CN104873985B - ADRB2 inhibitor is combined with Sorafenib and is preparing the application in treating liver-cancer medicine - Google Patents
ADRB2 inhibitor is combined with Sorafenib and is preparing the application in treating liver-cancer medicine Download PDFInfo
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- CN104873985B CN104873985B CN201510125237.7A CN201510125237A CN104873985B CN 104873985 B CN104873985 B CN 104873985B CN 201510125237 A CN201510125237 A CN 201510125237A CN 104873985 B CN104873985 B CN 104873985B
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Abstract
The present invention relates to pharmaceutical technology fields, and in recent years, beta 2 adrenoreceptor (ADRB2) and its signal path are received more and more attention in tumor prevention and Therapy study field.The present invention provides ADRB2 inhibitor to prepare the application in treating liver-cancer medicine, especially ADRB2 inhibitor can effectively inhibit liver cancer growth with Sorafenib combination, and can effectively inhibit the transplanting tumor formation efficiency of liver cancer cells, the progress of liver cancer is reduced, Clinical practice popularization is easy to.
Description
Technical field：
The present invention relates to pharmaceutical technology fields, and in particular to a kind of ADRB2 inhibitor is combined with Sorafenib and is controlled in preparation
Treat the application in liver-cancer medicine.
Background technology：
Primary hepatoma (hepatocellular carcinoma, HCC) is the fifth-largest kinds of tumor in the world,
It is number three in cancer related mortality reason.Hepatocellular carcinoma is common in developing country, is especially mainly in East Asia, Southeast Asia, too
Flat ocean basin and Sahara southern areas.In 626 reported, 000 patients with hepatocellular carcinoma, about 410,
000 is distributed in East Asia Region (wherein 346,000, China, 40,000, Japan).But due to invading and shifting before treatment
Incidence it is higher, the prognosis of hepatocellular carcinoma is poor.
Sorafenib is the multiple target point antitumor drug by Bayer and Onyx cooperative research and development, has and significantly inhibits tumour increasing
The effect with revascularization is grown, is first targeted drug for being used for clinical treatment hepatocellular carcinoma being approved by the FDA in the United States.Suo La
Fei Ni passes through to inhibiting protein serine/threonine RAF, the proliferation of tumour significantly to be controlled in ERK1/2 signal paths.This
Outside, Sorafenib also inhibits VEGF and PDGFR, to prevent tumour vascularization (Wilhelm SM, Carter C,
Tang L,Wilkie D,McNabola A,Rong H,,et al.:BAY 43-9006exhibits broad spectrum
oral antitumor activity and targets the RAF/MEK/ERK pathway and receptor
tyrosine kinases involved in tumor progression and angiogenesis.Cancer Res
2004,64(19):7099-7109.).But it is also easy to produce drug resistance using Sorafenib in liver cancer patient, therapeutic effect is also paid no attention to
Think.
With deepening continuously for research level, the abnormal effect in tumorigenesis of neuroendocrine system is more next
More attract attention.Wherein, beta-2 adrenoceptor wide expression is that adjusting body reply stress in most of mammalian cells
The critical function molecule of reaction process.According to structure and effect difference, beta-2 adrenoceptor can be divided into 3 three Asias β 1, β 2 and β
Type.The use (such as non-selective β 1, beta 2 adrenoreceptor inhibitor, Propranolol) of beta-2 adrenoceptor retarding agent helps
In the progression of disease for delaying breast cancer and patients with prostate cancer, improve patient's prognosis (Barron TI, Connolly RM, Sharp
L,Bennett K,Visvanathan K.Beta blockers and breast cancer mortality:a
population-based study.J Clin Oncol.Jul 12011；29(19):2635-2644.).
In recent years, beta 2 adrenoreceptor (ADRB2) and its signal path tumor prevention and Therapy study field by
More and more concerns.ICI-118,551 be specific beta 2 adrenoreceptor inhibitor, inhibits the effect of tumour also gradual
It is taken seriously.But effect of beta 2 adrenoreceptor (ADRB2) inhibitor in liver cancer treatment is still unclear, and there has been no texts at present
Offer report use in conjunction ADRB2 inhibitor and effect of the Sorafenib in treating liver cancer.
