TW201021808A - Composition containing tanshinone I and uses thereof - Google Patents

Abstract

The present invention provides antineoplastic composition comprising following formula: tanshinone I and monocyte- conditioned medium. The present invention also provides pharmaceutical composition containing tanshinone I useful for inhibiting angiogenesis and treating transcription factors AP-1 and NF- κB-related disorders, or for treating PDGF-β, VEGF and IL-8 pathway-related disorders.

Description

201021808 VI. Description of the invention: [Technical field to which the invention belongs]

H The present invention relates to a composition for antitumor comprising tanshinone I and a mononuclear cell conditioned medium. The present invention also relates to a composition comprising tanshinone I for use in the treatment of an angiogenesis-related disease. The present invention also provides for the use of tanshinone oxime for the manufacture of a disease for treating or inhibiting tumors, inhibiting angiogenesis, inhibiting transcription factor AP4 and nf_kB, and treating PDGF-β, or a vascular novel factor VEGF or IL_8 phase. The use of pharmaceuticals. [Prior Art] In the treatment of cancer, it is generally surgically removed and combined with adjuvant treatment, such as anticancer drugs and radiation therapy, depending on the condition. However, only some cancer patients can be treated with a diagnosis at the time of diagnosis. Most patients have already had cancer metastasis at the time of discovery. The chance of metastasis or recurrence after surgery is very high, and the side effects of many anticancer drugs are obvious. For example, oral mucosal inflammation and ulceration, nausea, vomiting, hair loss and inhibition of bone marrow function lead to infection and fever, and even other fatal complications. Cancer cell metastasis is the leading cause of death in cancer patients. In the complex process of cancer metastasis, the ability of cancer cells to invade is closely related to migration ability and cancer metastasis. Angiogenesis is a very important step in the growth, development and metastasis of tumors. There are no new blood vessels to supply oxygen and nutrients. Tumors cannot be grown by proliferation and proliferation alone. Generally, they are not long. The basement membrane decomposes, and the endothelial cells move into the crack space and grow, further proliferate and move, form a new long cell outside the lumen and then regenerate the basement membrane, and finally form a microvascular structure. Some literature also refers to the 201021808 macrophage cells (marc〇phage) that infiltrate into tumor tissues, which are stimulated to become molecules that provide the molecules needed to promote angiogenesis, destroy the matrix and the movement of tumor cells, and thus infiltrate the macrophages of tumor tissues. There is a significant association between cells and angiogenesis in tumors (〇no da/., Cancer C/zemoi/zer Pharmacol, 1999, 43: 69-71) ° The process of angiogenesis is affected by peripheral angiogenesis factors such as interleukins. _ 8 (Interleukin-8, IL-8), Vascular endothelial growth factor (VEGF) and fibroblast growth factor (bFGF) (Friesel ei α/., «/ , 1995, 9:919-925) and other influences. Previous studies have shown that tumor cells and stromal cells may be sources of angiogenic factors (C〇nn〇lly ei α/·, 1987, 144: 705-712), while IL-8 <new nomenclature: CXCL8 &gt It is the major angiogenic factor for non-small cell lung cancer (Smith et al., / 5 xp Meof, 1994, 179: 1409-1415); co-culture of fibroblasts with non-small cell lung cancer cells confirms tumor cells Both stromal cells and stromal cells are induced to produce IL-8 (Anderson α VIII, ® “π, Λα, 2000, 60: 269·72); other studies have also shown that mononuclear cells (Monocytes) are shared with non-small cell lung cancer cells. When cultured, cancer cells stimulate inflammatory cells to express IL-8 (White w α/., W (10) /, 2001, 166: 7549-5 5). Previous studies by the inventors also pointed out that non-small cell lung cancer cells and When co-cultured with Mocrophage, both of them will express IL-8 in large amounts (Chen ei a/., C&quot; « 2003, 9:729 - 737) 〇IL-8疋 belongs to CXC chemotactic hormone (CXC chemokine) a member of the family, the first chemotactic hormone that has been cloned (Matsushima et 201021808 al·, 1988), about 8.5kDa in size 'plays a very important role in the inflammatory response' mainly responsible for activation and neutrophils, eosinophlis, eosinophils (basophlis), single Nucleospheres, mast cells, and T and B lymphocytes migrate to areas of inflammation to participate in inflammatory responses (Strieter, RM, Am J Physiol Lung Cell Mol Physiol, 2002, 283:688-689) » And IL-8, in particular, has a strong chemotaxis for neutrophils. ® Looking at recent research data has fully clarified a concept:

The inflammatory response is an important key to tumorigenesis (Balkwill and Mant〇vani, 2001, 357: 539-45). Many cancers occur in areas that are infected, have irritating irritations and inflammation. As early as 1863, Rudolf Virchow noticed that these white blood cells in tumor tissue 'he assumed that the origin of cancer is in a chronic inflammation position' based on his hypothesis, pointing out that when irritants appear, and irritants The resulting tissue damage and subsequent inflammatory reactions promote cell thinning. Although it is known that Chu only knows that only the proliferation of cells does not cause cancer, cells continue to increase in the environment in which the immature cells contain inflammatory cells, growth factors, activated stromal cells, and DNA damage factors. $ increases the risk of developing a tumor. Nowadays, in-depth understanding of the mechanism of action in the micro-environment of tumors, an anti-inflammatory treatment method, steroid anti-inflammatory drugs can prevent breast cancer and cancer. Salvia miltiorrhiza has long been used in inflammatory cells and inflammatory cytokines and thereafter, for the development of cancer, there are many data pointed out: a variety of non-colon colorectal cancer, lung cancer, esophageal cancer and gastric treatment of coronary heart disease, myocardial infarction, liver Spleen 201021808 The traditional Chinese herbal medicine, which is swollen and mild in medicinal properties, has the function of promoting blood circulation, so it is also taken seriously for the purpose of inhibiting and treating tumors.

At present, the active ingredients extracted from the roots of Salvia miltiorrhiza are mainly classified into two types: fat-soluble Erzhuang and water-soluble phenolic acids. The former, such as tanshinone I, tanshinone n A, Tanshinone HB η b (tanshinone HB), cryptotanshinone (crypt 〇 tanshin〇ne) and danshen _ (miltirone), the latter such as salvianolic acid (saivian〇nc aeid), protocatechualdehyde (protocatechualdehyde), lithosperic acid (iith) 〇spermic acid), and squirrels (salviol) and so on. Tanshinone I is the main active ingredient extracted from the root of Salvia miltiorrhiza by ether or methylation. Its chemical name is: dimethylphenanthrene (1,2_b) furandione [l,6-Dimethylphenanthro(l,2-b)furan -10, 11- dione], having the structure shown by the following chemical formula i, molecular weight: 276 286 g/inol.

(I) Recent studies have shown that tanshinone I has anti-inflammatory activity (Kang, ei α/., Immunopharmacology, 2000, 49: 355-361) 'can inhibit the conversion of arachidonic acid to prostaglandins and is cytotoxic to tumor cells ( Ryu, Hu /απία Me丄, 1997, 63: 339-342), protects the heart vascular cells from the damage caused by ischemia (丫 &amp; § 1 to 〇 /., corpse / grasp 仏 from 6, 1989, 55 :51-54) and the activity of regulating chemical carcinogens Trp-Ρ-Ι and benzo[a]pyrene (Sato ei a/·, Mwki /?, 1992, 265: 149-154) and the like. Salvia miltiorrhiza 201021808 _ 03⁄4 I have no activity in inhibiting angiogenesis and have not been disclosed in any of the prior art documents. ^ Based on cancer... Therapeutic needs for new, effective, and low-side treatments. The use of tanshinone for anti-tumor and angiogenesis inhibition is worthy of further study. SUMMARY OF THE INVENTION There is a need for novel, effective, and low side-effect therapeutic drugs in the field of cancer treatment. Therefore, after long-term research and continuous beta test, the applicant developed anti-tumor and inhibited ash tube regeneration. SUMMARY OF THE INVENTION The present invention provides an antitumor composition comprising tanshinone I having the following chemical formula (I):

Reference (I); and a mononuclear cell conditioned medium. Wherein, the mononuclear cell conditioned medium can be prepared by the following steps: mixing a mononuclear bulb with a serum-free cell culture to form a mixed culture containing mononuclear cells. And the culture of the mononuclear cells. In a preferred embodiment of the invention, the nucleus cell condition is cultured. The 201021808 group includes, but is not limited to, interleukin-8 (IL-8), interleukin-4 (interleukin- 4. IL-4), interleukin-12 (IL-12) and combinations thereof. Preferably, the mononuclear cell line is a giant scorpion cell. More preferably, the macrophage cell line is a THP-1 cell-derived macrophage cell. According to the present invention, the tumor referred to is selected from the group consisting of non-small cell lung cancer, breast cancer, kidney cancer, soft tissue sarcoma, neuroendocrine tumor of lung sputum, cervical cancer, uterine cancer, head and Cervical cancer, glioma, prostate cancer, pancreatic cancer, lymphoma, melanoma, small cell lung cancer, ovarian cancer, colon cancer, esophageal cancer, gastric cancer, leukemia, and colorectal cancer. In a preferred embodiment of the invention, the antitumor composition further comprises a carrier. Preferably, the carrier is an aqueous solution. It is also mentioned that the anti-tumor composition just described is used for the manufacture of "medicine for tumors." It is preferable that the aforementioned anti-tumor medicine::: two: the range falls..1 The dose of tanshinone to 10 ° mg / kg ^ ^ medical music w is enclosed in 0.5 to 50 mg / kg two:: Salvia, 1 administration. More preferably, the medical haiku The present invention also provides a dose of tanshinone 1 at a dose of two::: a composition containing a chemical formula (1): a composition for inhibiting angiogenesis, which is used for inhibiting angiogenesis. In a preferred embodiment, or an excipient. Preferably, the method comprises: using a carrier, a diluent aqueous solution or an organic solution.

