of body weight, preferably Is 10-1000 mg, more preferably 20-500 mg, most preferably 50-150 mg.

The pharmaceutical compositions of the present invention may be formulated into tablets, capsules, granules, powders, syrups, solutions, emulsions, suspensions, injections, capsules, pills and the like according to methods known in the art. Pharmaceutically acceptable carriers suitable for use in formulating the pharmaceutical compositions of the invention include any and all solvents, dispersion media, fillers, solid carriers, aqueous solutions, coatings, fungicides, antibacterial agents, isotonics commonly used in the pharmaceutical arts. Agents, absorption retardants, and the like. The use of such carriers in pharmaceutically active ingredients is known in the art and described, for example, in Remington's Pharmaceutical Sciences , 18th edition, Mac Publishing, Pennsylvania, USA. Except incompatibility with the active ingredient, any carrier may be used to prepare the pharmaceutical composition of the present invention. Supplementary active ingredients can also be added to the pharmaceutical compositions. The proportion of the active ingredient combined with the pharmaceutically acceptable carrier can be readily determined empirically by those skilled in the art.

The pharmaceutical composition of the present invention can be administered orally or parenterally to an animal, preferably a human, in need of a differentiation effect on nerve cancer cells.

Example

Hereinafter, the present invention will be described in more detail with reference to Examples, but the present invention is not limited to these Examples.

I. Cell Culture

In an incubator containing 5% CO ₂ and maintained at 37 ° C., thermofluorination of L-glutamine (X50), penicillin (10,000 units / ml), streptomycin (10,000 μg / ml), 10% (vol / vol) SH-SY5Y cells were cultured in DMEM medium containing toasted fetal bovine serum (FBS).

II. Preparation and Processing of LC

LCs were lysed in DMEM medium and then the SH-SY5Y cells were seeded and 24 hours later the cells were first treated with LC. In this case, 0 days were indicated, and the control medium was treated with normal medium and the LC treated group was treated with LC at a corresponding concentration (0.1 to 1 μM) at two-day intervals. In addition, on the day of treatment with the medium, the corresponding morphological and biochemical differentiation experiments were performed together (2, 4, 6, 8, 10 days).

III. Growth inhibition

SH-SY5Y cells were seeded at 5 × 10 ⁴ cells / well in 24 well tissue culture plates. The medium treatment method of each group was as mentioned above. Live cells (viable cells) and dead cells were distinguished by trypan blue and measured using a hemacytometer. The cell proliferation inhibition rate of LC was calculated according to the following equation.

In the above formula,

Cn is the number of viable cells in the control group,

Tn is the number of viable cells in the sample (LC) treatment group.

IV. Morphological Differentiation Experiment

SH-SY5Y cells were seeded at 80 × 10 ⁴ cells / dish in 100 mm diameter Petri dishes. The medium treatment method of each group was as mentioned above. In this experiment, the number of neurites holding cells (%) and the length of neurites were measured as follows.

1.Number of neurites holding cells (%)

Several portions of a randomly selected 100 mm dish were taken with a phase contrast optical microscope (200 magnification), and then cells having neurites among 200 to 300 cells were measured.

2. Length of neurites

The cell number was measured and the length was measured after finding a part where the neurites were clearly observed.

V. Biochemical Differentiation Experiment

Nitrite (NO₂-), To measure the production of NO and L-citrulline and the activity of acetylcholine esterase (AChE), SH-SY5Y cells were subjected to 4.5 x 10 per 60 mm diameter Petri dish.⁴doggy Seeding with cell number.

NO ₂ Amount of-

Measurements were made using the Greries reagent method. Briefly, 150 μl of cell culture supernatant was placed in microtiter plate wells followed by 150 μl of grease reagent (0.2% naphthyl ethylenediamine in distilled ionized water and 2% sulfanilamide in 10% phosphoric acid). Absorbance was measured using a microplate reader at 540 nm after shaking in the dark at room temperature for 10 minutes. Calibration curves were prepared using sodium nitrite dissolved in DMEM. The concentration of NO ₂ − was determined by subtracting the absorbance value of the medium treated in each group from the supernatant absorbance value. Concentrations are expressed as nmol / mg (cell protein).

2. Production amount of NO

Total production of NO was measured using a NO analyzer (Antec Instruments, Inc., USA).

