CN109846866A - Application of the ent kauranoid class compound Parvifoline AA in pharmacy - Google Patents
Application of the ent kauranoid class compound Parvifoline AA in pharmacy Download PDFInfo
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Abstract
Application the present invention relates to ent-kaurene diterponoid as natural kill (natural killer, NK) cellular immunity sensitizer in preparation prevention and tumor immunotherapy drug.The invention demonstrates that ent-kaurene diterponoid Parvifoline AA increases liver cancer cells NKG2D ligand expression by targeting peroxiredoxins I and II (Prxs-I/II), to promote the identification and killing of NK cells against tumor cells.Compound pharmacological activity of the present invention is strong, and mechanism of action is apparent, safety and low toxicity, and stability is good, can be used as newtype drug, can also be used as the tool of research NKG2D ligand expression regulation associated signal paths.
Description
Technical field
The invention belongs to drug fields, lead more particularly to Ent-kaurane diterpenoid diterpene compound in biotechnology and medicine
Application in domain is used as tumour cell more particularly, to ent kauranoid class compound and sensitizer is immunized,
And its application in preparation of anti-tumor drugs.
Background technique
Tumour is to threaten the major disease of human health, clinical treatment mainly has three big means at present: operative treatment is put
Penetrate treatment and chemotherapy.However operative treatment is very limited for the oncotherapy effect for being easy to happen transfer, and chemotherapeutic
Object and radiation treatment while killing cancer cell, also generate different journeys to normal histocyte due to virulent side effect
The damaging action of degree causes the quality of life of therapeutic effect and patient to be affected.Immunotherapy of tumors is a kind of by swashing
Immune system that is living or transferring body itself, the therapeutic process for being identified and being killed to tumour cell is to be considered in recent years
The most possible treatment means for curing tumour.Natural kill (Natural killer, NK) cell is as in natural immune system
Main responsiveness lymphocyte plays important supervisory function bit in the generation, development and transfer process of control tumour, is
The first line of defence of antineoplastic immune.The activation of NK cell is mainly put down by between cell membrane surface Inhibitory receptor and activated receptor
The regulation of weighing apparatus, wherein NKG2D (Natural-killer group 2, member D) is more deep one of Recent study
Activating receptor, the NKG2D ligand on target cell are considered as the indicator of cellular stress state, under NKG2D ligand expression
It adjusts or missing is usually related to the immunologic escape of tumour.Therefore, the expression of tumour cell film surface NKG2D ligand is raised, is increased
The identification sensibility of NK cells against tumor cells is one of the available strategy based on NK cell tumour immunization therapy.
Natural products is derived from metabolism or the tunning of nature animal and plant and microorganism, more due to structure
Sample and novelty are the valuable source libraries of research, developing anti-tumor medicaments.Ent-kaurene diterponoid is main
From Labiatae Rabdosia plant, this kind of plant be used to prevent and treat inflammation, stomach civil usually as Chinese herbal medicine
The diseases such as enteric infection and cancer.It is previous research shows that ent-kaurane diterpenoid class compound (such as adenanthin, hair calyx
B prime, Oridonin etc.) it has broad application prospects in the prevention and treatment of the diseases such as tumour and inflammation.
So far, Ent-kaurane diterpenoid diterpene compound is as NK cellular immunity sensitizer and Ent-kaurane diterpenoid
There is not been reported in the application of preparation prevention and treatment tumour immunity adjusting drug for diterpene compound.
Summary of the invention
The purpose of the present invention is to provide entkaurane diterpenoids as NK cellular immunity sensitizer, and
The application in liver cancer immunity adjusting drug is prevented and treated in preparation.
Above-mentioned purpose to realize the present invention, the present invention provides following technical proposals:
Ent-kaurene diterponoid Parvifoline AA is in preparation prevention and immunization therapy hepatic carcinoma medicine
Application in object.
Ent-kaurene diterponoid Parvifoline AA is preparing answering in the immune sensitizer of tumour cell
With.
Application as mentioned, wherein the tumour is head and neck neoplasm, tumor in respiratory system, digestive system tumor, secretes
Urinary system tumour, skeletal system tumour, gynecological tumor, hematological system tumor, other types tumour.
Application as mentioned, wherein the head and neck neoplasm is thyroid cancer, nasopharyngeal carcinoma, meninx cancer, acoustic neurinoma, hangs down
Body tumor, carcinoma of mouth, craniopharyngioma, thalamus and brain stem tumor, angiogenic tumour or intracranial metastatic tumor；The respiratory system is swollen
Tumor is lung cancer；The digestive system tumor is liver cancer, gastric cancer, the cancer of the esophagus, colorectal cancer, the carcinoma of the rectum, colon cancer or cancer of pancreas；Institute
The Patients with Urinary System Tumors stated is kidney, bladder cancer, prostate cancer or carcinoma of testis；The skeletal system tumour is osteocarcinoma；It is described
Gynecological tumor be breast cancer, cervical carcinoma or oophoroma；The hematological system tumor is leukaemia, malignant lymphoma or multiple
Property myeloma；Other types tumour is malignant mela noma, glioma or cutaneum carcinoma.
Application as mentioned, wherein regulation of the compound Parvifoline AA by increase NKG2D ligand expression,
And then promote identification and the lethal effect of NK cells against tumor cells.
Application as mentioned, wherein generation of the compound Parvifoline AA by induced activity oxygen, to realize
The regulation of tumour cell NKG2D ligand expression.
