CN106692141A - Applications of berberine in preparing drugs used for treating stomach cancer - Google Patents

Abstract

The invention discloses novel applications of berberine in preparing drugs used for treating stomach cancer. It is confirmed by experiments that berberine is capable of inhibiting growth of human stomach cancer BGC-823 cells; the inhibition effect is related to BGC-823 autophagy induced by berberine; and critical regulating effect in inhibition of growth of human stomach cancer BGC-823 cells is realized by MAPKs family, Akt and mTOR protein. It is confirmed by in vivo experiments that berberine is capable of inhibiting the growth of tumor tissue. Berberine is low in toxicity and excellent in curative effect, so that application prospect of berberine in clinical treatment of stomach cancer is promising.

Description

Application of the jamaicin in treatment gastric cancer medicament is prepared

Technical field

The invention belongs to pharmaceutical technology field, it is related to jamaicin medical usage, and in particular to it is being controlled Treat the purposes of stomach cancer.

Background technology

Stomach cancer is most common malignant tumor of digestive tract, and Asia Japan, South Korea and China are stomach cancers high Hair area, the annual new cases of China about 400,000, account for the world always fall ill number of cases 42% (China is real With internal medicine journal, 2014,34 (4):408-415), 160,000 people are there are about every year die from stomach cancer.Early carcinoma of stomach Discovery rate it is low, the stomach cancers accepted for medical treatment of majority are advanced gastric carcinoma, and surgery alone is often difficult to effect a radical cure, and are changed Treatment plays a significant role in Comprehensive Therapy on Gastric Carcinoma, one of Main Means as treatment stomach cancer, but Therapeutic effect (Chinese clinical tumor, 2010,37 (3) still not fully up to expectations:171-175；Fudan Journal, 2002,29(4):323-325).Therefore, new efficient, low toxicity medicine is found to compel for treating stomach cancer In the eyebrows and eyelashes.

In recent years, the antineoplastic of plant origin is attracted attention again, according to statistics mesh Preceding antineoplastic about 30% is from natural resources and the derivative of native compound.It Not only there is unique physiologically active, preferable curative effect and relatively low toxicity, be even more that chemistry is closed Into, chemical modification provide novel and unique chemical constitution (Journal of Natural Products, 2007,70(3):461-477)。

Jamaicin (Berberine), also known as berberine, is from the ranunculaceae plant coptis (Coptidis Rhizoma a kind of isoquinoline alkaloid extracted in dry rhizome), is time-honored China One of conventional medicament, according to existing more than the 3000 years medicinal history of literature record, is clinically mainly used in Treatment enterogastric diseases.Recent study find, jamaicin autoimmune disease, diabetes, The fields such as anti-arrhythmia equally have therapeutic value (Arthritis＆Rheumatology, 2011, 63(4):949-959；European Journal of Pharmacology,2009,606(1-3):262-268； Carcinogenesis,2011,32(1):86-92).Additionally, jamaicin also has antitumor activity, it is many It is obvious that item research shows that jamaicin has to kinds of tumor cells such as breast cancer, colon cancer, liver cancer Inhibition (CHINA JOURNAL OF CHINESE MATERIA MEDICA, 2007,32 (10):881-934；Journal of Ethnopharmacology,1999(66):227-233；Journal of Cellular Biochemistry, 2010(109):329-338)。

Autophagy (Autophagy) is intracellular material composition to be degraded process using lysosome, is Eukaryotic institute is peculiar, and the degraded for being responsible for longevity albumen and some organelles is utilized.Autophagy is cell One kind reaction to internal and external environment pressure change, for maintaining protein metabolism balance and cellular environment steady Surely play an important role, autophagy also can induce cell death in some cases, it is considered to be difference In another apoptosis form (the II type journey of Apoptosis (I type programmed death) Sequence is dead).Tumour is frequently accompanied by the disorder of intraor extracellular environment, and autophagy is being maintained Cellular environment stable state aspect plays an important role.Therefore, autophagy is closely related with tumour.