Invention content：
The purpose of the present invention is to provide the new medical usages of ADRB2 inhibitor, and another object of the present invention is to carry
It is combined for ADRB2 inhibitor and Sorafenib and is preparing the application in treating liver-cancer medicine.
In order to overcome existing liver cancer treatment method to be difficult to that liver cancer growth and transfer, recurrence and Sorafenib is inhibited to control
The deficiencies of liver cancer drug resistance is high is treated, the present invention provides the novel medical uses of beta 2 adrenoreceptor (ADRB2) inhibitor.
The present invention provides beta 2 adrenoreceptor (ADRB2) inhibitor to prepare the application in treating liver-cancer medicine, special
It is not synergist or sensitizer of the beta 2 adrenoreceptor inhibitor as chemotherapy of hepatocellular carcinoma drug.
The chemotherapy of hepatocellular carcinoma drug is Sorafenib, cis-platinum or epirubicin etc..
Further, the present invention also provides beta 2 adrenoreceptor inhibitor to prepare answering in treating liver-cancer medicine
With the drug is that beta 2 adrenoreceptor inhibitor is combined with Sorafenib.
The present invention provides beta 2 adrenoreceptor inhibitor and Sorafenib to be combined in preparing treatment liver-cancer medicine
Using.
Beta 2 adrenoreceptor inhibitor of the present invention is the object for inhibiting or reducing ADRB2 genetic transcriptions (translation)
Matter, or the substance of ADRB2 receptor natural biological functions can be inhibited, such as ADRB2 is reduced by the methods of siRNA or shRNA
Genetic transcription；Using non-selective β 1, beta 2 adrenoreceptor inhibitor Propranolol or specific beta 2 adrenoreceptor suppression
Preparation ICI-118,551 inhibits ADRB2 receptor natural biological functions etc..
The present invention main technical schemes be：ADRB2 inhibitor is simultaneously administered patient with Sorafenib.
In the preferred embodiment of the present invention, the liver cancer includes hepatocellular carcinoma and cholangiocellular carcinoma.
In the preferred embodiment of the present invention, the ADRB2 inhibitor is oral preparation, injection or sustained release preparation etc..
In the preferred embodiment of the present invention, using ADRB2 inhibitor and Sorafenib as the administration of the drug of active ingredient
Mode includes intravenous injection, oral and topical administration etc..
In the preferred embodiment of the present invention, ADRB2 inhibitor can be with administering drug combinations with Sorafenib, or are separately administered.
The present invention confirms through cell experiment, ICI in the growth rate that can obviously slow down liver cancer cells for 24 hours, and this
Kind trend is gradually notable with the extension of time.
ICI can obviously inhibit Clone formation, and there are concentration dependents, prompt ICI that can actually inhibit tumour cell
Proliferation.
It interferes in liver cancer cells after ADRB2 expression, cell proliferation rate obviously slows down.
ICI combinations Sorafenib can obviously increase growth inhibitions of the Sorafenib to liver cancer cells, and this suppression
Level processed is higher than the sum of the cell inhibition being used alone caused by ICI or Sorafenib.
ICI is combined the liver cancer cells showed increased of generation apoptosis after Sorafenib, has prompted stronger fragmentation effect.
Use in conjunction ADRB2 inhibitor can significantly inhibit the growth of hepatocellular carcinoma transplantable tumor with Sorafenib.
The invention has the advantages that the method energy of this use in conjunction ADRB2 inhibitor and Sorafenib treatment liver cancer
Effectively inhibit liver cancer growth, and can effectively inhibit the transplanting tumor formation efficiency of liver cancer cells, reduce the progress of liver cancer, is easy to clinical
Application and popularizations.It can inhibit the proliferation and clonality of liver cancer cells using the method for the present invention, promote hepatoma cell apoptosis,
The growth for inhibiting liver cancer cells transplantable tumor, greatly strengthens sensibility of the liver cancer cells to Sorafenib, being easy to clinical expansion makes
With.
