KR20030066927A - Anticancer drug made of curcumin and 5-fluorouracil, and its clinical uses - Google Patents

Abstract

PURPOSE: An anticancer agent comprising curcumin and 5-fluorouracil having excellent anticancer effect on stomach cancer in capsule form is provided. The agent has a synergistic effect on the treatment of stomach cancer as compared to separate administration of curcumin and 5-fluorouracil. CONSTITUTION: The anticancer agent contains 150 to 800mg curcumin and 100 to 600mg 5-fluorouracil and is orally administered to a cancer patient. The curcumin inhibits proliferation of human stomach cancer cells in contrast to concentrations thereof, the proliferation inhibiting effect thereof is reversible. The concentration(5 to 50μM) of the curcumin shows that lower than a serum concentration(2 to 100μM) which can be reached by taking 160mg to 8g per a day.

Description

ANTICANCER DRUG MADE OF CURCUMIN AND 5-FLUOROURACIL, AND ITS CLINICAL USES} Curcumin and 5-Fluorouracil

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to anticancer agents, and more particularly to anticancer agents prepared by mixing curcumin and 5-fluorourouracil in capsule type and the use of the anticancer agents.

Gastric cancer, the most common cancer in our country, is already in progress at the time of diagnosis. In this case, try chemotherapy or radiation therapy, but the effect is not good and side effects are not so satisfactory treatment. Therefore, there is a need for a study of drugs that can improve the therapeutic effect by reducing the side effects of anticancer drugs or enhancing the effects of anticancer drugs when the anticancer agent and the mechanism of action are different.

One of these drugs is phenolic substances in many medicinal and edible plants, all of which are known to exhibit antioxidant and anti-inflammatory effects. Therefore, like the NSAIDS anti-inflammatory drugs used to prevent colon cancer by inhibiting inflammation, which is known to play an important role in the early stages of carcinogenesis, it is included in natural plants with anti-inflammatory effects that can inhibit carcinogenesis. The anti-cancer activity of phenolic substances has recently attracted attention, and one of these phenolic substances is curcumin.

Curcumin is the main ingredient of turmeric, a yellow spice extracted from the root of Curcuma longa Linn (Zingiberaceae), a plant belonging to the Ginger family of East India, and widely used in foods in India. It is a drug.

The present invention has been made in view of the above-mentioned general issues, and an object thereof is to provide an anticancer agent consisting of curcumin and 5-prourouracil and a use of the anticancer agent which can realize an excellent anticancer effect on cancer cells.

Another object of the present invention is to provide an anticancer agent consisting of curcumin and 5-fluorourouracil having an excellent effect on gastric cancer.

In a first aspect for achieving the object as described above, the present invention is characterized by an anticancer agent prepared in the form of a capsule by mixing kurukumin and 5-fluurouracil.

In addition, in the second aspect for achieving the above object, the present invention is a capsule form or oral method and non-regular method to the dosage of 150 ~ 800mg of curukumin and 100 ~ 600mg 5-Fluorouracil It is characterized by a method of treating gastric cancer by administering to.

1 to 6 are graphs showing the results of an anticancer agent consisting of curcumin and 5-fluurouracil in accordance with a preferred embodiment of the present invention.

Hereinafter, preferred embodiments of the present invention will be described in detail. In addition, it should be noted that in the following description, only parts necessary for understanding the anticancer agent of the present invention will be described, and descriptions of other parts will be omitted so as not to obscure the subject matter of the present invention.

In the present invention, the effect of curcumin (hereinafter referred to as "Curcumin") on the proliferation of human gastric cancer AGS (hereinafter referred to as "AGS cells") through the cell culture experiment, the existing anti-cancer drug 5-Pluuroyura The combined effect with yarn (hereinafter referred to as "5-FU") was investigated. In addition, in order to elucidate the mechanism of action in more detail, the ratio of each cell cycle following proliferation inhibition was analyzed using Flow Cytometry, which will be described in detail below.