3. NOS activity (L-citrulline production)

Improved methods to measure end products of nitric oxide in biological fluids: nitrite, nitrate, and S-nitrosothiols. Nitric Oxide. , Apr; 1 (2): 177-89, 1997, Marzinzig M, Nussler AK, Stadler J, Marzinzig E, Barthlen W, Nussler NC, Beger HG, Morris SM Jr, Bruckner UB to produce L-citrulline Was measured. Cells were separated from dishes by treatment with trypsin / EDTA and then washed 2-3 times with PBS. The cells were then sonicated on ice for about 15 seconds to make them ready for the experiment (cell lysates). 50 μl of urease (165 U / ml (water)) was added to 100 μl of cell lysate and incubated for 30 minutes. Thereafter, 50 µl of trichloroacetic acid (2.45 M) was added to remove the protein. The deproteinized sample was centrifuged at 15,000 g for 5 minutes, then 125 μl of supernatant was added to 375 μl of reaction mixture [40% (v / v) diacetylmonoxime (79 mM in 83 mM acetic acid) + 18% (v / v) mixed with antipyrine E (47.8 mM in water) + 42% (v / v) 15N H ₂ SO ₄ ] and reacted for 25 minutes at 96 ℃. After the reaction was completed, the mixture was left at room temperature to lower the temperature, and then centrifuged at 15,000 g for 5 minutes. 200 μl of the supernatant (1) was taken and stirred in (2) by adding an equivalent amount (200 μl) of dimethyl sulfoxide (DMSO) to the remaining liquid in the tube. The absorbances of (1) and (2) were measured at 450 nm using a microplate reader. The concentration of L-citrulline was calculated by the calibration curve created using citrulline in the same manner as described above.

4. Activity of Acetylcholine esterase (AChE)

(1) cell treatment

After washing the cells 2-3 times with PBS, the cells were separated from the dish using a rubber stick. After washing with PBS again 750 g was centrifuged. The supernatant was removed and then chilled Tris-HCl buffer (0.05 M, pH 7.5) (containing 1% (v / v) Triton X-100) in an amount approximately 4 times the remaining volume. Nuclei and unbroken cells were removed by centrifugation at 1000 g for 10 minutes at 4 ° C.

(2) AChE activity measurement

AChE activity is described by Elman et al., A new rapid colorimetric determination of acetylcholinesterase activity. Biochem Pharm ., 7: 88-95, 1961, George L. Ellan, K. Diane Courtney, Valentino Andres Jr, Robert M, Featherstone. Since AChE is known in two forms, cell membrane attachment type and release type, activity was measured and summed in both cell lysate and culture supernatant. 6 μl of cell lysate was added to 290 μl of medicament buffer containing 0.01% 5,5′-dithiobis (2-nitrobenzoic acid) (DTNB) dissolved in 0.05 M PBS, pH 7.4. The reaction was initiated by adding 6 μl of acetylthiocholine iodide to a final concentration of 5 × 10 ⁻⁴ M. The change in absorbance was measured at 405 nm for 10 minutes at 1.5 minute intervals. AChE activity values are presented as the average of changes made over 10 minutes.

<Experiment Result>

I. Proliferation Inhibition Rate

In order to confirm the effect of LC inducing cell death as anticancer activity, the number of viable cells was first measured by trypan blue exclusion assay. Cell proliferation of SH-SY5Y was inhibited in a concentration-dependent manner in the LC treated group (see FIG. 1). The proliferation inhibition rate of the cells measured for 2 days after LC treatment was 19.47% when treated with 0.1 μM LC and 30.71% when treated with 1 μM LC. After 4 days, the growth inhibition rate was almost the same. These data are presented as mean ± standard deviation (n = 5).

II. Morphological changes due to differentiation

The morphology of differentiated neurons is characterized by dendritic branching of neurites and outgrowth of neurites. The growth of neurites is the most obvious morphological marker of differentiation. Neurites in this experiment were defined as cytoplasmic processes that are more than twice the cell diameter.

Morphology of control and differentiated SH-SY5Y cells is shown in FIGS. 2A and 2B. These figures show the morphology of SH-SY5Y NB cells treated with fresh medium (control), 0.1 μM GSE and 1 μM GSE for 10 days. Cells were photographed every two days from the first day (day 0) using a phase contrast microscope. In these figures,? Is a neurites and? Is a conical proliferation structure. Undifferentiated SH-SY5Y cells were elliptical or polygonal with only neurites in some. In contrast, the cells treated with LC showed differentiated morphology after 2 days, forming the longest neurites and complex neural networks spanning several cells on the 10th day, with a conical proliferation structure at the ends. Control cells formed only a few neurocytoids over time.

1.Number of neurite-bearing cells (%)

200-300 cells were measured with a phase contrast optical microscope. Figure 3 shows the relative number of cells with neurites. Each column shown in the figure represents the mean ± standard deviation of neurites among 200 to 300 cells present in at least 10 microscope fields and 3 to 5 separate cultures, and the data represent mean ± standard deviation (n = 3) presented. In the cell group treated with LC, the number of cells with neurites already increased on the second day, and the cell group incubated with LC showed a significant increase for 10 days.

