CN101664403B

CN101664403B - Medicinal composition for treating cancer and signal transduction path thereof

Info

Publication number: CN101664403B
Application number: CN200810214388XA
Authority: CN
Inventors: 袁行修; 吴永昌; 张雪玲; 张芳荣
Original assignee: Individual
Current assignee: Individual
Priority date: 2008-09-05
Filing date: 2008-09-05
Publication date: 2012-01-04
Anticipated expiration: 2028-09-05
Also published as: HK1140677A1; CN101664403A

Abstract

The invention provides a medicinal composition for treating gynecologic cancer, prostatic cancer, bladder cancer and liver cancer, which contains flavonoids compounds of formula I. The flavonoids compounds have the activities of poisoning cancer cells, and arrest the cancer cells at S and G2/M periods by regulating and controlling cyclin of the cancer cells. Moreover, the flavonoids compounds regulate and control signal transduction paths of caspase-3, PARP, p-38 MAPK, JNK 1/2 and the like to induce the cell apoptosis. The flavonoids compounds can effectively inhibit xenoplastic transplantation cancer cells of naked mice, but have no side effect. The flavonoids compounds and platinum diaminodichloride have cooperative poisoning effect on the cancer cells.

Description

A kind of pharmaceutical composition and signal transduction path thereof that is used to treat cancer

Technical field

The present invention relates to a kind of pharmaceutical composition of treating cancer; Particularly relate to the pharmaceutical composition that comprises novel flavone compound-flavonoid plain (Protoapigenone); It is in order to cancers such as treatment gynecological cancer, carcinoma of prostate, and the present invention relates to said pharmaceutical composition the research of inductive cancerous cell signal transduction path.

Background technology

Flavone compound (flavonoid or bioflavonoid) is a kind of polyphenolic substance (polyphenolic compound), and it can suppress human cancer cell's growth.Flavone compound has the structure of phenyl benzopyrane ketone (phenylbenzopyrone), mainly comprises flavone (flavones), flavonol (flavanol), isoflavone (isoflavone), flavonol (flavonol), flavanone (flavanone) and flavanonol (flavanonol) ⁽¹⁹⁾Above-mentioned flavone compound can come to light in food plant, some medicinal plants and phytotherapy ^(11,20)Some flavone compound, for example apigenin (apigenin), genistein (genistein) and catechol (catechin) have been proved and have had the ability that suppresses ovarian cancer, breast carcinoma, colorectal cancer, carcinoma of prostate and blood cell growth ^{(1,2,4,6-8,22-23)}The biological activity of flavone compound comprises cell death inducing (apoptosis), suppresses cell cycle, suppresses growth, angiogenesis inhibiting (angiogenesis), antioxidation and above-mentioned active combination ^(12,16,19)These biological activitys reach through the conditioning signal pathway, and for example nuclear factor (nuclearfactor)-κ B (NF κ B), activator protein (activator protein)-1 or MAPK (mitogen-activated proteinkinases) are (MAPKs) ^(5,15,21)This means that flavone compound can be effective anticancer preparation.

The flavonoid element is a kind of novel flavone compound, has the structure suc as formula I, and its extraction is pteridophyta from native country, a kind of Taiwan---and the coarse wool Parathelypteris glanduligera (Kunze) Ching (Thelypteris torresiana, Gaud).The plain method for preparing of flavonoid is disclosed in No. the 094139201st, TaiWan, China patent application.In addition, the flavonoid element be proved to be to human liver cancer cell strain (Hep G2 and Hep3B), mankind mastopathy cell's strain (human breast adenocarcinoma cell line) (MCF-7), human adenocarcinoma of lung epithelial cell strain (human lung adenocarcinoma epithelial cell line) (A549) and mankind mastopathy cell's strain (MDA-MB-231) have the cell toxic action ⁽¹⁷⁾Yet,, whether above-mentioned patent and unexposed flavonoid element can suppress the growth of other cancerous cell, with and signal transduction path mechanism.Simultaneously, those skilled in the art also can't can be applicable to above-mentioned cancer by the flavonoid element, know the flavonoid element by inference and also can suppress the growth of other cancerous cell and have the other biological activity.

formula I

Gynecological cancer (genecological cancer) is the cancer that occurs in female sex organ, comprises the cancer of cervix uteri, fallopian tube (fallopian tubes), ovary, uterus, vagina and vaginal orifice.In the U.S., among annual 80000 new cases that diagnosed nearly half be uterus carcinoma.The risk of suffering from cancer increased with the age, and gene mutation or Inheritance also can increase risk.Common paclitaxel (paclitaxel;

) and carboplatin (paraplatin;

) effect is pretty good in the initial stage treatment of ovarian cancer late; Remission rate reaches 60-80%; But the recurrence chance is high, causes being higher than 70% sufferer death at last.

Carcinoma of prostate (prostate cancer) is a common malignancy, and is the relevant cause of the death of big cancer that is number three among the male in the U.S. ⁽¹⁰⁾Operation or radiation cure are the low main Therapeutic Method that arrives the carcinoma of prostate of moderate differentiation ⁽¹⁴⁾If cancerous cell is diffused into outside the pelvis, then there is not the method for efficacious therapy carcinoma of prostate.Need to rely on the growth of chemotherapy control cancer cell.Yet the chemotherapeutic agent of clinical use still has height toxicity to normal cell ⁽¹⁸⁾

Therefore, in order to solve the medication problem of gynecological cancer and carcinoma of prostate, add that the natural drug by plant extraction can overcome the degree of difficulty with the synthetic new drug of chemical synthesis process, flavone compound becomes the new selection of new drug development.

