Method for inhibiting cancer cell growth

Abstract

Disclosed is a method for inhibiting cancer cell growth in a subject in need thereof, comprising administering to the subject an effective amount of a compound from Antrodia camphorata, wherein the compound is represented by formula (I): ##STR00001##
wherein R1 is a hydrogen atom or an acetyl group; and a method of inhibiting cancer cell growth by using the compound, the cancer is selected from the group consisting of lung cancer, colon cancer, prostate cancer, liver cancer and breast cancer.

Claims

1. A method for inhibiting cancer cell growth in a subject in need thereof, comprising administering to the subject an effective amount of an isolated compound from Antrodia camphorata, wherein the compound is represented by formula (I): ##STR00005## wherein R1 is a hydrogen atom or an acetyl group.

2. The method as claimed in claim 1, wherein the cancer is selected from the group consisting of lung cancer, colon cancer, prostate cancer, liver cancer and breast cancer.

Description

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

(1) Antrodia camphorata Extract

(2) Antrodia camphorata fruiting bodies, mycelium or their mixture were provided (1.0 kg) and then extracted twice with an 10-fold ethanol solution to obtain two ethanol extracts. The ethanol extracts were concentrated to yield 230 g crude extract (LE-E). The crude extract was extracted three times with dichloromethane/water (1:1) to form a dichloromethane layer (LT-E-D, 102.6 g) and a water layer (LT-E-W, 127.4 g). Dichloromethane layer (6.0 g) was loaded to a layered silica gel column with hexane/dichloromethane (1:4), dichloromethane, and methanol/dichloromethane (5:95) to yield four layers, respectively ANCA-E-D-1, ANCA-E-D-2, ANCA-E-D-3, and ANCA-E-D-4.

(3) Anti Tumor Activity of Antrodia camphorata Extract

(4) Cell proliferation of A549 cell line (lung cancer), CT26 cell line (colon cancer), DU145 cell line (prostate cancer), HepG2 cell line (liver cancer), MDCK cell line (kidney from canine), PC3 cell line (prostate cancer), MDA-MB-231 cell line (breast cancer) and MCF-7 cell line (breast cancer) was assessed by MTT cell viability assay. The results are shown in tables 1-8.

(5) Above cell lines were cultured in determined medium for 24 hours. Proliferative cells were washed with PBS solution, treated with trypsin-EDTA (1), centrifuged at 1,200 rpm for 5 min, precipitated the cells and discarded supernatant. The cells were resuspended with 10 ml of fresh medium, and then loaded to 96-well plate. While the assay initiated, 0.01200 g/ml of Antrodia camphorata extract was added in each well, and the plate was incubated for 48 hours, at 37 C., 5% CO.sub.2. Each wall was added with 2.5 mg/ml MTT reagent in the dark. After 4 hours of reaction, 100 l lysis buffer were added to each wall to terminate the reaction. Finally, absorbances were read with an ELISA reader in the wavelength of 570 nm, so as to calculate the cell viability and half maximal inhibitory concentration (IC50). The experiment data was represented by meansSD. All data was statistically analyzed by paired-t test, and a P-value less than 0.05 was considered significant.

(6) TABLE-US-00001 TABLE 1 A549 cell line (lung cancer) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 200 +++ + +/ + +/ +/ +/ +/ ++++ 100 +++ + +/ + ++ +/ +/ +/ ++++ 50 +++ + ++ + ++ +/ +++ +++ +++ 25 +++ + ++ + ++ +/ +++ ++ ++++ 10 +++ + + ++ +++ +/ + + 1 +++ +++ + +++ +++ + + ++ 0.1 +++ +++ +++ +++ +++ ++ + +++ 0.01 +++ ++++ +++ +++ ++++ +++ +++ +++ 100

(7) TABLE-US-00002 TABLE 2 CT26 cell line (colon cancer) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 200 +++ +/ +/ +/ +/ +/ +/ +/ +++ 100 +++ +/ +/ +/ + +/ +/ +/ +++ 50 +++ +/ +/ +/ + +/ +/ + ++++ 25 ++++ +/ + + + +/ + + ++++ 10 +++ + + ++ ++ +/ + + 1 ++++ +++ + ++++ ++++ +/ + + 0.1 ++++ ++++ +++ ++++ +++ ++ + +++ 0.01 ++++ +++ +++ +++ +++ +++ +++ +++ 100

