JP5717129B2 - Anticancer agents combined with withanolide ingredients - Google Patents

Description

  The present invention relates to an anticancer agent having a high killing ability to tumor cells and a low toxicity to normal cells, which is formulated with a specific ratio of withanone and witherferin A which are constituents of Ashwagandha leaf extract as active ingredients.

Ashwagandha (scientific name: Withania somnifera; solanaceae) is one of the most commonly used medicinal herbs in Indian folk medicine Ayurveda and is known for its various therapeutic effects.
The present inventors have conventionally purified an ethanol extract (i-Extract) from the leaves of Ashwagandha and show that this component has a strong antitumor activity. In the study of biological activity by isolating the withanolide compound, we found a phenomenon that activates the tumor suppressor p53 specifically in tumor cells in the withanone (Withanone) contained in the components. Tumor effects were also confirmed (Patent Documents 1 and 2). The withanone has also been reported to extend normal cell life, protect normal cells from oxidative stress, protect DNA from UV damage and chemical toxicity (Patent Document 2).
On the other hand, withaferin A found in the above ethanol extract also shows tumor cell killing ability, but no tumor suppressive ability is found in vivo, and above all has strong toxicity to normal cells. There is a big drawback of doing. There has been an attempt to use Witherin A in combination with the peak 21 fraction containing a large amount of Withanone, but the normal cytotoxicity was considerably reduced, but the toxicity could not be completely lost (patent) Reference 1).
With an advantage that Withanon has extremely low toxicity to normal cells, it has a mild antitumor effect in vivo and is insufficient for use alone as an anticancer agent.
Therefore, there has been a demand for the development of an anticancer agent which is a combination of a plurality of withanolide compounds, has no toxicity to normal cells, and has a high cell growth inhibitory effect on cancer cells.

International Publication WO2005 / 083392 JP 2008-195704 A

  An object of the present invention is to provide an anticancer agent that has no toxicity to normal cells and has a high cell growth inhibitory effect on cancer cells by combining a plurality of withanolide compounds at a specific ratio.

In the present invention, paying attention to two withanolide compounds, wizzanone and witherferin A, contained in an ethanol extract (i-Extract) derived from Ashwagandha leaves, a composition in which the blending ratio of the two is varied is prepared. By analyzing the cell growth inhibitory ability against cancer cells as well as cytotoxicity against normal cells, the compounding ratios in which the synergistic effects of the respective components appear were found.
That is, in vitro, in the case of a composition containing Withanone (Formula 1 below) and Witherin A (Formula 2 below) in a weight ratio of 20: 1, there is no cytotoxicity to normal cells. It was confirmed that the epoch-making effect that the cell growth-inhibiting ability against cancer cells was strong was exhibited. Hereinafter, the composition is referred to as “WiNA-201”.
<Formula 1> Withanon:

<Formula 2> Witherin A:

Thus, as a combination of the two, the present inventors have found that there is an optimal numerical range that is not toxic to normal cells and has a strong cell growth inhibitory ability against cancer cells. Determines the maximum dose per body weight for the optimal ratio when administered in vivo (in vivo), taking into account the solubility and half-life of both ingredients, and witheron and witherferin A in a weight ratio of 2 : The case where the ratio of 1 was included was found to be the optimal numerical range in vivo. In vivo, in the case of a composition containing such a weight ratio of 2: 1 ("WiNA-21"), there is no toxicity to normal cells and tissues and a strong tumor cell growth inhibitory effect. Furthermore, it was possible to demonstrate an excellent anticancer agent function of cancer cell metastasis inhibitory effect as well as cancer cell invasive inhibitory effect.
Obtaining these findings completed the present invention.

That is, the present invention is specifically as follows.
[1] An anticancer agent comprising withanone and witherin A as active ingredients and containing both ingredients in a ratio of 2: 1 (weight ratio).
[2] The anticancer agent according to [1], which is administered as an injection.
An anticancer agent comprising Withanone and Witherin A as active ingredients and containing both ingredients in a ratio of 2: 1 (weight ratio).

  WiNA-201 of the present invention is nontoxic to normal cells under in vitro conditions and WiNA-21 under in vivo conditions, and is toxic specifically for cancer cells. Each of them is promising as an excellent anticancer agent because it has a remarkable antitumor activity under in vitro conditions and in vivo conditions, as well as weakening the invasion ability of cancer cells and having antimetastatic ability.