Therefore; The applicant is in view of in the known technology, because of the Drug resistance problem and research and develop the shortcoming that new chemical synthetic drug cost consideration is produced, through concentrated test and research; And in line with the spirit of working with perseverance; Finally visualize the present invention and " be used to treat the pharmaceutical composition and the signal transduction path thereof of cancer ", can overcome above-mentioned shortcoming, hereinafter will briefly describe.Have the list of references that the present invention quoted after this description:

Summary of the invention

The present invention is in order to treat gynecological cancer and carcinoma of prostate; With the flavone compound that goes out by plant extraction as pharmaceutical composition; Gynecological cancer cell, prostate gland cancer cell and transitional cell bladder carcinoma cell line are carried out in vitro tests (in vitro) and in vivo test (in vivo); Confirm the activity and the signal transduction path thereof of this flavone compound poisoning cancerous cell, promote this flavone compound clinical value.

The present invention provide a kind of treat cancer pharmaceutical composition, it comprises the flavone compound as shown in the formula I,

formula I

Wherein said cancer is selected from the group be made up of gynecological cancer, carcinoma of prostate, bladder cancer and hepatocarcinoma.

According to above-mentioned conception, gynecological cancer is selected from the group be made up of ovarian cancer, breast carcinoma and cervical cancer.

According to above-mentioned conception, ovarian cancer is MDAH-2774 or SKOV3 cell strain, and breast carcinoma is MDA-MB-468, MDA-C33A or T47D cell strain; Cervical cancer is the HeLa cell strain; Carcinoma of prostate is the LNCap cell strain, and bladder cancer is RT4 or T24 cell strain, and hepatocarcinoma is the Hep3B cell strain.

According to above-mentioned conception, said flavone compound extraction is from the pteridophyta of coarse wool Parathelypteris glanduligera (Kunze) Ching (Thelypteristorresiana) by name.

According to above-mentioned conception, said flavone compound is suppressed at S and G2/M cell cycle with cancerous cell.

According to above-mentioned conception, the expression of at least one cyclin of said flavone compound regulation and control cancerous cell comprises phosphorylation cell periodic protein B 1, cell periodic protein B 1, phosphorylation CdK2, CdK2 and Cdc25C.

According to above-mentioned conception, the caspase-3 (caspase-3) of said flavone compound regulation and control cancerous cell, gather ADP ribose polymerase (PARP), Bcl-xL and Bcl-2 albumen, cell death inducing.

According to above-mentioned conception, the p-38MAPK and c-Jun amino terminal kinases (JNK) 1/2 albumen of said flavone compound regulation and control cancerous cell, cell death inducing.

According to above-mentioned conception, this flavone compound also can with suitable Diaminodichloride (cis-diamminedichloridoplatinum) the associating therapeutic alliance cancer as shown in the formula II.

formula II

According to above-mentioned conception, said pharmaceutical composition comprises pharmaceutically acceptable carrier.

According to above-mentioned conception, the mammal that said flavone compound inhibition has cancer cell, said mammal comprises Mus and people.

The present invention relates to a kind of pharmaceutical composition of treating cancer in addition, and it comprises that the flavone compound as shown in the formula I reaches the suitable Diaminodichloride as shown in the formula II.

formula I

formula II

According to above-mentioned conception, said pharmaceutical composition suppresses the growth of ovarian cancer cell.

Description of drawings

Fig. 1 is the influence of the flavonoid element of variable concentrations to the colony formation of ovarian cancer cell.

The 2nd (A) figure and the 2nd (B) figure are respectively with the plain ovarian cancer cell (A) of handling of variable concentrations flavonoid and reached (B) 4 hours cell cycle distribution situation in 24 hours.

Fig. 3 is the S of the plain regulation and control of flavonoid and the protein expression sketch map of G2/M cell cycle.

Fig. 4 (A) is the plain inductive ovarian cellular apoptosis graphs of a relation of 10 μ M flavonoid.

Fig. 4 (B) is with plain 24 hours the inferior G1 cell cycle graph of a relation of ovarian cancer cell of handling of 10 μ M flavonoid.

Fig. 5 is the apoptotic protein expression sketch map of the plain regulation and control of flavonoid.

Fig. 6 (A) is the cell toxic effect of and along Diaminodichloride independent or Combined Treatment MDAH-2774 cell plain with flavonoid.

Fig. 6 (B) is independent or 24 hours the protein expression sketch map of Combined Treatment MDAH-2774 cell of the plain and suitable Diaminodichloride of flavonoid.

Fig. 7 is the plain growing state that suppresses MDAH-2774 ovarian tumor in the nude mouse of flavonoid.

Fig. 8 is the cut PARP protein expression sketch map in the ovarian tumor tissue.

Fig. 9 is the growth inhibited situation behind the plain processing of the variable concentrations flavonoid prostate gland cancer cell LNCap different time.

Figure 10 (A) and Figure 10 (B) are respectively with (A) annexinV-FITC test method(s) and reach (B) the plain inductive apoptosis of TUNEL test method(s) assessment flavonoid.

Figure 10 (C) is the protein expression sketch map of the plain cell death inducing of flavonoid.

Figure 11 (A) is the plain cell cycle distribution of handling

LNCap cell

6 and 12 hours of flavonoid.

Figure 11 (B) is plain LNCap cell 6 and the protein expression sketch map after 12 hours handled of variable concentrations flavonoid.

