CN104997760A - Double-function anti-androgen drug and uses thereof - Google Patents
Double-function anti-androgen drug and uses thereof Download PDFInfo
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Abstract
The present invention relates to a double-function anti-androgen drug, wherein the chemical formula of the anti-androgen drug is C15H12O4, the molecular weight is 256.25, the structural formula is represented by a formula (1), and the compound represented by the formula (1) concurrently has double activity of AR antagonism and Stat3 signal transduction inhibiting. According to the present invention, the compound represented by the formula (1) concurrently has double activity of AR antagonism and Stat3 signal transduction inhibiting; and the experimental results show that the anti-androgen mechanism of the drug is different from the anti-androgen mechanisms of the known anti-androgen drugs such as flutamide and bicalutamide, and the drug can be used as the anti-androgen drug, the prodrug or the precursor compound for prostate cancer treatment.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, the application in especially a kind of androgen antagonist medicine and before the treatment row gland disease, be specifically related to a kind of difunctional androgen antagonist medicine and uses thereof.
Background technology
Cancer is high No. second, the human health " killer " of mortality rate, and the natural anti-cancer drugs of development high-efficiency low-toxicity is one of great scientific and technological task of solving emphatically of current and even following necessary for human.Now conventional chemical anti-tumor drugs also exists the larger shortcoming of toxic and side effects, and limit clinical practice, the natural antitumor medicine therefore developing low toxicity has manifested huge potentiality, and day by day becomes focus and the focus of Chinese scholars research.
Carcinoma of prostate (Prostate Cancer, PCa) is the modal malignant tumor of male reproductive system, and especially in elderly men, sickness rate is the highest.In China, although comparatively European and American developed countries' carcinoma of prostate morbidity and mortality rate low, present the trend of maintaining sustained and rapid growth in recent years, within 2012, become the tumor that Chinese male genitourinary system sickness rate is the highest.
Along with the further investigation to correlation molecule mechanism in PCa course of disease evolution process, the signal path of a series of regulation and control PCa microenvironment, androgen receptor and Bone tumour etc. and key molecule become the important goal that targeted therapy is intervened.The study hotspot that to take AR as targeted therapy PCa be after androgen deprivation therapy (ADT), the medicine of a series of Targeted-control AR is also proved has good anti-PCa effect, AR antagonist mainly flutamide and the bicalutamide of current clinical practice, belong to non-steroid class androgen antagonist, but these two kinds of medicines play an important role at treatment early prostate cancer, Late efficary is poor, also may can produce antiandrogen withdrawal syndrome.In addition if the new drug Abiraterone acetate (Abiraterone Acetate) of U.S. FDA approval listing in 2011 is by suppressing the key enzyme-CYP450c17 in androgen synthesis, reduce androgen levels.The nuclear translocation of MDV-3100 AR capable of blocking of Medivation company of Zai You U.S. recent development and the combination with DNA thereof, play anti-androgen therapy effect, these two kinds of medicines all can make patient's prostate specific antigen (PSA) level reduce, and effectively extend the survival of patients phase.But although AR has facilitation to most of PCa cell proliferation to have research to point out at present, AR has inhibitory action to PCa stem cell (CSCs) and epithelium basal cell propagation; The disappearance that AR expresses also can activate the Stat3 signal of PCa cell, promotes PCa cell proliferation.Therefore also there is the probability impelling PCa disease in the medicine of single targeting AR, relative to single targeted therapy, Mutiple Targets medicine has more advantage, the article that Nature in 2012 delivers adopts poly pharmacology Mutiple Targets strategy to resist cancer, the more single target drug of Mutiple Targets medicine more effectively can affect the complication system of cell, improves therapeutic effect.Up to now, there is no the medicine report about having AR antagonism and Stat3 suppression dual-use function simultaneously.
Summary of the invention
For solving the problems of the technologies described above, the invention provides a kind of difunctional androgen antagonist medicine and uses thereof, this androgen antagonist medicine has AR antagonism simultaneously and suppresses Stat3 intracellular signaling double activity.
For achieving the above object, technical scheme of the present invention is as follows:
A kind of difunctional androgen antagonist medicine, the chemical formula of described androgen antagonist medicine is C
15h
12o
4, molecular weight is 256.25, and structural formula is such as formula shown in (1):
Formula (1) compound has AR antagonism simultaneously and suppresses the double activity of Stat3 intracellular signaling.In the present invention, by formula (1) compound referred to as HYLY008.