Comparison of tumor cell growth inhibitory effect and normal cell toxic effect at various blending ratios of Withanone and Witherin A. [After 30 hours of incubation, only with Wizanone and Withaferin A 20: 1 (WiNA-201) shows that tumor cell growth is suppressed under in vitro conditions and that there is no toxicity to normal cells. ] Inhibition of migration of human glioblastoma cells (YKG1). [Migration of the surrounding cells (= repair of cell layer) after 8 hours on YKG1 cells that had scratched the cell layer was compared. Of i-Extract (ethanol extract), Withaferin A, Withanon and WiNA-201, WiNA-201 most effectively delayed migration under in vitro conditions. ] Inhibition of human fibrosarcoma cell (HT1080) infiltration. [By measurement in Boyden chamber assay. Inhibition of invasive ability was observed in all of Witherin A, Withanone and WiNA-201, but WiNA-201 showed the strongest effect under in vitro conditions. ] Inhibition of human fibrosarcoma cell (HT1080) infiltration. [By QCM ^TM cell invasion assay kit. It was quantitatively shown that WiNA-201 exhibited the strongest invasive ability suppression effect under in vitro conditions. ] Inhibition of adhesion between human fibrosarcoma cells (HT1080) and fibronectin. [Measured by cell adhesion assay. Witherin A, withanone and WiNA-201 all inhibited cell adhesion to fibronectin, but WiNA-201 showed the most potent effect under in vitro conditions. ] Toxicity test by intravenous injection to mice. [Weighs mice after injecting WiNA-21 etc. into the tail vein of nude mice. As judged by body weight, WiNA-21 was not toxic to mice. ] The potent anticancer effect of WiNA-21 in the nude mouse tumor assay. [The growth of a human fibrosarcoma cell line (HT1080) under subcutaneous transplantation of nude mice showed a strong cancer suppressive effect in the WiNA-21 injection group compared to the control group. ] Tumor volume change in nude mouse tumor assay. [Two tumor volumes in each of four mice in the WiNA-21 injection group and the comparative control group mice were averaged, and changes in tumor volume were examined. ] Inhibition of human fibrosarcoma cells under in vivo conditions. [Lung tissue 5 weeks after WiNA-21 was administered intraperitoneally to human fibrosarcoma cell line 8 days after intravenous injection in nude mice and when phosphate buffer / saline was used (control) Observe cancer cell metastasis to Little tumor metastasis is observed in the WiNA-21 injection group. ] WiNA-21 (in vivo) as well as WiNA-201 (in vitro) increases the cell adhesion protein NCAM-140. [A: Human neuroblastoma cell line was cultured for 48 hours in the presence of Witherin A, Withanone or WiNA-201, and the expression levels of various NCAMs were confirmed. B: In vivo tumor formation experiment using HT1080 cell line. Comparison of various NCAM expression levels using WiNA-21 in HT1080 cell tumors made subcutaneously in nude mice. C: Comparison of expression levels of NCAM-140 and PSA-NCAM using WiNA-201 in HT1080 cell culture. ] Strong expression induction of NCAM and suppression of PSA-NCAM expression by WiNA-201. [WiNA-201 was added to HT1080 cell culture medium and the expression of NCAM and PSA-NCAM was examined by immunostaining. It shows that PSA-NCAM decreased while WiCAM-201 administration increased NCAM. ] Inhibition of matrix metalloproteinase MMP2 expression by WiNA-21 (in vivo) and WiNA-201 (in vitro). A significant decrease in MMP2 protein was observed with the administration of WiNA-201 and WiNA-21. [A: Witherferin A, withanone or WiNA-201 was added to the culture medium of the YKG1 cell line and cultured, and the expression level of MMP2 protein was examined by Western blot. B: MMP2 protein expression level in the culture medium of HT1080 cell line. C: MMP2 protein expression level in HT1080-derived tumors subcutaneously in nude mice. ] Reduction of ERK of signaling protein by WiNA-21 and WiNA-201. [A significant decrease in ERK was observed with the administration of WiNA-201 and WiNA-21. A: Examination of the expression level of ERK in the culture solution of YKG1 cell line. B: ERK expression level in the culture medium of HT1080 cell line. C: ERK expression level in HT1080-derived tumors subcutaneously in nude mice. ]