Figure 12 (A) and Figure 12 (B) are the protein expression sketch map of the plain LNCap of processing of flavonoid cell.

Figure 13 (A) handles the LNCap cell with SB203580 or SP600125 earlier, again with plain 1 hour the protein expression sketch map of cell of handling of flavonoid.

Figure 13 (B) handles the LNCap cell with SB203580 or SP600125 earlier, again with plain 12 hours the survivaling cell ratio chart of cell of handling of flavonoid.

Figure 14 (A) and Figure 14 (B) are that p38MAPK inhibitor SB203580 and JNK1/2 inhibitor SP600125 reach (B) effect of cell cycle arrest for the plain inductive apoptosis of (A) flavonoid.

Figure 15 (A) weakens the expression sketch map of p38MAPK and JNK1/2 for p38MAPK and the narrow spectrum siRNA of JNK1/2.

Figure 15 (B) suppresses apoptotic ratio for p38MAPK siRNA and JNK1/2siRNA.

Figure 15 (C) is that p38MAPK and JNK1/2 siRNA suppress the plain inductive apoptotic cell cycle distribution graph of a relation of flavonoid.

Figure 15 (D) is that p38MAPK and JNK1/2 siRNA suppress the plain inductive expression of cellular proteins sketch map of flavonoid.

Figure 16 (A) is time and the tumor magnitude relationship figure of lumbar injection flavonoid element to the nude mice of xenotransplantation LNcap cell.

Figure 16 (B) is lumbar injection low dosage and the high dose flavonoid element protein expression sketch map (Figure 16 (B)) to cut PARP, p-p38MAPK and the p-JNK1/2 of the nude mice tumor of prostate of xenotransplantation LNcap cell

Figure 17 (A) to Figure 17 (C) be plain cancerous cell (A) RT4, (B) T24 and (C) the growth inhibited situation behind the Hep3B different time handled of variable concentrations flavonoid.

Figure 18 is the plain cell cycle distribution situation that suppresses human hela cell strain HeLa of flavonoid.

The specific embodiment

" a kind of pharmaceutical composition and signal transduction path thereof that is used to treat cancer " that the present invention carried can fully be understood by following description of test, can be those skilled in the art reference is provided, but be merely example, do not limit the scope of the present invention.

Cytologic experiment

One, experiment material:

The plain extraction of flavonoid is from whole strain coarse wool Parathelypteris glanduligera (Kunze) Ching ⁽¹⁷⁾, and be dissolved in dimethyl sulfoxine (dimethylsulfoxide, DMSO) in.During experiment, add cultured cells with 1000 times of dilutions.

Gynecological cancer cell strain used in the present invention is as follows: (1) human ovarian cancer cell's strain (immortalized non-cancer human breast epithelial cellline) (MDAH-2774 and SKOV3), breast carcinoma cell strain (MDA-MB-468, MDA-C33A and T47D), cervical cancer cell strain (HeLa and C33A) and the non-cancer human mammary epithelial cell of immortalization strain (MCF-10A); It preserves center (American Type Culture Collection available from U.S.'s typical strain; ATCC; Manassas; And be incubated in the DMEM-F12 culture medium of having added 10% hyclone and penicillin (penicillin)/streptomycin (streptomycin)/amphotericin B (amphotericin B) VA); And the human ovarian cancer superficial epithelium of (2) immortalization cell strain (human ovarian surfaceepithelial; HOSE) (HOSE6-3 and HOSE11-12); It is provided by the Cao Shihua of Hong Kong University professor (Prof.GSW Tsao); And be incubated at the MCDB105 that added 10% hyclone and penicillin/streptomycin/amphotericin B and M199 mixed culture medium (mixing of 1:1 volume ratio) (Sigma, St.Louis, MO) in.

The human benign prostatic JEG-3 (LNCap) that the present invention uses is preserved the center available from U.S.'s typical strain, is incubated in the RPMI1640 culture medium of having added 10% hyclone and penicillin/streptomycin/amphotericin.The LNCap cell is handled and is analyzed so that the flavonoid of 2.5,5 and 10 μ M is plain respectively.In some experiment, the LNCap cell was handled 1 hour with p38MAPK inhibitor SB203580 or JNK1/2 inhibitor SP600125 earlier before the plain processing of flavonoid.

Two, experimental technique:

1.XTT method is grown in cell proliferation (XTT cell proliferation assay) and choosing:

Each hole kind at 96 porose discs goes into 7 * 10 ³Individual cell; Again with the flavonoid of variable concentrations (2.5,5 and 10 μ M)

plain handle

12,24 and/or 48 hours after; (Sigma, St.Louis MO) measure the plain cytotoxicity of flavonoid with XTT cell proliferation detection method (XTT cellproliferation assay).Light absorption value when measuring wavelength 490nm and 650nm with the ferment immunity analysis instrument, and with OD ₄₉₀-OD ₆₅₀Value is calculated 50% inhibition concentration (IC ₅₀) ⁽³⁾In addition,, handled cell 3 hours so that the flavonoid of variable concentrations is plain in order to measure the plain effect of long-time use flavonoid, again with fresh culture cultured cell 14 days to form cell colony (colony), at last with crystal violet with cell dyeing and observe.

2. cell survival test:

Different cell cycle S KOV3 cell culture are in 6 cm cell culture dishs, and behind the plain treatment S KOV3 of the flavonoid cell with various dose (0,5 or 10 μ M/ml), collecting cell also mixes with the 1:1 volume ratio with trypan blue.The apoptotic cell that is colored with microscope examination did.