In a preferred embodiment of the present invention, comprise further, formula (1) compound is used for the treatment of carcinoma of prostate.
In a preferred embodiment of the present invention, comprise further, the mode that formula (1) compound is used for the treatment of carcinoma of prostate is: androgen antagonist medicine, prodrug or lead compound.
In a preferred embodiment of the present invention, comprise further, formula (1) compound can suppress the activity of prostate cancer cell line LNCaP and PC-3 cell, and it suppresses the effect of LNCaP cell and PC-3 cytoactive to show as: concentration dependent and time dependence.
The suppression prostate cancer cell line LNCaP of formula (1) compound (HYLY008) and the activity of PC-3 cell are studied, result shows: under identical action time, raise with HYLY008 concentration, cell viability reduces, compared with matched group, HYLY008 significantly can suppress the growth vigor of LNCaP and PC-3, and has obvious concentration dependent.For LNCaP cell, the IC50 of HYLY008 effect 24h and 48h is 49.34 μMs respectively, 24.53 μMs, and for PC-3 cell, the IC50 of HYLY008 effect 24h and 48h is 37.21 μMs respectively, 19.59 μMs.25, the HYLY008 of 50,100 μMs is with the increase of action time, and cell viability is remarkable reduction compared with matched group, in obvious time dependence.
In a preferred embodiment of the present invention, comprise further, formula (1) compound can affect the cycle of prostate cancer cell line LNCaP and PC-3 cell, its effect affecting the cycle shows as: have retardation to the S phase of LNCaP cell and PC-3 cell, and effect has time dependence.
The impact of formula (1) compound (HYLY008) on carcinoma of prostate LNCaP and PC-3 cell cycle is studied, and result shows: there is retardation the S phase of HYLY008 to LNCaP and PC-3 cell, and its effect is in regular hour dependency.
In a preferred embodiment of the present invention, comprise further, formula (1) compound can affect prostate cancer cell line LNCaP and PC-3 apoptosis, formula (1) compound is to LNCaP cell and PC-3 cytosis after 48 hours, the percentage ratio of LNCaP apoptotic cell rises to 46.87% from the percentage ratio that 6.41% rises to 17.49%, PC-3 apoptotic cell from 3.76%.
Formula (1) compound (HYLY008) is studied the apoptotic impact of carcinoma of prostate LNCaP and PC-3, flow cytomery result shows: after HYLY008 effect 48h, the percentage ratio of LNCaP apoptotic cell rises to 17.49% from 6.41%, the percentage ratio of PC-3 apoptotic cell rises to 46.87% from 3.76%, illustrates that HYLY008 has significantly apoptosis-induced effect to prostate gland cancer cell.
In a preferred embodiment of the present invention, comprise further, formula (1) compound on prostate cancer LNCaP emiocytosis PSA has influence: formula (1) compound reduces LNCaP emiocytosis PSA level, and it significantly can be lowered the expression of PSA mRNA level in-site and protein level and be dose dependent.
HYLY008 is to the influence research of prostate cancer cell line LNCaP secretion PSA, and result shows: 50 μMs
Formula (1) compound (HYLY008) acts on the level that LNCaP obviously can reduce emiocytosis PSA, and compared with matched group PSA 113.63ng/ml, after effect 48h, PSA drops to 41.55ng/ml.Using 50 μMs of Bic as positive control, after all acting on LNCaP cell 48h, the effect of 50 μMs of HYLY008 downward emiocytosis PSA is suitable with the effect of 50 μMs of Bic.In addition, HYLY008 significantly can lower the expression of PSA mRNA level in-site and protein level and be dose dependent, under 0.1 μM of R1881 stimulates, the PSA of LNCaP cell significantly raises, but under the intervention of HYLY008, have obvious antagonism to the up-regulated of PSA, the PSA level of 0 group or control group is standardized as 1.0 all.
In a preferred embodiment of the present invention, comprise further, formula (1) compound suppresses the expression of AR in LNCaP cell to have influence to being in harmonious proportion under AR signal: formula (1) compound can be lowered the expression of AR albumen and be dose dependent.