(8) TABLE-US-00003 TABLE 3 DU145 cell line (prostate cancer) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 200 +++ +/ +/ +/ +/ +/ +/ +/ +++ 100 +++ +/ +/ +/ + +/ +/ +/ ++++ 50 ++++ +/ + +/ + +/ + + +++ 25 +++ +/ + +/ +/ +/ + + ++++ 10 ++++ +/ +/ +/ +/ +/ +/ +/ 1 +++ + +/ ++ +++ +/ +/ +/ 0.1 +++ +++ + +++ +++ +/ +/ ++ 0.01 +++ +++ +++ +++ +++ ++ ++ +++ 100

(9) TABLE-US-00004 TABLE 4 HepG2 cell line (liver cancer) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 200 +++ +/ +/ +/ +/ +/ +/ +/ +++ 100 +++ +/ +/ +/ + +/ +/ +/ +++ 50 +++ +/ +/ +/ + +/ +/ + ++++ 25 +++ +/ + + ++ +/ + + ++++ 10 +++ + + ++ +++ +/ +/ + 1 +++ ++ + +++ ++++ +/ + + 0.1 +++ +++ ++ ++++ +++ + + +++ 0.01 +++ +++ ++ ++++ ++++ ++ ++ +++ 100

(10) TABLE-US-00005 TABLE 5 MDCK cell line (kidney from canine) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 200 +++ +++ +/ ++ + +/ +/ +/ +++ 100 +++ +++ +/ +++ ++++ +/ +/ +/ ++++ 50 +++ +++ +++ +++ +++ +/ + ++ +++ 25 +++ +++ +++ +++ +++ + + +++ ++++ 10 +++ +++ +++ +++ +++ + +++ +++ 1 +++ +++ +++ +++ +++ + +++ ++ 0.1 +++ +++ +++ +++ +++ +++ +++ +++ 0.01 ++++ +++ ++++ +++ +++ ++++ +++ ++++ 100

(11) TABLE-US-00006 TABLE 6 PC3 cell line (prostate cancer) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 200 +++ +/ +/ +/ +/ +/ +/ +/ ++++ 100 ++++ +/ +/ +/ + +/ +/ +/ +++ 50 ++++ +/ + +/ + +/ + + ++++ 25 ++++ +/ + +/ +/ +/ +/ +/ +++ 10 +++ +/ +/ + +/ +/ +/ +/ 1 ++++ + +/ +++ +++ +/ +/ +/ 0.1 ++++ +++ + +++ +++ +/ +/ +++ 0.01 ++++ ++++ +++ ++++ ++++ ++ ++ +++ 100

(12) TABLE-US-00007 TABLE 7 MDA-MB-231 cell line (breast cancer) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 100 ++ ++ +/ + +/ +/ +/ +/ +++ 50 +++ + +/ + ++ +/ +/ ++ +++ 25 +++ + ++ ++ ++ +/ ++ ++ ++++ 10 +++ ++ ++ ++ ++ + ++ ++ ++++ 1 +++ ++ ++ +++ +++ + + ++ 0.1 +++ +++ +++ +++ +++ ++ ++ +++ 0.01 +++ +++ +++ +++ +++ +++ +++ +++ 0.001 +++ +++ +++ +++ +++ +++ +++ +++ 100

(13) TABLE-US-00008 TABLE 8 MCF-7 cell line (breast cancer) Dose ANCA- ANCA- ANCA- ANCA- ANCA- ANCA- 3 days g/ml DMSO ANCA-E E-D E-W E-D-1 E-D-2 E-D-3 E-D-4 Cell Blank 100 ++ ++ +/ ++ +++ +/ +/ +/ ++++ 50 ++ ++ ++++ ++ +++ +/ ++++ ++++ ++++ 25 +++ ++ +++ ++ ++ +++ ++++ +++ +++ 10 ++++ + ++ ++ +++ ++ +++ +++ +++ 1 +++ ++ ++ +++ +++ + ++ ++ 0.1 ++++ +++ ++ +++ +++ ++ ++ +++ 0.01 ++++ +++ +++ +++ +++ +++ ++ +++ 0.001 ++++ ++++ +++ ++++ +++ +++ +++ +++ 100