Application as mentioned, wherein the compound Parvifoline AA passes through targeting peroxiredoxins I and
II, to realize the regulation of tumour cell NKG2D ligand expression.
Application as mentioned, wherein the compound Parvifoline AA passes through up-regulation liver cancer cells film surface
NKG2D ligand, to realize NK cell to the identification sensibility of liver cancer cells.
Application as mentioned, wherein the compound Parvifoline AA passes through up-regulation liver cancer cells film surface
NKG2D ligand, to realize NK cell to the killing sensibility of liver cancer cells.
Compared with prior art, the present invention has following excellent benefit:
Entkaurane diterpenoids of the present invention can be used as NK cellular immunity sensitizer preparation prevention and immunization therapy
Tumour medicine.First demonstration that compound Parvifoline AA can significantly raise Hepatocellular carcinoma cell line, HepG2,
The transcript and expression of Huh7 film surface ULBP1, ULBP3 and MICA/B, and dose dependent is presented.To normal liver cell L-02
NKG2D ligand expression do not influence.Parvifoline AA is by promoting the generation of intracellular ROS to raise ULBP1, ULBP3
With the transcript and expression of MICA/B.Protein spectrum the result shows that, Parvifoline AA be by with Prxs-I/II
The site Cys173/172 combines, to inhibit the activity of Prxs-I/II, eventually leads to the rising of ROS level.NKG2D matches body surface
The up-regulation reached, can significantly promote NK cell in vivo/outside to the identification of liver cancer cells and killing.Above data shows
Parvifoline AA pharmacological action is strong, and pharmacological mechanism is apparent, and it is one that safety with higher, which has good medical value,
The immune hypersitization medicine of the treatment liver cancer of a great development and application prospect.
Entkaurane diterpenoids of the invention can be administered orally or without mouth, and dosage is because of drug difference
And have nothing in common with each other, for adult, daily 1-1000mg is proper.
When oral administration, make compound and conventional medicinal adjuvant such as excipient, disintegrating agent, binder, lubrication first
The mixing such as agent, antioxidant, coating agent, colorant, aromatic, surfactant, is made into granule, capsule, tablet etc.
Form administration；It can be administered in the form of injection, infusion solution or suppository etc. when non-oral administration.When preparing above-mentioned preparation, it can be used
Conventional preparation technique.
Detailed description of the invention
Fig. 1 shows the chemical structure of Parvifoline AA；
A in Fig. 2 shows influence of the Parvifoline AA for the NKG2D ligand expression of Hepatocellular carcinoma cell line.
It is handled cell 24 hours with 20 μM of Parvifoline AA, each NKG2D ligand of flow cytometer detection cell membrane surface (ULBP1,
ULBP2, ULBP3 and MICA/B) expression；
B in Fig. 2 shows influence of the Parvifoline AA for the NKG2D ligand expression of hepatocellular carcinoma H22.With 20
μM Parvifoline AA handle cell 24 hours, each NKG2D ligand of flow cytometer detection cell membrane surface (ULBP1, ULBP2,
ULBP3 and MICA/B) expression；
C in Fig. 2 shows influence of the Parvifoline AA for the NKG2D ligand expression of liver cancer cells Huh7.With 20 μ
The Parvifoline AA of M is handled cell 24 hours, each NKG2D ligand of flow cytometer detection cell membrane surface (ULBP1, ULBP2,
ULBP3 and MICA/B) expression；
D in Fig. 2 shows influence of the Parvifoline AA for the NKG2D ligand expression of normal liver cell L-02.
It is handled cell 24 hours with 20 μM of Parvifoline AA, each NKG2D ligand of flow cytometer detection cell membrane surface (ULBP1,
ULBP2, ULBP3 and MICA/B) expression；
Fig. 3 shows the influence of NKG2D ligand transcription of the Parvifoline AA to Hepatocellular carcinoma cell line.With difference
The Parvifoline AA of concentration is handled SMMC-7721 cell 24 hours, and fluorescence quantitative RT-RCR detects each NKG2D ligand
The transcriptional level of (ULBP1, ULBP2, ULBP3 and MICA/B)；
A in Fig. 4 shows that SMMC-7721 cell after Parvifoline AA processing, stimulates the activation degree of NK cell.
SMMC-7721 cell is after 20 μM of Parvifoline AA is handled 24 hours, with NK92 cell with 5:1 (NK92:SMMC-
Cell number ratio 7721=5:1) is incubated for 4 hours altogether, the specific expressed molecule CD56 of flow cytometer detection NK cell membrane surface and
The expression of reactivity molecule CD107 α；
After B in Fig. 4 shows the function of Activating receptor NKG2D of closing NK cell in advance with NKG2D specific antibody,
Stimulation activation degree of the SMMC-7721 cell handled through Parvifoline AA to NK cell.NK92 cell is with NKG2D antibody
After 37 DEG C are closed 30 minutes, 24 hours SMMC-7721 cells are handled with 5:1 with the Parvifoline AA through 20 μM
(NK92:SMMC-7721=5:1) cell number ratio is incubated for 4 hours altogether, specific expressed point of flow cytometer detection NK cell membrane surface
The expression of sub- CD56 and reactivity molecule CD107 α；
A in Fig. 5 shows influence of the Parvifoline AA to SMMC-7721 cytotoxic activity.With various concentration
After Parvifoline AA is handled SMMC-7721 cell 24 hours, it is thin to SMMC-7721 that mtt assay detects Parvifoline AA
The cytotoxic activity of born of the same parents.IC50=33.16 μM；
B in Fig. 5 shows NK92 cell to the killing activity of the SMMC-7721 cell handled through Parvifoline AA.