Formula (1) compound (HYLY008) is lowered AR signal and is suppressed the expression of AR in LNCaP cell to be studied, result shows: 20,40 or 80 μMs of HYLY008 significantly can lower ARmRNA expression in LNCaP cell, be adjusted downward to 25% respectively, 50% and 85%, the effect of 50 μMs of HYLY008 downward AR is more obvious than positive control drug Bic.And under the induction of 0.1 μM of R1881, AR mrna expression does not add 30% than by R1881 induction group, and after intervention with 10,25 and 50 μMs of HYLY008, the expression of the ARmRNA of 60%, 75% and 80% can be lowered respectively.We can lower the expression of AR albumen by protein immunoblot discovery HYLY008 and be dose dependent further
In a preferred embodiment of the present invention, comprise further, formula (1) compound suppresses the expression of Stat3 in LNCaP cell to being in harmonious proportion under Stat3 signal: formula (1) compound is obviously lowered Stat3mRNA and Stat3 protein expression and is dose dependent.
Formula (1) compound (HYLY008) is lowered Stat3 signal and is suppressed the expression of Stat3 in LNCaP cell to be studied, result shows: 20,40 or 80 μMs of HYLY008 intervene LNCaP cell 24 hours, Stat3mRNA and Stat3 protein expression is obviously lowered and is dose dependent; Adopt 0.1 μM of R1881 induced LNCaP cell 12 or after 24 hours, Stat3 protein level increases 45% and 78% respectively, show to stimulate can regulate Stat3 signal in prostate gland cancer cell by forward by synthetic androgen.Under the intervention of HYLY008, Stat3 pulls back to the expression of negative control group, and 50 μMs of HYLY008 can obviously lower Stat3 protein expression, declines and is about 70% of R1881 induction group
In a preferred embodiment of the present invention, comprise further, formula (1) compound can suppress nude mice prostate gland cancer cell PC-3 growth of xenografted.
Study the suppression nude mice prostate gland cancer cell PC-3 growth of xenografted of HYLY008, result shows: the growth of the obvious Tumor suppression of HYLY00850mg/kg/day energy from the 13rd day, and the gross tumor volume of this group is 40% of matched group volume; Continue administration until the 28th day, 25mg/kg/day and 50mg/kg/day administration group gross tumor volume is (538.4 ± 98.6) mm
3(368.6 ± 62.7) mm
3, and the gross tumor volume of matched group is (992.0 ± 158.9) mm
3; The weight of administration group tumor is respectively (0.36 ± 0.05) g and (0.24 ± 0.06) g, and matched group is (0.63 ± 0.12) g; In process of the test, we carry out gravimetry to Mouse Weight and spleen tissue, find that administration group and matched group do not have significant difference, tentatively point out HYLY008 under dosage 50mg/kg/day not have toxic and side effects to mice.
The invention has the beneficial effects as follows:
One, formula of the present invention (I) compound H YLY008 have AR antagonism simultaneously and suppress the intracellular signaling double activity of Stat3; Experimental result provides the androgen antagonist mechanism of this medicine, and it is different from known androgen antagonist medicine flutamide and bicalutamide, and medicine of the present invention can be used for the treatment of carcinoma of prostate as androgen antagonist medicine, prodrug or lead compound.
Two, the present invention is by about the experimental data of the purposes of formula (I) compound H YLY008, demonstrates the effect that it is applied in row adenocarcinoma medicine before the treatment, has filled up the blank that AR antagonism and Stat3 suppress the medicine of dual-use function.
Accompanying drawing explanation
In order to be illustrated more clearly in the technical scheme in embodiment of the present invention technology, be briefly described to the accompanying drawing used required in the description of embodiment technology below, apparently, accompanying drawing in the following describes is only some embodiments of the present invention, for those of ordinary skill in the art, under the prerequisite not paying creative work, other accompanying drawing can also be obtained according to these accompanying drawings.
Fig. 1 is the inhibitory action of HYLY008 to carcinoma of prostate LNCaP, PC-3 cells growth activity is concentration-dependent result figure;
Fig. 2 is the inhibitory action of HYLY008 to carcinoma of prostate LNCaP, PC-3 cells growth activity is the result figure of time dependence;
Fig. 3 is the period profile figure of LNCaP, PC-3 cell under HYLY008 effect;
Fig. 4 is that HYLY008 blocks LNCaP, PC-3 cell cycle in the S phase and in time dependence (n=3) result figure;
Fig. 5 be HYLY008 on carcinoma of prostate LNCaP, PC-3 percentage of cerebral apoptosis affect result figure;
Fig. 6 is the effect diagram that HYLY008 expresses prostate cancer cell line LNCaP PSA;
Fig. 7 is the effect diagram that HYLY008 expresses prostate cancer cell line LNCaP AR;
Fig. 8 is the effect diagram that HYLY008 expresses prostate cancer cell line LNCaP Stat3;
Fig. 9 is the effect diagram that HYLY008 grows Human Prostate Cancer PC-3 Cell Line transplanted tumor in nude mice.
