CN105030785A - Applications of Promethazine in preparing anti-liver cancer and/or colorectal carcinoma and/or lung cancer products - Google Patents
Applications of Promethazine in preparing anti-liver cancer and/or colorectal carcinoma and/or lung cancer products Download PDFInfo
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- CN105030785A CN105030785A CN201510386589.8A CN201510386589A CN105030785A CN 105030785 A CN105030785 A CN 105030785A CN 201510386589 A CN201510386589 A CN 201510386589A CN 105030785 A CN105030785 A CN 105030785A
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Abstract
The invention discloses applications of Promethazine in preparing anti-liver cancer and/or colorectal carcinoma and/or lung cancer products. Medicine Promethazine approved by FDA and CFDA is subjected to tumor drug repositioning, drugs with indication not including tumor are subjected to screening based on different cell lines (tissue types) and mutation sites of tumor, the novel application of Promethazine in resisting liver cancer and/or colorectal carcinoma and/or lung cancer is discovered, and new use for approved drug is realized.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the application of a kind of Promethazine in the anti-hepatocarcinoma of preparation and/or colon cancer and/or pulmonary carcinoma product.
Background technology
Tumor threatens the most common of human health to be also the most serious a kind of disease, and research and development high efficiency anti-tumor medicine is most important to the life cycle extending patient.In recent years, along with the develop rapidly of cancer genomics and molecular pharmacology, the research and development of new type antineoplastic medicine achieve good achievement, but because new drug development has high input, cycle, the long bottleneck that waits cannot overcome, and the feature such as tumor individual inheritance variation is large, caused many traditional anti-tumor medicine effects not add, new drug is expensive, and side effect is not bright.
The researcheres such as BarabasiAL were published in " NatureReviewsGenetics " opinion in 2011 the article pointed out, the molecular network analysis carried out based on GWAS result of study and mutual work group (interactome) strategy is expected to disclose the new drug target of complex disease and molecular marker, and the understanding that final formation is machine-processed to disease incidence and therapeutic scheme is brand-new.Even more noteworthy, medicine reorientation (drugrepositioning) research finds, GWAS studies the tumor susceptibility gene of locking and has protein-protein interaction (protein-proteininteraction with it, PPI) gene more easily becomes medicine indirectly target spot, and this finds that there is and helps explain that the mechanism of action of existing medicine and guides the research and development of new drug.2014, show in the paper that the researcheres such as OkadaY are delivered on " Nature " in 101 tumor susceptibility genes of the rheumatoid arthritis that GWAS result of study confluence analysis obtains and have 98 direct or indirect targets being used for treatment medicine for treating rheumatoid arthritis at present, but also studied by medicine reorientation, they have also found that the granted medicine for other indications of dozens of also can be used for treating rheumatoid arthritis.
Summary of the invention
This research is just by integrating the drug data base that in the cancer gene spectrum CancerGeneCensus of cancer group Cosmicversion72 data base and protein interaction STRINGversion10 data base and DrugBankversion4.2, FDA ratifies, the candidate's reorientation medicine obtained, examination experiment is carried out to tumor cell line, screens the antitumor drug made new advances.The candidate tumor doing tumor cell line screening suppresses medicine to see as follows:
Nicardipine,Promethazine,Estrone,Promethazine,Sunlidac,Promethazine,Etonogestrel,Levonorgestrel,Mesalazine,Indomethacin,Sulfasalazine,Balsalazide,Irbesartan,Ibuprofen,Isoprenaline,PentosanPolysulfate。
An object of the present invention is to provide the novelty teabag of Promethazine.
Promethazine provided by the invention is preparing the application in Hepatoma therapy and/or colon cancer and/or pulmonary carcinoma product.
Second object of the present invention is to provide the novelty teabag of Promethazine.
The invention provides the application of Promethazine in preparation suppression hepatocarcinoma and/or colon cancer and/or proliferation of lung cancer cells product.
3rd object of the present invention is to provide the novelty teabag of Promethazine.
The invention provides the application of Promethazine in preparation reduction hepatocarcinoma and/or colon cancer and/or lung carcinoma cell IC50 value product.
In above-mentioned application, described hepatoma carcinoma cell is HUH-7; Described colon cancer cell is LOVO, and described lung carcinoma cell is NCI-H2141.
In above-mentioned application, described product is medicine or test kit.
4th object of the present invention is to provide a kind of product.
Product provided by the invention, its active component is Promethazine; Described product has following at least one function:
1) Hepatoma therapy and/or colon cancer and/or pulmonary carcinoma;
2) hepatocarcinoma and/or colon cancer and/or proliferation of lung cancer cells is suppressed;
3) hepatocarcinoma and/or colon cancer and/or lung carcinoma cell IC50 value is reduced.