S 201021808 The present invention also provides a use of a tanshinone I having the formula (I) for the manufacture of a medicament for treating or inhibiting an angiogenesis-related disease or disorder. Preferably, the aforementioned medicinal product for treating or inhibiting an angiogenesis-related disease or disorder is preferably a dose of between 范 波 A 至 至 至 至 至 至 至 至 至 至 至 至 _ ' ' ' ' ' ' ' ' ' ' ' The pharmaceutical product is administered in a dose ranging from w to between. More preferably, the medical treatment is based on the administration of a dose of tanshinone I at a dose ranging from 1 to 20 mg/kg. β I The neovascular related diseases or disorders according to the present invention are selected from the group consisting of non-small cell lung cancer, breast cancer, and kidney cancer (10) Tumm〇r), neuroendocrine tumor of lung tissue of soft tissue sarcoma, cervical cancer, uterine cancer, head and neck cancer, glioma (neur〇) Gii〇nia), month | J prostate cancer, pancreatic cancer 'lymphoma, meanc nia), small cell lung cancer (small ® cel1 bronch〇genic cancer) , ovariai cancer, colonic carcinoma, esophageal carcinoma, stomach carcinoma, leucemia, colorectal carcinoma, neoplastic disease, re Restenosis, rheumatoid arthritis, Crohn's disease, diabetic retinopathy, Psoriasis, endometriosis, macular degeneration, neovascular glaucoma, and obesity 201021808 (adiposity) ° The present invention also provides a method for inhibiting menstruation A five-tube nascent factor-induced cell migration composition comprising salvia miltiorrhiza _ j of formula (1). An angiogenic factor according to the invention means any growth factor which can induce angiogenesis, including but not limited to: vascular endothelial growth factor (VEGF), platelet-derived growth factor-β (platelet- Derived growth factor-β 'PDGF-β), basic fibroblast gr〇wth fact (m, ❿ bFGF), IL-8, tumor necrosis factor (tumor necr〇sis fact〇ra, TNF a) And a growth growth factor-β (TGF-β). In a preferred embodiment of the invention, the composition for inhibiting angiogenesis-induced cell migration further comprises a Carrier, diluent or excipient

The present invention also provides a pharmaceutical composition for treating a disease associated with a transcription factor Αρ·ι and nf_kB, which comprises tanshinone I having the chemical formula (1). 「 The "transcription factor Ap-1 and NF-κΒ related diseases" include, but are not limited to, inflammatory and tissue repair diseases, including asthma (asthma) and chronic obstructive pneumonia (chr〇nic 〇bstructive pulm〇) Nary disease, COPD) inflammatory bowel disease, rheumatoid arthritis; osteoarthritis and fibroidosis; dermatitis, including allergic dermatitis ), dry skin (ps〇riasis) and skin damage caused by ultraviolet rays; autoimmune diseases, including systemic lupus erythematosus; multiple sclerosis (muhiple scler〇sis); cognac 10 201021808 arthritis (psoriatic arthritis) ); ankylosing spondylitis; ather 〇 sclerosis; tissue and device S rejection; Alzheimer's disease; stroke; glomerulonephritis; , including Hodgkins disease; and specific viral infections, including dengue virus infections.

In a preferred embodiment of the invention, the pharmaceutical composition for treating a disease associated with transcription factor AP-1 and NF-icB further comprises a carrier, diluent or excipient. The present invention also provides a use of tanshinone I for the manufacture of a medicament for treating a disease associated with the transcription factor AP-1 and NF-κΒ. In a preferred embodiment of the present invention, the pharmaceutical preparation for treating a disease associated with the transcription factor AP-1 and NF-κΒ is in a dose ranging from 〇.丨 to 1〇〇mg/kg. Tanshinone is administered by a worker. Preferably, the pharmaceutical product is more preferably a dose of tanshinone which falls between 〇5 and 5 〇mg/kg, and the medical product is in a range of up to mg. A dose of /kg between tanshinone I is administered. The present invention also provides a pharmaceutical composition for treating a disease of PDGF_p, a new factor vegf or a river-directed signal transmission path, which comprises tanshinone I having the chemical formula (1). According to the present invention, the "PDGF_p, vascular new factor vegf or μ target signal transmission path disease" includes, but is not limited to: porridge-like dying =!; inflammatory and traumatic repair diseases, including asthma and chronic Resistance: joint: (C0PD), bronchiolitis, viral liver fibrosis, rheumatism "human Helicobacter pylori infection induced stomach (four) inflammation; osteoarthritis and fibrosis 201021808 • Wig-like degenerative disease; dermatitis 'including allergic dermatitis , skin damage caused by psoriasis and ultraviolet rays; autoimmune diseases, including systemic lupus erythematosus; ^ chronic myeloproliferative diseases; multiple sclerosis; dry arthritis; ankylosing spondylitis; diabetes; renal glomerulonephritis; Cancer, neoplastic disease, restenosis, rheumatoid arthritis, Crohn's disease, diabetic retinopathy, psoriasis Endometriosis, endetriosis (macular ❹ degenerati〇n), neovascular glaucoma (neovascular gla) Uc〇ma) and adiposity. In a preferred embodiment of the invention, the pharmaceutical composition for treating a disease associated with PDGF-β, angiogenic factor VEGF or IL-8-related signaling pathways, Further comprising a carrier, diluent or excipient. The invention also provides the use of tanshinone I for the manufacture of a medicament for the treatment of a PDGF-β, a vascular novel factor VEGF or IL-8 related signal transmission pathway. In a preferred embodiment of the present invention, the aforementioned medical system for treating diseases of PDGF-β, blood b new factor VEGF or IL-8 related signal transmission pathway falls within a range of 〇_丨 to 丨〇〇mg/ The dose of tanshinone is administered between kg, preferably. The pharmaceutical product is administered in a dose of tanshinone ranging from 0.5 to 5 mg/kg. More preferably, the pharmaceutical product is administered. The administration of tanshinone I at a dose ranging from 1 to 20 mg/kg. DETAILED DESCRIPTION OF THE INVENTION According to the present invention, heparin protein is aggregated with type IV collagen (c〇llagen type IV) and Xenopus Sugar (heparan sulfate proteoglycan), inner 12