2. Length of neurites

As the cells for measuring the length of the neurites, only those which can reliably confirm the neurites without being crowded were selected. The total neurites length of the cells was much longer in the cells treated with LC compared to the control (see FIG. 4). However, although the measurements were taken every other day, the rate of increase of neurite length in the same LC concentration group was not very fast. Some areas on the photographs were selected to estimate neurite length in cells, and the results were presented as mean ± standard deviation (n = 3).

III. Biochemical Changes According to Differentiation

NO ₂ Amount of-

After addition of 0.1-1 μM of LC to the medium, the amount of NO ₂ − produced in SH-SY5Y cells increased and decreased after peaking around 4 days of culture (see FIG. 5). NO ₂ − production in control cells was lower than in LC treated cell groups throughout the observation period. The N0 ₂ -production on day 4 was 19.39 ± 3.44 in the control group, 35.71 ± 4.21 in the 0.1 μM LC treated group and 45.63 ± 3.78 in the 1 μM LC treated group. NO ₂ − production in the control is presumed to be because low levels of NO ₂ − may act as signal transduction molecules of tumor cell proliferation. The NO ₂ − concentration in the cell supernatant cultured in this experiment was measured with a grease reagent, and the data were presented as mean ± standard deviation (n = 5).

2. Production amount of NO

NO has recently been found to play an important role in signal transduction. This free radical gas is produced from L-arginine through a complex reaction catalyzed by NOS as shown in Scheme 2 above. NO diffuses freely through the cell membrane, but its life is very short, several seconds or less. This is because the reactivity is very active. Thus, NO is suitable for acting as an intermediate signaling molecule between cells and between surrounding cells.

As can be seen from FIG. 6, NO was produced in LC-treated SH-SY5Y cells. The maximum value (ie maximum yield) was observed two days after treatment. The value was 31.15 ± 3.77 in the control group, 94.75 ± 14.48 in the LC treatment group of 0.1 μM and 101.37 ± 9.83 in the LC treatment group of 1 μM. Thereafter, NO production in LC-treated SH-SY5Y cells decreased little by little, but even on day 10, more NO was produced in the LC-treated group than in the control group. The NO analyzer was used to detect the total NO (NO _2- + NO _3- ) production in this experiment, and the data were presented as mean ± standard deviation (n = 5).

3. Activity of NOS

Analysis of L-citrulline levels in cultured cells demonstrated that an increase in L-citrulline concentration suggests an increase in NOS activity.

NOS activity was measured in cell lysates at 2 day intervals after adding 0.1-1 μM of LC to the medium (see FIG. 7). Measurement data are presented as mean ± standard deviation (n = 5). Enzyme activity of LC-treated SH-SY5Y cells increased significantly over 10 days compared to control cells, peaked on day 2, and then decreased. Day 2 NOS activity was 149.75 ± 12.22 in the control group, 280.98 ± 27.7 in the LC treated group of 0.1 μM, 247.6 ± 24.79 in the LC treated group of 1 μM. L-Citrulline is a 1: 1 stoichiometric product of NO synthesized by NADPH dependent NOS. However, as shown in Figures 6 and 7, the concentrations of these two materials are different. This is because NO is an unstable gas having a short half-life of about 5 to 10 seconds in the extracellular space.

4. Activity of AChE

Since AChE is considered a marker for the final differentiation of neurons, AChE activity was measured as a biochemical indicator of SH-SY5Y cell differentiation by LC. AChE is mostly present in the cell membrane, but in some cells it is released into the medium. Therefore, the amount of AChE present in the cell membrane and the amount of AChE present in the culture supernatant were measured together. Cells treated with LC began showing AChE activity already on day 2 after treatment (see FIG. 8). Here AChE activity is the mean ± standard deviation of 3 to 5 measurements according to separate experiments. The AChE activity of LC treated cells during the entire measurement period was about twice that of the control group. The highest activity appeared on day 8.

NB is a tumor that appears well in children. [Prognostic factor in neuroblastoma. Cancer., Jun 1; 59 (11): 1853-9, 1987, Evans AE, D'Angio GJ, Propert K, Anderson J, Hann HW. It is believed to be derived from neuroblasts that cannot differentiate. NB is a malignant tumor, but it is also relatively degenerative. NB cells may also differentiate into benign ganglion cell tumors. An interesting therapeutic approach for NB is the use of drugs that induce final differentiation of tumor cells in vivo to stop cell proliferation. NB cell numbers in mice and humans are very important models for studying cell differentiation mechanisms. Cell differentiation of these cell lines has been described in NGF, cAMP, hypothalamic factor, anthracycline, RA, PMA {Research on the differentiation of human and murine neuroblastoma cells. Oncology. , 48 (3): 196-201, 1991, Busse E, Bartsch O, Kornhuber B], as well as natural extracts such as resveratrol and polyphenol extracts. However, the mechanism of differentiation by these substances has not yet been clarified.