3. cell cycle and inferior G1 phase are analyzed:

Handled ovarian cancer cell respectively 24 hours and prostate

gland cancer cell

6 or 12 hours with the flavonoid of variable concentrations plain (0,2.5,5 and 10 μ M); With trypsin trypsin) collecting cell and with 70% ethanol fixed cell 1 hour; Clean 2 times with phosphate buffer solution (PBS) again; And be suspended in iodate third ingot (propidium iodide, PI)/ribonucleic enzyme A (RNaseA) solution in 30 minutes.Mechanism of action through iodate third ingot dyeing DNA; With flow cytometer (FACScan flow cytometry, BectonDickinson, San Jose; CA) distribution of detection cell cycle and inferior G1, and with CellQuest software (BS Biosciences) analytical data.

4.Annexin the V apoptosis is analyzed:

This experimental principle is when the apoptosis process, to detect early stage apoptotic cell with annexin V.With ovarian cancer cell and prostate gland cancer cell respectively with the plain co-cultivation of 10 μ M flavonoid in the chamber slide 3 hours, the PBS with ice cleans cell 2 times earlier, again in 25 ℃ to contain annexin V-FITC (1mg/ml; Strong Biotech, Taipei, Taiwan) and 4 ', the mixed solution dyeing of 6-diamidine-2-phenylindone (Sigma) 15 minutes is cleaned cell 2 times with PBS again, the cell that is colored with fluorescence microscope.

5. terminal deoxynucleotidyl transferase mediates dUTP breach end-labelling (TUNEL) test method(s):

The TUNEL test method(s) is to detect DNA whether to fragment into fragment.With plain prostate

gland cancer cell

12 or 24 hours of handling of the flavonoid of variable concentrations (2.5,5 and 10 μ M), again with DeadEnd colorimetry TUNEL system (Promega, Madison, TUNEL dyeing WI).Step is: behind the plain processing of flavonoid prostate gland cancer cell; Cancerous cell was fixed in 4% the metaformaldehyde (paraformaldehyde) 30 minutes, and more fixed cell and digoxin gripped the terminal deoxynucleotidyl transferase catalytic reaction of recombinating under the moist environment that the dUTP that closes and mixture of ribonucleotides place 37 ℃ jointly.Afterwards, add stop bath again in 37 ℃ of effects 15 minutes.Clean cell with PBS, place diaminobenzidine (DAB) solution again and reaction 15 minutes in the dark place.The cell that is colored with microscopic examination.Count 1,000 cell learning the ratio of TUNEL positive cell, and calculate the apoptotic index in later stage.

6. immunoblotting assay:

The plain cell of handling 24 hours of flavonoid that will pass through variable concentrations (0,2.5,5 and 10 μ M) cleans 2 times, with EBC buffer solution (50mM Tris (pH7.6), 120mM NaCl, 0.5%NonidetP-40,1mM beta-mercaptoethanol, 50mM NaF and 1mM Na ₃VO ₄) break cell, and measure protein concentration with Bio-Rad protein reagent box.Resolve total protein of cell with polyacrylamide (polyacrylamide SDS) gel, the Western blot that will be resolved again is to nitrocellulose membrane.With the two anti-Recognition Protein that specificity bonded resists, peroxidase indicates, (Amersham, NJ USA) detect albumen intensity with enhanced chemiluminescence detection system more in regular turn.

7. nude mice model:

And have among the 0.1ml PBS 2 * 10 ⁶Individual MDAH-2774 human ovarian cancer cell (or 1 * 10 ⁶Individual LNCap prostate gland cancer cell) the right oxter of female nude mice (Foxnlun/Foxnlnu) in age in subcutaneous injection to 6 week.When tumor is visible (about 3 * 3mm), with the nude mice random packet, and every other day lumbar injection flavonoid element or control group.In this group of xenotransplantation MDAH-2774 ovarian cancer cell, control group, low dose group and high dose group are respectively PBS, (this dosage is equal to the IC of MDAH-2774 to every gram nude mice body weight 0.069 μ M flavonoid element ₅₀1/10th) and every gram nude mice body weight 0.69 μ M flavonoid plain (this dosage is equal to the IC of MDAH-2774 ₅₀); In this group of xenotransplantation LNCap prostate gland cancer cell, control group, low dose group and high dose group are respectively PBS, (this dosage is equal to the IC of LNCap to every gram nude mice body weight 0.37 μ M flavonoid element ₅₀Ten minutes one) and every gram nude mice body weight 3.7 μ M flavonoid plain (this dosage is equal to the IC of LNCap ₅₀).Measure the tumor size and measure body weight with caliper weekly, and with formula " width ²* length/2 " the calculating gross tumor volume.The nude mice of this group of xenotransplantation LNCap prostate gland cancer cell is being used flavonoid plain five all backs with dark anesthesia execution, and gets blood sample immediately.With Sysmex XE-2100 (TOA Medical Electronics; Kobe, Japan) counting nude mice blood cell number is with Beckman LX20 (Beckman-coulter; Fullerton; USA) measure blood urea nitrogen, creatinine, aspartic transaminase (asparatate aminotransferase, AST) and alanine aminotransferase (alanine aminotransferase, ALT) content.

8. the siRNA clpp gene subtracts (siRNAknockdown):

This experimental principle is with narrow spectrum siRNA (small interfering RNA; SiRNA) (Santa Cruz Biotechnology; Inc.) strike the p38MAPK and the JNK1/2 that subtract the LNCap cell and express, it is to use Lipofectamine2000 (Invitrogen) to carry out transfection (transfection) and accomplishes.The LNCap cell culture after 48 hours, with the plain LNCap cell of handling of flavonoid, is regathered the cell that is processed and analyzes.