«I 201021808 . Extracellular matrix gel of eniactin and laminin

(Matrigel)-based in vitro invasiveness assay confirmed that tanshinone I and tanshinone Π A can significantly inhibit CL15 cells in the presence of THP-1 cell-conditioned media at low concentrations (human) Invasive ability of lung cancer cell line] Among them, tanshinone I can reduce the high degree of invasion of cl1_5 lung cancer cells. From the experimental results of in vitro cell migration assay, it was confirmed that under the stimulation of THP-1 cell conditioned medium It can promote the migration ability of CLl_5 lung cancer cells®, and the effective components of Salvia miltiorrhiza Bge.: tanshinone j, tanshinone π Α and cryptotanshinone can effectively inhibit the migration ability of CL 1 -5 lung cancer cell lines in the presence of THpq cell conditioned medium at low concentration. Among them, the inhibitory effect of tanshinone I is preferably up to 64%. Through gelatin zym〇graphy analysis, it is understood that tanshinone I, tanshinone oxime and cryptotanshinone have inhibitory gelatinase induced by ΤΗρ"} cell conditioned medium ( Gelatinase) activity. The results of the present study confirmed that tanshinone 1 can effectively inhibit tumor growth Increasing the angiogenesis of the blood vessels and preventing the metastasis of cancer cells. The results of previous studies by the present inventors also showed that the density of macrophage in tumor tissues was significantly positively correlated with the gene expression of IL_8 and the number of microvessels, and patients with paralysis There is a significant negative correlation between the survival rate and the co-culture of CL1-5 lung cancer cells with macrophages, which will increase the expression of il_8 gene several times. Some anti-inflammatory drugs such as aspirin (aSpirm), PDTC, dexamethasone, and celecoxib have the ability to inhibit the expression of lung cancer cells compared to the _8 gene after co-culture (Chen G/·, c 心如如 and 2〇〇3, 9 729_737). 13 201021808 It was found in the experimental results of the present invention that tanshinone I and salvia miltiorrhiza, in the presence of ΤΗΡ-1 cell conditioned medium, can inhibit the expression of IL-8 mRNA associated with cancer metastasis, and with the concentration of Salvia miltiorrhiza var. Increased and decreased. Among them, tanshinone has the strongest inhibitory ability, and can reduce the expression of IL_8 gene by 80% at a concentration of 10 Mg/ml. The regulation of il_8 gene expression is including transcription As well as the post-transcriptional hierarchy, the molecular regulation of IL-8 gene transcriptional activation is a complex process. Previous studies have confirmed that this sequence of IL_8 gene 5, the lateral region © from -133 to -i In the region where IL_8 gene expression is important and has a strong regulatory ability (Mukaida speaks y, Μ卯265:21 128-21 133; Harant et al., J Biol Chem^ l9g6^ 271:26954-26961 ) ' contains the binding sequences of transcription factors: Api, nfil6 and NF-kB. Previous studies by the present inventors have shown that the promotion of IL-8 gene expression after co-culture of lung cancer cells with macrophages is regulated by the nf_kB pathway. In the experiments of the present invention, from the results of the next luciferase reporter gene assay, we found that the IL_8 gene expression of lung adenocarcinoma cells induced by macrophages requires at least one transcription factor of AIM, NF_IL6 and nf_kB. Mutual synergy, while NF-κΒ is the most important transcription factor affecting IL-8 gene expression; NF_IL6 is a synergistic activator. From the results of this study, it was also confirmed that the regulation of the cancer metastasis-associated gene IL-8 by Salvia miltiorrhiza var. sinensis is in the transcriptional level, and the IL_8 gene expression is reduced by affecting the pathway of NF-kB and ΑΡ-1. The regulation of the transcriptional activity of the promoter usually increases the ability of the transcription factor to bind to the DNA. In the past studies, it was pointed out that 'tanshinone I inhibits the expression of IL-12 by macrophages via the ability to inhibit NF-kB 14 201021808 DNA binding, Furthermore, the inflammatory response is inhibited (Kang BY et al., Immunopharmacology, 2000, 49, 355-361). In addition, tanshinone I was also found to inhibit the DNA binding ability of AP-1, which is increased by the treatment of cytotoxic acid (TPA) in the fibroblast cell line (NIH 3T3) of mice, thereby inhibiting cell growth (Park S a/., 5w). //· ATorea/t CTzem. Soc., 1999, 20: 925-928). In the experiments of the present invention, electrophoretic shift analysis (EMS A) was used to analyze the activity of tanshinone I and tanshinone oxime on the promoter of NF-κΒ and AP-1 binding IL-8 gene, and the results of the present invention were first. It was confirmed that tanshinone I directly inhibited the binding ability of NF-κΒ and AP-1 to the IL-8 gene promoter, which in turn affected the expression of IL-8 gene. Signal-transfer studies on platelet-derived growth factor-β (PDGF-β) have shown that platelet-derived growth factor binds to receptors on endothelial cell membranes, and activated PDGF receptors then activate She and Shb, two SH2 domain proteins, activated She will initiate Ras-Rafl-MAPK pathway to promote cell proliferation, and activated Shb will promote cell migration via PI3K initiation of Rac/cdc42 pathway recombinant cytoskeleton (Hooshmand-Rad ei a/·, Ce// Λα., 2000, 257: 245-254), while the activated PDGF receptor also activates the protein-glucuronite esterase-1 containing the Src homology 2 (SH2) structural domain [ Src homology 2 (SH2) domain-containing protein tyrosine phosphatase substrate 1; SHPS -1 ; also known as protein tyrosine phosphatase, non-receptor type-1 (non-receptor) Type substrate-Ι)], activated SHPS-1 and then activated SHP-2 initiates FAK-paxillin and Rho pathway recombinant cytoskeleton promotion 15 201021808 Cell attachment and movement (Inagaki α/·, 乂, 2000, l9:6721- 6731; Oshima ei α/_, Factory 5 still 2002, 519··1-7), induced vascular neovascularization. VEGF is a giyCOprotein, usually in the form of a homodimer, sometimes in the form of a heterodimer, and different forms of dimers can induce different signals. The signal transduction cascade thus has a different physiological meaning (Cross ei α/·, 7 &gt; 6) heart «SW·, 2003, 28: 488-494).