After 20 μM of Parvifoline AA is handled SMMC-7721 cell 24 hours, incubated altogether from NK92 cell with different cell proportions
It educates 4 hours, it is living to the killing of SMMC-7721 cell that Non-Radioactive Cytotoxicity kit detects NK cell
Property.Wherein in function enclosed experiment, NK92 cell is closed 30 minutes, corresponding kind at 37 DEG C in advance with NKG2D specific antibody
Belong to the IgG of source animal as negative control；
A in Fig. 6 shows influence of the Parvifoline AA to NK92 cell activity.NK92 cell is through 20 μM
After Parvifoline AA is handled 24 hours, the specific expressed molecule CD56 of flow cytometer detection NK92 cell membrane surface and reactivity
The expression of molecule CD107 α；
B in Fig. 6 shows that Parvifoline AA influences the expression of NK92 cell activation receptor NKG2D.NK92 is thin
Born of the same parents are after 20 μM of Parvifoline AA is handled 24 hours, the table of flow cytometer detection NK cell membrane surface Activating receptor NKG2D
Change up to level；
A in Fig. 7 shows influence of the Parvifoline AA to SMMC-7721 intracellular reactive oxygen level.20 μM
After Parvifoline AA is handled SMMC-7721 cell 24 hours,Green kit detects intracellular ROS water
It is flat；
B in Fig. 7 shows that Parvifoline AA promotes the ROS generated to SMMC-7721 cell membrane surface NKG2D ligand
The influence of expression.After the N-acetyl-L-cysteine (NAC) of 5mM is pre-processed or is not handled SMMC-7721 cell 1 hour,
Parvifoline AA add processing 24 hours, each NKG2D ligand of flow cytometer detection cell membrane surface (ULBP1, ULBP3 and
MICA/B expression)；
A in Fig. 8 shows the Parvifoline AA horizontal influence to GSH in the cell.SMMC-7721 cell is with difference
The Parvifoline AA of concentration is handled 24 hours, and then by cell cracking, GSH reagent box for detecting content detects intracellular GSH
Level.Compound Isoforretin A is as positive control；
B in Fig. 8 shows influence of the Parvifoline AA in extracellular levels to GSH.The compound of various concentration
Parvifoline AA, Adenanthin and Isoforretin A are incubated with the ratio of 5:1 and 10:1 with 20 μM of GSH altogether respectively
It educates 2 hours, the level of intracellular GSH is then detected with GSH reagent box for detecting content；
A in Fig. 9 shows Parvifoline AA to the activity influence of peroxidase Peroxiredoxins I.Slow
Fliud flushing (50mM HEPES buffer, pH7.0；1mM EDTA) in be separately added into 250 μM β-NADPH, 0.04U TrxR and
0.1U hTrx-1, total volume are 180 μ L.Then the Prxs-I albumen (final concentration of 640nM) that addition total volume is 10 μ L+no
With the Parvifoline AA (1 μM, 2 μM, 5 μM or 10 μM) of concentration) mixture.It is eventually adding the H of 10 μ L2O2(final volume is
100 μM) starting reaction.Continue the oxidation level of 10 minutes detection β-NADPH under 340nM wavelength using microplate reader；
B in Fig. 9 shows Parvifoline AA to the activity influence of peroxidase Peroxiredoxins II.?
Buffer (50mM HEPES buffer, pH7.0；1mM EDTA) in be separately added into 250 μM of β-NADPH, 0.04U TrxR with
And 0.1U hTrx-1, total volume are 180 μ L.Then the Prxs-II albumen (final concentration of 640nM) that addition total volume is 10 μ L+
The Parvifoline AA (1 μM, 2 μM, 5 μM or 10 μM) of various concentration) mixture.It is eventually adding the H of 10 μ L2O2(final volume is
100 μM) starting reaction.Continue the oxidation level of 10 minutes detection β-NADPH under 340nM wavelength using microplate reader；
A in Figure 10 shows the first mass spectrometric figure that Parvifoline AA modifies the 173rd Cys of Prxs-I.The weight of 2 μ g
Histone Prxs-I and 10 μM of Parvifoline AA is incubated at room temperature 2 hours, then runs SDS-PAGE glue, and coomassie is bright
After indigo plant dyeing, after methanol/glacial acetic acid decoloration, destination protein band is cut down, carries out LC-MS/MS detection to determine egg
White decorating site；
B in Figure 10 shows the second order ms figure that Parvifoline AA modifies the 173rd Cys of Prxs-I.The weight of 2 μ g
Histone Prxs-I and 10 μM of Parvifoline AA is incubated at room temperature 2 hours, then runs SDS-PAGE glue, and coomassie is bright
After indigo plant dyeing, after methanol/glacial acetic acid decoloration, destination protein band is cut down, carries out LC-MS/MS detection to determine egg
White decorating site；
A in Figure 11 shows molecular target of the Prxs-I as Parvifoline AA, regulates and controls the expression of NKG2D ligand.