9. immunohistochemical analysis:

Tissue samples is fixed with 10% formaldehyde or 4% metaformaldehyde ⁽²⁶⁾After, cut into slices again, dewater, with the wax embedding.The sample slice thickness is 3m or 4m; Carry out immunostaining with haematoxylin-Yihong or with monoclonal antibody; Again with (horseradishperoxidase) (Dako DenmarkA/S of Universal LAB+ test kit/HRPO (HRP); Glostrup Denmark) demarcates, or with the contrary dyeing of haematoxylin (hematoxylin).In the microscopically observation of cell.

Three, experimental result:

1. with the plain ovarian cancer cell that suppresses of flavonoid:

See also and see table 1, be the cytotoxicity of flavonoid element the different carcinoma cell strain.In table 1, the flavonoid element has the strongest cytotoxicity to ovarian cancer cell (MDAH-2774 and SKOV3), but lower to the cytotoxicity of the human ovarian cancer superficial epithelium cell strain of immortalization (HOSE6-3 and HOSE11-12).Identical result also can be corresponding to breast carcinoma cell strain (MDA-MB-468 and T47D) and the non-cancer human mammary epithelial cell of immortalization strain (MCF-10A).

In addition, see also Fig. 1, the influence that the colony of ovarian cancer cell is formed for the flavonoid element of variable concentrations.In Fig. 1, along with the plain concentration of flavonoid increases, the ratio that MDAH-2774 and SKOV3 colony form is low more.Can be known that by table 1 and Fig. 1 the flavonoid element has toxicity and can suppress its growth ovarian cancer cell MDAH-2774 and SKOV3, but the non-ovarian cancer epithelial cell of immortalization is not had toxicity, the representation class flavone has optionally cytotoxicity to ovarian cancer cell.

Table 1, flavonoid element are to the cytotoxicity of different cell strains

^aAverage ± the standard deviation of three independent experiments

See also Fig. 2 (A), for handling 24 hours cell cycle distribution situation of ovarian cancer cell with variable concentrations flavonoid element.In Fig. 2 (A), the flavonoid element makes significantly and gets into S and the MDAH-2774 in G2/M cycle and the increase of SKOV-3 cell, and along with the plain concentration of flavonoid increases, the cell that gets into S and G2/M cell cycle also increases thereupon.See also Fig. 2 (B), be ratio with plain treatment S 4 hours cell cycle distribution of KOV3 cell of variable concentrations flavonoid and survivaling cell.In Fig. 2 (B), behind the plain cell of treatment S KOV3 respectively, reduce by 23.5% and 45.4%, 20.3% and 34.7% respectively at the outgrowth SKOV3 cell quantity of S, G2 and M cell cycle with 5 and 10 μ M flavonoid, and 44.8% and 70.5%.

See also Fig. 3, be the S of the plain regulation and control of flavonoid and the protein expression sketch map of G2/M cell cycle.In Fig. 3, the p-Cdk2 of MDAH-2774 and SKOV3 cell, Cdk2, p-cell periodic protein B 1 (ser ¹⁴⁷) and cell periodic protein B 1 expressing quantity increase along with the plain concentration of flavonoid and descend, and p-Cdc25C (ser ²¹⁶) expression then rises.Because belonging to, Cdk2, cell periodic protein B 1 and Cdc25C suppress and the albumen of regulating cell cycle, in the research in the past, and Cdc25C (Ser ²¹⁶) phosphorylation be bonded to the albumen of 14-3-3 family, and be terminated in the Cytoplasm, and prevented jejune mitosis ⁽⁹⁾And the phosphorylation of cell periodic protein B is that the entering M cycle is required, and needs the Cdc25C effect ⁽²⁴⁾Can know the plain Cdc25 (Ser that increases of flavonoid by Fig. 3 ²¹⁶) phosphorylation, and reduce Cdk (Thr ¹⁶¹), cell periodic protein B 1 (Ser ¹⁴⁷) expression.Therefore can know by Fig. 2 (A), Fig. 2 (B) and Fig. 3; The flavonoid element has significant cytotoxicity to SKOV3 at S and G2/M cycle; The flavonoid element not only makes the cell cycle arrest of SKOV3 in S and G2/M cycle, also in these cycles the SKOV3 cell is caused bigger cytotoxicity.Its cell cycle is stagnated in expressing through the cyclin of regulation and control ovarian cancer cell really that flavonoid is plain.

Apoptotic qualitative assessment principle is to migrate to cell surface with the Phosphatidylserine (phosphatidylserine) that annexinV-FITC reagent detects kytoplasm microcell (cytoplasmic leaflet).Seeing also Fig. 4 (A), is the plain inductive ovarian cellular apoptosis graphs of a relation of 10 μ M flavonoid.In Fig. 4 (A), compare with matched group, with the plain apoptosis of handling ovarian cancer cell 3 hours with the SKOV3 that causes 24.5% MDAH-2774 and 34.2% respectively of 10 μ M flavonoid.See also Fig. 4 (B), for handling 24 hours inferior G1 cell cycle graph of a relation of ovarian cancer cell with 10 μ M flavonoid element.In Fig. 4 (B), along with the plain concentration of flavonoid increases, the inferior G1 peak value of MDAH-2774 and SKOV-3 cell also increases thereupon.