1. Materials and Methods

(1) material

curcumin, 5-FU and dimethyl sulfoxide (DMSO) were purchased from Sigma Corporation (St. Louis, MO, USA), RPMI 1640, fetal bovine serum (FBS), 0.05% trypsin-0.02% EDTA and 100 units / ml penicillin Streptomycin was purchased from GIBCO (USA). Curcumin solution was added to each well 10μl, dissolved in DMSO solution when added immediately used, and in all experiments the same amount of 0.4% DMSO solution was added to the control group instead of curcumin. The solution containing 5-FU was added to each well 10μl, dissolved immediately in PBS when added, and the same amount of PBS was added to the control.

(2) cell culture

AGS cells were used for the experiment while being cultured and cultured from the Korea Cell Line Bank (Seoul National University), and the cell culture was used CO ₂ incubator (Sanyo, model MCO96, Japan). AGS cells were cultured in 37%, 5% CO ₂ incubator using RPMI 1640 containing 100 units / ml penicilin-streptomycin and 10% FBS. Each cultured cancer cell was refeeded 2-3 times a week, washed with PBS in 6-7 days, and then detached with 0.05% trypsin-0.02% EDTA. The separated cells were centrifuged and placed in the cultured cancer cells, and mixed well so that the cancer cells were evenly distributed by pipette, and the cells were divided and subcultured every 6 to 7 days. Each passage number was recorded during subculture, and when the passage number was 10 or more times, new cancer cells were removed from the liquid nitrogen tank and re-cultured.

(3) curcumin AGS cell proliferation inhibitory effect

After washing AGS cells in the proliferative phase with PBS, the cells attached with 0.05% trypsin-0.02% EDTA enzyme were separated and centrifuged, and the accumulated cancer cells were mixed well so as to evenly disperse the 10 × 10 ⁴ cells / Seed at well concentration and incubated for 24 hours. After confirming that the cancer cells had adhered to the plate, curcumin was added to 3 wells at concentrations of 1 μM, 5 μM, 10 μM, 25 μM, and 50 μM in medium containing 10% FBS (triplicate), 37 ° C., and 5%. Incubated in a CO ₂ incubator. After 2 and 4 days from the day of sample treatment, the adherent cells were separated by 0.05% trypsin-0.02% EDTA enzyme, and the number of each cell was measured by hemocytometer, and the number of cells was 0.4% DMSO solution. The cancer cell proliferation inhibitory effect was observed in comparison with the number of cells.

(4) Interaction between curcumin and 5-FU on proliferation of AGS cells

In order to understand the interaction between curcumin and 5-FU on proliferation of AGS cells, AGS cells in the proliferative phase were washed with PBS, centrifuged after separation of cells attached with 0.05% trypsin-0.02% EDTA enzyme, The cancer cells were mixed well to be evenly dispersed, seeded at 10 × 10 ⁴ cells / well in 6 well plates and incubated overnight. After confirming that the cancer cells adhered to the plate, control, curcumin 5 μM, curcumin 10 μM, 5-FU 0.1 μg / ml, 5-FU 0.3 μg / ml, 5-FU 0.1 μg / ml + Curcumin 5 μM, 5-FU 0.1 μg / ml + curcumin 10 μM, 5-FU 0.3 μg / ml + curcumin 10 μM divided into 8 wells (triplicate), incubated in 37 ° C, 5% CO ₂ incubator It was. After 4 days from the date of sample treatment, adherent cells were isolated from 0.05% trypsin-0.02% EDTA enzyme. Thus, the number of isolated cells was measured by a hemocytometer to observe the cancer cell proliferation inhibitory effect of each group compared to the control group.

(5) Reversibility of curcumin's AGS cell proliferation inhibitory effect

In order to investigate the reversibility of curcumin's proliferation inhibitory effect on AGS cells, AGS cells in the proliferative phase were washed with PBS, and then changed to a medium containing 0.05% trypsin-0.02% 10% FBS, followed by EDNA enzyme-attached cells. Separated. After centrifugation of the isolated AGS cells, the integrated cancer cells were mixed well to be evenly dispersed, seeded at 2 × 10 ⁴ cells / well in a 6 well plate and incubated for 24 hours. After confirming that the cancer cells had adhered to the plate, change to medium containing 10% FBS and add 10 μM of curcumin to 3 wells, and add 0.4% DMSO solution to the other 3 wells as a control, and again in 37 ℃, 5% CO ₂ incubator. After 24 hours of incubation, the cells were counted by changing to medium containing 10% FBS (day 0), and the cells proliferated on day 1, 3, 5 and 7 were then 0.05% trypsin-0.02% EDTA. Each cell number was separated by an enzyme and measured by a hemocytometer. The number of cells thus measured was compared between the two groups to observe the reversibility of the cancer cell proliferation inhibitory effect.