1. Method for Producing “WiNA-21” or “WiNA-201” of the Present Invention After obtaining an alcohol extract (i-Extract) obtained by extracting and concentrating Ashwagandha leaves with alcohol (aqueous alcohol), Patent Document 1 Alternatively, the following withanone (Formula 1) and witherferin A (Formula 2) are purified according to the method of 2. While both powders can be mixed and administered at a weight ratio of 2: 1 or 20: 1, it is preferable to use them as a composition in which 10 mg / ml is dissolved in DMSO solvent in advance. In the examples of the present invention, it was used as a WiNA composition.
When “2: 1 (weight ratio)” is used, it means rounding to 2: 1, so it includes “2.4 to 1.5: 1”, that is, “24:10 to 15:10”. And preferably 22:10 to 18:10. Similarly, “20: 1 (weight ratio)” includes “20.4 to 19.5: 1”, that is, “204: 10 to 195: 10”, preferably 202: 10 to 198: 10.
<Formula 1> Withanon
<Formula 2> Witherin A

2. Effect of WiNA-201 in vitro and optimal blending ratio of withanolide compound in vivo based on it (WiNA-21)
In vitro administration of withanolide compounds, in the case of a composition containing withanone (formula 1) and withferrin A (formula 2) in a weight ratio of 20: 1, cells to normal cells There is an epoch-making effect that it has no toxicity and has a strong ability to suppress cell growth against cancer cells (WiNA-201).
Thus, when both were combined in a specific ratio, it became clear that there was a numerical range in which there was no toxicity to normal cells and the ability to suppress cell growth was strong for cancer cells.
Therefore, as an actual anticancer agent, the optimum blending ratio was determined even when administered in vivo. In other words, the dose in vivo is the amount relative to the weight of the individual, and the behavior in vivo such as solubility and half-life does not always match that in vitro, so it is necessary to optimize the dose.
At that time, in the past, only ingestion and local injection were used at the time of collecting in vivo data. However, in this example, mice were used in this example because the actual anticancer drug administration form is often administered into the bloodstream (intravenous injection). Attempted injection into the bloodstream. Therefore, the maximum dose for body weight (kg) of mice that can be injected into the tail vein was examined. With respect to the body weight (kg) of mice, witherone-1 mg and witherferin A-0.5 mg are the maximum doses. I understood it. An injection containing 2: 1 by weight of Withanone and Witherin A was manufactured and injected into the tail vein of mice ("WiNA-21"). It was found that the toxicity to cells and tissues was extremely low.
And when this "WiNA-21" was applied to tumor cells transplanted subcutaneously in nude mice, it was confirmed not only a strong tumor cell growth inhibitory effect, but also a cancer cell invasion inhibitory effect, a cancer cell metastasis inhibitory effect Excellent anticancer drug function was demonstrated.