SMMC-7721 cell transfecting wild type Prxs-I expression plasmid or its 173rd site mutation Cys173-Ala plasmid (Prxs-
IM), empty plasmid is as transfection control.After transfection 24 hours, cell is handled with 20 μM of PARVIFOLINE AA.After 24 hours,
The expression of flow cytometer detection cell membrane surface NKG2D ligand；
B in Figure 11 shows the generation of Prxs-I regulation ROS, to induce the expression of NKG2D ligand.SMMC-7721 turns
Dye handles cell after shPrxs-I or shScramble plasmid 24 hours, with 5mM NAC.The flow cytometer detection cell after 24 hours
The expression of film surface NKG2D ligand；
C in Figure 11 shows molecular target of the Prxs-I as Parvifoline AA, regulates and controls the expression of NKG2D ligand.
After SMMC-7721 is transfected shPrxs-I or shScramble plasmid 24 hours, cell is handled with 20 μM of PARVIFOLINE AA.24
After hour, the expression of flow cytometer detection cell membrane surface NKG2D ligand；
A in Figure 12 shows Parvifoline AA to the time effect of ERK and AKT phosphorylation level.SMMC-7721 is thin
Born of the same parents are handled with 20 μM of Parvifoline, respectively at 0,1,3,6 and 12 hour collection cell, are tested after cracking for WB, respectively
Have detected the AKT and ERK of total amount AKT and ERK and phosphorylation state.Actin is used to be used as internal reference；
B in Figure 12 shows Parvifoline AA to the concentration gradient effect of ERK and AKT phosphorylation level.Respectively with
The Parvifoline AA of 0,5,10,20 and 40 μM of concentration handles SMMC-7721 cell, and cell is collected after 3 hours, is used after cracking
It is tested in WB, has detected the AKT and ERK of total amount AKT and ERK and phosphorylation state respectively.Actin is used to be used as internal reference；
C in Figure 12 shows ERK phosphorylation inhibitor U0126 and AKT phosphorylation inhibitor MK2206 to Parvifoline
The influence of AA.20 μM of Parvifoline AA and U0126 or MK-2206 handle SMMC-7721 cell simultaneously.After 3 hours, collect
Cell tests for WB after cracking, has detected the AKT and ERK of total amount AKT and ERK and phosphorylation state respectively.Actin is used
In as internal reference；
D in Figure 12 shows that ERK phosphorylation inhibitor and AKT phosphorylation inhibitor regulate and control Parvifoline AA
The influence of NKG2D ligand expression.SMMC-7721 cell is handled with 20 μM of Parvifoline AA, while handling U0126 (uplink)
Or MK-2206 (downlink), after 24 hours, collect cell, carry out FCM analysis cell membrane surface ULBP1, ULBP3 and
The expression of MICA/B；
E in Figure 12 shows influence of the PARVIFOLINE AA to ROS-ERK signal path.5mM NAC pre-processes SMMC-
After 7721 1 hours, 20 μM of PARVIFOLINE AA are added or solvent continues with 3 hours, collects cell, is used for after cracking
WB experiment, has detected the AKT and ERK of total amount AKT and ERK and phosphorylation state respectively.Actin is used to be used as internal reference；
F in Figure 12 shows the schematic diagram of mechanism of ParvifolineAA regulation NKG2D ligand expression；
A in Figure 13 shows Parvifoline AA in the different plant tumor model of mouse to the growth inhibition effect of liver tumour.
1 × 10 is injected under every C57/BL6j mouse right axillary6A Hepa1-6 cell.After 6 hours of cell inoculation, mouse is divided at random
At three groups, intraperitoneal administration carried out to mouse by per kilogram 5 or 10 milligrams of dosage, successive administration 12 days.Measurement mouse is negative daily
The volume of lotus tumour, data are indicated with the mean+/-standard error of 4 mouse mouse gross tumor volumes；
B in Figure 13 shows Parvifoline AA in the different plant tumor model of mouse to the inhibiting effect of liver tumour weight.
After administration, tumor tissues is taken to weigh, data are indicated with the mean+SD of 4 mouse tumor knurl weights；
C in Figure 13 shows influence of the Parvifoline AA to tumor tissue size.After administration, tumor tissues is taken to clap
According to；
D in Figure 13 shows influence of the Parvifoline AA in the different plant tumor model of mouse to mouse weight.It is right daily
The weight of mouse is weighed, and weight value is indicated with the mean+/-standard error of 4 mouse；
E in Figure 13 shows influence of the Parvifoline AA in the different plant tumor model of mouse to mouse spleen weight.It gives
After medicine, spleen tissue is taken to weigh, data are indicated with the mean+SD of 4 mouse tumor knurl weights；
F in Figure 13 shows influence of the Parvifoline AA in the different plant tumor model of mouse to mouse spleen form.It gives
After medicine, spleen tissue is taken to take pictures；
G in Figure 13 shows that Parvifoline AA turns mouse liver tumour NKG2D ligand in the different plant tumor model of mouse
The influence of record.After mouse tumor tissue is shredded with eye scissors, with pancreatin and dispase digestion at individual cells, a part is for extracting
Total RNA, after reverse transcription is at cDNA, the mRNA level in-site of fluorescence quantitative PCR detection Rae-1, Mult and H60；
H in Figure 13 shows that Parvifoline AA matches body surface to mouse liver tumour NKG2D in the different plant tumor model of mouse
The influence reached.Another part tumor tissues derived cell is used for the expression of flow cytometer detection cell membrane surface Rae-1, Mult and H60；
I in Figure 13 shows that Parvifoline AA kills load tumor NK cells in mice in the different plant tumor model of mouse and lives
The influence of property.After load tumor mouse spleen takes out, after being shredded with eye scissors, with pancreatin and dispase digestion at individual cells, use
After the method for magnetic bead yin choosing isolates and purifies NK cell, it is divided into two parts, a part carries out the killing activity inspection of NK cell at once
It surveys, for Yac-1 as target cell, the ratio (E:T) of effector cell and target cell is respectively 2.5:1,5:1 and 10:1；
J in Figure 13 shows that Parvifoline AA is active to load tumor NK cells in mice in the different plant tumor model of mouse
It influences.Another part NK cell is tested for degranulation, the expression of FCM analysis NK cell membrane surface CD49b and CD69；
K in Figure 13 shows CD69 in Figure 13 J+NK cell percentages statistical result.Wherein*P < 0.05,**p≤
0.01 He***P≤0.001 indicates statistical significant difference.
Specific embodiment
With reference to the accompanying drawing, essentiality content of the invention is further illustrated with the embodiment of the present invention, but not with
This limits the present invention.