MTOR (Mammalian target of rapamycin, mTOR) kinases is amino acid, ATP With the receptor of hormone, cell growth has important regulative, is the negativity regulation and control point of autophagy Son, and play " entrance guard (gatekeeper) " effect (Current Opinion in Cell Biollogy, 2005, 17(6):596-603).MTOR kinases can by directly or indirectly promote Apg13 phosphorylations, and Prevent it from being combined with Apg1, so as to suppress the generation of autophagy；It can also directly suppress ATG1 The activity of kinases suppresses generation (the Molecular Biology of the Cell, 2009,20 of autophagy: 1991-2003).Rapamycin (Rapamycin) is played and suppressed by suppressing the activity of mTOR P70S6 (p70 Ribosomal S6 kinases) is active and induces autophagy to occur effect (Cancer, 2004,100 (4):657-666；Nature Reviews Cancer,2004,4(5):335-348).MTOR kinases is located at Akt/PKB downstreams, the growth factor signal approach that it mediates Akt/PKB is integrated with autophagy approach To together, hinge as autophagy regulation (Current Medicinal Chemistry, 2013,20 (15): 1923-1945)。

PI3K is a protease family for participation diphosphoinositide, in cell development, is divided Play an important roll in change and breeding.I types PI3K is the negative regulator of autophagy, and it can phosphoric acid Change 4- phosphoric acid phosphatidylinositols (PtdIns4P) and 4,5- diphosphonic acid phosphatidylinositols (PtdIns [4, 5] P2), generation 3,4- diphosphonic acid phosphatidylinositols (PtdIns [3,4] P2) and 3,4,5- triphosphoric acid phosphorus Acyl inositol (PtdIns [3,4,5] P3), then in conjunction with Akt/PKB, and activates Akt/PKB and presses down Generation (the Trends in Cell Biology, 2010,20 of autophagy processed:355-362).Type III PI3K is certainly Positive regulator bitten, with the process that its substrate PI3P promotes autophagy, the inhibitor 3-MA of type III PI3K The formation of autophagosome precursor can effectively be suppressed, the early stage rank that type III PI3K is excited in autophagy is illustrated Section plays an important roll.In mammal, type III PI3K can also be formed with anchorin p150 Compound, and this compound is combined regulation and control autophagy (Embo Journal, 2000,19 (21) with Beclin 1: 5720-5728).Beclin 1 is the gene homologous with yeast Apg6 found in breast cancer cell (Autophagy,2007,3:28-31), be uniquely find and clone so far mammal " from Bite gene ", positioned at human chromosome 17q21, participate in the formation of autophagosome, and formation with tumour, Embryonic development is closely related with the generation of disease.

Mitogen activated protein kinases (Mitogen-activated protein kinased, MAPK) It is a histone serine/threonine kinases, positioned at the end position eventually of endochylema signal transduction pathway, by carefully Extracellular stimulus signal is transduceed to cell and its core, cause cell biology react (such as cell propagation, Differentiation, conversion and apoptosis etc.) during there is vital effect.The signal of MAPK turns Approach is led mainly to be made up of three kinds of kinases：MAPK, mapk kinase (MEK or MKK) with MAPKK kinases (MEKK).MEKK activates MEK, and then MEK is by the junket of MAPK Propylhomoserin and threonine residues phosphorylation, and then activated (Physiological Reviews, 2012,92: 689-737).It is extracellular signal-regulated kinase (extracellular to study at present more Signal-regulated kinase, ERK), c-Jun nitrogen ends kinases (c-Jun NH2-terminal kinase, ) and p38 kinases (Genes＆Cancer, 2013,4 (9-10) JNK:342-359).

Although the current pharmacological research on jamaicin is a lot, so far, for barberry Alkali anti-gastric cancer is acted on and the research of mechanism is few.

The content of the invention

It is an object of the invention to provide a kind of new application of jamaicin, especially treatment stomach is being prepared Application in cancer drug；The structure of the jamaicin is：

Its minimum effective dose for being used to treat stomach cancer is 14 μM.

It is dead to there is autophagy in jamaicin induction human gastric cancer.