Abnormal expression of VEGF can cause many human diseases, such as cardiac vascular disease, pulmonary edema, inflammatory response, tumor growth and metastasis, and other abnormal physiological phenomena are related to VEGF expression (Tammela is α/., 乂Car Mountain· 〇.fine·, 2004, 65: 550-563.). The tanshinone I of the present invention has the ability to inhibit cell thinning, cell survival, cell migration, and angiogenesis induced by the pDGF_p, vEGF, and IL-8 pathways. Further, in the animal model of the present invention, administration of the pharmaceutical composition of the present invention inhibits tumor growth, inhibits tumor angiogenesis, inhibits formation of lung tumors, and prevents tumor cell metastasis. EXAMPLES The present invention will be exemplified by the following examples, which are not intended to limit the description of the invention disclosed above. These embodiments may be modified and modified to make the experimental materials of the present invention Method ~ does not depart from the invention 4 of the present invention. A. Cell culture: CL1_5 highly invasive human lung cancer cell line (Lung 16 201021808 adenocarcinoma cell line) (Chu et al., Am J Respir Cell Mol Price/., 1997, 17: 353-60 And the monocyte cell line THP-1 (ATCC TIB202; ATCC, Manassas, VA), cultured in 10% fetal bovine serum (FBS), 100 mg/ml streptomycin, 100 U/ Ml penicillin (peniciiiin) and 0.450/. Glucose RPMI 1640 medium. All cells were cultured at 37 ° C, 20% oxygen and 5% carbon dioxide. B. THP-1 cell conditioned medium: β monocyte strain ΤΗΡ-1 was stimulated by adding phorbol myristate acetate (PMA) (Sigma) after 24 hours by adding 3·2 X 1〇-7Μ, phorbol myristate acetate (PMA) Differentiated into macrophages, cultured for another 24 hours, and then removed for another 24 hours to remove the effects of PMA, then washed 3 times with PBS, added to RPMI 1640 without jk clear for 24 hours, and the supernatant was collected as THP. -1 cell conditioned medium. C. Animal rearing: Female immunodeficiency mice (semie combined immunodeficient. _ mice, SCID mice) were purchased from the National Taiwan University Animal Center, and each was housed in a sterile stainless steel cage. The animal room was controlled at room temperature, and both feed and drinking water were supplied. Adequate 'to be immunized to the whole rat five weeks after the start of animal testing. D. Reagents: The sources of the drugs used in the following examples are as follows: purified tanshinone j, tanshinone n A, and cryptotanshinone are purchased from Jiuding Biotech; and dexamethasone were purchased from Sigma. The above drugs were dissolved in dimethyl sulfoxide (DMSO) at a concentration of 17 201021808 1 mg / mi, and stored in _2 (rc storage standby. Danshenone, tanshinone oxime and crypto-danshen _ single-component is the use of standards to LC - MS method for quantification. Example 1 Evaluation of the active ingredient of Salvia miltiorrhiza Bunge against CL1_5 human lung adenocarcinoma cells in order to evaluate the toxicity of Salvia miltiorrhiza active ingredient to CL 1 -5 human lung adenocarcinoma cell line 'In this example The CL1_5 cells were treated with 〇, 丨, 1〇, 2〇, 40, 80 &amp; 16〇_ml of Danshen 嗣1, Danshen _ΠΑ and Hidden Danshen Steel for 24 hours, using trypan blue dye Inverted microscope was used to evaluate the viability of CL 1 -5 cells. After comparing the values obtained by cell counting with the control group, the results in the panel of Figure A showed that the control group was 丹pg/ml of tanshinone I, Danshen Gang HA and Hidden Danshen _ treatment 'and the percentage of dead cells, tanshinone I, tanshinone Π A and cryptotanshinone can affect the survival rate of cL1_5 cells at low concentration. The LDw dosages of tanshinone, tanshinone nA and cryptodansin are: 80 pg/m Bu 60 pg/ml and 30 Pg/ml, LI^. The doses were: 1〇pg/ml, 10 pg/ml and 5 pg/ml» after the 'LD10 dose to detect the effect of Salvia miltiorrhiza active ingredient on the proliferation rate of CL1-5 lung cancer cell lines. Prepare 3 96-well culture plates, each plate 3 has attached lxl〇4 CL1-5 lung cancer cells and add anti-inflammatory drugs respectively. Dexamethasone (5 pg/ml) and Salvia miltiorrhiza Active ingredient: Danshenone (1 〇Mg/ml), tanshinone oxime A (10 pg/ml) and cryptotanshinone (5 μ§/ηι1) or RPMI 1640 'treated for 24, 48 and 72 hours, using tetrazolium blue colorimetric assay (ΜΤΤ analysis The proliferation rate of CL 1 -5 lung cancer cells was analyzed. The results showed that the first map showed that ' CL1-5 lung cancer cell line untreated bite added 5 pg/ml dexamethasone (Dexa), 10 pg/ml tanshinone j (Τ1) ), 18 201021808 10 pg/ml tanshinone oxime A (T2A) and 5 pg/ml cryptotanshinone (Cryp) were treated for 24, 48 and 72 hours respectively, and the proliferation ability of CL1-5 lung cancer cells was determined by MTT assay (Lee wa /., Mol_ Cancer Therap., 2008, 7:3527-3538.) Tanshinone I inhibited the growth of 27% CL 1-5 lung cancer cells at 72 hours. Example 2 utilized the fine running ability (j./j viiro cell migration assay) To understand the effect of the active constituents of Salvia miltiorrhiza on the cell migration ability of CL1-5 human lung adenocarcinoma cells © Culture CL1-5 lung cancer cells on 24-well plates until the monolayer cells are evenly distributed Full plate. Carefully scrape a three-mm cell-free trajectory in the center of the plate with a blue blue tip, immediately rinse (except) any unattached cells with culture medium, then re-add RPMi 1 64 〇 or contain different drugs THP-1 cell conditioned medium (10 pg/ml tanshinone;!, 1〇pg/ml tanshinone π A and 5 pg/ml cryptotanshinone and 5 pg/ml dexamethasone) dM indicates) continue to culture the cells. At different time points (〇, 24 hours), observe cell movement and growth and take a photo record. After 24 hours, the cells were fixed and stained with crystal violet, and the number of cells that migrated into the trajectory was counted. Results As shown in the second panel, culture of CL1-5 lung cancer cell lines using THpq cell conditioned medium (CM) alone doubled the migration capacity of cli_5 lung cancer cells, 5 짧1, the anti-inflammatory drug dexamethasone (Dexa), And salvia miltiorrhiza active ingredients: 1〇pg/ml Danshen_gong (Τ1), 1〇μ§/πι1 tanshinone (Τ2Α) and 5叩/, such as cryptotanshinone (Cryp) can be inhibited in the presence of sputum cell conditioned medium (CM) The migration ability of CL1_5 lung cancer cell line, among which tanshinone 1 has the best inhibitory effect (64%). 19 201021808 Example 3 Using In vitro cell invasion assay To understand the effect of the active constituents of Salvia miltiorrhiza on the cell migration ability of CL1-5 human lung adenocarcinoma cells. First, extracellular matrix gel (Matrigel, 2.5 mg) /ml; BD Biosciences Discovery Labware, Bedford, MA) was incubated on a 8 μm film (Corning Costar Corporation, Cambridge, MA) in a Transwell® culture dish for 60 minutes at 37 °C. The cells of CL 1 -5 lung cancer cells were beaten with TG-EDTA (trysin-EDTA), and the supernatant was removed by centrifugation, and the drug was not contained or contained (5 pg/ml dexamethasone, 1 and 10 Mg). /ml Tanshinone I, 1 and 10 pg/ml tanshinone π A and 0.5 and 5 pg/ml cryptotanshinone) RPMI 1640 re-dispersed cells '1xl 06 cells were seeded in Transwell® covered with a matrix gel On the culture dish, 6 μM of RPMI 1640 containing 10% FBS was added to the wells under the Transwell® culture dish; after overnight incubation, the medium was removed, and the covered matrix gel was cleaned with a cotton swab and added with methanol. After the cells, the cells were stained by Liu s stain (Hand sel ® Technol〇gies, Inc., Taipei, Taiwan) and observed under a phase-contrast microscope to calculate the number of cells passing through the matrix-containing film. And compare the ability and percentage of cells passing through the membrane under the treatment or treatment of the active ingredient of Salvia miltiorrhiza. All experiments were repeated three more times independently. Results As shown in the third panel, culture of CL 1 -5 lung cancer cell lines in ΤΗΡ-1 cell conditioned medium (CM) increased the invasive ability of CL 1 -5 lung cancer cells by 40%. In addition, the active components of Salvia miltiorrhiza Bunge I (1〇1 Kg/ml), tanshinone jj a (1〇Mg/mi, 1 pg/mi) and cryptotanshinone (5 pg/ml, 0.5 pg/mi) and antibiotics were added. The inflammatory drug dexamethasone (5 μ§/ιη1), of which 20 .201021808 tanshinone I combined with ΤΗΡ-1 cell conditioned medium can exert effects on CL 1 ·5 lung cancer cells and reduce their invasive ability. Example 4 Analysis of the effect of active constituents of Salvia miltiorrhiza on the secretion of MMP protein in CL1-5 lung cancer cell lines by gelatin zymography In the experiment of the present invention, CL1-5 lung cancer cell lines were RPMI 1640 and THP- 1 cell conditioned medium containing 5 pg/ml dexamethasone, 10 pg/ml tanshinone I, 10 pg/ml tanshinone HA, 5 pg/ml cryptotanshinone or ❹ 3 pg/ml tanshinone I, 3 pg/ml tanshinone oxime A and 1 · 7 pg / ml cryptotanshinone mixture (Mix) in ΤΗΡ - 1 cell conditioned medium was cultured for 24 hours, after which the collection medium was analyzed by gelatin zymography and analyzed by gelatin protein electrophoresis. The metal-binding protease has gelatinase activity. The detailed steps are as follows: The supernatant (culture medium) collected for 24 hours is cultured, and 20 μΐ and 10 μΐ 2χ sample buffer solution (2x sample buffer: 125mM Tris-HCl © ρΗ6·8, 4% SDS, 20%) Glycolol) Mix well and prepare 10% SDS-PAGE with 1 mg/ml gelatin: 10% gel [1% gelatin, 10% Acrylamide/bis, 375 mM Tris-HCl pH 8_8, 0.1% SDS, 0.5 〇/ Ammonia persulfate, 0.05% TEMED] and 3% stack (3% Acrylamide/bis, 125 mM Tris-HCl pH 6.8, 0.1% SDS, 0.5% ammonium persulfate, 0.05% TEMED), colloid After solidification, it was placed in an electrophoresis tank, and the mixed sample was injected into a colloid, and electrophoresis was carried out at 100 V for 2 hours. Carefully remove the colloid. Wash the buffer solution (wash buffer: 5 OmM Tris-HCl ρΗ7·5, 2.5% Triton X-100) at room temperature for 15 minutes 21 201021808 2 times and replace with activated buffer solution (&amp;&lt;^丫&amp;^〇11131^化1*:5 0〇1^1丁1^-HC1 pH7.5, 150mM NaCM, 100mM CaCl2, 〇.〇5 % NaAzide) Oscillation at 37°C 18- 2 hours. Carefully remove the colloid and stain it with 5% 5% of Coomassie-blue for 30 minutes' and then de-stain for 1 hour with destain solution [100/glacial acetic acid, 30% methanol] And take a photo record. The fourth area B is a colloidal photograph' and the fourth area a is based on the result of the b area. The image is analyzed by ImageJ software (NIH), and the obtained data is plotted as 'as shown' 'tanshinone I, tanshinone oxime The combination of cryptotanshinone and THp_ i cell conditioned medium can inhibit the activity of matrix metalloproteinases (MMPs). Example 5 Effectiveness of Salvia miltiorrhiza Active ingredient for inhibiting IL_8 mRNA of CL1-5 lung cancer cells CL1-5 lung cancer cell line was cultured in ΤΗΡ-1 cell conditioned medium or simultaneously with different concentrations of Salvia miltiorrhiza active ingredient: Tanshinone 1 (〇1 , i and 10 pg/ml), tanshinone oxime A (0.1, 1 and 1 〇 yg/mi) and cryptotanshinone® (〇.〇5, 〇·5 and 5 Mg/ml). After 24 hours of culture, the total amount of RNA extracted from the cells was converted into cDNA by reverse transcription reaction to immediately quantify the polymerase chain reaction, using IL-8 specific primers (SEQ ID N: 5 and SEQ ID NO: 6). And the TATA b〇x binding pr〇tein (TBP) specific primers (SEQ ID N: 7 and SEQ ID NO: 8) were used to analyze the expression levels of IL-8 mRNA under various treatments. The relative relationship between the gene expression of the experimental group and the control gene (TBP gene) in the experimental group is as follows: -ACT=-[CTInterst- CTTBP] where CT is defined as exceeding a certain - fixed threshold, which can be detected Firefly 22 201021808 The number of PCR cycles of light. The ratio of gene expression between the two is defined as follows: 2_Λ K (K: constant). As shown in the fifth figure, the combination of tanshinone I and tanshinone oxime and ΤΗρ_丨 cell conditioned medium inhibited the expression of the cancer metastasis-associated gene IL_8 A induced by the conditioned medium of Τηρ_1 cells alone. The effect is more pronounced' and decreases with the increase in the concentration of Danshen and I. Among them, tanshinone oxime has the strongest inhibitory ability, and the il-8 gene expression can be reduced at a concentration of 10 pg/ml. β Example 6 uses transient transfection and luciferase reporter gene assay to determine the effect of active constituents of Salvia miltiorrhiza on the activity of cancer metastasis-associated gene IL-8 promoter in order to detect the active constituents of Salvia miltiorrhiza Effect on the IL_8 promoter of cancer metastasis-related genes> The IL_8 gene-specific primer was synthesized using the online bioinformatics database, covering approximately 178 base pairs upstream of the 5' end, including transcription factors: AP-1, NF-IL6, NF-κΒ binding sequence, this genomic fragment will be cloned into the luciferase-containing reporter gene vector (pGL-basic luciferase ❹ reP〇rter-3 vector, Promega, Madison, WI), and confirmed by sequencing . In addition, the transcription factor binding sequence is mutated by a synthetic special primer, and the mutated IL-8 promoter sequence is cloned into a vector to detect which transcription factor binding sequence is subjected to macrophage conditions. Affected by the marcophage-conditioned medium, the combination of different transcription factors in the sequence mutation and the normal sequence is shown in the seventh panel A region, and the colored squares represent mutations in the sequence, and IL-8 wt, πιΚίΡ - κιβ, mAP_l and mNFIL-6 are not (Yao ei a/·, Ce// 2005, 32: 540-547). 23 201021808 The vector containing the IL-8 gene transcriptional regulatory region was cotransfected with the pSV-β-galactosidase vector (Promega) using LipofectAMINETM to the CL1-5 cell line. After 24 hours, mediums with different drug concentrations were then added: tanshinone I (T1-10 and 1 pg/ml), tanshinone oxime A (T2A-10 and 1 pg/ml) and cryptotanshinone (Cryp-5 and 0.5 pg/) Continue to culture the cells with or without dosing. After 24 hours of culture, the luciferase activity (Dual-light Luciferase Reporter Gene Assay System, Tropix) was analyzed to confirm the o transcriptional activity of the IL-8 promoter. The above experiments included both untransfected and transfection control vector (pGL-3 Control vector) as a negative control group and detection of β-galactosidase activity to normalize transfection efficiency between experiments. (transfection efficiency), and do 3 repetitions. The method of cotransfection, luciferase and β-galactosidase reporter gene assay and luminescence detector detection are as follows: A. Co-transfection (cotransfection): © Co-transfection was performed using LipofectAMINETM plus reagent (Invitrogen, Gaithersburg, MD). 1 x 105 cells were cultured in 6-well plates for 16 to 24 hours, and when the cells were grown to 7 to 8 minutes, they were taken out for cell transfection. Prepare two mixtures in a 1.5πι1 microcentrifuge tube (2 samples): buffer a = 600 μΐ serum-free medium + 12 μL Lipofectamine; buffer Β = 600 μΐ serum-free medium + 8 ΐ ΐ plus reagent + 2 pg β - gal and 6 pg of IL-8p-Luc or pGL3-LUC plastids, each mixed uniformly and allowed to react at room temperature for 15 minutes. Buffer B - one drop was gently added to buffer A, mixed at 24 201021808 and then placed in a warm reaction for 20 minutes. The cultured cells were taken out, the supernatant was aspirated, and gently washed once with a little serum-free medium. Add 2·4 ml of serum-free medium, add 600 μM mixture (Α+Β) one by drop, and gently shake the plate to make the agent even. The plate was placed in a 37 C, 5% C 2 incubator and allowed to stand for 4 to 6 hours. The culture plate was taken out, and the medium of the sigma solution was aspirated and replaced with 3 ml of serum-containing RPMI 丨 64 培养基 medium, and further cultured at 37 ° C for 16 to 24 hours to restore the cells and allow the plastid to replicate. Thereafter, coculture and medicinal treatment were carried out for 24 hours. B. Luciferase and β-galactosidase reporter gene assay: This experiment uses the Dual-light Luciferase Reporter Gene Assay System (Tropix, Bedford, MA), which contains the following reagents:

Lysis solution (100 mM potassium phosphate pH 7.8, 0.2%

Triton X-100) ‘ Stored in 4〇c; Buffer A, powdered, first dissolved in 5ml ddl^O, stored in 4t:, i weeks, or packed in 2〇&lt;t,

1 year; Galacton-Plus substrate: for ΐοοχ, saved at 4. 〇; Before use, 'diluted with Buffer B; Buffer b, powder, first dissolved in 22 ml ddH2 〇, stored at 4 ° C, 1 week, or sub-packaged, stored in - 20C '1 year; Light Emission Accelerator-Π 4°C. The procedure for the luciferase and β-galactosidase reporter assays is to prepare a lysis solution, remove the required amount, and add DTT (to make the final concentration about 〇·5 mM, but not more than 〇 5 mM). Transfected cells were removed, the medium was aspirated and gently washed twice with ice-cold 1X PBS. Add cells containing 〇·5 mM DTT 25.201021808 Dissolve the solution to cover the cells (usage: 250 Ml/6 〇mm culture dish), scrape the cells with a cell scraper, and aspirate them into a microcentrifuge tube. At the low temperature of '12, 〇〇〇rPm, centrifuge for 2 minutes. The supernatant was aspirated and immediately tested at -80 °C. C · Cold light detector detection: Before the measurement, the cold light meter should be turned on first and warmed up for 3 minutes. A. Take the amount of f and put it back at room temperature. Dilute the GalaCt〇n_Plus substrate (1〇〇X) to IX with BUffer B. 10 μl of cell lysate (lysate) was added to 25 Ml of BufferA. After mixing the homologous sentences, (10) ^ buffer containing β of Galacton_Plus substrate was added within minutes, and luciferase chemical luminescence was measured immediately. After 3 to 60 minutes, 1 〇〇 μΐ Light Emission Accelerator-Π was added and the β-galactosidase chemical luminescence was measured immediately. For the analysis of the transcriptional activity of the active ingredient of Salvia miltiorrhizae for the IL_8 promoter, the fruit is shown in Section 7B. THIM cell conditioned medium (cm) increased luciferase activity by approximately 1〇, while adding Danshen ❹ Example 7 elMeasured by electrophoretic mobility shift assay (EMSA) The effect of the active ingredient of Salvia miltiorrhiza on the regulation of Jiang 8 gene is in this example, combined with the conditioned medium of ΤΗΡ·1 cells alone or with the following active ingredients of Salvia miltiorrhiza: 5 Mg/ml Salvia miltiorrhiza (τι), ι〇叫Μ Tanshinone oxime A (T2A) or 5 μ§/Γη1 丹 丹 dry (dryp), (the combination is shown in the area of Figure A of Figure 8) co-cultured for 24 hours of nuclear extract extracted from CLi_5 cancer cells (nuclear protein) (5盹), with 2 called labeled lMg/ml) and tanshinone u A (1〇/〇11) significantly inhibited the transcriptional activity of the Jiang 4 promoter. 26 201021808 NF-κed probe (Dig-labeled NF-κΒ probe) was mixed and homogenized for 40 minutes, and the following electrophoretic mobility shift assay was performed. ° Based on the 5' upstream transcription factor AP of IL-8 gene The -1 and NF-κΒ binding sequences were used to design and synthesize probes of AP-1 and NF-κΒ. The experimental procedures were as follows: (1) Synthesis of oligonucleotides (〇11§〇1111(:16〇1) ;丨(163): The synthetic nucleotide sequence used is shown in Table 1 below, in which the partial sequence in the sequence of the sequence is the transcription factors AP-1 and NF- upstream of the 5' end of IL-8. Β binding sequence. Table 1 Synthesis of oligonucleotide sequence AP-1 5,-AAAAGTGTGAT^ICTCA|GGTTTGTGA Two ACT| F: ^a]GGTTTG-3, (SEQ ID NO: 1)

AP-1 5,-AAACAAACCTGAGTCATCACAAACCTGAGTCA r: TCACACT-3' (SEQ ID NO: 2) NFkB 5'-ΑΑΑΑΑΊ^[ΓΟΟΑΑΤΤΤ&lt;:(:|(:ΟΧΟΑΑΤ€〇[Γ〇αΑ| F: lATTTCC|CCCGA-3 ' (SEQ ID NO: 3)

NFkB 5,-AAATCGGGGGAAATTC.CACGATTCGGGGGAAA R; TTCCACGATT-35 (SEQ ID NO: 4) (2) Oligonucleotide adhesion (annealing): Take AP-1 and NF-κΒ probe forward (F) and reverse Mix uniformly to (R) 2.5 nmole. The oligonucleotide was opened at 95 ° C for 10 minutes to open the secondary structure of the oligonucleotide. After the adhesive reaction was further carried out at 65 ° C for 2 hours, it was allowed to slowly cool down, at least for 20 minutes, and after being cooled to room temperature, it was stored at -20 ° C. (3) Label the probe with Dig: 27 201021808 Adhesive oligonucleotide (in an approached 1.5 ml microcentrifuge tube, add 2 μΐ 1 OX dATP, dCTP, dGTP (2 mM) ' 2 μΐ

Dig dUTP (1 mM), 2 μΐ Klenow and 0.5 μΐ BSA (1 mg/ml),

And add ddHjO to 20 to mix evenly, and react in a 37〇c water bath for 3 to 4 hours. The reaction was stopped by the addition of 0.2 M EDTA (1 to 2 μM). After heating at 75 ° C for 15 minutes, it was stored at -20 〇C for use. (4) Purification of the probe (using the probe Quant G-50 column):

Prepare a 15 ml centrifuge tube with a 2.0 ml microcentrifuge tube in the middle. Remove the G-50 column and mix it first. Be careful not to create bubbles. Then break the lower column and loosen the lid. Place it in a 15 ml centrifuge tube with a 2.0 ml microcentrifuge tube in the middle. 〇Xg, centrifuge for 1 minute. Remove the column, add 45 μl of IX TE buffer, carefully mix evenly. Do not create bubbles, then put back into the 15 ml centrifuge tube. Centrifuge for 2 minutes at 7 〇〇 xg. Remove the G_5 column and the microcentrifuge tube and place the new one.