Embodiment 1:
The preparation of Parvifoline AA:
Dry Rabdosia rubescens medicinal material (Isodonrubescens (Hemsley) H.Hara) blade-section 12.5Kg is taken to crush, with
70% acetone/water solution room temperature cold soaking extracts 3 times, and coarse extract is concentrated under reduced pressure to obtain in combined extract, and coarse extract plus water are suspended,
It is successively extracted with ethyl acetate and n-butanol.Ethyl acetate portion is through silica gel column chromatography (chloroform/acetone=1:0；9:1；8:2；7:
3；6:4；1:1, gradient elution) obtain 7 parts: Fr.A-G.Wherein, Fr.C (206g) is through silica gel column chromatography (petroleum ether/acetone
=1:0 → 0:1, gradient elution) obtain 5 parts: Fr.C1-Fr.C5.Fr.C2 purify through semi-preparative HPLC (RP-C18,
MeOH/H2O=60:40), compound Parvifoline AA (20.8mg) is obtained.
Parvifoline AA, Molecule Formula:C20H26O5；MS=346；white amorphous solid
；1H NMR(C5D5N, 500MHz): δ 1.14 (1H, overlap, H-1a), 2.27 (1H, br d, J=13.7Hz, H-1b), 1.13
(1H,overlap,H-2a),1.45(1H,overlap,H-2b),1.14(1H,overlap,H-3a),1.29(1H,br d,J
=13.0Hz, H-3b), 2.12 (1H, s, H-5 β), 1.83 (1H, dd, J=13.6,5.5Hz, H-9 β), 2.53 (1H, m, H-
11a), 1.48 (1H, overlap, H-11b), 1.59 (1H, m, H-12a), 2.39 (1H, m, H-12b), 3.17 (1H, d, J=
9.4Hz,H-13α),5.82(1H,s,H-14α),6.21(1H,s,H-17a),5.40(1H,s,H-17b),1.35(3H,s,Me-
18),0.87(3H,s,Me-19),4.50(1H,br.s,H-20),5.84(1H,br.s,HO-14),8.02(1H,br.s,HO-
20)；
13C NMR(C5D5N,125MHz):δ24.3(t,C-1),19.2(t,C-2),41.0(t,C-3),33.3(s,C-
4),62.1(d,C-5),211.8(d,C-6),90.2(s,C-7),60.2(s,C-8),58.3(d,C-9),45.9(s,C-10),
20.2(t,C-11),32.0(t.C-12),43.2(d,C-13),73.7(d,C-14),204.7(s,C-15),152.8(s,C-
16),116.8(t,C-17),34.8(q,C-18),23.1(q,C-19),81.1(d,C-20)。
Parvifoline AA selectively promotes the expression of liver cancer cells film surface NKG2D ligand.
Present invention application FCM analysis method have detected Parvifoline AA to liver cancer cell lines SMMC-7721,
The influence of tri- plants of cell membrane surface NKG2D ligands (ULBP1, ULBP2, ULBP3 and MICA/B) of HepG2 and Huh7 expression, and
Influence to normal liver cell L-02.As a result, it has been found that Parvifoline AA significantly raises SMMC-7721, HepG2 and Huh7 tri-
Strain cell membrane surface ULBP1, ULBP3 and MICA/B) expression (Fig. 2A, 2B, 2C), and to normal liver cell L-02 film surface
NKG2D ligand expression is without influence.
Embodiment 2:
The transcription of Parvifoline AA promotion Hepatocellular carcinoma cell line cell NKG2D ligand.
The method of present invention application fluorescence quantitative RT-RCR has detected Parvifoline AA to SMMC-7721 cell
The influence of NKG2D ligand transcription.After dosing acts on 24 hours, intracellular total serum IgE, after reverse transcription is at cDNA, fluorescence are extracted
Quantitative RT-PCR detects the transcriptional level of each NKG2D ligand (ULBP1, ULBP2, ULBP3 and MICA/B).As a result, it has been found that
Parvifoline AA has concentration dependent to the transcription facilitation of ULBP1, ULBP3 and MICA/B.
Embodiment 3:
Parvifoline AA promotes NK92 cell by the expression of up-regulation SMMC-7721 cell membrane surface NKG2D ligand
Activity.