See also Fig. 5, be the apoptotic protein expression sketch map of the plain regulation and control of flavonoid.Because cut PARP albumen is apoptotic sign, and by the caspase-3 activation, and caspase-3 is regulated and control by the Bcl-2 family protein.So in Fig. 5, plain Bcl-2 and the Bcl-xL that reduces ovarian cancer cell of flavonoid expresses, activation caspase-3 and cut off complete PARP and inducing cell moves towards apoptosis.In addition, the flavonoid element can increase Bad and Bax expression.

In the clinical research in the past; Along the Diaminodichloride be a kind of common, based on the cancer therapy drug or the chemotherapeutics of platinum, comprise sarcoma (sarcoma), small cell lung cancer, ovarian cancer, lymphatic cancer and germ cell tumor cancers such as (germ cell tumor) in order to treatment.Can formation combine or mutual bonded platinum complexes with DNA in cell along the Diaminodichloride, it is dead finally to open apoptosis program or automated cell.Therefore, plain and suitable Diaminodichloride is mixed into pharmaceutical composition with flavonoid, studies this pharmaceutical composition and whether has collaborative (synergistic) cell toxic effect.See also Fig. 6 (A), be the cell toxic effect of and along Diaminodichloride independent or Combined Treatment MDAH-2774 cell plain with flavonoid.In Fig. 6 (A), the flavonoid of 1 and 2.5 μ M plain (being called for short PA at this) suppresses 24.96% and 53.36% cell growth respectively, and the suitable Diaminodichloride of 5 and 10 μ M (being called for short CIS at this) also suppresses 28.48% and 36.91% cell growth respectively.When 1 μ M flavonoid element is combined into pharmaceutical composition with 5 and 10 μ M along the Diaminodichloride respectively, then can suppress 39.73% and 55.40% cell growth respectively.When the plain concentration of flavonoid increases to 2.5 μ M and when being combined into pharmaceutical composition with 5 and 10 μ M along the Diaminodichloride respectively, then can suppress 72.02% and 82.65% cell growth respectively.Hence one can see that, and the flavonoid element not only can suppress the growth of ovarian cancer cell separately, also can be combined into pharmaceutical composition with suitable Diaminodichloride, shows stronger poisoning cell ability.

formula II

See also Fig. 6 (B), be independent or 24 hours the protein expression sketch map of Combined Treatment MDAH-2774 cell of the plain and suitable Diaminodichloride of flavonoid.In Fig. 6 (B); With plain without flavonoid and handle along the Diaminodichloride; Or the group of handling with the plain or suitable Diaminodichloride of flavonoid separately compares; Can induce complete PARP to be cut off with 5 μ M along the cell that the Diaminodichloride handles through 1 μ M flavonoid is plain, and the activity of activation caspase-3.

See also Fig. 7, be the plain growing state that suppresses MDAH-2774 ovarian tumor in the nude mouse of flavonoid.In Fig. 7, to compare with the matched group of using PBS, the flavonoid element of low dosage and high dose can suppress the intravital ovarian tumor growth of nude mice significantly.See also table 2, for body weight, full blood count and the blood biochemical test result of the plain lumbar injection nude mice of flavonoid after 7 weeks.Can find that in table 2 lumbar injection flavonoid element can't damage hematopoietic potential, liver function, the renal function of nude mice significantly.

After above-mentioned nude mice execution, win its intravital MDAH-2774 tumor, carry out immunoblotting and immunohistochemical analysis, assess its apoptosis situation.See also Fig. 8, be the cut PARP protein expression sketch map in the ovarian tumor tissue.In Fig. 8, cut PARP is expressing in the plain ovarian tumor tissue of handling through flavonoid, but in the control group, does not express.In addition, the flavonoid element is induced the apoptosis of xenotransplantation MDAH-2774, and the nucleus of cut PARP expression (nuclear expression) is obviously increasing (result does not show) through the plain tumor tissues of handling of flavonoid.

2. with the plain prostate gland cancer cell that suppresses of flavonoid:

See also Fig. 9, be the growth inhibited situation behind the plain processing of the variable concentrations flavonoid prostate gland cancer cell LNCap different time.In Fig. 9, the ability of the plain LNCap of inhibition of flavonoid cell growth is along with time and the plain concentration of flavonoid increase and increases.Can obtain maximum inhibition effect, its IC in 48 hours with plain processing of flavonoid ₅₀Value is 3.7 ± 0.2 μ M.And present contraction state (result does not show) through the plain cell of handling 12 and 24 hours of flavonoid.

See also Figure 10 (A), for assess the plain inductive apoptosis of flavonoid with the annexinV-FITC test method(s).In Figure 10 (A), after 3 hours, the ratio of annexinV-FITC positive cell increases to 24.2 ± 1.3% with the plain LNCap cell of handling of 10 μ M flavonoid, and matched group has only 2.7 ± 1.0%.See also Figure 10 (B), for assess the plain inductive apoptosis of flavonoid with the TUNEL test method(s).In Figure 10 (B), the ratio of TUNEL positive cell is along with time and the plain dosage of flavonoid increase and increases.See also Figure 10 (C), be the protein expression sketch map of the plain cell death inducing of flavonoid.In Figure 10 (C), cut PARP and cut caspase-3 protein expression increased along with the plain dosage of flavonoid and action time.