(6) Cell cycle analysis using flow cytometry

Curcumin was analyzed twice by flow cytometry to determine the stage of cell cycle in AGS cells. Cells cultured for 4 days in medium containing 10 μM of curcumin were washed with PBS solution and fixed with 70% ethanol at 4 ° C. for 30 minutes. After fixation, RNA was treated with RNase A (Sigma, USA) and stained with DNA intercatalating dye propidium iodide (Sigma, USA) to compare cell cycles using the Becton Dickinson FACStar Flow Cytometer and Becton Dickinson Cell Fit Software. It was.

(7) statistical analysis

For the analysis of the experimental results, Duncan's multiple range test in ANOVA was considered meaningful when the p-value was less than 0.05.

2. Results

(1) Curcumin AGS cell proliferation inhibitory effect

The number of AGS cells on the 2nd day after addition of curcumin 1 μM was (65 ± 2) × 10 ⁴ , and the number of AGS cells in the control group with 0.4% DMSO only was (63 ± 3) × 10 ⁴ , between the curcumin 1 μM addition group and the control group. There was no difference in the degree of proliferation. And this trend appears to be the fourth il sexual AGS cells in the group with addition of 1 μM curcumin was no significant difference as compared with (183 ± 7) x10 ⁴ as in the control group (184 ± 6) x10 ^4.

If the addition of curcumin concentration of 5 μM, the drug can AGS cells at 2 days after the addition of (55 ± 2) eoteuna as x10 ⁴ fewer AGS cells than (63 ± 3) x10 ⁴ of the control number Statistical significance was not on the other hand, after the drug is added on day 4, the number of drug addition group cells (121 ± 3) x10 ⁴ as significantly higher than x10 ⁴ (183 ± 7) of the control group proliferation was inhibited (p <0.05).

This curcumin AGS cell proliferation inhibitory effect was prominent in proportion to the concentration, and as the concentration was increased to 10 μM, 25 μM and 50 μM, the proliferation was significantly inhibited in proportion to the concentrations on the 2nd and 4th days. That is, at 5 μM, the number of cells at day 4 was (121 ± 3) x10 ^4, which was significantly lower than that of the control group (183 ± 7) x10 ⁴ , showing a 33.9% growth inhibition effect, and at 10 μM as well. The number of AGS cells on day 2 and day 4 was (38 ± 4, 90 ± 3) × 10 ⁴ , respectively, and its proliferation was significantly suppressed compared to that of control group (63 ± 3, 183 ± 7) × 10 ⁴ , and on day 2 39.7 % And 50.9% of the growth inhibitory effect on day 4. This tendency is more pronounced at higher concentrations: (8 ± 1, 14 ± 1) x10 ⁴ for 25 μM and (2 ± 1, 0) x10 ⁴ for 50 μM (63 ± 3 , 183 ± 7) x10 ⁴ , the proliferation was significantly inhibited. At 25 μM, the proliferation inhibitory effect was 92.4% compared to the control group. In particular, at the concentrations of 25 μM and 50 μM, the number of cells on the second day after curcumin addition was (8 ± 1, 0) x10 ⁴ and (2 ± 1, 0) x10 ⁴ , respectively, rather than the number of cells initially divided (10x10 ⁴ ). After 4 days incubation at 50 μM concentration, almost no cancer cells were seen in the medium. On the other hand, this effect will be clearly understood by Table 1 and FIG.

{Table 1} Effect of curcumin on AGS cells after 2 days and 4 days treatment.