Jamaicin is by suppressing Akt and p38MAPK and downstream mTOR signal paths induction people BGC-823 Cells autophagy.

Jamaicin can substantially reduce the growth of BGC-823 cells transplanted tumor in nude mice, and toxicity is smaller.

Described medicine can be made enteron aisle with the carrier for pharmaceutically receiving by this formulation art known method Or non-bowel combines the formulation of medicine.Dosage form mainly include liquid preparation, granule, tablet, Electuary, capsule and pill, capsule, pill or injection.

Application of the jamaicin of the present invention in antineoplastic is prepared has the beneficial effect that： (1) jamaicin can substantially suppress human gastric cancer propagation.(2) with clinical anti-gastric cancer Medicine 5 FU 5 fluorouracil is compared, and jamaicin has obvious antitumous effect in vivo and toxicity is relatively low. Therefore, jamaicin has preferable clinical antitumor agents application prospect.

Brief description of the drawings

Fig. 1 is the shadow that jamaicin (BBR) grows to human gastric cancer transplanted tumor in nude mice Ring, wherein, Figure 1A is influences of the BBR to transplanted tumor in nude mice volume；Figure 1B is BBR to naked The influence of mouse body weight；

Fig. 2 is growth inhibition effects of the BBR to human gastric cancer；

Fig. 3 is that BBR induces human gastric cancer dead and autophagy, wherein, Fig. 3 A are The cell death of phase contrast microscope observation BBR inductions；Fig. 3 B are lured for fluorescence microscope BBR The cell autophagy led；Fig. 3 C are the ratio of flow cytometry quantitative analysis BBR Induces Autophagies； Fig. 3 D are influences of the protein immunoblotting method detection BBR to autophagy correlative protein expression；

Fig. 4 is the human gastric cancer death that autophagy inhibitor 3-MA suppresses BBR inductions；

Fig. 5 is the human gastric cancer autophagy that Akt suppresses BBR inductions, wherein, figure 5A is the influence for pre-processing Akt inhibitor MK2206 to BBR inducing cell deaths；Fig. 5 B are Influences of the pretreatment Akt inhibitor MK2206 to BBR Induces Autophagies；Fig. 5 C are BBR Influence to Akt and p-Akt protein expressions；

Fig. 6 is the human gastric cancer autophagy that MAPKs suppresses BBR inductions, wherein, Fig. 6 A are pretreatment ERK, JNK, P38 inhibitor (PD98059, SP600125 and SB203580) Influence to the cell death of BBR inductions；Fig. 6 B for pretreatment PD98059, SP600125 and The influence of the cell autophagy that SB203580 is induced BBR；Fig. 6 C are BBR to ERK, JNK With the influence of P38 protein expressions；

Fig. 7 is the human gastric cancer autophagy that mTOR suppresses BBR inductions, wherein, Fig. 7 A are the influence for pre-processing the cell death that Rapamycin is induced BBR；Fig. 7 B are pre- The influence of the cell autophagy that treatment Rapamycin is induced BBR；Fig. 7 C are BBR to mTOR And the influence of p-mTOR protein expressions；

Fig. 8 is the mTOR activity of Akt, p38 mediation BBR inductions.

Specific embodiment

The present invention can be illustrated by following implementation method.

Embodiment 1：Influence of the jamaicin to human gastric cancer Nude Mouse Model

1 experimental technique

BGC-823 cells are cultivated to exponential phase, pancreatin digestion in RPMI-1640 culture mediums After be centrifuged, with PBS cushioning liquid adjust to cell concentration be 5 × 10⁷/mL.24 nude mices are taken, In every right side oxter subcutaneous vaccination oncocyte liquid 0.2mL of mouse, raise in SPF clean rooms. Treat that tumour is long to 100mm³When, animal is randomly divided into four groups, every group 6.Specific packet and Application process is, control group：The μ L of PBS 100 of 5%DMSO；Positive controls：5-FU 20 mg/kg；Jamaicin low dose group：10mg/kg/ days；Jamaicin high dose group：22.5mg/kg/ days. Intraperitoneal injection, is administered once for every 2 days, successive administration 2 weeks, dynamically observes swollen during experiment Knurl growing state is simultaneously weighed, and daily with vernier caliper measurement tumour major diameter (a) and minor axis (b), is used After medicine 14 days, mice with tumor is put to death, peel off knurl body, the tumor tissues that will be stripped out are weighed.According to formula Calculate gross tumor volume and gross tumor volume inhibiting rate：Mean tumor volume (mm³)=ab²/2；Gross tumor volume Inhibiting rate (%)=(1- experimental groups mean tumor volume/negative control group mean tumor volume) × 100%.