Description of the drawings
Fig. 1 is the schematic diagram that the present invention inhibits liver cancer cell growth using ADRB2 inhibitor, and wherein A is that ICI inhibits liver cancer
The figure of cell proliferation rate, B are the photo that ICI inhibits liver cancer cells Clone formation, and C is that ICI inhibits liver cancer cells Clone formation
Experiment clone's quantity counts figure；
Fig. 2 is the schematic diagram of present invention interference ADRB2 expression inhibiting liver cancer cell growths；
Fig. 3 is the growth schematic diagram of use in conjunction ADRB2 inhibitor of the present invention and liver cancer after Sorafenib, and wherein A is
SMMC-7721 cells as a result, B is HepG2 cells as a result, C is the result of HCC-LM3 cells；
Fig. 4 is that use in conjunction ADRB2 inhibitor of the present invention and the clonality of liver cancer cells after Sorafenib are illustrated
Figure, wherein A are the photo of Clone formation, and B is that clone formation number purpose counts figure；
Fig. 5 is the apoptosis ratio schematic diagram of liver cancer cells after use in conjunction ADRB2 inhibitor and Sorafenib of the present invention；
Fig. 6 is the schematic diagram of use in conjunction ADRB2 inhibitor of the present invention and liver cancer cells transplanting efficiency after Sorafenib.
Specific implementation mode：
Below in conjunction with specific embodiment, the invention will be further described.It should be understood that following embodiment is merely to illustrate this
Invention is not for restriction the scope of the present invention.
ICI-118,551 (hereinafter referred to as ICI) are specific beta 2 adrenoreceptor inhibitor, are purchased from Sigma companies,
Article No. I127-5MG.
Propranolol is non-selective β 1, and beta 2 adrenoreceptor inhibitor is purchased from Sigma companies, article No. P0884-1G.
Sorafenib (Sorafenib) is purchased from LC laboratories, article No. S-8502.
Hepatocellular carcinoma cells system SMMC-7721, HepG2, PVTT, HMCC-LM3 are purchased from cell institute of the Chinese Academy of Sciences.
Embodiment 1：
Liver cancer cell lines SMMC-7721, HepG2 and PVTT are passed to 96 orifice plates respectively, per 3000, hole cell, each
Cell is divided into DMSO and two groups of ICI, and every group sets 5 secondary orifices.It was calculated as 0 moment after cell is adherent, while each group is given respectively
DMSO or 10 μM of ICI.Culture medium is sopped up in 0,24,48,72,96h respectively, is added and is detected with the CCK8 of DMEM diluted 10%
Reagent, 37 DEG C of incubators are incubated the absorbance value at detection 450nm after 1h.It is bent that growth is drawn with the absorbance value that each time point is surveyed
Line.
By A in Fig. 1 it is found that ICI is in the growth rate that can obviously slow down liver cancer cells for 24 hours, and this trend with
The extension of time and gradually significantly.
In order to further prove that ICI can inhibit the proliferation of liver cancer cells, We conducted cell clone experiments.It is specific real
It applies as follows：MHCC-LM3 cells are passed in 6 orifice plates, per 500, hole cell.It is divided into 10 μM of DMSO, ICI and 20 μM 3 of ICI
Group, every group sets 3 secondary orifices.After cell is adherent, each group changes the culture medium containing respective concentration drug into.Cell changes liquid one per 3d
Secondary, 15d absorbs culture medium, and with twice of normal saline flushing, violet staining 5min is added, absorbs crystal violet, then use physiology
Normal saline washing 2 times, takes pictures, and calculates and form clone's number in each hole.Every group of each secondary orifices Clone formation number is counted, and is drawn
Count block diagram.
By B in Fig. 1 it is found that ICI can obviously inhibit Clone formation, and there are concentration dependents, and Fig. 1 C are to Clone formation
Number, which counts, proves that ICI can actually inhibit the proliferation of tumour cell.