Curcumin (μM) Cell number × 10 ⁴ 2 days 4 days 0 63 ± 3 ^a 183 ± 7 ^a One 65 ± 2 ^a 184 ± 6 ^a 5 55 ± 2 ^ae 121 ± 3 ^b 10 38 ± 4 ^be 90 ± 3 ^c 25 8 ± 1 ^cf 14 ± 1 ^d 50 2 ± 1 ^df 0 ^e

(2) Interaction between curcumin and 5-FU on proliferation of AGS cells

At 4 days of culture, the number of AGS cells in the control group was (186 ± 7) x10.⁴AGS cell number of the group to which 5-FU 0.1 μg / ml was added was (138 ± 5) x10.⁴And The number of cells in the group to which curcumin 5 μM was added was (119 ± 5) x10⁴Both groups significantly inhibited cell proliferation than the control group, and this tendency was more pronounced in the remaining five groups, which significantly inhibited cell proliferation compared to the control group (p <0.05). The number of AGS cells in the 5-FU 0.1μg / ml group was (138 ± 5) x10.⁴Was The number of cells in the curcumin 5 μM group was (119 ± 5) x10⁴as 5-FU0.1μg / ml and curcumin 5μM showed similar growth inhibition effect. This is clearly shown in Table 2.

TABLE 2 Effects of curcumin (μM) and / or 5-fluorouracil (μg / ml) on 4 days treatment of AGS cells.

Cell number × 10 ⁴ control 186 ± 7 ^a curcumin 5 119 ± 5 ^bij curcumin 10 77 ± 7 ^cj 5FU 0.1 138 ± 5 ^di 5FU 0.3 97 ± 3 ^ej 5FU 0.1 + curcumin 5 10 ± 2 ^fk 5FU 0.1 + curcumin 10 0 ± 0 ^gk 5FU 0.3 + curcumin 5 4 ± 2 ^hk

On the other hand, as shown in the graph of Figure 2, look at the effect of curcumin on the inhibitory effect of 5-FU, AGS cell number of the group to which 5-FU 0.1μg / ml and curcumin 5 μM was added (10 ± 2) x10⁴(138 ± 5) x10 of the group to which only 5-FU 0.1μg / ml was added as⁴And (119 ± 5) x10 of the group added only curcumin 5 μM⁴The number of cells was significantly lower than that of (p <0.05).

As shown in FIG. 3, this tendency is more marked when the concentration of curcumin is increased. When 10 μM of curcumin is added in combination with 0.1 μg / ml of 5-FU, cells are added to the medium on day 4 of culture. The proliferation inhibitory effect was so clear that there was no.

On the other hand, as shown in Figure 4 shows a similar tendency when increasing the concentration of 5-FU, the cell number is (4 ± 2) x 10 when added in combination of 5-FU 0.3μg / ml and curcumin 5μM⁴(97 ± 3) x10 of the group to which only 5-FU 0.3 μg / ml was added⁴or (119 ± 5) x10 of curcumin 5 μM alone⁴The cell number was significantly lower than that of the control group (p <0.05).

In addition, as shown in FIG. 5, the growth inhibition rate of the cells showed a growth inhibition rate of 95% compared to the control group in the group in which 5-FU 0.1 μg / and curcumin 5 μM were added together, and 5-FU 0.1 μg / Much lower than the combined inhibition rate (26 + 36 = 62%) in the ml alone group and the curcumin 5 μM alone group, the two drugs showed distinct synergism. This trend was also observed when the concentration of curcumin was increased to 10 μM, and similar results were obtained when the concentration of 5-FU was increased to 0.3 μg / ml.

(3) Reversibility of curcumin AGS cell proliferation inhibitory effect

In an experiment to investigate the reversibility of curcumin's anti-proliferative activity, the control group was not added with curcumin, the curcumin was incubated for 24 hours, curcumin was removed, and curcumin was added thereto. When divided into groups and observed, 24 hours after inoculation, curcumin was added, followed by incubation for 24 hours. There was no difference as 5.5 ± 0.5) × 10 ⁴ and (5.5 ± 0.5) × 10 ⁴ , and the number was smaller than that of the control group (9.0 ± 1.0) × 10 ⁴ , but there was no statistical significance. not remove the curcumin after 3 days, that is, the number of the removed group of inoculation day 4 cells (25.0 ± 2) of the group that did not remove the curcumin as x10 ⁴ (16.5 ± 1.5), but quite frequent than the x10 ⁴ statistical significance It was significantly less than (36.5 ± 2.5) × 10 ⁴ of the control group, but there was no statistical significance.

