CN106581009A - Composition having synergistic antitumor effect - Google Patents

Abstract

The present invention discloses a composition having a synergistic antitumor effect, wherein the composition comprises cryptotanshinone and curcumol. According to the present invention, the research results show that the combined use of cryptotanshinone and curcumol can produce the special antitumor molecular mechanism, wherein curcumol can eliminate the AKT activation effect produced by the mTOR inhibition of cryptotanshinone so as to provide the synergistic effect, the significant antitumor effect is provided, and the obvious adverse reaction does not exist; and the antitumor composition provides significant treatment effects on breast cancers and prostate cancers.

Description

A kind of compositionss with synergistic antitumor effect

Technical field

The invention belongs to drug world, and in particular to a kind of compositionss with antitumor action.

Background technology

Cancer has become the serious commonly encountered diseases and frequently-occurring disease for threatening human life, and sickness rate is into the trend of cumulative year after year.China's cancer mortality accounts for the 2nd of the various diseases cause of the death, has occupied first place in city.Cancer not only causes physiological huge injury to patient, while psychologically, mentally also bringing severe trauma to patient, and causes the heavy burden of family and society.

At present, the selectivity of the big multipair inhibiting tumour cells of antitumor drug for clinically using is not strong, and systemic administration toxicity is larger, and while anticancer grows, normal cell growth is also suppressed.Therefore, it is still an extremely urgent and important global task to research and develop the novel antitumor drug of high-efficiency low-toxicity.In recent years, substantial amounts of Clinical and experimental study proves, many Chinese medicine compound and many compositions from Chinese medicine play the role of good in terms for the treatment of and prevention of tumour and rehabilitation, have been increasingly becoming people's concern and the emphasis studied.

Cryptotanshinone is a kind of effective ingredient in salviamiltiorrhizabung.Modern pharmacological research shows that cryptotanshinone has the effect such as antiinflammatory, antibacterial, induction liver drug enzyme, and significantly improves cardiovascular and cerebrovascular disease activity.However, recently for the research of cryptotanshinone is concentrated on its antitumor action.Many researchs confirm that cryptotanshinone shows the effect of obvious anticancer propagation in vitro, and this is relevant with its inducing apoptosis of tumour cell, promotion tumor cell differentiation.Find in our further investigations to cryptotanshinone antitumor mechanism, cryptotanshinone can be by suppressing mTOR (mammalian target of rapamycin, mammal rapamycin target protein) signal transduction pathway, so as to suppress cyclin Cyclin D and Rb, induction of cell cycle arrest is in the G0 phases, ultimately result in cell death (Cancer Prevention Research.2010,3 (8)：1015-1025).

MTOR is an important kinases in the whole process key links such as cellular metabolism, growth, propagation are adjusted.Interference suppresses mTOR that the growth retardation of cell especially cancerous cell can be caused then dead, thus an important target being also acknowledged as in the middle for the treatment of of cancer.Current mTOR inhibitors Rapamycin (rapamycin) and the like have been formed and have been applied in clinical antineoplastic, but single Rapamycin analog does not obtain intended effect to the treatment of most of tumors in clinical trial.

With going deep into for research, it is found that mTOR signal paths have feedback activation mechanism：Suppress the multiple protein signal paths related to growth and proliferation of cell is promoted of mTOR energy feedback activation, including PI3K/AKT, MAPK/ERK, Mnk/eIF4E.And it is considered that these feedback activation mechanisms weaken the efficiency of mTOR inhibitors, this is also its clinical effect not up to the reason for being expected.Thus researcher proposes then can should effectively prevent the generation of this feedback mechanism simultaneously using mTOR inhibitors and other protein signal pathway inhibitors, so as to play more preferable antitumor action (Chinese clinical tumor, 2011,38 (24)：1597-1599).

The content of the invention

The technical problem to be solved is to provide a kind of compositionss with synergistic antitumor effect, and the composition is clear and definite, stable curative effect.

A kind of compositionss for strengthening antitumor action with collaboration, as made by following raw materials in part by weight medicine proportioning：

4 parts of cryptotanshinone, 6 parts of curcumenol.

Preferably, it is by made by following weight is than proportioning：5 parts of cryptotanshinone, 5 parts of curcumenol.

Preferably, it is by made by following weight is than proportioning：6 parts of cryptotanshinone, 4 parts of curcumenol.