Present invention application FCM analysis technology, the specific expressed molecule CD56 of detection NK cell membrane surface and activation
The expression of property molecule CD107 α.The Activating receptor of NK92 cell is closed in advance or do not closed to NKG2D specific antibody
After the function of NKG2D (37 DEG C, 30 minutes), the SMMC-7721 handled through Parvifoline AA and NK92 cell are with 5:1
(NK92:SMMC-7721=5:1) cell number ratio is incubated for altogether, the expression water of flow cytometer detection CD56 and CD107 α after 4 hours
It is flat.Streaming as a result, it has been found that, the SMMC-7721 handled through Parvifoline AA can remarkably promote NK92 compared with solvent control
Cell activation, the expression of CD107 α significantly improve and (are increased to 50.4% from 23.8%) (Fig. 4 A).And the specificity of NKG2D
Antibody closed NK92 cell in advance, even if being incubated for altogether with the SMMC-7721 handled through Parvifoline AA, activation degree
Also considerable decrease (only 15%) (Fig. 4 B).Illustrate the SMMC-7721 cell membrane surface NKG2D promoted by Parvifoline AA
The up-regulation of ligand expression can mediate NK92 cell to pass through the activity of NKG2D receptor-ligand Interaction enhanced NK92 cell.
Embodiment 4:
Parvifoline AA enhances NK92 cell by the expression of up-regulation SMMC-7721 cell membrane surface NKG2D ligand
Killing activity.
Present invention application MTT method detects influence of the Parvifoline AA to SMMC-7721 cytotoxic activity.With difference
After the Parvifoline AA of concentration is handled SMMC-7721 cell 24 hours, mtt assay detects Parvifoline AA to SMMC-
The cytotoxic activity of 7721 cells.As a result, it has been found that IC of the Parvifoline AA to SMMC-7721 cell50For 33.16 μM of (figures
5A).And Parvifoline AA is 20 μM to the effective concentration that SMMC-7721 cell NKG2D ligand expression regulates and controls, and is far below
IC50Value.Lactic dehydrogenase release experiment discovery, by the SMMC-7721 cell that PARVIFOLINE AA is handled, to NK92 cell
Identification and killing show stronger sensibility, and within the scope of centainly effect target ration, with effect target ration
Increase, NK92 cell is to the killing activity of SMMC-7721 cell also linear incremental trend.And NKG2D receptor is special
Property antibody closing after, NK92 cell is inhibited (Fig. 5 B) by most of to the killing activity of SMMC-7721 cell.Illustrate NK92
What the interaction that the cytotoxic activity of cell is to rely on NKG2D-NKG2D ligand was realized.
Embodiment 5:
Parvifoline AA does not directly affect the activity of NK92 cell
Present invention application FCM analysis method detects Parvifoline AA to the direct activity influence of NK92 cell.
After 20 μM of Parvifoline AA is handled NK92 cell 24 hours, specific expressed point of flow cytometer detection NK92 cell membrane surface
The expression of the expression and Activating receptor NKG2D of sub- CD56 and reactivity molecule CD107 α.As a result, it has been found that
Parvifoline AA has no effect on the expression (Fig. 6 A) of the sub- CD56 of NK92 cell membrane surface and reactivity molecule CD107 α,
The expression of NKG2D is not influenced.Illustrate that Parvifoline AA does not directly affect the activity of NK92 cell.
Embodiment 6:
Parvifoline AA is by promoting the generation of the intracellular ROS of SMMC-7721 to adjust NKG2D ligand expression.
The present invention usesGreen kit detects intracellular ROS level.20 μM of Parvifoline AA
After processing SMMC-7721 cell 24 hours, the level of the intracellular ROS of flow cytometer detection.As a result, it has been found that Parvifoline AA energy
Remarkably promote the level of intracellular ROS, and the use of ROS scavenger NAC, the level (Fig. 7 A) of ROS can be significantly reduced.And it is right
The NKG2D ligand of SMMC-7721 cell membrane surface carries out synchronized detection, finds the expression of ULBP1, ULBP3 and MICA/B
Positive correlation is presented in horizontal with ROS, illustrates that Parvifoline AA is the life by promoting the intracellular ROS of SMMC-7721
At adjusting NKG2D ligand expression.
Embodiment 7:
GSH in the combination cell of the part Parvifoline AA.
Present invention application GSH content kit detect Parvifoline AA in the cell/outside and the combination of GSH.SMMC-
7721 cells are handled 24 hours with the Parvifoline AA of various concentration, then by cell cracking, GSH reagent box for detecting content
Detect the level of intracellular GSH.Compound Isoforretin A is as positive control.As a result, it has been found that Parvifoline AA exists
In conjunction with having part with GSH into the cell, but it is far below the combination (Fig. 8 A) of compound Isoforretin A and GSH.And extracellular
In experiment, compound Parvifoline AA, Adenanthin the and Isoforretin A of various concentration are respectively with 5:1 and 10:
1 ratio and 20 μM of GSH are incubated for 2 hours altogether, and the level of intracellular GSH is then detected with GSH reagent box for detecting content.As a result
It was found that in conjunction with Parvifoline AA has part with GSH in the cell, but far below compound Adenanthin,
The combination (Fig. 8 B) of Isoforretin A and GSH.
Embodiment 8:
The activity of Parvifoline AA inhibition peroxidase Peroxiredoxins I/II.
The activity of present invention application Trx-TrxR system detection Peroxiredoxins I/II.In buffer (50mM
HEPES buffer, pH7.0,1mM EDTA) in be separately added into 250 μM of β-NADPH, 0.04U TrxR and 0.1U hTrx-
1, total volume is 180 μ L.Then Prxs-I or Prxs-II albumen (final concentration of 640nM)+difference that total volume is 10 μ L is added
The Parvifoline AA (1 μM, 2 μM, 5 μM or 10 μM) of concentration) mixture.It is eventually adding the H of 10 μ L2O2(final volume is 100 μ
M) starting reaction.Continue the oxidation level of 10 minutes detection β-NADPH under 340nm wavelength using microplate reader.As a result, it has been found that
The activity of Parvifoline AA presentation dose-dependent inhibition Prxs-I/II.