2 experimental results

As shown in figure 1, compared with control group, the tumour growth of each group is subject to different degrees of the suppression System.Jamaicin tumor-inhibiting action high and low dosage is approached with 5-FU.In drug treatment, 5-FU Nude mice body weight can be substantially reduced, and jamaicin has no significant effect for nude mice body weight, and nude mice essence God is in good condition, grow, diet, activity it is normal, have no obvious adverse reaction, point out small Bark of a cork tree alkali has obvious inhibiting effect to transplanted tumor in nude mice, and compared with 5-FU, jamaicin toxicity is relatively low.

Embodiment 2：Growth inhibition effect of the jamaicin to human gastric adenocarcinoma BGC-823 cells

1 experimental technique

BGC-823 cells are inoculated in containing 10% hyclone, 1 × 105U/L penicillin and 100g/L In the RPMI-1640 culture mediums of streptomysin, 5%CO₂Incubator, cultivates at 37 DEG C.

By the BGC-823 cells of exponential phase, with every hole, 8 × 104 are inoculated in 96 orifice plates, In 37 DEG C of 5%CO₂24h is cultivated in incubator.Add final concentration to be respectively 14,21,32,48, 72nd, 108 μM of jamaicin, 3 multiple holes of every group of setting, while setting negative control.Put 37 DEG C 5%CO₂Cultivate 6 in incubator respectively, 12,24,36,48h.100 μ L 5mg/mL are added per hole MTT, after continuing to cultivate 2.5h, carefully discards nutrient solution in hole, and the μ of DMSO 150 are added per hole L, micro oscillator vibration 10min Shi formazans are completely dissolved, and enzyme mark is utilized under 490nm wavelength Instrument determines each hole light absorption value (A) value, and inhibiting rate is calculated according to formula below：

Inhibiting rate (%)=[A₄₉₂(control)-A₄₉₀(sample)]/[(A₄₉₀(control)-A₄₉₀ (blank)] × 100%

2 experimental results

From Figure 2 it can be seen that the induction BGC-823 cells that jamaicin is capable of time and dose dependent are dead Die.14-108 μM of jamaicin has obvious inhibitory action to BGC-823 cell growths.48 IC50 values are 31.7 μM during h.32 μM of jamaicins act on BGC-823 cell 48h, suppression Rate processed can reach 51.33%, and as extended durations of action substantially increases.

Embodiment 3：Jamaicin induces BGC-823 cell autophagies

1 experimental technique

By BGC-823 cells with every hole 2 × 10⁵The density of individual cell is inoculated in 6 orifice plates, training Add concentration to act on 24h for 32 μM of jamaicins after supporting 24h, use inverted microscope observation of cell Form.

By BGC-823 cells with every hole 5 × 10⁵The density of individual cell is inoculated in 24 orifice plates, training After adding autophagy specific inhibitor 3-MA to anticipate l h after foster 24h, jamaicin effect is added To 48h, nutrient solution is removed, washed once with PBS, add the MDC of 0.05mM to be kept away in 37 DEG C Light dyes l h, is 380nm in excitation wavelength, and launch wavelength is under the fluorescence microscope of 525nm Take pictures.

By BGC-823 cells with every bottle 4 × 10⁵Individual cell sub-bottle, adds certain density after 24h Jamaicin, or 3-MA pretreatment 1h are added, continue to cultivate to the specified time.Cell is collected, is used PBS once, adds the MDC of 0.05mM in after 37 DEG C of lucifuge dyeing l h, using streaming Cytometric Analysis autophagy ratio.