Embodiment 2：
Liver cancer cell lines PVTT is passed to 12 orifice plates, cell density is 50% or so.After cell is adherent, slow virus will be compareed
Or each 15 μ L of shADRB2 viruses in two disturbance sites are added separately in cell culture medium suspension, are shaken up.Liquid is changed after 8h,
A concentration of 5 μM of puromycin is added afterwards for 24 hours to continue to cultivate, incoming 6 orifice plates, continue to cultivate when cell density is 100%.It is logical
After crossing RT-PCR identification interference effects, cell is passed to 96 orifice plates, per 3000, hole cell.It was calculated as 0 moment after cell is adherent,
Culture medium is sopped up in 0,24,48,72,96h respectively, the CCK8 detection reagents with DMEM diluted 10% are added, 37 DEG C of incubators are incubated
Educate the absorbance value at detection 450nm after 1h.Growth curve is drawn with the absorbance value that each time point is surveyed.
As shown in Figure 2, it interferes in liver cancer cells after ADRB2 expression, cell proliferation rate obviously slows down.
Embodiment 3：
Liver cancer cell lines SMMC-7721, HepG2 and HMCC-LM3 are passed to 96 orifice plates, per 5000, hole cell.Wait for cell
It is divided into four groups after adherent, every group sets 5 secondary orifices, and each group changes the culture medium containing following drug into respectively：
1)DMSO；
2)ICI 20μM；
3)Sorafenib 20μM+DMSO；
4)Sorafenib 20μM+ICI 20μM。
It is put into the incubation of 37 DEG C of incubators and sops up culture medium afterwards for 24 hours, be added the CCK8 detection reagents with DMEM diluted 10%, 37
DEG C incubator is incubated the absorbance value at detection 450nm after 1h.On the basis of the absorbance value of first group of detection, it is thin to calculate 2-4 groups
The inhibiting rate of born of the same parents draws growth inhibiting block diagram.
From the figure 3, it may be seen that ICI combinations Sorafenib can obviously increase growth inhibitions of the Sorafenib to liver cancer cells,
And this suppression level is higher than the sum of the cell inhibition being used alone caused by ICI or Sorafenib.
Embodiment 4：
Liver cancer cell lines MHCC-LM3 is passed to 6 orifice plates, 500 cells are passed per hole.It is put into 37 DEG C of incubators to be incubated 1 week, wait for
Cell is divided into 3 groups after growing up to macroscopic clone, and every group sets 3 secondary orifices.Each group changes the culture containing 5 μM of Sorafenib into
Simultaneously following drug is added simultaneously respectively in base, makes to contain 10 μM of DMSO, ICI or 20 μM of ICI in culture medium.
Cell is put into 37 DEG C of incubators to be incubated, is changed the liquid once per 3d, and is kept in fresh culture containing as before
The drug of concentration.Culture medium is absorbed after 2 weeks, with twice of normal saline flushing, violet staining 5min is added, absorbs crystal violet,
Normal saline flushing is used again 2 times, taken pictures, and calculate and form clone's number in each hole.Every group of each secondary orifices Clone formation number is counted,
And draw counting block diagram.
As shown in Figure 4, compared to Sorafenib groups are applied alone, ICI, which is combined Sorafenib, can significantly inhibit liver cancer cells
Clonality prompts ICI that can enhance the effect of the inhibition liver cancer of Sorafenib.
Embodiment 5：
Liver cancer cell lines MHCC-LM3 is passed to 6 orifice plates, per Kong Chuanyue 2 × 105A cell.It is divided into four after cell is adherent
Group, every group sets 3 secondary orifices, and each group changes the culture medium containing following drug into respectively：
1)DMSO；
2)ICI 20μM；
3)Sorafenib 20μM+DMSO；
4)Sorafenib 20μM+ICI 20μM。
It is put into 37 DEG C of incubator incubations and sops up culture medium afterwards for 24 hours, cell dissociation is got off with pancreatin, physiological saline is used in combination to suspend
Centrifugation, sops up supernatant, stays cell precipitation after in 1.5mlEP pipes.The apoptosis detection reagent prepared (Annexin V+PI) is added
To suspension cell in the EP pipes containing cell precipitation, it is incubated 15min under the conditions of room temperature is protected from light, terminate liquid is then added, it is upper
Formula cell instrument detects.