Detailed description of the invention
Below in conjunction with the accompanying drawing in the embodiment of the present invention, be clearly and completely described the technical scheme in the embodiment of the present invention, obviously, described embodiment is only the present invention's part embodiment, instead of whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art, not making the every other embodiment obtained under creative work prerequisite, belong to the scope of protection of the invention.
Embodiment 1
The object of the present embodiment is suppression LNCaP cell in order to prove formula (1) compound and HYLY008 and PC-3 cytoactive.
In the present embodiment, cell viability adopts multi-functional microplate reader (Synergy HT, Bio-tech, USA) measure, to take the logarithm respectively after cell culture trophophase cell dissociation, centrifugal, abandon supernatant, blow and beat resuspended, the every hole 1 × 10 of LNCaP (purchased from Chinese Academy of Sciences's Shanghai cell bank)
4, the every hole 5 × 10 of PC-3 (biomedical research institute of University Of Suzhou is so kind as to give)
3individual cell passes in 96 well culture plates, every hole 100 μ l, stays a hole not add cell as blank.
After cell attachment, inhale and abandon old culture medium, grouping administration, 37 DEG C, 5%CO
2cultivate 12,24,48 and 72h respectively, abandon culture supernatant, every hole adds not containing the RPMI-1640100 μ l of FBS, 5mg/mlMTT 10 μ l, continue to cultivate 4h, supernatant abandoned by back-off plate, every hole adds DMSO150 μ l, vibration, makes the crystallization of bluish violet formazan dissolve completely, and multi-functional microplate reader detects absorbance (A) at 490nm place.
Calculate: cell viability (Cell Viability)=(A
experimental group-A
blank)/(A
negative control-A
blank).Result is as table 1, and shown in Fig. 1 and 2, HYLY008 suppresses the vigor of human prostata cancer LNCaP and PC-3 cell.Table 1:HYLY008 concentration dependent suppresses human prostata cancer LNCaP cell and PC-3 cells growth activity
Result is: as shown in table 1, under identical action time, raises with HYLY008 concentration, cell viability reduces, compared with matched group, HYLY008 significantly can suppress the growth vigor of LNCaP and PC-3, and has obvious concentration dependent (as shown in Figure 1).As shown in Figure 2, for LNCaP cell, the IC50 of HYLY008 effect 24h and 48h is 49.34 μMs respectively, 24.53 μMs, and for PC-3 cell, the IC50 of HYLY008 effect 24h and 48h is 37.21 μMs respectively, 19.59 μMs.25, the HYLY008 of 50,100 μMs is with the increase of action time, and cell viability is remarkable reduction compared with matched group, in obvious time dependence.
Embodiment 2
The object of the present embodiment proves the impact on LNCaP, PC-3 cell cycle in carcinoma of prostate.
In the present embodiment, trophophase cell dissociation of taking the logarithm, centrifugal, abandon supernatant, blow and beat resuspended, the every hole 5 × 10 of LNCaP
5, the every hole 2.5 × 10 of PC-3
5individual cell passes in 6 orifice plates, every hole 2ml.
After cell attachment, inhale and abandon old culture medium, grouping administration, 37 DEG C, 5%CO
2cultivate 0 respectively, 12,24,48h, digestion, collecting cell, washes 1 time with pre-cooling PBS, adds pre-cooling 70% ethanol, fixed cell, and 4 DEG C are spent the night.Next day, sample is centrifugal, abandons fixative, washes 1 time with pre-cooling PBS, and each sample adds 0.5ml PI/RNase Staining Buffer, and room temperature lucifuge hatches 15min, and in 24h, flow cytometer detects.As shown in Figures 3 and 4, there is retardation the S phase of HYLY008 to human prostata cancer LNCaP and PC-3 cell to result, and its effect is in regular hour dependency.
Embodiment 3
The object of the present embodiment proves that HYLY008 is on the apoptotic impact of carcinoma of prostate LNCaP, PC-3.