3. Characteristics of “WiNA-201” and “WiNA-21” of the present invention The compositions “WiNA-201” and “WiNA-21” of the present invention have the following characteristics.
(1) WiNA-201 and WiNA-21 are toxic specifically for cancer cells. There is no toxicity to normal cells. (Fig. 1, Fig. 6)
(2) It is harmless even if WiNA-21 is intravenously injected into mice.
The non-toxicity of WiNA-21 in vivo was confirmed by 10 days of mouse tail vein injection. (Fig. 6)
(3) WiNA-201 exhibits an antitumor effect in vitro. (Figure 1)
(4) WiNA-21 exhibits an antitumor effect in vivo.
The strong anticancer suppressive activity of WiNA-21 was confirmed by nude mouse tumorigenesis assay. (Figs. 7 and 8)
(5) WiNA-201 weakens the invasive ability of cancer cells.
The inhibitory effect of WiNA-201 on the ability to infiltrate cancer cells was observed by observing the effect of inhibiting the migration ability of cancer cells (Fig. 2), invasion assay using Boyden chamber (Fig. 3), and quantification using QCM ^TM cell invasion assay kit (Fig. 4). ).
(6) WiNA-201 and WiNA-21 have anti-metastatic ability of cancer cells.
The anti-metastatic potential of WiNA-21 was confirmed by a tumor metastasis assay. (Fig. 9)
In addition, the fact that WiNA-201 and WiNA-21 increased NCAM-140, a cell adhesion protein, indicates that WiNA is strongly involved in the induction of anti-metastatic effects, and that PSA-NCAM also has a decreasing effect. Show increased cell adhesion and decreased metastasis. (Figs. 10 and 11)
(7) WiNA-201 weakens the adhesion of cancer cells to the extracellular matrix.
The inhibitory effect of WiNA-201 on the ability to adhere to the extracellular matrix (fibronectin) of cancer cells was confirmed by a cell adhesion assay. (Fig. 5)
(8) WiNA-201 increases the expression of cell adhesion proteins in cancer cells.
WiNA-201 and WiNA-21 show an increasing action of the cell adhesion protein NCAM-140. This indicates that WiNA is strongly involved in the induction of antimetastatic effects.
Although the relationship between the cell adhesion protein NCAM and tumor regression is already known, in this Example 8, the increase in NACM-140 is seen from the fact that WiNA-201 and WiNA-21 increase NACM-140. Was strongly associated with causing tumor shrinkage. (FIG. 10A, B, FIG. 11)
In addition, WiNA-201 and WiNA-21 are expected to be effective as NCAM-140 expression enhancers and inhibitors.
(9) WiNA-201 and WiNA-21 decrease the expression of metalloprotease MMP2 in cancer cells. WiNA-201 and WiNA-21 are matrix metalloproteases (MMPs) that are involved in angiogenesis to supply oxygen and nutrients to tumors. It was demonstrated to suppress the expression of MMP-2, which is the target of the drug. That is, WiNA-201 and WiNA-21 can be expected to have an effect as an MMP-2 expression inhibitor. (Fig. 12)
(10) WiNA-201 and WiNA-21 decrease cancer cell signaling protein ERK. That is, WiNA-201 and WiNA-21 can be expected to have an effect as an ERK expression inhibitor. (Fig. 13)

4). The present invention relates to an “anticancer agent” comprising “WiNA-21” as an active ingredient. The term “anticancer agent” as used herein refers to a human or mammal. It refers to a pharmaceutical composition having anticancer activity or antitumor activity when administered orally or parenterally, which can be used for the prevention, treatment or treatment of cancer.
The administration method of the pharmaceutical composition of the present invention is not particularly limited, and can be oral, nasal, parenteral, pulmonary, transdermal, transmucosal, etc., and can be in various dosage forms. However, administration by injection is particularly suitable. For example, for oral administration, it can be tablets, capsules, etc., and various pharmaceutically acceptable carriers, such as excipients, binders, disintegrants, lubricants, flavoring agents, coloring agents. May include, but is not limited to, agents, sweeteners, flavoring agents, solubilizers, suspending agents, emulsifiers, coating agents, vitamin C, antioxidants.
The dosage of the pharmaceutical composition of the present invention varies depending on the age, weight and symptoms of the human being administered, but generally 1 mg to 1000 mg, preferably 100 mg to 500 mg is used as an adult daily dose. Can be divided into several doses. It can also be administered in combination with other anti-tumor agents and treatments.
The composition of the present invention can be in the form of a food or dietary supplement, or in the form of a quasi-drug or cosmetic.
In the case of “WiNA-201”, the blood, lymphocytes, dendritic cells, etc. of the patient are taken out of the living body and administered together with other immunoactive agents, cytokines and the like in vitro.

The present invention will be described in more detail in the following examples, but the present invention is not limited thereto.
In addition, the description items in the documents cited in the present specification are incorporated as descriptions in the present specification. Unless otherwise specified, known methods such as Molecular Cloning 3rd edition Sambrook J et al., Cold Spring Harbor Laboratory Press, 2001, Cell Engineering Supplement "Bio-Experiment Illustrated" (Shyujunsha, 2001), etc. And can be carried out according to the instructions of commercially available reagents and kits.