Remove 2 already oligonucleotides and place in a Klenow buffer, i ^ microcentrifuge tube into a 15 ml centrifuge tube. Remove the 20 μM probe and add 30 μΐ 0.5Χ buffer to mix well. Drop the g_5 tube into the center of the bead, drop the G-50 column back into the 15 ml centrifuge tube, centrifuge at 700 xg for 2 minutes to purify the probe and collect it in a microcentrifuge tube. Store at -2 〇 °C spare. (5) EMSA analysis: Two sheets of 1.5 mm 6% polypropylene were prepared by mixing 6 ml 30% acrylamide, 3 ml 50% glycerol, 3 ml 10X TBE, 18 ml ddH20, 90 μΐ 10% AP and 90 μΐ TEMED. P〇iyacryiamide gel [native gel]. After coagulation, the neutral gel was placed in an electric bath 28 201021808 in a bath. Pre-run for 30 minutes at 4 ° C with ice-cold 5 5X TBE buffer (45 inM Tris_HCl, 45 mM boric acid, 1 mM EDTA). Remove 6 pg of nuciear extract and add 4 ^ binding buffer (20% glyCer〇l, loo mM TriS-HCl pH8:0, 300 mM KC1, 25 mM MgCl2, 5 mM DTT), 1 μΐ p〇ly d(IC) (1 gg/pl), 2 μΐ competitor (probe with unlabeled Dig) (200X) and supplement ddH2〇 to 18 μb mixed evenly at room temperature 2 〇 to 30 minutes” Add 2μ1 probe (1 ng/μΐ), mix well, and let stand at ® room temperature for 20 to 30 minutes. The mixture was poured into a colloidal well, and electrophoresed at 120 C for 180 minutes at a low temperature of 4 C. Prepare 2 sheets of paper larger than colloid and 1 piece of nylon film (nylon membraneMRoche Molecular Biochemicals, Mannheim, Germany), according to: sponge (filter paper - Whatman 3MM paper) - nylon film - gel - paper - Haijin The order is stacked from bottom to top, placed in the smashing clamp, and then placed in a tumbling tank containing 0.5X TBE buffer, and smashed at 300 °C for 70 minutes at a low temperature of 4 °C. The probe and probe in the colloid were spun down onto the nylon membrane with the nuclear egg white complex. The twisted nylon film was taken out, washed once with 0.5X TBE buffer, 'the film was placed in a box and dried at 55 ° C' and irradiated with ultraviolet rays to fix the probe on the film. Subsequently, the pretreated nylon film was subjected to blocking treatment by Northern blotting (Hong a/·, Jw Ce//).

Mol Biol, 2000, 23: 355-363), add anti-Dig-AP antibody (1:10,000, Roche Molecular Biochemicals, Mannheim, Germany), shake slowly for 30 minutes at room temperature, with CDP-Star (Tropix, Bedford, MA) ) For chemical luminescence, the reaction was carried out for 5 minutes. Tablet in the dark room 29 201021808 and rinse the film, observe the results, the results are shown in Figure A of Figure 8. The combination of THP-1 cell conditioned medium alone or with the active ingredient of Salvia miltiorrhiza: 5 pg/ml tanshinone (τι), 1〇pg/ml tanshinone π A (T2A) and 5 pg/ml cryptotanshinone (Cryp) As shown in the area of panel b, the intranuclear protein (5 pg) extracted from CL1-5 cancer cells cultured for 24 hours was mixed with 2 ng of Dig-labeled Ap-1 probe. The result of performing electrophoretic mobility shift assay for 40 minutes is shown in Figure VIII, panel b. From the results of Panels A and B in Figure 8, tanshinone I (Τ1) has an inhibitory effect on NF-kB and αρ" binding to IL-8 gene promoter compared to tanshinone ΠΑα (Τ2Α) and cryptotanshinone (Cryp). . Example 8 Animal Test of Antitumor Effect of Tanshinone I 8-1 Effect of Tanshinone I on Tumor Growth of Rats Lunging Lung Cancer Cells 5x1 0 CL 1 -5 cancer cells were suspended in 200 μM RPMI 1 640, respectively A total of 30 female immunocompromised rats (SCID) were injected subcutaneously to 5 weeks. Three days after the injection of cancer cells, 1 〇〇μΐ of PBS (as negative control group), tanshinone j (丨mg/kg) dissolved in 100 pl PBS, and tanshinone were administered by intraperitoneal administration every day; [(1 Mg/kg) and tanshinone Liao A (1 mg/kg) dissolved in 100 μΐ PBS, 100 μΐ of THP-1 cell conditioned medium (CM) (as control group), tanshinone I (1 mg/kg) dissolved in 100 μιη THP-1 cell conditioned medium and tanshinone I (1 mg/kg) and tanshinone π A (1 mg/kg) were dissolved in 1〇〇μ1 ΤΗΡ-1 cell conditioned medium. After the cancer cells were injected, the old gas was sacrificed for a day and the tumor was taken out to measure its weight and size, and a photograph was taken. 30 201021808 - The results are shown in Annexes 1 and 9 and Figure 9 is an annex. The results of the data are drawn. The pharmaceutical composition of the tanshinone I plus THP-1 cell conditioned medium of the present invention can make the tumor obviously Decrease, and the longer the tumor is applied, the smaller the tumor, and the tumor of the mouse is about 85% smaller than the control group at 12 days of application. The effect of 8-2 tanshinone on tumor angiogenesis in mice with lung cancer cells was determined by the above-mentioned effect of "8-1 tanshinone" on the growth of tumors in mice with lung cancer cells, and sacrificed 15 days after injection of cancer cells. After taking the mouse and taking out the weight and size of the tumor, and taking a photo record, the tumor was soaked in formalin and fixed into tissue wax blocks. Then the sections were stained with CD_3丨 antibody for immunohistochemical staining, and the number of tumors was observed under a microscope. As shown in Annex 2, the average number of color (brown) per solid field of the immunohistochemical staining of CD_31 antibody was calculated by 10 fields, and the results are shown in the tenth figure. The medical composition of tanshinone I plus ❹THP 1 cell conditioned medium can make the number of blood vessels in the tumor of the mouse in the experimental group for 12 days (tanshinone I (1 mg/kg) is dissolved in 1〇〇μ1 THP] cell conditioned medium) [Please refer to Annex II (b)] than the control group (only THP-i cell conditioned medium) [Please refer to Annex II (a) for a significant reduction. 3 Tanshinone I for lung In the cells of mice, the effect of cancer cell metastasis is based on the above-mentioned "8-1 Tanshinone oxime on tumor growth in mice with lung cancer cells." The mice were sacrificed 15 days after the injection of cancer cells and the ', 'in' weights were removed. After the size and photographing, the mouse's lungs 31 • 201021808 were also taken out and soaked in formalin fixed, made into a tissue wax block from &gt; A ,, and then sliced with H&amp;E stained mouse lung cancer screening agency r, Shift, ', σ is' under the microscope, select 10 fields of view to calculate the average number of cancerous spine and metastasis nodes in each field of view, as shown in Annex III. Shown, the tanshinone of the present invention

The pharmaceutical composition of I plus THP_1 cell conditioned medium can make the lungs of mice transfer, and the number of eyes is the same as that of the control group (only THIM cell conditioned medium is applied). Please refer to Annex III (4) for a significant reduction in 'and even no nodules in the lungs. It is apparent to those skilled in the art that various modifications and variations can be made without departing from the scope and spirit of the invention. Although the present invention has been described in terms of specific details, it must be understood that the present invention should not be unduly limited to the specific details. In fact, the various modifications that are obvious to those skilled in the art are also covered by the following claims. Example 9: Transient transfection and luciferase rep〇rter gene assay were used to determine the effect of active constituents of Salvia miltiorrhiza on the VEGF promoter activity of cancer metastasis-associated genes. For the effect of the VEGF promoter of the cancer metastasis-related gene, the VEGF promoter (Chau, β/.20〇2, 21:8817-29) provided by Dr. An was colonized on the reporter gene containing luciferase ( pGL-Basic luciferase reporter-3 vector, Promega

Madison, WI), and confirm it in sequence. The vector containing the VEGF promoter is used with the pSV-β-galactosidase vector (Promega) 32 201021808

LipofectAMINETM was co-transfected (c〇transfection) to CL1-5 cell line for 24 hours, then added medium containing different drug concentrations··tanshinone i (T1 lOpg/ml), tanshinone π A (T2A 10Mg/ml) and hidden The cells were cultured with tanshinone (Cryp 5 pg/ml) or unmedicated. Analysis of luciferase activity after 24 hours of culture (Dual-light Luciferase Reporter Gene Assay