On the other hand, the number of cells in the curcumin-removed group was (81.0 ± 3.0) x10 ⁴ at 5 days after curcumin was removed, which was not significantly different from that of the control group (113.5 ± 4.5) x10 ^4. Compared to (30.5 ± 2.5) x10 ⁴ in the untreated group, the number of cells was significantly higher (p <0.05), indicating that curcumin's proliferation inhibitory effect was significantly reduced. This tendency was more pronounced at the 8th day of inoculation, which was 7 days after the curcumin removal, and the number of cells in the curcumin-removed group was (139.0 ± 3) x10 ⁴ compared to the 5th day than the (51.5 ± 2.5) x10 ^{4 in the} group not curcumin By showing a greater difference (p <0.05), it was clearly shown that curcumin's proliferation inhibitory effect was reversible. This is clearly shown in Table 3 and FIG. 6.

Table 3 Reversibility of the effects of curcumin (10 μM) on the growth of AGS cells.

Cell number × 10 ⁴ 0day 2days 4days 6days 8days Control 2 9.0 ± 1.0^a36.5 ± 2.5^a113.5 ± 4.5^a143.5 ± 3.5^a 4.5 ^† 4.1 3.1 1.6Curcumin removed^*2 5.5 ± 0.5^a25.0 ± 2^a81.0 ± 3.0^a139.0 ± 3^b 2.8 4.5 3.2 1.7Curcumin 2 5.5 ± 0.5^a16.5 ± 1.5^a30.5 ± 2.5^c51.5 ± 2.5^c 2.8 3.0 1.8 1.7

In addition, referring to Table 3, the proliferation rate of the day inoculated with AGS cells and the second day of inoculation at day 1 after the addition of curcumin was 2.8 in the curcumin-added group, which was much lower than 4.5 of the control group. The proliferation rate between 1 and 3 days after curcumin removal was 3.0 in the group without curcumin removal, which was significantly lower than 4.1 in the control group, whereas 4.5 in the group without curcumin showed no significant difference from the control group. In addition, there was no significant difference between the control group and the removed group as 3.1 and 3.2, respectively, in the growth rate between 3 and 5 days of curcumin removal, that is, between 4 and 6 days of inoculation. It was significantly lower than the group removed, indicating that the action of curcumin is reversible.

(4) Cell cycle analysis using flow cytometry

As shown in Table 4, cell cycle measurements were performed using gastric cancer cells from the group cultured for 4 days in a medium containing curcumin 10 μM and the control group cultured without curcumin. In the control group, the G0-G1 group was 50.85%, S. group was 26.82%, G2-M group was 21.95%, 20.05% G0-G1 group was added in curcumin 10 μM group, 19.66% in S group and 60.46% in G2-M group. As the number of cells in was much higher, cell cycle progression at the G2-M phase was blocked, indicating that cell proliferation was inhibited.

Table 4 The effect of curcumin (10 μM) on cell cycle distribution in AGS cells after 4 days of incubation at 37 ° C

Sample (μM) % of cells G0 / G1 S G2 / M Control (0) 50.85 26.82 21.95 Curcumin (10) 20.05 19.66 60.46

3. Consideration

In the present invention, the proliferation of AGS cells was significantly inhibited in the group treated with curcumin compared to the control group (p <0.05), and this inhibitory effect was clearly observed when the dose of curcumin was 5 μM, and the concentration was gradually increased to 50 μM. The anti-proliferative effect was also proportional to the concentration. These results observed in the present invention showed the same results as in the report of Moragoda et al., Curcumin has a proliferation inhibitory effect on gastric cancer cells as well as other human cancer cells, curcumin is useful as a therapeutic agent for gastric cancer patients It strongly suggested the possibility of being written.

The daily intake of curcumin that can be administered orally to humans has been reported from 160mg / day to 1 ~ 8gm / day, and the concentration that can be reached in the blood at this intake is exactly Can be estimated from 2 μM to 100 μM. This concentration is similar to or much higher than 5 μM in which the anti-proliferative effect of AGS cells is shown in the present invention, and when the oral dose of curcumin is administered orally to an actual patient, the anti-proliferative effect of gastric cancer can be sufficiently exerted. In addition, urcumin can be administered intraperitoneally, and curcumin derivatives are being developed recently, further supporting that curcumin can be usefully used as a therapeutic agent for stomach cancer.