Preferably, it is by made by following weight is than proportioning：7 parts of cryptotanshinone, 3 parts of curcumenol

Above-mentioned active component can be extracted by prior art or be prepared, it is also possible to directly commercially.

Above-mentioned active component prepares the compound combination for the treatment of tumor by being simply mixed according to formula ratio.

Application of the aforementioned pharmaceutical compositions in antitumor drug is prepared.

Synergistic antitumor acts on new mechanism：Suppress the multiple protein signal paths related to growth and proliferation of cell is promoted of mTOR energy feedback activation, including PI3K/AKT, MAPK/ERK, Mnk/eIF4E, this is the main cause of such compound antitumor limited efficacy.And cryptotanshinone can suppress mTOR paths, and also there is inhibitory action to MAPK/ERK and Mnk/eIF4E, but while activate PI3K/AKT.Therefore, in the present composition, cryptotanshinone combination suppresses the curcumenol of AKT, then, while mTOR is suppressed, prevent feedback mechanism from activating, and produces synergistic antitumor effect.

Description of the drawings

Fig. 1 explanations：The impact that cryptotanshinone is individually expressed to MCF-7 cells AKT with combination curcumenol.

Specific embodiment

According to following embodiments, the present invention may be better understood.However, as it will be easily appreciated by one skilled in the art that be merely to illustrate the present invention described by embodiment, and should not also without limitation on the present invention described in detail in claims.

Embodiment 1：

A kind of compositionss with antitumor action, it is obtained by the component mix homogeneously of following parts by weight：

6 parts of cryptotanshinone

4 parts of curcumenol

Embodiment 2：The antitumor action of the present composition

1. experimental technique

1.1. material and reagent

Cryptotanshinone, curcumenol (Xi'an and continuous heavy rain biological engineering company limited)；Calf serum (Lanzhou people's marine growth Engineering Co., Ltd, AKT, p-AKT (Santa Cruz, CA, USA)；β-tubulin；MTT(sigma)；1640 culture medium (Gibico) 1.2. cell lines, cell culture and animal

MCF-7 cells are provided with safety key lab by Jiangsu Province's herbal medicine efficacy.Human umbilical vein epithelial cell (HUVEC) carries out primary separation from Placenta Hominiss.Cleaning grade Balbc nude mices, Shanghai Slac Experimental Animal Co., Ltd., credit number：SCXK (Shanghai) 2012-0002；Feeding environment：22 ± 2 DEG C of temperature and relative humidity (55 ± 10%, illumination 12-hr illumination (8：00-20：00) with the circulation of 12-hr lucifuges, free water and diet.

1.3.MTT

The cell for taking exponential phase of growth makes individual cells suspension, is inoculated in 96 orifice plates with 104, every hole cell, per 100 μ l of pore volume.Flat board is put into 37 DEG C of CO2 (5%) incubators 24 hours.5 concentration of test-compound add 96 orifice plates, and 37 DEG C, are incubated 48h in the incubator containing 5%CO2 and 100% humidity.Supernatant is sucked carefully, PBS is gently washed, and abandons supernatant.Add per hole the fresh RPMI RPMI-1640s of 180 μ l, MTT PBS to be made into 5mg/ml solution, add 20 μ l per hole, 37 DEG C incubate 4 hours.Supernatant is suctioned out, 150 μ l DMSO is added per hole, is shaken up, microplate reader determines optical density at 570nm.Different IC50 values of the compound to MCF-7 and DU145 cells are calculated according to data.

1.4.Western Blot

Cell protein quality sample is obtained：2×10⁵B16BL6 cells are inoculated in six orifice plates, add many concentration of test samples, inhale and abandon culture fluid, add sample-loading buffer (50 mM Tris, pH 7.2 after effect 48h after 24h；150 mM NaCl；1%sodium deoxycholate；0.1%SDS；1%Triton-X 100；10 mM NaF；1 mM Na₃VO₄；Proteaseinhibitor), cell pyrolysis liquid, ultrasound 15 seconds 2 times, 100 DEG C of heat denatured albumen are collected.

Electrophoresis：Running gel (12%) is prepared, SDS-PAGE is carried out.