Embodiment 9:
The site the Cys173 covalent bond of Parvifoline AA and Prxs-I.
Modification of the present invention using the method detection Parvifoline AA of LC-MS/MS to Prxs-I albumen.2μg
Recombinant protein Prxs-I and 10 μM of Parvifoline AA be incubated at room temperature 2 hours, then run SDS-PAGE glue, examine horse
After the dyeing of this brilliant blue, after methanol/glacial acetic acid decoloration, destination protein band is cut down, carries out LC-MS/MS detection with true
Determine the decorating site of albumen.First mass spectrometric and the second order ms discovery of Prxs-I, Parvifoline AA covalent bond Prxs-I
The 173rd Cys (Figure 10 A, 10B).
Embodiment 10:
Parvifoline AA regulates and controls SMMC-7721 cell NKG2D ligand by the site Cys173 of targeting Prxs-I
Expression.
The present invention has cloned the Prxs-I plasmid of wild type and the plasmid of the 173rd site mutation (Cys-Ala) first
(Prxs-IM), low-quality grain (shPrxs-I) is struck and for Prxs-I.SMMC-7721 cell transfecting wild type Prxs-I table
Up to plasmid or its 173rd site mutation Cys173-Ala plasmid (Prxs-IM), empty plasmid is as transfection control.It is small to transfect 24
Shi Hou handles cell with 20 μM of Parvifoline AA.After 24 hours, the expression of flow cytometer detection cell membrane surface NKG2D ligand
It is horizontal.As a result, it has been found that wild type Prxs-I's is overexpressed significant sex reversal Parvifoline AA to the upper of NKG2D ligand
It adjusts, and the overexpression of Prxs-IM has no effect on Parvifoline AA to the regulating and controlling effect (Figure 11 A) of NKG2D ligand.When
When Prxs-I is individually struck low, ULBP1, ULBP3 and MICA/B are increased in the expression conspicuousness of cell membrane surface；If
The expression that scavenger NAC, the NKG2D ligand of ROS is handled while Prxs-I strikes low is then inhibited (Fig. 4 .8E to a certain extent
With attached drawing 6)；And PARVIFOLINE AA is added while Prxs-I is struck low and handles cell, the expression of NKG2D ligand is then in
Reveal the effect (Figure 11 B, 11C) of collaboration superposition.The above result shows that PARVIFOLINE AA is by acting on Prxs-I/II
Target spot is to regulate and control the expression of NKG2D ligand.
Embodiment 11:
Parvifoline AA adjusts the expression of NKG2D ligand by activation ERK.
The method of present invention application WB has detected Parvifoline AA in SMMC-7721 cell to ERK and AKT phosphorylation
Influence.20 μM of Parvifoline AA handles SMMC-7721 cell, puts collect cell in different times, cracks, into
Row WB detection, as a result, it has been found that, with gradually increasing for Parvifoline AA processing time, the phosphorylation level of ERK and AKT are in
Reveal variation, wherein after Parvifoline AA is handled 3 hours, the most significant (figure of the phosphorylation elevated levels of ERK and AKT
11A).The processing time is scheduled on 3 hours, Parvifoline AA concentration dependent enhances the phosphorylation level (figure of ERK and AKT
11B).Then we apply ERK phosphorylation inhibitor U0126 and AKT phosphorylation inhibitor MK2206 as tool molecule, as a result
It was found that U0126 and MK2206 can significantly inhibit the phosphorylation (Figure 11 C) of ERK and AKT respectively.U0126 and MK2206 is pre- respectively to be located
For reason after SMMC-7721 cell 3 hours, Parvifoline AA continues with cell, and Flow cytometry is used after 24 hours
The expression of NKG2D ligand.As a result, it has been found that U0126 can significantly reverse Parvifoline AA to make the expression regulation of NKG2D ligand
With, and MK2206 does not influence the expression of Parvifoline AA regulation NKG2D ligand.Illustrate that Parvifoline AA is logical
It crosses activation ERK and adjusts NKG2D ligand expression.
Embodiment 12:
Parvifoline AA passes through the growth of the activity suppression tumour of activation NK cell in vivo.
Application mouse Transplanted tumor model of the present invention evaluates the internal anti-tumor activity of Parvifoline AA.Immune strong
The different plant tumor model of Hepa1-6 rat liver cancer is established in full C57/BL6j mouse, is given using the method for intraperitoneal injection by weight
The dosage daily administration of 5mg/kg and 10mg/kg.Gross tumor volume is measured daily, and mouse weight is monitored.It 12nd day will
Mouse dislocation is put to death, and tumor tissue and spleen tissue are then stripped.Digestion is carried out to tumor tissue and is dispersed into individual cells, a part is used
In the mRNA transcriptional level of fluorescence quantitative RT-RCR detection NKG2D ligand, a part is used for the table of flow cytometer detection NKG2D ligand
It reaches；NK cell in spleen tissue is purified using the method for magnetic bead sorting.As a result, it has been found that compared with solvent control,
Parvifoline AA administration group conspicuousness inhibit Hepa1-6 transplantable tumor volume and weight, and show certain dosage according to
Rely property (Figure 13 A, 13B, 13C).And mouse weight, compared with solvent control, Parvifoline AA administration group is not shown
Difference (Figure 13 D).However the spleen tissue of solvent control group is compared with PARVIFOLINE AA processing group, hence it is evident that enlargement (figure
13E, 13F), illustrate that PARVIFOLINE AA has certain activation and protective effect to the intracorporal immune system of mouse.With it is molten
Agent control group is compared, and Parvifoline AA can significantly raise the transcription of Hepa1-6 cell NKG2D ligand and table in tumor tissues
Up to (Figure 13 G, 13H).And the NK cells in mice of Parvifoline AA administration group has stronger function and killing activity (figure
13I, 13J, 13K).Illustrate that Parvifoline AA is the expression by promoting tumour cell film surface NKG2D ligand in vivo,
To enhance the immunologic cytotoxicity activity of NK cell, and then control the growth of tumour.