In the present invention, the combination administration of 5-FU and curcumin showed a tendency to synergistically inhibit the growth of AGS cells, and this synergy has not been reported until this time, thereby curcumin alone as well as the most potent therapeutic agent of gastric cancer When combined with 5-FU, which is one of the most suggestive, it could be a more potent treatment.

On the other hand, when curcumin was removed from the curcumin-added group, there was no significant difference between the control group and the degree of proliferation, indicating that curcumin's proliferation inhibitory effect on AGS was reversible. It is not due to the fact that it means that it has a certain mechanism of action that affects the cell proliferation process. This possibility can be seen more clearly in the results of cell cycle analysis, suggesting that curcumin inhibits cell proliferation by blocking the cell cycle in the G2-M phase, which has been demonstrated in other reports.

Inhibition of protein kinase C, inhibition of EGF-receptor tyrosine kinase, and expression of c-jun, c-fos, and c-myc are the mechanisms by which curcumin inhibits the proliferation of cancer cells. And the like). In particular, curcumin inhibits the activation of NFkappaB by inhibiting IkappaB kinase and, as a result, lowers the action of Cyclooxygenase-2 (COX-2) and inducible andnitric oxide synthase (iNOS). It is in the spotlight as a potent cancer preventive and anti-inflammatory drug such as an inhibitor, and it is thought that more extensive and in-depth studies on all these mechanisms will be needed.

In the above results, curcumin inhibited gastric cancer cell proliferation in proportion to the concentration, and when combined with the conventional anticancer 5-FU, the curcumin showed significant proliferation inhibition compared to the curcumin or 5-FU alone group, and in proportion to the concentration The synergistic effect suggested that curcumin alone could be a more potent treatment in combination with 5-FU. The anti-proliferative action was reversible, blocking the cell cycle at the G2-M phase, indicating that curcumin is a drug with a certain mechanism of action, and more systematic and in-depth studies should be conducted. It became.

4. Conclusion

curcumin inhibited the proliferation of human gastric cancer cells in proportion to its concentration, and its proliferation inhibitory effect was reversible and blocked the G2-M cell cycle. Curcumin concentration (5 ~ 50 μM) applied to the present invention tends to be lower than the serum concentration (2 ~ 100 μM) range that can be reached by ingestion of 160mg ~ 8g per day, the allowable amount of curcumin, curcumin As a result, it can be useful for the treatment of gastric cancer patients. When curcumin and 5-FU were administered in combination, there was a marked synergistic effect, which showed that curcumin could be used as a good treatment for gastric cancer when used alone and in combination with 5-FU. Further animal studies and clinical studies suggest that curcumin may be used in the treatment of gastric cancer, as well as other 5-FU anticancer drugs.

As described above, an anticancer agent consisting of curcumin and 5-fluorourouracil according to an embodiment of the present invention achieves many effects as follows.

First, curcumin inhibited the proliferation of human gastric cancer cells in proportion to its concentration, and its proliferation inhibitory effect was reversible and blocked the G2-M cell cycle. In particular, the concentration of curcumin (5-50 μM) applied in the present invention tends to be lower than the range of serum concentration (2-100 μM) that can be reached by ingestion of 160mg-8g per day, which is the allowable amount of curcumin. The high dose has the effect of being useful for the treatment of gastric cancer patients.

Secondly, when curcumin and 5-fluurouracil are used in combination, there is a marked synergistic effect, so curcumin can be used as a good therapeutic agent for gastric cancer when administered in combination with 5-fluurouracil alone. Showed.

Claims (6)

Anticancer agent made by mixing kurukumin and 5-prurourouracil. [Claim 2] The anticancer agent of claim 1, wherein the anticancer agent is prepared in the form of a capsule. A method of treating gastric cancer by administering a combination of curukumin and 5-fluorourouracil simultaneously. The method of claim 3, The curcumin 150 ~ 800mg and 5-Fluorouracil in a dose of 100 ~ 600mg administration method. The method according to claim 3 or 4, The curcumin and 5-fluorourouracil are administered by an oral method. The method according to claim 3 or 4, The curukumin and 5-fluorourouracil are administered in a capsule form.