(14) The symbols used in tables respectively represent: 025% cell viability: +/; 2550% cell viability: +; 5075% cell viability: ++, 75100% cell viability: +++, >100% cell viability: ++++. The solvent used herein is DMSO, whose IC50 value is 2.34%, which means when the drug is diluted to contain 2.34% of DMSO would cause 50% cell death. In this experiment, when the drug concentration was diluted to 100 l/ml, DMSO concentration was 0.5%. ANCA-E, ANCA-E-D, ANCA-E-W, ANCA-E-D-1, ANCA-E-D-2, ANCA-E-D-3, and ANCA-E-D-4 are different extracts.

(15) According to the results shown in above tables, ANCA-E-D-2, ANCA-E-D-3, ANCA-E-D-4 can preferably inhibit the survival of various tumor cells. For example, in comparison to the other extracts, ANCA-E-D-2 and ANCA-E-D-3 preferably inhibit survival of A549 cell line (lung cancer), CT26 cell line (colon cancer), DU145 cell line (prostate cancer), HepG2 cell line (liver cancer), MDCK cell line (kidney from canine), PC3 cell line (prostate cancer), MDA-MB-231 cell line (breast cancer) and MCF-7 cell line (breast cancer). Though the effect of ANCA-E-D-4 is lower than ANCA-E-D-2 and ANCA-E-D-3, it still has a moderate inhibition effect thereof. Accordingly, above extracts can be used for treating cancers, such as lung cancer, colon cancer, prostate cancer, liver cancer and breast cancer, and the effective compounds contained in those extracts also can be purified.

(16) Purification of Antrocamol LT1 and Antrocamol LT2 from Antrodia camphorata Extracts

(17) According to the above results, ANCA-E-D-2 and ANCA-E-D-3 were subjected to C18 reverse-phase HPLC columns for purification. For ANCA-E-D-3 purification, a fraction collected at 18.75 min (80% MeOH/H20) was concentrated to yield a novel compound Antrocamol LT1 (150 mg). For ANCA-E-D-2 purification, a fraction collected at 25.10 min (80% MeOH/H20) was concentrated to yield another novel compound Antrocamol LT2 (170 mg). The structures of the novel compounds were determined as follow.

(18) Antrocamol LT1 was a transparent aqueous product, the molecular formula was determined as: C.sub.24H.sub.38O.sub.5; 4-hydroxy-5-[9-hydroxy-3,7,11-trimethyldodeca-2,6,10-trienyl]-2,3-dimethoxy-6-methyl-cyclohex-2-enone; molecular weight: 406.

(19) 1H-NMR Spectral Data of Antrocamol LT1: .sup.1H-NMR (400 MHz, CDCl.sub.3): 1.12 (3H, d, J=7.2 Hz), 1.61 (3H, s), 1.64 (3H, s), 1.66 (3H, s), 1.68 (3H, s), 1.72 (1H, m), 1.98-2.30 (8H), 2.51 (1H, dq, J=11.6, 7.2 Hz), 3.62 (3H, s), 4.02 (3H, s), 4.33 (1H, d, J=2.8 Hz), 4.35 (1H, dt, J=9.2, 4.0 Hz), 5.09 (1H, d, J=8.4 Hz), 5.14 (1H, t, J=7.2 Hz), 5.15 (1H, t, J=7.2 Hz); .sup.13C-NMR (100 MHz, CDCl.sub.3): 012.17 (q), 15.95 (q), 16.19 (q), 18.13 (q), 25.72 (q), 25.93 (t), 26.78 (t), 39.41 (t), 39.98 (d), 43.29 (d), 47.94 (t), 58.81 (q), 60.48 (q), 65.35 (d), 67.24 (d), 121.64 (d), 127.64 (d), 128.42 (d), 132.03 (s), 134.99 (s), 135.97 (s), 137.42 (s), 160.82 (s), 197.15 (s).