In the present embodiment, trophophase cell dissociation of taking the logarithm, centrifugal, abandon supernatant, blow and beat resuspended, the every hole 5 × 10 of LNCaP
5, the every hole 2.5 × 10 of PC-3
5individual cell passes in 6 orifice plates, every hole 2ml.
After cell attachment, inhale and abandon old culture medium, grouping administration, 37 DEG C, 5%CO
2cultivate 0 respectively, 24,48h, pancreatin (not containing EDTA) digestion, collecting cell, pre-cooling PBS (containing 2%FBS) washes 1 time.
Illustrate according to test kit, 1 × Binding buffer 100 μ l re-suspended cell, adds Annexin V-FITC5 μ l, after room temperature lucifuge hatches 15min, add PI 5 μ l, room temperature lucifuge hatches 15min, add 1 × Binding buffer 400 μ l, in 1h, flow cytometer detects.
Result as shown in Figure 5, HYLY008 studies the apoptotic impact of carcinoma of prostate LNCaP and PC-3, flow cytomery result shows: after HYLY008 effect 48h, the percentage ratio of LNCaP apoptotic cell rises to 17.49% from 6.41%, the percentage ratio of PC-3 apoptotic cell rises to 46.87% from 3.76%, illustrates that HYLY008 has significantly apoptosis-induced effect to prostate gland cancer cell.
Embodiment 4
The object of the present embodiment is the impact proving that HYLY008 expresses prostate cancer cell line LNCaP PSA.
In the present embodiment, trophophase cell dissociation of taking the logarithm, centrifugal, abandon supernatant, blow and beat resuspended, the every hole 1 × 10 of LNCaP
5pass in 24 orifice plates, every hole 1ml.After cell attachment, inhale and abandon old culture medium, grouping administration, 37 DEG C, 5%CO
2cultivate 0 respectively, 12,24,48h, collect every hole culture supernatant ,-80 DEG C of preservations are to be measured, avoid multigelation.With reference to PSA ELISA kit operating instruction: add standard substance, quality-control product or sample 100 μ L/ hole, add enzyme labelled antibody 50 μ L/ hole, mixing, shrouding, hatches 1h for 37 DEG C; Wash plate, repeat 3 times; Add developer A liquid 50 μ L/ hole, developer B liquid 50 μ L/ hole, mixing, shrouding, 37 DEG C of lucifuge colour developing 15min; Finally add stop buffer 50 μ L/ hole, in 10min, detect A value in multi-functional microplate reader at 450nm place; Production standard curve, calculates testing sample PSA concentration.
The method of qRT-PCR is adopted to detect the expression of PSA mRNA in LNCaP cell in addition.To take the logarithm trophophase cell dissociation, centrifugal, abandon supernatant, blow and beat resuspended, the every hole 5 × 10 of LNCaP
5pass in 6 orifice plates, every hole 2ml.After cell attachment, inhale and abandon old culture medium, grouping administration, 37 DEG C, 5%CO
2cultivate 24h, digestion, PBS washes 1 time, cell total rna to be extracted.Every 1 × 10
6cell concentration, adds 1mlTrizol, fully blows and beats; Incubated at room 5min, adds pre-cooling chloroform 200 μ L/1ml Trizol, thermal agitation 15s, incubated at room 5min; 4 DEG C of centrifugal 12000g, 15min; Draw upper strata, about 400-600 μ L/1mlTrizol; Add the isopropyl alcohol 500 μ L/1ml Trizol of pre-cooling, put upside down mixing, incubated at room 10min; 4 DEG C of centrifugal 12000g, 10min, obtain white gels sample precipitation and RNA; Abandon supernatant, with pre-cooling 75% ethanol 1ml/EP, washing RNA; 4 DEG C of centrifugal 7500g, 5min, abandon supernatant, air at room temperature drying 15 ~ 30min; Add pre-cooling RNase-free Water and dissolve RNA; Survey RNA concentration with Nanodrop, becoming cDNA by meeting the total serum IgE reverse transcription of purity requirement A260/A280 between 1.8-2.0, finally carrying out qRT-PCR amplification.