(Example 1) Examination of blending ratio of two kinds of withanolides Withanone and Witherin A were selected as two kinds of withanolides, and the optimal combination of both in vitro was examined.
The inventors of the present invention have previously studied a combination of Wizanone and Witherin A in a 1: 1 ratio (WiNA-11), which is toxic to normal cells and is not suitable as an active ingredient of an anticancer agent. I have already confirmed that (not shown).
Therefore, with respect to the culture solution (3 × 10 ⁵ cells / 0.2 ml) of human osteosarcoma cells (U20S strain) and normal cells (TIG-1 strain), Withanone 5.0 μg / ml, Witherin A0.250 μg / ml (WiNA -201 [20: 1]), Withanone 2.0 μg / ml, Witherin A 0.4 μg / ml (WiNA-102 [10: 2]), Withanone 5.0 μg / ml, Witherin A 0.250 μg / ml (WiNA-201 [20: 1]) is added, and a photograph after further culturing for 30 hours is shown (FIG. 1). As a control, 2 μl of DMSO was added.
The 20: 1 combination of Withanone and Witherin A (WiNA-201) kills cancer cells but is harmless to normal cells. On the other hand, the 10: 2 combination of Wizanon and Witherin A (WiNA-102) and the 10: 3 combination of Withanone and Witherin A (WiNA-103) are toxic to normal cells.
From the above results, it was determined that the optimal blending ratio of Withanone and Witherin A in vitro was 20: 1. Since “WiNA-201” is expected to be an excellent anticancer agent having high anticancer activity without toxicity to normal cells, details of the effect of “WiNA-201” as an anticancer agent will be described in the following examples. To consider.

(Example 2) Infiltration suppression of human glioblastoma cells
In order to confirm the ability of WiNA-201 to suppress invasion of human glioblastoma cell lines, the cell migration ability was observed by “wound healing assay”. Human glioblastoma cells (YKG1) cells were cultured in a 6-well culture dish, and when the cells approached 100% confluence, the cell layer was uniformly damaged (20 gauge needle).
i-Extract, Witherin A, Withanone and WiNA-201 were added to the culture solution at 2 µg / ml, 0.25 µg / ml, 5 µg / ml, 5 µg / ml + 0.25 µg / ml, respectively, and cell movement after 8 hours. Was observed with a phase-contrast microscope. The movements of the control and withanolide added cells were photographed over time. A control (comparison control group) to which 2 μl of DMSO was added was used. In the control, the surrounding cells migrated to the wound in about 8 hours, but migration to the wound (= repair of the cell layer) was delayed in cells treated with i-Extract, withanone or WiNA-201 in the culture medium. Of these, WiNA-201 most effectively delayed migration (Figure 2).

(Example 3) Infiltration suppression of human fibrosarcoma cells (3-1) Invasion assay using Boyden chamber
The inhibitory effect of WiNA-201 on the invasion ability of human fibrosarcoma cell line HT1080 was examined by invasion assay using Boyden chamber (Corning Inc., Corning, NY).
The mechanism of the Boyden chamber is as follows. The culture dish is separated by an upper and lower polycarbonate filter membrane. This membrane has an 8 μm hole, and Matrigel (50 μl of 5 mg / ml) is added and left at 37 ° C. for 2 hours to gel. 1 × 10 ⁶ cells are suspended in 100 μl of serum-free medium and poured onto the upper layer of the gelled Matrigel. The lower layer of the culture dish is filled with a culture solution (600 μl) containing fibronectin (5 μg / ml) as a cell adhesion substrate. In this state, the chamber is left at 37 ° C. for 24 hours. Cells having invasive ability decompose Matrigel, pass through the filter membrane, and move from the upper layer to the lower layer. After completion of the culture, the cells remaining on the upper layer of the filter membrane are removed with a cotton swab. Cells infiltrating the lower layer are fixed by exchanging the culture medium with methanol / acetone (1: 1) and stained with crystal violet. Stained cells are dried, removed from the culture dish and photographed under a microscope.