(20) Antrocamol LT2 was a transparent aqueous product, the molecular formula was determined as: C.sub.26H.sub.40O.sub.6; 4-acetoxy-5-[9-hydroxy-3,7,11-trimethyldodeca-2,6,10-trienyl]-2,3-dimethoxy-6-methyl-cyclohex-2-enone; molecular weight: 448.

(21) 1H-NMR Spectral Data of Antrocamol LT2: .sup.1H-NMR (400 MHz, CDCl.sub.3): 1.18 (3H, d, J=7.2 Hz), 1.54 (3H, s), 1.64 (3H, s), 1.67 (3H, s), 1.69 (3H, s), 1.72 (1H, m), 1.80-2.40 (8H), 2.50 (1H, dq, J=11.6, 7.2 Hz), 3.65 (3H, s), 3.98 (3H, s), 4.36 (1H, m), 5.10 (1H, t, J=6.8 Hz), 5.12 (1H, d, J=8.0 Hz), 5.20 (1H, t, J=6.4 Hz), 5.72 (1H, t, J=3.2 Hz); .sup.13C-NMR (100 MHz, CDCl.sub.3): 12.80 (q), 15.96 (q), 16.09 (q), 18.14 (q), 20.93 (q), 25.72 (q), 26.19 (t), 26.76 (t), 39.47 (t), 41.25 (d), 42.98 (d), 48.12 (t), 59.65 (q), 60.67 (q), 65.53 (d), 68.98 (d), 120.74 (d), 127.42 (d), 128.25 (d), 131.74 (s), 134.70 (s), 137.31 (s), 137.56 (s), 158.21 (s), 169.73 (s), 196.84 (s).

(22) TABLE-US-00009 TABLE 9 MDA- MB- MDCK CT26 A549 HepG2 PC3 DU-145 231 MCF-7 g/ml g/ml g/ml g/ml g/ml g/ml g/ml g/ml ANCA-E >200 10 10 10 1 1 20.28 1.21 >100 ANCA-E-D 100 1 1 1 0.1 0.1 30.72 0.97 35.03 4.32 ANCA-E- 25 0.1 0.1 0.1 0.1 0.1 26.53 1.82 30.85 1.19 D-3 Antrocamol >10 0.070 0.006 0.093 0.003 0.014 0.001 0.057 0.002 0.057 0.009 0.98 0.05 0.99 0.08 LT1 Antrocamol >10 0.80 0.03 1.06 0.22 0.59 0.02 0.69 0.06 0.91 0.08 1.03 0.05 0.95 0.11 LT2

(23) The half maximal inhibitory concentration (IC50) is a measure of the effectiveness of a compound in inhibiting biological or biochemical function. This quantitative measure indicates how much of a particular drug or other substance (inhibitor) is needed to inhibit a given biological process (e.g. cell apoptosis-inducing activity of a compound) by half. As shown in table 9, novel compounds Antrocamol LT1, Antrocamol LT2, and extracts of ANCA-E, ANCA-E-D and ANCA-E-D-3 had a predominant anti cancer activity for various cancers, such as lung cancer, colon cancer, prostate cancer, liver cancer and breast cancer. In the future, based on the present invention, these novel compounds and extracts can be further developed to anti cancer drugs.

(24) All of the references cited herein are incorporated by reference in their entirety.

(25) The foregoing description of the exemplary embodiments of the invention has been presented only for the purposes of illustration and description and is not intended to be exhaustive or to limit the invention to the precise forms disclosed. Many modifications and variations are possible in light of the above teaching.

(26) The embodiments and examples were chosen and described in order to explain the principles of the invention and their practical application so as to enable others skilled in the art to utilize the invention and various embodiments and with various modifications as are suited to the particular use contemplated. Alternative embodiments will become apparent to those skilled in the art to which the present invention pertains without departing from its spirit and scope. Accordingly, the scope of the present invention is defined by the appended claims rather than the foregoing description and the exemplary embodiments described therein.

(27) Some references, which may include patents, patent applications and various publications, are cited and discussed in the description of this invention. The citation and/or discussion of such references is provided merely to clarify the description of the present invention and is not an admission that any such reference is prior art to the invention described herein. All references cited and discussed in this specification are incorporated herein by reference in their entireties and to the same extent as if each reference was individually incorporated by reference.