Further to prove that jamaicin induction BGC-823 cells occur autophagy, using Western blot The expression of autophagy GAP-associated protein GAP after method detection jamaicin effect.BGC-823 cells with 8 × 10⁵In blake bottle, overnight incubation is the density kind of individual/mL after concentration is added after adherent 24h 32 μM of jamaicins, cell is collected after effect different time, is cracked using protein lysate ice bath Supernatant is collected in 30min, centrifugation, and determining albumen with Bio-Rad protein assay reagent contains Amount.Sample is separated with 10%SDS- polyacrylamide gel electrophoresises, and goes to nitrocellulose On film.Primary antibody overnight incubation, TBST is washed 3 times, and secondary antibody is incubated 2h, and TBST is washed 3 times, ECL luminous substrates develop the color, film for medical X-ray radiography exposure.

2 experimental results

From Fig. 3 A, using inverted phase contrast light microscope, can be observed with after barberry alkali process Cell there occurs obvious morphological change.Culture is attached at during BGC-823 cell normal growths Bottom of bottle face, in regular fusiformis or cerioid.After 32 μM of jamaicin effect 48h, cell number Mesh is significantly reduced, and occurs substantial amounts of vacuole in cytoplasm.

From Fig. 3 B, observed under fluorescence microscope, when MDC is dyeed, normal cell Uniform green fluorescence is sent, after adding 32 μM of jamaicin function cells 48h, is gone out in endochylema The MDC of existing many bright greens raises particle, it was demonstrated that the increase of autophagy lysosome.Flow cytometry knot Fruit also show, with the increase of jamaicin action time, peak is moved to right, into the cell of M1 by Cumulative to add, i.e., MDC staining positive cells time dependence increases (Fig. 3 C).Add 3-MA pre- After treatment, the fluorescent grain of the MDC induced by jamaicin is significantly reduced, MDC positive cell rates I.e. autophagy ratio is also substantially reduced, so as to demonstrate the autophagy (figure that 3-MA inhibits jamaicin to induce 3B).Additionally, we have detected autophagy GAP-associated protein GAP Beclin 1 and LC3 with Western blot methods Expression.Add jamaicin after, Beclin 1 expression dramatically increase, the types of LC3 I to II conversion also Substantially rise, and after adding 3-MA, the increase of Beclin l expression and the conversion of LC3 are pressed down System, further demonstrates jamaicin and induces BGC-823 cells to there occurs autophagy (Fig. 3 D) really.

Embodiment 4：3-MA suppresses the BGC-823 cell deaths of jamaicin induction

1 experimental technique

To investigate effect of the autophagy in the BGC-823 cell deaths that jamaicin is induced, 3-MA is added After pretreatment, growth inhibition effect of the jamaicin to cell is detected using mtt assay.

2 experimental results

From fig. 4, it can be seen that compared with jamaicin independent role group, the cell growth suppression of 3-MA pretreatments Rate processed is substantially reduced, and shows that autophagy promotes jamaicin induction BGC-823 cell deaths.

Embodiment 5：Akt suppresses the cell autophagy of jamaicin induction

1 experimental technique

In order to determine effects of the Akt in jamaicin induction BGC-823 cell autophagies, Akt is introduced Inhibitor MK2206 pretreatment cells.Detect that growth of the jamaicin to cell presses down using mtt assay Make and use.After MDC dyeing, the autophagy ratio of cell after being acted on using flow cytomery jamaicin Example.

Western blot methods (with the method for embodiment 3)

2 experimental results

From Fig. 5 A, the inhibitor MK2206 pretreatment cells of Akt are added, with jamaicin list Only effect group is compared, and MK220 can make the cell inhibitory rate and MDC positive cells that jamaicin is induced Ratio substantially increases, and this shows the cell autophagy that Akt inhibits jamaicin to induce, so as to promote thin Born of the same parents are survived.Western blot results show that phospho-Akt is with the extension of jamaicin action time Reduce, and the expression of Akt then remains unchanged；Pretreatment 3-MA can substantially reverse phospho-Akt The reduction (Fig. 5 B) of protein expression.Prompting Akt suppresses the autophagy of jamaicin induction, autophagy process Lower the activity of Akt.