Transferring film：(wet method transfer) film process：Cut out in advance and an equal amount of filter paper of adhesive tape and pvdf membrane, 10min in immersion transferring film buffer.Transferring film：Membrane-transferring device is put well by the order of carbon anode plate, 2 metafiltration paper, pvdf membrane, gel, 2 metafiltration paper, negative electrode carbon plate from bottom to up successively, filter paper, gel, pvdf membrane Accurate align, and each step goes bubble removing, upper pressure implant to fill it up with transferring film liquid.Switch on power, constant pressure 24V shifts 3h.After transferring film terminates, deenergization takes the film out.

Immunoreation：Film is washed, 5% defatted milk powder coating buffer coating 1h washes film.One is added to resist, 4 DEG C of placement more than 12h.Film being washed again, adding corresponding two to resist, 4 DEG C of placement more than 12h wash film.Film adds ECL nitrite ions, light reaching the film in magazine, developing machine developing fixing.

1.5. mouse entity models of tumor growth

The good MCF-7 breast cancer cells of adherent growth are digested with 0.25% pancreatin, plus supernatant is abandoned in 800 rpm of culture fluid centrifugations, then with serum-free medium suspension tumor cell, adjust cell number.Then in the subcutaneous place's inoculated tumour cell suspension 0.2ml of every right side of mice groin (about 10⁶It is individual), inoculation next day starts gastric infusion, and continuous 28 days, daily observation measured the subcutaneous place's tumour growth situation of mice groin.Administration puts to death mice after terminating, and strips separation tumor tissue, with scales/electronic balance weighing, records knurl weight.

2. experimental result

2.1. the impact that the present composition is bred to MCF-7 cells and AKT is expressed

Find out from 1 result of table, the alone IC50 values to MCF-7 and DU145 cells of cryptotanshinone are respectively 3.01ug/ml and 1.04 μ g/ml, and the alone IC50 values to MCF-7 and DU145 cells of curcumenol are respectively 9.50 μ g/ml and 13.43 μ g/ml.And 1.25 μ g/ml and 0.67 μ g/ml after cryptotanshinone and curcumenol combination, are respectively to the IC50 values of MCF-7 and DU145 cells, its IC50 value has more obvious reduction, and the effect of two medicines collaboration anticancer after illustrating to be combined is strengthened.

Fig. 1 results show that cryptotanshinone (CPT) is alone can substantially to activate AKT, the expression of p-AKT is substantially increased；Curcumenol (curcumol) then has obvious inhibiting effect to the expression of p-AKT；Equally, the expression of p-AKT is reversed to suppress by activation after cryptotanshinone is combined with curcumenol.Illustrate that curcumenol essentially eliminates cryptotanshinone by suppressing mTOR and AKT that feedback causes activation, and then enhance the antitumor action of cryptotanshinone.(effect of the cryptotanshinone to mTOR can be found in document Cancer Prevention Research.2010, and 3 (8)：1015-1025)

1. cryptotanshinone of table is independent and is combined IC50 value of the curcumenol to MCF-7 and DU145 cells

2.2. impact of the present composition to nude mice model tumour growth

The tumor weight of 2 result of table display alone group of mice of cryptotanshinone is compared with model group significant difference (p ＜ 0.05), and alone group of mice of curcumenol compares then no significant difference with model group.Cryptotanshinone is heavy with the mouse tumor of curcumenol combination group significantly less than bright group (p ＜ 0.01).These results suggest that cryptotanshinone suppresses tumour growth effect obvious with curcumenol combination, and effect is better than alone group of cryptotanshinone.

2. cryptotanshinone of table is independent and is combined impact of the curcumenol to nude mice model tumour growth

Compare with model group, * p ＜ 0.05, * * p ＜ 0.01.

3. conclusion

Result above shows that curcumenol can significantly increase inhibitory action of the cryptotanshinone to MCF-7 and DU145 cancerous cell, while promoting cryptotanshinone to suppress nude mouse xenograft in body tumour growth.And this suppresses the expression of p-AKT related to curcumenol, curcumenol suppresses AKT to eliminate the effect that cryptotanshinone activates AKT, thus enhances the antitumor action of cryptotanshinone.The two combination, can significantly increase the anti-tumor activity of cryptotanshinone.

Claims (3)

1. it is a kind of with synergistic antitumor effect compositionss, it is characterised in that containing chemical composition：Cryptotanshinone And curcumenol (curcumol) (cryptotanshinone).

2. in compositionss according to claim 1 the content ratio (mass ratio) of cryptotanshinone between 40%-70% Between.

3. tumor according to claim 1 is breast carcinoma and carcinoma of prostate.