See also Figure 11 (A), handle the cell cycle distribution of

LNCap cell

6 and 12 hours for flavonoid is plain.In Figure 11 (A), the flavonoid element makes the LNCap cell be stuck in S and G2/M cell cycle, has suppressed the carrying out of cell cycle program.See also Figure 11 (B), handle LNCap cell 6 and the protein expression sketch map after 12 hours for the variable concentrations flavonoid is plain.In Figure 11 (B), the expression of Cdk2 is along with the plain dosage of flavonoid and increase action time and increase.After the flavonoid element is handled 6 hours, activatory p-cell periodic protein B 1 (Ser ¹⁴⁷) the expression reduction; Yet, after the flavonoid element is handled 12 hours, activatory p-cell periodic protein B 1 (Ser ¹⁴⁷) and cell periodic protein B 1 expression all reduce.After the flavonoid element is handled 6 and 12 hours, not activatory p-Cdc25C (Ser ²¹⁶) expression all increases.

Some flavone compound (comprising (-)-epigallocatechin gallate (a kind of catechin is called for short EGCG) and apigenin) its active anticancer that comes to light is relevant with the MAPK control path ^(13,25)Therefore, whether also can participate in the MAPK control path through the plain LNCap cell of handling of flavonoid is also studied.See also Figure 12 (A) and Figure 12 (B), be the plain protein expression sketch map of handling the LNCap cell of flavonoid.In Figure 12 (A) and Figure 12 (B), will cause the phosphorylation of p38MAPK and JNK1/2 to increase significantly in 1 hour with the plain LNCap cell of handling of flavonoid.And KAPK kinases (MKK) 3/6 and MKK4 are the kinases at the p38MAPK and the JNK1/2 upper reaches, and the phosphorylation of MKK3/6 and MKK4 (is p-MKK3/6 (ser ^189/270) and p-MKK4 (thr ²¹⁶)) also therefore increase.In addition, proteic whole expression of MKK3/6, MKK4, p38MAPK and JNK1/2 do not receive the plain influence of handling of flavonoid, the phosphorylation of ERK1/2 also unaffected (result does not show).

For the activation of assessing p38MAPK and JNK1/2 the plain inductive apoptosis role of flavonoid; Earlier p38MAPK inhibitor SB203580 and JNK1/2 inhibitor SP600125 were handled the LNCap cell respectively 1 hour, again with the plain LNCap cell (Figure 13 (A)) of handling of flavonoid.When SB203580 and SP600125 exist respectively, will be blocked by plain inductive p-38MAPK of flavonoid and JNK1/2 phosphorylation (being activation).See also Figure 13 (B), for handling the LNCap cell respectively with SB203580 and SP600125 earlier, again with plain 12 hours the survivaling cell ratio chart of cell of handling of flavonoid.In Figure 13 (B); Through SB203580 and SP600125 handles respectively, again with the survival rate of the plain LNCap cell of handling of flavonoid all than only the survival rate through the plain LNCap cell of handling of flavonoid is high, deactivating of expression p38MAPK and JNK1/2 can make by the plain LNCap cell proliferation that suppresses of flavonoid.

Further whether the activation of research p38MAPK and JNK1/2 relates to by plain inductive apoptosis of flavonoid and cell cycle arrest.See also Figure 14 (A) and Figure 14 (B), be p38MAPK inhibitor SB203580 and JNK1/2 inhibitor SP600125 effect for plain inductive apoptosis of flavonoid and cell cycle arrest.In Figure 14 (A), SB203580 and SP600125 can reduce because of the plain inductive apoptotic cell ratio of flavonoid.In addition, SB203580 and SP600125 can suppress cut caspase-3 protein expression.In Figure 14 (B), SB203580 reduces because of plain inductive S of flavonoid and G2/M cell cycle arrest significantly, but SP600125 does not have such ability.When SB203580 exists, can make p-Cdc25C (Ser ²¹⁶) express and reduce, Cdk2 expresses increase; And SP600125 is not to influence (result does not show) with the plain inductive cell cycle arrest of flavonoid significantly.

For further research p38MAPK and JNK1/2 strike the expression (Figure 15 (A)) that subtracts p38MAPK and JNK1/2 with p38MAPK and the narrow spectrum siRNA of JNK1/2 (siRNA) respectively the plain inductive apoptotic role of flavonoid.Moreover p38MAPK siRNA and JNK1/2siRNA all can suppress the plain inductive apoptosis (Figure 15 (B)) of flavonoid.See also in Figure 15 (C), p38MAPK siRNA optionally suppresses because of plain inductive S of flavonoid and G2/M cell cycle arrest, however the JNK1/2siRNA such ability of tool not.This mechanism is through regulation and control Cdk2 and not activatory p-Cdc25C (Ser ²¹⁶) express and reach (Figure 15 (D)).The above results is consistent with the result of the 14th figure.

For whether the test class flavone can suppress the growth of prostate gland cancer cell in vivo, with the right oxter of LNCap cell skin injected to nude mice, and plain with the lumbar injection flavonoid, the analysis classes flavone suppresses the ability of tumor proliferation.See also Figure 16 (A), be time and the tumor magnitude relationship figure of lumbar injection flavonoid element to the nude mice of xenotransplantation LNcap cell.In Figure 16 (A), in injection 5 weeks of back, the average tumor size of high dose and low dosage (is respectively 1153.0 ± 218.7mm ³And 1876.9 ± 428.6mm ³) significantly than the big or small (3409.8 ± 704.8mm of the average tumor of control group ³) little.And do not cause the damage of significant hematopoietic potential, liver function, renal function.There is not tangible body weight difference (table 3) between high dose, low dosage and control group yet.In addition, the protein expression of cut PARP, p-p38MAPK and the p-JNK1/2 of low dose group and high dose group is all than control group high (Figure 16 (B)).Unphosphorylated p38MAPK and JNK1/2 protein expression do not change (result does not show) because of the plain effect of flavonoid.