System, Tropix) to confirm the transcriptional activity of the VEGF promoter. The above experiment also included the untransfected and only the transfection control group vector (pGL-3 Control vector) as a negative control group, and the β-galactosidase (p_ © galactosidase) activity was detected to standardize the transfection efficiency between experiments. (transfection efficiency) 'And do 3 repetitions. The method steps of co-transfection (c〇transfecti〇n), luciferase and β-galactosidase reporter gene assay, and luminescence detector detection are as follows: A Cotransfection: Co-transfection was performed using LipofectAMINETM plus reagent (Invitrogen, Gaithersburg, MD). 1 χ ι〇5 cells were cultured in a 6-well culture plate and cultured for 16 to 24 hours. When the cells were grown to 7 to 8 minutes, they were taken out and subjected to cell transfection. Prepare two mixtures in a 1.5 ml microcentrifuge tube (2 samples): buffer a = 600 μ1 serum-free medium + 12 μL Lipofectamine; buffer Β = 6 μμ serum-free medium + 8 μΐ plus reagent + 2 β - gai and 6 pg of IL-8p-Luc or pGL3-Luc plastids, each mixed uniformly and then allowed to stand at room temperature for 5 minutes. The buffer B-drop was gently added to the buffer a, mixed well, and left to stand at room temperature for 20 minutes. The cultured cells were taken out, the supernatant was aspirated, and gently washed once with a little serum-free medium. Add 33 201021808 2·4 ml medium containing no clear liquid, add 600 μιη mixture (Α+Β) 滴 by drop. Gently shake the plate to make the agent even. Place the plate at 37 °C, 5 °/. In the incubator of C〇2, let stand for 4 to 6 hours. The culture plate was taken out, and the medium containing the mixed solution was aspirated, replaced with 3 ml of serum-containing RPMI丨64〇 medium&apos; and left to stand at 37 ° C for 16 to 24 hours to restore the cells and allow the plasmid to replicate. Thereafter, coculture (c〇culture) and dosing treatment were carried out for 24 hours. B. Brucine enzyme and β-galactosidase reporter gene test:

This experiment uses the Dual-light Luciferase Reporter Gene Assay System (Tropix, Bedford, MA), which contains the following reagents:

Lysis solution (100 mM potassium phosphate ρΗ7·8, 0.2% Triton X-100), stored at 4〇c; Buffer a, powdered, first dissolved in 5ml ddH2〇, stored at 4°C,! Weeks, or sub-packaged in _2〇β(:, 1 year; Galacton-Plus substrate: 100X, stored at 4°C; diluted with Buffer B before each use; Buffer B, powdered , first dissolve back in 22 ml ddH2 〇, store at 4 ° C, 1 week, or store in -20 C, 1 year; Light Emission Accelerator-Π, stored at 4 ° C. Fluorescence twist and β- The procedure for the galactosidase reporter assay is to prepare a lysis solution, remove the required amount, and add DTT (so that the final concentration is about 〇·5 mM, but not more than 〇.5. The transfected cells were removed, the medium was aspirated and gently washed twice with ice-cold 1X PBS. The cells were covered with a cell cold solution containing 0.5 mM DTT (usage: 25 μl/6 〇mm culture dish). The cell scraper scrapes the cells and sucks them into a microcentrifuge tube. Centrifuge for 2 minutes at 4, 〇 low temperature, 34 201021808 12,000 rpm. Pipette the upper, and then take the upper/month liquid and measure or save immediately. At -80 ° C. C. Cold light detector detection: Before the measurement, the cold light meter should Turn on 'warming for 3 〇 minutes. Buffer a and B take the required amount and put it back at room temperature, and (5) such as (10) ^ (100X) diluted to lx with Buffer B. Take 1 μμ1 cell lysate (iysate) 25 WBufferA, mix well, add 1〇〇μΐ buffer containing β of Galacton-Plus substrate in 1 minute, measure luciferin® enzyme chemical luminescence immediately, etc. After 30 to 60 minutes, add 100 μΐ Light Emission Accelerator-Π 'Immediate determination of β_galactosylase chemical luminescence. The results of the transcriptional activity analysis of the active ingredient of Salvia miltiorrhiza Bunge for VEGF promoter are shown in Figure 12. ΤΗΡ-1 cell conditioned medium (CM) will cause A significant increase in photozyme activity, while the addition of tanshinone I (10 pg/ml) significantly inhibited the transcriptional activity of the VEGF promoter. Example 10 Effectiveness of Salvia miltiorrhiza active ingredient for inhibiting VEGF-A mRNA in CL1-5 lung cancer cells Analysis ❿ CLl-5 lung cancer cell line was cultured in THP-1 cell conditioned medium or at the same time added different concentrations of Salvia miltiorrhiza active ingredients: tanshinone I (10 pg / ml), tanshinone π A (10 pg / ml) and cryptoglucoside I (5 pg/ml). After 24 hours of incubation, total RNA yield cancer cell extract was transferred into the cDNA reverse transcription reaction, real time quantitative polymerase chain reaction to analyze the performance of the various processes of the amount of VEGF-A mRNA. The relative relationship between the gene expression of the experimental group and the TATA box binding protein is as follows: -△ CT= -[CTInterst - CTTBP] ' 35 201021808 where ct is defined as exceeding a certain fixed threshold After that, the number of PCR cycles of firefly light can be detected. The ratio of gene expression is defined as follows: 2 - △ CT χ κ (K: constant). The results are as shown in Fig. 13 'Compared with the combination of THP4 cell conditioned medium alone, the combination of tanshinone and THPd cell conditioned medium inhibited the expression of angiogenesis-related gene VEGF_A mRNA induced by tanshinone. „A(T2A) and crypto-salt _(cry) did not produce the ability to inhibit the expression of VEGF_A mRNA. Salvia miltiorrhiza _ι reduced the expression of VEGF_A gene by 5〇% when the concentration was 1〇μ§/ιη1. Example 11 Effectiveness of Salvia miltiorrhiza Active ingredient for inhibiting PDGF-β mRNA of CL15 lung cancer cells CL1-5 lung cancer cell lines were cultured in cell conditioned medium or simultaneously with tanshinone I (1〇Mg/ml). The total amount of RNA extracted from the cells was converted into eDNA by reverse transcription reaction, and the expression of PDGF_p mRNA under various treatments was analyzed by real-time polymerase chain reaction. The gene expression of the experimental group and the control group (TATA) were observed. The relative relationship of the amount of performance of the box binding protein is as follows: -ACT= ~[CTInterst- CTTBP] /, medium, CT 为 is the number of PCR cycles that can detect fluorescence after a certain fixed threshold The ratio of gene expression of the two is defined as follows: 2_Λ CTx K (K: constant). The results are shown in Figure 14. Compared with the conditioned medium containing ΤΗΡ-1 cells alone, the conditions of Salvia miltiorrhiza and THp_i cells The combination of the medium inhibited the expression of the angiogenesis-related gene pDGF-p mRNA, and the expression of PDGF-β gene was reduced by (10)% 36 201021808 at a concentration of 丨〇. Explanation] The first area A shows the evaluation of the cytotoxic dose of tanshinone I, tanshinone jjA and cryptotanshinone on CL 1 -5 lung cancer cells at 24 hours, while the B area shows that Danshen 阙I is thinner on CL 1 -5 lung cancer cell lines. The effect of the rate. The second panel shows the effect of tanshinone 1 on the migration of CL1_5 lung cancer cell lines.

The third panel shows the effect of tanshinone 1 on the invasion ability of CLl-5 lung cancer cell lines. The fourth panel shows that the Danshen sign + J 1 sr CL1-5 lung cancer cell line secreted ΜΜΡ egg * in the 帛 帛 Β 帛 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 为 第四 第四 第四 第四 第四 第四 第四 第四The data is obtained by plotting. The fifth panel shows the effects of Salvia miltiorrhiza _! and Salvia miltiorrhiza on THP-i cell conditions in CL1_5 lung cancer cells. The nutrient-induced ... floating performance 2 shows that tanshinone 1 is related to cancer metastasis...