  On the gelled matrigel of the human fibrosarcoma cell line (HT1080) prepared as described above, 2 μl, 0.25 μg / ml, 5 μg / ml, and 5 μg / ml of control DMSO, Witherin A, Withanone and WiNA-201, respectively. ml + 0.25μg / ml was added, cultured at 37 ° C for 24 hours, cells infiltrated below the membrane were fixed with methanol / acetone (1: 1), stained with crystal violet, dried and photographed . Inhibition of invasive ability was observed in all of Witherin A, Withanon and WiNA-201, but WiNA-201 showed the strongest effect. (Figure 3)

(3-2) Quantification with QCM ^TM cell invasion assay kit
The inhibitory effect of WiNA-201 on the invasion ability of the HT1080 cell line was further quantified by fluorescence measurement using a QCM ^™ cell invasion assay kit (QCM ^™ kit from Millipore). The cells were cultured in a 6-well culture dish for 48 hours and then used for experiments.
Like the Boyden chamber, this kit also separates the upper and lower layers of the culture dish with an extracellular matrix layer. If the cells poured into the upper layer have invasive ability, the extracellular matrix is decomposed and moved to the lower layer. Wet the extracellular matrix layer with 300 μl of warm serum-free medium for 15 to 30 minutes. Then, remove about 250 μl of the culture without breaking the membrane, and add cells (250 μl, 1 × 10 ⁶ cells / ml). The lower layer is filled with a culture solution containing 10% fetal bovine serum. Add 2 μl, 0.25 μg / ml, 5 μg / ml, 5 μg / ml + 0.25 μg / ml of control DMSO, witherferin A, withanone and WiNA-201 to the culture solution, respectively, in this state at 37 ° C. for 24 hours. After culturing, the upper culture solution is removed. Remove the invasion chamber insert, soak in warm cell detachment solution (225 μl) and leave at 37 ° C. for 30 minutes. The released cells were then lysed with an NP40 cell lysate and stained with a nucleic acid stain (CyQuant GR ^R dye, Molecular Probes) (15 minutes at room temperature). The number of stained cells was counted with a plate reader (480/520 nm filter). (Fig. 4)
As a result, suppression of invasive ability was observed in all of Witherin A, Withanone and WiNA-201, but WiNA-201 showed the strongest inhibitory action.

Example 4 Cell Adhesion Assay In this experiment, the inhibitory effect of WiNA-201 on the ability of human fibrosarcoma cells (HT1080) to adhere to the extracellular matrix constituent factor (fibronectin) is examined.
The HT1080 strain was cultured in a 6-well culture dish for 48 hours and then used for the experiment. A 96-well culture dish was coated with fibronectin at room temperature for 2 hours and then blocked with 0.5% BSA / PBS for 30 minutes. After suspending in a serum-free culture solution so that the number of cells becomes 5 × 10 ⁵ cells / ml, 100 μl of cells are placed on the above setting, and witherferin A, withanone and WiNA-201 are each 0.25 μg / ml. The cells were cultured at 37 ° C. for 45 minutes at 5 μg / ml, 5 μg / ml + 0.25 μg / ml. Thereafter, the culture solution was carefully removed, the culture dish was washed three times with PBS, and the number of cells adhered to the bottom was counted. To measure the number of cells, an MTT colorimetric assay was performed. Add 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazoliumbromide (MTT) (0.5 mg / ml) to the culture and leave it for 4 hours. Formazane is dissolved and turns reddish purple. FIG. 5 shows the results of measuring the absorbance at 550 nm with a plate reader (Wallac).
Witherferin A, withanone and WiNA-201 all inhibited cell adhesion to fibronectin, but WiNA-201 showed the most potent action.

(Example 5) Toxicity test by WiNA-21 injection In order to inject into the tail vein of mice, the maximum dose with respect to body weight (kg) of mice capable of tail vein injection of withanone and withferrin was examined. It was found that with the body weight (kg), witherone-1 mg and witherferin A-0.5 mg were the maximum doses.
WiNA-21 (withanone-1 mg / kg body weight + witherferin A-0.5 mg / kg body weight) was injected into nude mice into the tail vein. Each time 200 μl was administered at daily intervals for 10 days. For comparison, 200 μl of control (PBS), witherin A (0.5 mg / kg body weight) and withanone (1 mg / kg body weight) were similarly administered. Mice were weighed every 3 days. Feeding and behavior were recorded daily (Figure 6).
As a result, mice administered Witherin A showed clear toxicity that appetite decreased and body weight decreased, but WiNA-21 showed a steady weight gain similar to Withanone and showed no toxicity to mice. It was.