Embodiment 13:
Compound Parvifoline AA or other entkaurane diterpenoids are taken, routinely method adds injection
Water, refined filtration can be made into injection after encapsulating sterilizing.
Embodiment 14:
Compound Parvifoline AA or other entkaurane diterpenoids are taken, aseptic injection is dissolved in
It in water, is filtered with sterile funnel, packing, it is sterile after frozen drying to seal up to powder-injection.
Embodiment 15:
Compound Parvifoline AA or other entkaurane diterpenoids are taken, routinely method is equipped with various
Pharmaceutic adjuvant can be made into tablet.
Use compound Parvifoline AA or other entkaurane diterpenoids as pharmaceutical activity at
Point, use several excipient as the adjunct ingredient for preparing composition of medicine tablet, proportion is made every and contains according to a certain percentage
The tablet samples of drug ingedient 1-100mg, table 1 provide the formula rate of conventional tablet.
Certain amount compound Parvifoline AA or other entkaurane diterpenoids and excipient is auxiliary
Material is prepared into various dose tablet formulation (such as table 1): several excipients uniformly being mixed with bulk pharmaceutical chemicals, 1% hydroxyl first is added
Soft material is made in base sodium cellulosate solution in right amount, and sieving granulation, wet grain is dried and whole grain of being sieved, and magnesium stearate is added and talcum powder is mixed
Close uniformly rear tabletting to obtain the final product.
The bulk pharmaceutical chemicals and accessory formula of 1 Ent-kaurane diterpenoid diterpene compound composition of medicine tablet of table
Claims (9)
1. ent-kaurene diterponoid Parvifoline AA is in preparation prevention and immunization therapy hepatic carcinoma drug
In application.
2. ent-kaurene diterponoid Parvifoline AA is preparing answering in the immune sensitizer of tumour cell
With.
3. application as claimed in claim 1 or 2, it is characterised in that the tumour be head and neck neoplasm, tumor in respiratory system,
Digestive system tumor, Patients with Urinary System Tumors, skeletal system tumour, gynecological tumor, hematological system tumor, other types tumour.
4. application as claimed in claim 3, it is characterised in that the head and neck neoplasm is thyroid cancer, nasopharyngeal carcinoma, meninx
Cancer, acoustic neurinoma, hypophysoma, carcinoma of mouth, craniopharyngioma, thalamus and brain stem tumor, angiogenic tumour or intracranial metastatic tumor；Institute
The tumor in respiratory system stated is lung cancer；The digestive system tumor is liver cancer, gastric cancer, the cancer of the esophagus, colorectal cancer, the carcinoma of the rectum, knot
Intestinal cancer or cancer of pancreas；The Patients with Urinary System Tumors is kidney, bladder cancer, prostate cancer or carcinoma of testis；The skeletal system
Tumour is osteocarcinoma；The gynecological tumor is breast cancer, cervical carcinoma or oophoroma；The hematological system tumor be leukaemia,
Malignant lymphoma or Huppert's disease；Other types tumour is malignant mela noma, glioma or cutaneum carcinoma.
5. application as claimed in claim 1 or 2, it is characterised in that the compound Parvifoline AA passes through increase
The regulation of NKG2D ligand expression, and then promote identification and the lethal effect of NK cells against tumor cells.
6. application as claimed in claim 5, it is characterised in that the compound Parvifoline AA passes through induced activity oxygen
Generation, to realize the regulation of tumour cell NKG2D ligand expression.
7. application as claimed in claim 5, it is characterised in that the compound Parvifoline AA passes through targeting
Peroxiredoxins I and II, to realize the regulation of tumour cell NKG2D ligand expression.
8. application as claimed in claim 5, it is characterised in that the compound Parvifoline AA is thin by up-regulation liver cancer
The NKG2D ligand on after birth surface, to realize NK cell to the identification sensibility of liver cancer cells.
9. application as claimed in claim 5, it is characterised in that the compound Parvifoline AA is thin by up-regulation liver cancer
The NKG2D ligand on after birth surface, to realize NK cell to the killing sensibility of liver cancer cells.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111135159A (en) * | 2019-12-27 | 2020-05-12 | 广东省林业科学研究院 | Application of diterpene compound in preparation of tyrosinase inhibitor |
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| CN107206100A (en) * | 2014-10-27 | 2017-09-26 | 弗罗里达中央大学研究基金会 | The method and composition of NK |
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| CN107254439A (en) * | 2009-12-29 | 2017-10-17 | 加米达细胞有限公司 | Strengthen the method for NK propagation and activity |
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| CN111135159A (en) * | 2019-12-27 | 2020-05-12 | 广东省林业科学研究院 | Application of diterpene compound in preparation of tyrosinase inhibitor |
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