NO, considered a cytotoxic agent and a mediator of cell death, is also associated with several differentiation mechanisms. In other words, NO induces cell differentiation in the promyelocytic cell line HL-60 and enables the differentiation by the retinoic acid of human monocyte line U-937. In addition, it has already been reported that the differentiation of human NB cell line SK-N-BE is associated with an increase in NOS (Retinoic acid-induced differentiation in a human neuroblastoma cell line is associated with an increase in nitric oxide synthesis. J Cell Physiol. , Jan; 174 (1): 99-106, 1998, Ghigo D, Priotto C, Migliorino D, Geromin D, Franchino C, Todde R, Costamagna C, Pescarmona G, Bosia A].

The above experimental results show that LC induces differentiation of human NB cell line SH-SY5Y, and NO produced by iNOS and nNOS in these differentiated cells ultimately inhibits cell proliferation by LC, growth of neurites, AChE It is necessary to induce activation. Referring to Figure 1 it can be seen that the proliferation of SH-SY5Y cells by LC treatment. This is due to the antioxidant action of LC. In carcinogenesis, free radicals play an important role in the proliferation stage of cancer cells. On the other hand, antioxidant enzymes, superoxide dismutase (SOD), catalase, and glutathione reductase, reduce the levels of free radicals to levels suitable for normal physiological environments. Grape seed extract, proanthocyanidin, which has a radical scavenging effect, and polyphenol, a tea extract, also exhibit cytotoxic and proliferative effects on cancer cells as in the experimental results of the present invention.

Morphological characteristics of differentiated NB cells are defined as the formation of neuronal processes, ie neurites. Cells treated with LC showed morphology with neurites (see FIGS. 2A and 2B). Complex neurites can be observed on day 10. During this period, the production of biochemical factors NO ₂ −, NO production and NOS activity increased with morphological changes. The levels of these biochemical factors were on average 2.1 times higher for the 0.1 μM LC treated group and 3.25 times higher for the 1 μM LC treated group compared to the control. The production of L-citrulline is stoichiometrically equal to the total production of NO, but the activity of NOS in cell lysates is not the same as the actual production of NO in intact cells. There are two reasons for this. One is due to the production of S-nitrosothiol (RSNO), another group of derivatives of NO. As a result of the study by Margin Jig M et al., The level of RSNO in cell culture supernatant of cytokine-stimulated humans was 3.7 ± 0.1 μM, while RSNO was not substantially detected in control culture (<0.15 μM). The other reason is that L-citrulline recycles to L-arginine. However, the efficiency at which citrulline recycles to arginine at the biosynthetic enzyme level of arginine varies from cell type to cell type.

Factors affecting the growth rate or direction of the nervous system include neurotransmitters such as cell substrate components, adhesion molecules, growth factors, acetylcholine, etc. {Regulation of acetylcholinesterase expression during neuronal differentiation. J Biol Chem. Feb 23; 271 (8): 4410-6, 1996, Coleman BA, Taylor P.}. Acetylcholine esterase (AChE), produced by hydrolysis of acetylcholine, is directly related to the differentiation of neurons. Attempts have been made to determine the effect of AChE on the growth of neurites, but this has not provided a successful outcome. In the present invention, AChE activity was found to increase during the differentiation of SH-SY5Y induced by LC (see FIG. 8). It was also found that the linear increase in neurite outgrowth and the relative number of neurite-containing cells were nearly identical to AChE activity. This result is consistent with a study by Koenigsberger et al., Which demonstrated that the increase in AChE activity is related to the level of neurites growth and differentiation markers.Neurite differentiation is modulated in neuroblastoma cells engineered for altered acetylcholinesterase expression. J Neurochem. , Oct; 69 (4): 1389-97, 1997, Koenigsberger C, Chiappa S, Brimijoin S]. Although AChE is mainly present in the cell membrane, AChE activity was also measured in the medium in this experiment. Thus, in summary, LC induced differentiation of human NB cell line SH-SY5Y cells and produced NO and L-citrulline (NOS activity). It also increased the activity of AChE, the final marker of neuronal differentiation. These results demonstrate that LC is useful as a differentiator of human NB cells.

Claims (1)

A neuroblastoma (NB) cell differentiator comprising leucocyanidine as an active ingredient.