(Example 6) Nude mouse tumor formation assay In this experiment, the growth inhibitory effect of human fibrosarcoma cell line (HT1080) under subcutaneous transplantation of nude mice by WiNA-21 was compared between the WiNA-21 injection group and the control group. investigated.
Balb / c nude mice (4 weeks old female) were purchased from a vendor (Claire Japan) and habituated to the animal facility for 1 week. 3 × 10 ⁶ human fibrosarcoma cell lines (HT1080) were suspended in 0.2 ml culture medium and injected subcutaneously into the lower abdomen (two sites per animal). After the appearance of a small tumor (8 days later), the tumor mice were randomly divided into groups of 4 mice. The control group receives phosphate buffered saline (PBS). In the test group, WiNA-21 (withanone-1 mg / kg body weight + witherferin A-0.5 mg / kg body weight) was administered five times every other day by tail vein injection. Tumor growth and animal health were monitored daily.
As a result, in the control group, many of the tumors formed a huge wet tumor, but in the WiNA-21 injection group, there were some mice in which the tumor almost disappeared, and the tumor growth rate of other mice may be slow. Observed (FIG. 7). In FIG. 7, a photograph was taken on the day shown in the figure.
At the same time, nude mouse transplanted tumors in the WiNA-21 injection group and the comparative control group were measured with calipers, and the volume of the subcutaneous tumor was calculated as V = (length × width × thickness) / 2. Average values of 4 mice were plotted (FIG. 8).
The above results show the strong cancer inhibitory action of WiNA-21.

(Example 7) Tumor metastasis assay In this experiment, the metastasis inhibitory effect of WiNA-21 on human fibrosarcoma cell line (HT1080) in vivo is examined.
In the metastasis assay, 3 × 10 ⁵ human fibrosarcoma cell lines (HT1080) were suspended in 0.2 ml of culture medium and injected into the tail vein. After 8 days, 8 mice are randomly divided into 2 groups. As in Example 6 above, phosphate buffered saline was administered to the control group, and WiNA-21 was administered to the test group 5 times every other day (tail vein injection). Mice were euthanized after 5 weeks and observed for cancer cell metastasis to lung tissue. (Fig. 9)
In the WiNA-21 injection group, little or no tumor metastasis was observed.

(Example 8) Induction effect of neural cell adhesion factor (NCAM) by WiNA-201 and WiNA-21 (8-1) Western blotting operating conditions Immunoblotting used in this example and the following examples (Western blotting) ) Was performed under the following conditions.
RIPA buffer was used for cell lysis. The amount of protein was measured by the BCA method, 20 mg of protein was separated by SDS-PAGE, and transferred to Immobilon-P membrane (Millipore) with a semi-dry transfer instrument. The antibodies used for immunoblotting are as follows. NCAM antibody (AbCys SA), MMP2 antibody (Santa Cruz), phosphorylated ERK antibody, phosphorylated p38 antibody (Cell Signaling), and actin antibody (Chemicon International). Horseradish peroxidase-conjugated rabbit IgG antibody was used as the secondary antibody, and ECL kit was used for color development (GE Healthcare).

(8-2) In vitro induction of neuronal cell adhesion factor (NCAN) Human neuroblastoma cell line (IMR32 line), Withaferin A, Withanon and WiNA-201, 0.25 μg / ml, 5 μg / ml and 5 μg / The cells were cultured at 37 ° C. for 48 hours in the presence of ml + 0.25 μg / ml. Withanon and WiNA-201 strongly induced NCAM-140, a cell adhesion protein. WiNA-201 induced NCAM-180, whereas WiNA-201 exclusively induced NCAM-140. There was no effect on the synthesis of NCAM-120. (FIG. 10A).
An increase in the cell adhesion protein NCAM-140 indicates that WiNA is strongly involved in inducing antimetastatic effects.

(8-3) NCAM induction experiment in tumor (in vivo) formed by HT1080 cell line By the same operation as in Example 6, a tumor of HT1080 cells was made subcutaneously in nude mice. NCAM expression after 18 days in the tumor injected with Witherin A, Withanone and WiNA-21 was observed by the immunoblotting method described in (8-1) above. Tumors injected with WiNA-21 had higher NCAM-140 protein levels than the control group. Similar results were obtained with Witherin A. On the other hand, NCAM-120 was decreased in tumors injected with Withanone and WiNA-21. In conclusion, WiNA-21 causes tumor shrinkage in vivo and increases the cell adhesion protein NCAM-140. (FIG. 10B).
Although the relationship between cell adhesion by the cell adhesion protein NCAM and tumor regression is already known, in this Example 8, WiNA-201 and WiNA-21 reduced the tumor size by increasing NACM-140. It is suggested that it was caused.