Effect of sex Figure 7 shows the design of the m-reporter gene vector; and B = the transcriptional regulatory region of the fluorescent fluorescein report gene analysis results THIM cell conditional culture: Figure 8 area A shows salvia miltiorrhiza Inhibition; Figure 8 b / binding to the IL-8 gene; inhibition of the IL-8 gene promoter f shows that the danshenone I on the AP-i junction ninth panel shows the effect of tanshinone oxime on &gt; growth. ^The mice that know lung cancer cells are swollen. 37.201021808 The tenth panel shows the effect of tanshinone i on tumor angiogenesis in mice that have lung cancer cells. The eleventh panel shows the effect of tanshinone I on the metastasis of cancer cells in mice that have been administered lung cancer cells. Figure 12 shows the effect of tanshinone I on the VEGF promoter activity of the angiogenesis-related gene. Figure 13 shows the effect of tanshinone I and tanshinone oxime on the expression of VEGF-A mRNA 诱发 induced by THP-1 cell conditioned medium in CL1-5 lung cancer cells. Figure 14 shows the effect of tanshinone I on the expression of PDGF-β mRNA induced by conditioned medium of CL1-5 lung cancer cells via THP-1 cells. [Explanation of main component symbols] (None) [Simple description of the attachment] Annex I shows the effect of tanshinone I on the tumor growth of mice that are administered with lung cancer cells. Annex II shows the effect of tanshinone I on tumor angiogenesis in mice that have been administered lung cancer cells. Annex III shows the effect of tanshinone I on cancer cell metastasis in mice that have been administered lung cancer cells. 38 201021808 Sequence Listing &lt;110〉 National Chung Hsing University &lt;120&gt; Composition containing tanshinone I and its use &lt;130〉 Nil &lt;160〉 8 &lt;170&gt; Patentln version 3.4 &lt;210〉 &lt;211&gt;;212&gt;〇&lt;213&gt; 1 39 DNA homo sapiens AP-1 F &lt;400> 1 aaaagtgtga tgactcaggt ttgtgatgac tcaggtttg 39 &lt;210〉 2 &lt;211> &lt;212〉 &lt;213&gt; 36 DNA homo sapiens AP- 1 R &lt;400〉 2 aaacaaacct gagtcatcac cctgagtcat cacact 36 &lt;210&gt;&lt;211&gt; Q &lt;212&gt;&lt;213> 3 45 DNA homo sapiens NF-kB F &lt;400> 3 aaaaaaaatc gtggaatttc ccccgaatcg tggaatttcc cccga 45 &lt ;210&gt; 4 &lt;211&gt;&lt;212&gt;&lt;213&gt; 42 DNA homo sapiens NF-kB R &lt;400> 4 aaatcggggg aaattccacg attcggggga aattccacga tt 42 &lt;210&gt; 5 &lt;211&gt; 21 201021808 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt; IL-8-F &lt;400&gt; 5 ctcttggcag ccttcctgat t &lt;210&gt; 6 &lt;211&gt; 28 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;;220> φ &lt;223&gt; IL-8-R &lt;400〉 6 tatgcactga Catctaagtt ctttagca &lt;210&gt; 7 &lt;211&gt; 20 &lt;212&gt; DNA &lt;213&gt; artificial sequence&lt;220&gt;&lt;223&gt; TATA box binding protein-F &lt;400&gt; 7 cacgaaccac ggcactgatt ❹ &lt;210〉 8 &lt;211&gt; 22 &lt;212&gt; DNA &lt;213&gt;Artificial sequence&lt;220&gt;&lt;223&gt; TATA box binding protein-R &lt;400&gt; 8 ttttcttgct gccagtctgg ac

Claims (1)

201021808 VII. Patent application scope: 1. An anti-tumor composition comprising: tanshinone I having the following chemical formula (I): (I); and Φ a mononuclear cell conditioned medium. 2. The anti-tumor composition of claim 1, wherein the mononuclear cell conditioned medium is prepared by the following steps: mixing a mononuclear bulb with a serum-free cell culture medium, To form a mixed culture containing mononuclear cells; and culturing the mixed culture for a period of time, the cells are removed from the mixed culture to obtain the mononuclear cell conditioned medium. 3. The anti-tumor composition according to claim 2, wherein the mononuclear cell conditioned medium comprises a cytokine selected from the group consisting of: interleukin _8, · &quot; » IT δ (interleukln_8, IL 8), interleukin-4 (IL-4), interleukin-1, interleukin-12 (IL-12) and combinations thereof. 4 · For example, the anti-tumor composition of the second application of the patent scope, wherein the mononuclear cell line is a macrophage. 5. For example, the anti-tumor composition of the patent application scope ###贝贝, wherein the python cell line is derived from THP-1 cells. 6. For example, the macrophages of the first to the ninth king of the patent application. The anti-tumor group 201021808 of the above-mentioned item is wherein the tumor is selected from the group consisting of non-small cell lung cancer, breast cancer, kidney cancer, soft tissue sarcoma, neuroendocrine tumor of the lung, cervical cervix Cancer, uterine cancer, head and neck cancer, glioma, prostate cancer, pancreatic cancer, lymphoma, melanoma, small cell lung cancer, nest cancer, colon cancer, esophageal cancer, gastric cancer, leukemia, and colorectal cancer. 7_ The anti-tumor composition according to item 6 of the Shenqing patent scope, which further comprises a carrier. 8' The antitumor composition of claim 7, wherein the carrier is an aqueous solution or an organic solution. 9. The use of the antitumor composition according to any one of claims 1 to 8 of the invention, for the manufacture of a medicament for antitumor. The use according to claim 9, wherein the pharmaceutical product is administered in a dose ranging from 0.1 to 100 mg/kg of tanshinone J. 11. The use according to claim 9, wherein the medicine is administered with a dose of tanshinone J. ranging from 0.5 to 50 mg/kg. The application described in claim 9 of the patent application, wherein the medicine is a dose of tanshinone which falls between 1 and 20 mg/kg. A composition for inhibiting ash tube regeneration, comprising tanshinone oxime having the following formula (I): , 201021808 ο ° The composition of claim 1, further comprising one; inhibiting angiogenesis ^ 1 2 3 carrier, diluent or thief as in claim 13. The composition of ❹ is further encapsulated for inhibiting angiogenesis - as in the patent application No. 13^5H-liquid. The composition of the angiogenesis, the further step comprising: : = is described for the inhibitory group. 3 early nuclear cell condition conditioning 17. The application of the (4) (4) anti-tumor composition of the early nuclear cell conditioned medium is prepared by the following steps: mixing a nucleated nucleated cell with a serum-free cell culture medium, To form a mixed culture containing mononuclear cells; and culturing the mixed culture for a period of time, removing cells from the mixed culture to obtain the mononuclear cell conditioned medium. The anti-tumor composition according to claim 17, wherein the mononuclear cell conditioned medium comprises a cytokine selected from the group consisting of interleukin-8 (interleukin- 8, IL-8), interleukin-4 (IL-4), interleukin-12 (IL-12) and 2 combinations thereof. 3 1 9. The antitumor composition according to claim 16, wherein the singular mononuclear cell line is a giant scorpion cell. The anti-tumor composition according to claim 19, wherein the python cell line is a ΤΗΡ-1 cell-derived macrophage. 2 1. Salvia miltiorrhiza _ I is used for the manufacture of a medicament for treating or inhibiting an angiogenesis-related disease or disorder, wherein Salvia miltiorrhiza _ I has the following chemical formula (1): (1). 22, such as applying for a patent model to fall within a range of 〇 I. The use of the invention of claim 21, wherein the dosage of the medicine is between 1 and 100 mg/kg of tanshinone. The use of the medicine according to claim 21, wherein the pharmaceutical product falls within the range of 0.5 to Dan of 5 〇 mg / kg dose; ... medication. The use of 21, wherein the dose of the drug is between mg/kg of tanshinone I 24. If the scope of the patent application is in the range of 1 to 2, the drug is in the range of 25. 2, as in the scope of the patent application 22 to 24 ... wherein the group of angiogenesis-related diseases or disorders: atherosclerosis; Inflammatory and traumatic repair: asthma and chronic obstruction (4) inflammation (c〇PD), bronchiolitis, viral liver fibrosis, Hi arthritis, pyloric screw __; osteoarthritis and fibrosis; Including allergic dermatitis,] 201021808 skin damage caused by skin and ultraviolet rays; autoimmune diseases, including systemic lupus erythematosus; chronic myeloproliferative diseases; multiple sclerosis; dry phlegm; ankylosing spondylitis; Renal glomerulonephritis; and cancer; neoplastic disease, resten〇sis, rheumatoid arthritis, Crohn's disease, diabetic retinopathy Retin〇pathy), psoriasis (psoriasis), neurite endometriosis (end〇metrj〇sis), degenerative disease (macular degeneration), neovascular green . Eye square (neovascular giaucoma) and obesity (adiposity) 26. - species for inhibiting factor-induced angiogenesis by cell migration composition 'comprising tanshinone I has the following chemical formula ⑴ of: (I) The composition for inhibiting vascular neonatal factor-induced cell migration according to claim 26, which further comprises a carrier, a diluent or an excipient. A pharmaceutical composition for treating a disease associated with NF-κB, a transcription factor AP-1, comprising tanshinone I having the following chemical formula (1): (I) 201021808 29·If the scope of application for patent application is 28th, +, defensive, the medical composition described in the article, the progress is 3, a carrier, a diluent or a excipient. 30.~ For the use of tanshinone I, for the treatment of the transcription factor AP-1蛊 &quot; related diseases of the drug it, 1 + &amp; μ column chemical formula (1): Use # #中丹参嗣! Have the next 31. The use of claim 3, wherein the pharmaceutical system is administered at a dose ranging from 0.1 to 100 mg/kg of tanshinone I. 32. The use according to claim 30, wherein the medical mouthwash X is in the range of 〇. 5 to 5 的 of the dose of Danshen. 33. The use of claim 30, wherein the pharmaceutical is administered in a dose ranging from 1 to 20 mg/kg of tanshinone I. 34. A pharmaceutical composition for treating a disease of an angiogenic factor PdgF_P, VEGF or a _8 related signal transmission pathway, comprising a salvia having the following chemical formula (I) _ j : 201021808 Ο (I) 〇 35. The pharmaceutical composition of claim 34, which further comprises a carrier, diluent or excipient. Salvia miltiorrhiza _ I is used in the manufacture of pharmaceuticals for the treatment of diseases of the ash tube neonatal factor or 1L_8 related signal transmission pathway, wherein Salvia miltiorrhiza _ I has the following chemical formula (j): 〇 ❹ 38. If the scope of the patent application is 36, the product is used in the range - macro / η. Danshen at a dose between 0.^50mg/kg... 39. The strain described in item 36 of the patent application is a net-poor application, wherein the medicine falls from 1 to 20 mg/ The dose of the drug between kg. ...in the Danshen Brewing II vote VIII, schema: (such as the next page)