Amplification program is: 95 DEG C, 5min → (95 DEG C, 15s → 60 DEG C, 60s → 72 DEG C, 30s) 40 circulation, and using β-actin as reference gene, the relative expression quantity of genes of interest calculates according to-△ △ CT, and wherein the primer sequence of PSA is:
Forward：5’-GTTGTCTTCCTCACCCTGTCCG-3’；
Reverse：5’-AGCAAGATCACGCTTTTGTTCCTG-3’；
The primer sequence of β-actin is:
Forward:5’-TGACGTGGACATCCGCAAAG-3’；
Reverse:5’-CTGGAAGGTGGACAGCGAGG-3’。
In addition, the method for Western Blotting is adopted to detect the expression of PSA albumen in LNCaP cell.To take the logarithm trophophase cell dissociation, centrifugal, abandon supernatant, blow and beat resuspended, the every hole 1 × 10 of LNCaP
6pass in 25cm
2culture bottle.After cell attachment, inhale and abandon old culture medium, grouping administration, 37 DEG C, 5%CO
2cultivate 24h, scrape cell, 1200rpm, centrifugal 5min, abandons supernatant, washes 1 time with pre-cooling PBS; Add appropriate RIPA lysate, thermal agitation 5 ~ 10min, ice bath 30min; 4 DEG C of centrifugal 12000g, 10min, retain supernatant ,-80 DEG C of preservations, testing protein concentration.Protein concentration is detected by BCA method.Add 5 × Loadingbuffer, 100 DEG C, 5min albuminous degeneration, fast cold on ice.
Carry out SDS-PAGE electrophoresis to the protein sample of degeneration, transfer printing albumen is to pvdf membrane subsequently, transferring film condition 250mA, 2.5h, then closes with in 5% defatted milk, and room temperature shakes 1.5h at a slow speed, and primary antibodie is hatched, and 4 DEG C are spent the night; TBST washes film 3 times, 10min/ time; Two anti-hatch, and room temperature shakes 1h at a slow speed; TBST washes film 3 times, 10min/ time, the imaging of Odyssey infrared imaging system.
HYLY008 is to the influence research of prostate cancer cell line LNCaP secretion PSA, result as shown in Figure 6, show: 50 μMs of HYLY008 act on the level that LNCaP obviously can reduce emiocytosis PSA, compared with matched group PSA 113.63ng/ml, after effect 48h, PSA drops to 41.55ng/ml.Using 50 μMs of Bic as positive control, after all acting on LNCaP cell 48h, the effect of 50 μMs of HYLY008 downward emiocytosis PSA is suitable with the effect of 50 μMs of Bic.In addition, HYLY008 significantly can lower the expression of PSA mRNA level in-site and protein level and be dose dependent, under 0.1 μM of R1881 stimulates, the PSA of LNCaP cell significantly raises, but under the intervention of HYLY008, has obvious antagonism to the up-regulated of PSA.The PSA level of 0 group or control group is standardized as 1.0 all.
Embodiment 5
The object of the present embodiment is the impact proving that HYLY008 expresses prostate cancer cell line LNCaP AR.
In the present embodiment, adopt the method for qRT-PCR to detect the expression of AR mRNA in LNCaP cell, concrete grammar is with qRT-PCR part in embodiment 4, and wherein the primer sequence of AR is:
Forward：5’-CCTATCCCAGTCCCACTTGTGTCA-3’；
Reverse：5’-TACTTCTGTTTCCCTTCAGCGGC-3’。
In addition, the method for Western Blotting is adopted to detect the expression of AR albumen in LNCaP cell; Concrete grammar is with Western Blotting part in embodiment 4.
HYLY008 lowers AR signal and suppresses the expression of AR in LNCaP cell to be studied, result is as shown in Figure 7: 20,40 or 80 μMs of HYLY008 significantly can lower ARmRNA expression in LNCaP cell, be adjusted downward to 25% respectively, 50% and 85%, the effect of 50 μMs of HYLY008 downward AR is more obvious than positive control drug Bic.And under the induction of 0.1 μM of R1881, AR mrna expression does not add 30% than by R1881 induction group, and after intervention with 10,25 and 50 μMs of HYLY008, the expression of the AR mRNA of 60%, 75% and 80% can be lowered respectively.
Further by protein immunoblot find HYLY008 can lower AR albumen expression and in dose dependent.
Embodiment 6
The object of the present embodiment is the impact proving that HYLY008 expresses prostate cancer cell line LNCaP Stat3.