Can know by The above results, but, increase cut PARP and caspase-3 and express, final cell death inducing with plain prostate gland cancer cell LNCap activating P 38 MAPK and the JNK1/2 signal transduction path handled of flavonoid.The present invention has confirmed that also the activity of p38MAPK and JNK1/2 can be suppressed and suppressed by narrow spectrum siRNA by its inhibitor SB203580 and SP600125 respectively, reduces apoptosis and reduce cut caspase-3 active.Therefore, the present invention has confirmed that p38MAPK and JNK1/2 play an important role at the plain inductive apoptosis of flavonoid.

In the present invention, p38MAPK that is activated and JNK1/2 cause cell cycle arrest in G1/S or G2/M cycle, and it is to reach through increasing not activatory p-Cdc25C (Ser216) and minimizing p-cell periodic protein B 1 (Ser147) and Cdk2.

3. with plain other cancerous cell that suppress of flavonoid:

Except ovarian cancer and carcinoma of prostate, the present invention is an object of study with bladder cancer, hepatocarcinoma and cervical cancer also, and whether research flavonoid element also can suppress the growth of these cancerous cell.At first, the flavonoid element is to human transitional cell papilloma (human urinary bladder transitional cell papilloma) RT4 (ATCC number: HTB-2), T24 (ATCC number: HTB-4) and human liver cancer cell strain Hep3B (ATCC number: cytotoxicity (IC HB-8064) ₅₀) be respectively 5.105 ± 3.25 μ M, 5.52 ± 0.598 μ M and 1.86 ± 0.656 μ M.See also the 17th (A), 17 (B) and 17 (C) figure, handle cancerous cell (A) RT4, (B) T24 and (C) the growth inhibited situation behind the Hep3B different time for the variable concentrations flavonoid is plain.In Figure 17 (A); 4 μ M and 8 μ M flavonoid are plain to be handled RT4 cell 24 and all can reduce the survivaling cell ratio in 48 hours; Yet the plain survivaling cell ratio of low concentration (0.5,1 and 2 μ M) flavonoid surpasses 100%, infers the growth of the plain meeting of low concentration flavonoid irritation cell.In Figure 17 (B) and Figure 17 (C), the flavonoid element presents dose-dependent effect and time-dependent effect to the growth inhibited of T24 and Hep3B cell.

See also Figure 18, be the cell cycle distribution situation of flavonoid element human hela cell strain HeLa.In Figure 18, the flavonoid element can be stuck in the G2/M cycle with the HeLa cell, with the cell division of blocking-up HeLa cell, and makes the HeLa cell get into apoptosis.

In sum; The present invention is with plain ovarian cancer cell, prostate gland cancer cell, transitional cell bladder carcinoma cell line, HCC and the cervical cancer cell handled of flavonoid; Effective cell death inducing, and confirm its signal transduction path, and pass through animal experiment; Confirm that the flavonoid element does not have significant liver toxicity, nephrotoxicity, hematotoxicity to nude mice, mean that the flavonoid element can become to be applied to people or other mammiferous chemotherapeutic agents.In addition, flavonoid element of the present invention can with unite and process pharmaceutical composition along the Diaminodichloride, reach effective treatment of cancer effect.Flavonoid element of the present invention can be processed the pharmaceutical dosage form that can transmit medicine with pharmaceutically acceptable carrier.The estimable innovative design of the real genus of the present invention, dark tool industrial value is filed an application in accordance with the law.

The present invention can make various modifications by those skilled in the art, but does not break away from protection scope of the present invention.

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Claims

1. pharmaceutical composition of treating ovarian cancer; It comprises that the flavone compound as shown in the formula I reaches the suitable Diaminodichloride as shown in the formula II; The concentration of wherein said flavone compound is between 1 μ M to 2.5 μ M, and said suitable Diaminodichloride's concentration is between 5 μ M to 10 μ M.

formula I

formula II

2. pharmaceutical composition according to claim 1, wherein said ovarian cancer be MDAH-2774 and SKOV3 cell strain one of them.

3. pharmaceutical composition according to claim 1, wherein said flavone compound is got by the pteridophyta extraction.

4. pharmaceutical composition according to claim 3, wherein said pteridophyta are the coarse wool Parathelypteris glanduligera (Kunze) Ching.

5. pharmaceutical composition according to claim 1, wherein said flavone compound is suppressed at S cell cycle or G2/M cell cycle with cancerous cell.

6. pharmaceutical composition according to claim 5; Wherein said flavone compound is regulated and control the expression of at least one cyclin of said cancerous cell, and said at least one cyclin comprises phosphorylation cell periodic protein B 1, cell periodic protein B 1, phosphorylation CdK2, CdK2 and Cdc25C.

7. pharmaceutical composition according to claim 5, wherein said flavone compound are regulated and control caspase-3, PARP, Bcl-xL and the Bcl-2 albumen of said cancerous cell, induce said cancer cell-apoptosis.

8. pharmaceutical composition according to claim 5, wherein said flavone compound are regulated and control the p-38MAPK and the JNK1/2 albumen of said cancerous cell, induce this cancer cell-apoptosis.

9. pharmaceutical composition according to claim 1, wherein said pharmaceutical composition comprises pharmaceutically acceptable carrier.

10. pharmaceutical composition according to claim 1, wherein said ovarian cancer is from the people.