Embodiment 6：MAPKs suppresses the cell autophagy of jamaicin induction

1 experimental technique

In order to investigate effect of the MAPKs families in jamaicin induction BGC-823 cell autophagies, Introduce the corresponding inhibitor of ERK, JNK and p38MAPK (PD98059, SP600125 and SB203580) pretreatment cell.Growth inhibition effect of the jamaicin to cell is detected using mtt assay. After MDC dyeing, the autophagy ratio of cell after being acted on using flow cytomery jamaicin.

Western blot methods (with the method for embodiment 3)

2 experimental results

As shown in fig. 6, suppressing ERK, JNK and p38MAPK can significantly improve barberry The inhibitory rate of cell growth of alkali induction and MDC positive rates.Additionally, Western blotting methods Detection MAPKs protein contents change, find phospho-ERK, phospho-JNK, Phospho-p38MAPK is gradually decreased with the extension expression of jamaicin time, pre-processes 3-MA The reduction that can substantially reverse phosphorylated protein to express, and the protein content of unphosphorylated form is constant, Test result indicate that autophagy suppresses the activity of ERK, JNK and p38MAPK.

Embodiment 7：MTOR suppresses the cell autophagy of jamaicin induction

1 experimental technique

In order to determine effects of the mTOR in jamaicin induction BGC-823 cell autophagies, introduce The inhibitor Rapamycin pretreatment cells of mTOR.Detect jamaicin to cell using mtt assay Growth inhibition effect.After MDC dyeing, cell after being acted on using flow cytomery jamaicin Autophagy ratio.

Western blot methods (with the method for embodiment 3)

2 experimental results

As shown in fig. 7, compared with jamaicin independent role group, pre-processing mTOR inhibitors Rapamycin can make jamaicin induce cell inhibitory rate and MDC positive rates substantially increase. Western blot results display phospho-mTOR is reduced with the extension of jamaicin action time, And the expression of mTOR is not changed in.Pretreatment 3-MA can substantially reverse phospho-mTOR eggs The reduction of white expression.

Embodiment 8：The mTOR activity of Akt, p38 mediation jamaicin induction

1 experimental technique

Influence of the Akt and MAPKs signal paths to mTOR is further investigated, Akt is introduced respectively And inhibitor MK2206, SB203580, PD98059 and SP600125 of MAPKs families are pre- Treatment cell.

Western blot methods (with the method for embodiment 3)

2 experimental results

As shown in figure 8, inhibitor MK2206, SB203580 of Akt or p38MAPK is added, The phospho--mTOR levels that can induce jamaicin are further reduced；And add ERK or JNK Inhibitor PD98059, SP600125, phospho-mTOR levels are but without significant changes. Above test result indicate that, Akt and p38MAPK is located at mTOR upstreams, regulation mTOR Activity.

Claims (7)

1. application of the jamaicin in treatment gastric cancer medicament is prepared.

2. application according to claim 1, it is characterised in that jamaicin induces human gastric cancer There is autophagy, and autophagy induction BGC-823 cell deaths in BGC-823 cells.

3. application according to claim 1, it is characterised in that jamaicin is by suppressing Akt With p38MAPK and downstream mTOR signal paths induction BGC-823 cell autophagies.

4. application according to claim 1, it is characterised in that the jamaicin suppresses The proliferation activity of tumour cell in BGC-823 cell subcutaneous transplantation knurls.

5. application according to claim 1, it is characterised in that jamaicin minimum effective dose It is 14 μM.

6. the application according to claim 1-5 any one, it is characterised in that the jamaicin Clinically acceptable preparation is constituted with pharmaceutically acceptable carrier.

7. application according to claim 6, it is characterised in that the preparation includes liquid system Agent, granule, tablet, electuary, capsule and pill, capsule, pill or injection.