(8-4) NCAM induction experiment in HT1080 cell culture medium
Using the HT1080 cell culture solution, the same culture as in (8-2) was performed. In HT1080 cell culture medium, the increase of NCAM-140 by WiNA-201 was observed most strongly. On the other hand, PSA-NCAM with long-chain polysialic acid (PSA) often found in developing neurons was decreased. (FIG. 10C).
Here, a decrease in PSA-NCAM indicates an increase in cell adhesion and a decrease in metastasis.

(Example 9) Observation of NCAM expression induction in tumor cells using immunostaining method In this example, the induction of NCAM expression in tumor cells of WiNA-201 is observed using the immunostaining method. Specifically, it was performed under the following conditions.
The HT1080 cell line is cultured and adhered on a coverslip submerged in a 12-well culture dish. The culture conditions at that time were the same as in Example 1 above, and Withaferin A, Withanone and WiNA-201 in the culture solution were 0.25 μg / ml, 5 μg / ml, 5 μg / ml + 0.25 μg / ml, respectively. It has been added.
After incubation for 48 hours, wash with phosphate buffered saline (PBS) and fix with cold methanol / acetone (1: 1) for 5 to 10 minutes. The fixed cells are washed with PBS and then immersed in 0.2% Triton X-100 / PBS for 10 minutes to make a hole in the cell membrane. Block with 2% bovine serum albumin / PBS, and react with NCAM antibody (Santa Cruz Biotechnology) or PSA NCAM antibody (Cell signaling). As a secondary antibody, Alexa488-conjugated goat anti-rabbit IgG antibody is used (Molecular probes). Wash 2 or 3 times with 0.2% Triton X-100 / PBS and seal with mounting solution. For observation, a fluorescence microscope of Carl Zeiss was used.
The expression of NCAM and PSA-NCAM in cells added with WiNA-201 and the control group was examined by immunostaining. NSA increased with WiNA-201 administration, while PSA-NCAM decreased. (Fig. 11)

(Example 10) Inhibition of expression of matrix metalloprotease MMP2 In this example, human neuroblastoma cell line (IMR32 line) and human fibrosarcoma cell line (HT1080) are known as factors that promote angiogenesis. Among the matrix metalloproteases MMPs, the inhibitory effect of WiNA-201 on MMP-2 expression, which is considered to be most closely related to tumor growth, was examined by the Western blotting method described in Example (8-1).
YKG1 and HT1080 cell lines were cultured (culture conditions are the same as in Example 1), witherferin A, withanone or WiNA-201 was added to the culture solution, and the expression of MMP2 protein after 48 hours of culture was examined. . A significant decrease in MMP2 protein was observed in both YKG1 and HT1080 cell lines supplemented with WiNA-201. (Fig. 12A, B)
On the other hand, in the HT1080 cell line grown subcutaneously in nude mice in the same manner as in Example 6, administration of WiNA-21 caused a significant decrease in MMP2 as in the case of adding WiNA-201 in vitro. (FIG. 12C).

(Example 11) Inhibition of expression of extracellular signal-regulated kinase (ERK)
ERK is a signaling molecule that is involved in a large number of signal transduction pathways, and is thought to be involved in cancer cell signal transduction because it is thought to regulate cell differentiation and proliferation. Yes. In this example, the inhibitory effect of WiNA-201 on ERK expression was performed in the same manner as in Example 10 using the YKG1 strain and the HT1080 cell line.
As a result, a decrease in ERK protein was observed when WiNA-201 was added to the culture solution of YKG1 and HT1080 cell lines. However, even when the same experiment was performed with Witherin A or Withanone, the expression of ERK was not changed (FIGS. 13A and 13B). Similar results were observed in HT1080-derived tumors subcutaneously in nude mice administered with WiNA-21 (FIG. 13C).

Claims (2)

  An anticancer agent comprising Withanone and Witherin A as active ingredients and containing both ingredients in a ratio of 2: 1 (weight ratio). The anticancer agent according to claim 1 , which is administered as an injection.