As shown in Figure 5, ICI, which is combined Sorafenib, can obviously increase the hepatoma cell apoptosis caused by Sorafenib
Ratio, and the liver cancer that the ratio of caused hepatoma cell apoptosis is higher than caused by exclusive use ICI or Sorafenib is thin
The sum of born of the same parents' apoptosis ratio.
Embodiment 6：
It is digested with pancreatin after liver cancer cell lines SMMC-7721 cultures to sufficient density, is used in combination physiological saline to be resuspended, carefully
After born of the same parents count, by resuspension 3 × 10 in every 100 μ l physiological saline6Cell is resuspended in the amount of a cell, and cell is squeezed into nude mice skin
Under, per 100 μ l of side.After 2 weeks, nude mice is divided into 4 groups according to nude mouse tumor growing state, every group of tumor average volume is made to connect
Closely.
4 groups of nude mices are handled as follows respectively：
1)CTL；
2) Propranolol 1mg/l is added in drinking water；
3) intraperitoneal injection Sorafenib 50mg/kg/d；
4) Propranolol 1mg/l+ intraperitoneal injection Sorafenib 50mg/kg/d are added in drinking water.
It measures a diameter of tumor within every 4 days, after calculating each group gross tumor volume, draws tumor growth curve figure.
As shown in fig. 6, Propranolol (non-selective β 1, beta 2 adrenoreceptor inhibitor) can inhibit the growth of tumour,
And Propranolol can be obviously promoted inhibiting effect of the Sorafenib to tumour growth, and the two has tumor inhibition effect folded
Add effect.
The preferred embodiment of the invention is illustrated above, but the invention be not limited to it is described
Embodiment, those skilled in the art can also make various equivalent under the premise of without prejudice to the invention spirit
Modification or replacement, these equivalent modifications or replacement are all contained in the application claim limited range.
Claims (1)
1. beta 2 adrenoreceptor inhibitor is combined with Sorafenib and is preparing the application in treating liver-cancer medicine, 2 kidneys of β
Upper parathyrine acceptor inhibitor is that " ICI-118,551 ", the liver cancer is hepatocellular carcinoma, and active ingredient is only in the drug
For " ICI-118,551 " and Sorafenib.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201510125237.7A CN104873985B (en) | 2015-03-20 | 2015-03-20 | ADRB2 inhibitor is combined with Sorafenib and is preparing the application in treating liver-cancer medicine |
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| CN201510125237.7A CN104873985B (en) | 2015-03-20 | 2015-03-20 | ADRB2 inhibitor is combined with Sorafenib and is preparing the application in treating liver-cancer medicine |
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Non-Patent Citations (5)
| Title |
|---|
| beta-Adrenoreceptor antagonists reduce cancer cell proliferation, invasion, and migration;Ozlem Darcansoy Iseri等;《Pharmaceutical Biology》;20140715;第52卷(第11期);第1375页左栏第2段，第1376页右栏第4段，第1377页右栏第1段，1379页左栏第1段，表1-2，图3 * |
| Effects of the combined administration of propranolol plus sorafenib on portal hypertension in cirrhotic rats;M. D’Amico等;《Am J Physiol Gastrointest Liver Physiol》;20120308;第302卷(第10期);G1191-G1198页 * |
| Non-selective beta-blockers may reduce risk of hepatocellular carcinoma:a meta-analysis of randomized trials;Maja Thiele等;《Liver International》;20150206;第52卷(第11期);第2009-2016页 * |
| The mitogenic effectors of isoproterenol in human hepatocellular carcinoma cells;AIHUA YUAN等;《ONCOLOGY REPORTS》;20100101;第23卷(第1期);第151-157页 * |
| Vicus Therapeutics Announces Phase II Data Demonstrating Overall Survival Benefit with VT-122 Plus Sorafenib in Patients with Advanced Hepatocellular Carcinoma;MORRISTOWN, N.J;《PRWEB》;20140905;正文第1-2和5-6段 * |
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