In the present embodiment, adopt the method for qRT-PCR to detect the expression of Stat3mRNA in LNCaP cell, concrete grammar is with qRT-PCR part in embodiment 4, and wherein the primer sequence of Stat3 is:
Forward:5’-TGCTGACCAACAATCCCAAGAATG-3’；
Reverse:5’-TTTAGCCCATGTGATCTGACACCC-3’。
In addition, the method for Western Blotting is adopted to detect the expression of Stat3 albumen in LNCaP cell.Concrete grammar is with Western Blotting part in embodiment 4.As shown in Figure 8, the expression of HYLY008 to LNCaP cell Stat3 has obvious downward effect to result.
HYLY008 lowers Stat3 signal and suppresses the expression of Stat3 in LNCaP cell to be studied, and result shows: 20,40 or 80 μMs of HYLY008 intervene LNCaP cell 24 hours, Stat3mRNA and Stat3 protein expression is obviously lowered and is dose dependent; Adopt 0.1 μM of R1881 induced LNCaP cell 12 or after 24 hours, Stat3 protein level increases 45% and 78% respectively, show to stimulate can regulate Stat3 signal in prostate gland cancer cell by forward by synthetic androgen.Under the intervention of HYLY008, Stat3 pulls back to the expression of negative control group, and 50 μMs of HYLY008 can obviously lower Stat3 protein expression, declines and is about 70% of R1881 induction group.
Embodiment 7
The object of the present embodiment proves that the growth of HYLY008 to Human Prostate Cancer PC-3 Cell Line transplanted tumor in nude mice has inhibitory action.
In the present embodiment, set up PC-3 Nude Mouse Model, trophophase PC-3 cell of taking the logarithm, digestion, counting, cell density 1 × 10
7only, subcutaneous vaccination is in BALB/c nude mice right fore axillary fossa in 6 week age for individual/ml, 0.2ml/.Within every two days, observe once, become tumor after one week, use vernier caliper measurement gross tumor volume, grow to average about 50mm to gross tumor volume
3, PC-3 tumor bearing nude mice is divided into 3 groups at random by gross tumor volume, and often organizing 8, is Control group respectively, HYLY00825mg/kg group and HYLY00850mg/kg group.HYLY008 powder is dissolved in the normal saline containing 20%1,2-propylene glycol, is mixed with 25,50mg/kg medicinal liquid, the normal saline of Control group containing 20%1,2-propylene glycol.Lumbar injection, successive administration 28 days, every day period measures nude mice body weight, as the index of nude mice health status; Within three days, measure gross tumor volume once: V (mm
3)=0.5 × L × W
2(L: tumor major diameter; W: tumor minor axis); Administration is after 28 days, and disconnected neck is put to death, and peels off every nude mouse tumor tissue, and normal saline cleans, and takes pictures by group arrangement; Each tumor is weighed respectively.
Study the suppression nude mice prostate gland cancer cell PC-3 growth of xenografted of HYLY008, result such as Fig. 9 shows: the growth of the obvious Tumor suppression of HYLY00850mg/kg/day energy from the 13rd day, and the gross tumor volume of this group is 40% of matched group volume; Continue administration until the 28th day, 25mg/kg/day and 50mg/kg/day administration group gross tumor volume is (538.4 ± 98.6) mm
3(368.6 ± 62.7) mm
3, and the gross tumor volume of matched group is (992.0 ± 158.9) mm
3; The weight of administration group tumor is respectively (0.36 ± 0.05) g and (0.24 ± 0.06) g, and matched group is (0.63 ± 0.12) g; In process of the test, we carry out gravimetry to Mouse Weight and spleen tissue, find that administration group and matched group do not have significant difference, tentatively point out HYLY008 under dosage 50mg/kg/day not have toxic and side effects to mice.
Confirmed by the experimental result of embodiment 1-7, formula of the present invention (I) compound H YLY008 has AR antagonism simultaneously and suppresses the intracellular signaling double activity of Stat3; Experimental result provides the androgen antagonist mechanism of this medicine, and it is different from known androgen antagonist medicine flutamide and bicalutamide, and medicine of the present invention can be used for the treatment of carcinoma of prostate as androgen antagonist medicine, prodrug or lead compound.
The experimental technique of unreceipted actual conditions in above-described embodiment, usually conveniently condition as people such as Li Yan, Zhang Jian, in the condition described in Cytobiology and molecular biology common experimental technology (2009), or according to the condition that manufacturer advises.Unless otherwise indicated, in literary composition use reagent from Hyclone company of the U.S., Shanghai Xi Tang Science and Technology Ltd., Shanghai Shen work biology, Wei Ao bio tech ltd, Shanghai, Japanese Takara company, Beijing Quanshijin Biotechnology Co., Ltd, traditional Chinese medicines reagent company limited, Sigma Co., USA, Applied biosystems company of the U.S., percentage ratio and number are calculated by weight.
To the above-mentioned explanation of the disclosed embodiments, professional and technical personnel in the field are realized or uses the present invention.To be apparent for those skilled in the art to the multiple amendment of these embodiments, General Principle as defined herein can without departing from the spirit or scope of the present invention, realize in other embodiments.Therefore, the present invention can not be restricted to these embodiments shown in this article, but will meet the widest scope consistent with principle disclosed herein and features of novelty.
Claims (10)
1. a difunctional androgen antagonist medicine, is characterized in that, the chemical formula of described androgen antagonist medicine is C
15h
12o
4, molecular weight is 256.25, and structural formula is such as formula shown in (1):
Formula (1) compound has AR antagonism simultaneously and suppresses the double activity of Stat3 intracellular signaling.
2. the purposes of difunctional androgen antagonist medicine according to claim 1, is characterized in that, formula (1) compound is used for the treatment of carcinoma of prostate.
3. the purposes of difunctional androgen antagonist medicine according to claim 2, is characterized in that, the mode that formula (1) compound is used for the treatment of carcinoma of prostate is: androgen antagonist medicine, prodrug or lead compound.
4. the purposes of difunctional androgen antagonist medicine according to claim 2, it is characterized in that, formula (1) compound can suppress the activity of prostate cancer cell line LNCaP and PC-3 cell, and it suppresses the effect of LNCaP cell and PC-3 cytoactive to show as: concentration dependent and time dependence.
5. the purposes of difunctional androgen antagonist medicine according to claim 2, it is characterized in that, formula (1) compound can affect the cycle of prostate cancer cell line LNCaP and PC-3 cell, its effect affecting the cycle shows as: have retardation to the S phase of LNCaP cell and PC-3 cell, and effect has time dependence.
6. the purposes of difunctional androgen antagonist medicine according to claim 2, it is characterized in that, formula (1) compound can affect prostate cancer cell line LNCaP and PC-3 apoptosis, formula (1) compound is to LNCaP cell and PC-3 cytosis after 48 hours, the percentage ratio of LNCaP apoptotic cell rises to 46.87% from the percentage ratio that 6.41% rises to 17.49%, PC-3 apoptotic cell from 3.76%.
7. the purposes of difunctional androgen antagonist medicine according to claim 2, it is characterized in that, formula (1) compound on prostate cancer LNCaP emiocytosis PSA has influence: formula (1) compound reduces LNCaP emiocytosis PSA level, and it significantly can be lowered the expression of PSA mRNA level in-site and protein level and be dose dependent.
8. the purposes of difunctional androgen antagonist medicine according to claim 2, it is characterized in that, formula (1) compound suppresses the expression of AR in LNCaP cell to have influence to being in harmonious proportion under AR signal: formula (1) compound can be lowered the expression of AR albumen and be dose dependent.
9. the purposes of difunctional androgen antagonist medicine according to claim 2, it is characterized in that, formula (1) compound suppresses the expression of Stat3 in LNCaP cell to being in harmonious proportion under Stat3 signal: formula (1) compound is obviously lowered Stat3mRNA and Stat3 protein expression and is dose dependent.
10. the purposes of difunctional androgen antagonist medicine according to claim 2, is characterized in that, formula (1) compound can suppress the growth of nude mice prostate gland cancer cell PC-3 transplanted tumor.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1156024A (en) * | 1996-02-02 | 1997-08-06 | 南京中医药大学 | Antiarrhythmic containing isoliquirtin |
| CN1961889A (en) * | 2005-11-09 | 2007-05-16 | 北京大学 | Application of isoliquiritigenin in preparation of medicament for preventing and/or treating depression |
| CN101152165A (en) * | 2007-09-06 | 2008-04-02 | 武汉大学 | Antineoplastic isoliquirtigenin tablet |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1156024A (en) * | 1996-02-02 | 1997-08-06 | 南京中医药大学 | Antiarrhythmic containing isoliquirtin |
| CN1961889A (en) * | 2005-11-09 | 2007-05-16 | 北京大学 | Application of isoliquiritigenin in preparation of medicament for preventing and/or treating depression |
| CN101152165A (en) * | 2007-09-06 | 2008-04-02 | 武汉大学 | Antineoplastic isoliquirtigenin tablet |
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