In the said goods, described hepatoma carcinoma cell is HUH-7; Described colon cancer cell is LOVO, and described lung carcinoma cell is NCI-H2141.
In the said goods, described product is medicine or test kit.
Experiment of the present invention proves, the present invention is to FDA, the granted medicine Promethazine of CFDA carries out tumour medicine reorientation, the cell line (organization type) different according to tumor and mutational site are not that anti-tumor drug screens to indication, find the anti-hepatocarcinoma of Promethazine and/or colon cancer and/or this novelty teabag of pulmonary carcinoma, realize old medicine and newly use.
Accompanying drawing explanation
Fig. 1 is 96 well culture plate medicine sieve medicine template distributions.
Fig. 2 is that Promethazine is responsive to hepatocarcinoma; EC50=41.9877; IC50=31.3712; R
2=0.9988.
Fig. 3 is that Promethazine is responsive to colon cancer; EC50=18863738.7272; IC50=33.4520; R
2=0.9164.
Fig. 4 is that Promethazine is responsive to pulmonary carcinoma; EC50=33.9362; IC50=34.6324; R
2=0.9670.
Detailed description of the invention
The experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
In following embodiment, medicine to be measured is Promethazine, and its chemical structural formula is as follows:
it is drugbank product, and catalog number is DB01069.
The source of the hepatoma carcinoma cell HUH-7 below in embodiment and colon cancer cell LOVO, Proliferation of Human Ovarian Cell SKOV-3 and maxicell lung carcinoma cell NCI-H661, lung carcinoma cell NCI-H2141 is as follows:
Main instrument below in embodiment and consumptive material
DMSO(fromSigma,Cat.No.D4540)
The saturating Tissue Culture Plate (fromCorning, Cat.No.3610) in the 96-well white end
CellTiterGlo test kit (fromPromega, Cat.No.G7573)
Doxorubicin positive drug (fromMCE, Cat.No.HY-15142)
FetalBovineSerum(fromGibco,Cat#10099141)
100mm culture dish (fromCorning, Cat#430167)
RPMI-1640medium(fromGibco,Cat#A1049101)
DMEMmedium(fromGibco,Cat#11995081)
DMEM/F12medium(fromGibco,Cat#11330057)
EMEMmedium(fromGibco,Cat#10370021)
Mutidrop384 cell liquor separator (Thermo, Cat#5840150)
The multi-functional plate reading machine (PerkinElmer, Cat#2300-001M) of EnSpire
Embodiment 1, CELLTITER-GLO detect the anti-hepatocarcinoma of Promethazine and/or colon cancer
One, the preparation of orifice plate to be measured
1, plating cells
A) complete medium needed for each cell is prepared.
B) before experiment starts, confirm the medicine sieve cell name be marked on 100mm culture dish, culture medium and generation time, the information such as generation, guarantee that experiment is errorless.
C) attached cell operation refer step d) to i), suspension cell operation refer step j) to l).
D) vacuum pump is utilized to draw cell culture medium during sterile working.
E) with the aseptic PBS solution rinse cell surface layer of 2ml, then vacuum pump sucking-off PBS waste liquid is used.
F) 1ml0.25% (w/v) Trypsin-0.038% (w/v) EDTA solution digestion cell is added gently to culture dish, mix gently several under, solution covers cell surface layer, under inverted microscope, observation of cell digestion situation, stops trypsinization effect when cell will come off.
G) in culture dish, add 5ml37 DEG C of preheated complete medium, blow and beat cell gently with pipet, make it split away off bottom culture dish.
H) this cell suspension is transferred in 15ml or 50ml sterile centrifugation tube, the centrifugal 5min of 1000rpm.
I) vacuum pump sterile working sucking-off supernatant culture medium is utilized.Add 5ml37 DEG C of preheated complete medium re-suspended cell precipitation, blow and beat mixing gently.
J) blow and beat cell gently with pipet, make it split away off completely bottom culture dish.
K) this cell suspension is transferred in 15ml or 50ml sterile centrifugation tube, the centrifugal 5min of 1000rpm.
L) vacuum pump sterile working sucking-off supernatant culture medium is utilized.Add 5ml37 DEG C of preheated complete medium re-suspended cell precipitation, blow and beat mixing gently.
M) count cell suspension with cell counter, adjustment cell suspension carries out plating cells experiment to proper density fishplate bar.
N) HUH-7 cell and LOVO cell are carried out according to the method described above, obtain HUH-796 porocyte culture plate and LOVO96 porocyte culture plate respectively.
HUH-7 cell, complete medium is DMEM, is life product, 11995081, fishplate bar density (cells/well) is 6000.
LOVO cell, complete medium is DMEM, is life product, 11995081, fishplate bar density (cells/well) is 8000.
2, medicine Promethazine to be measured prepares and dosing (200X final concentration):
1) medicine Promethazine motherboard preparation to be measured
A) with DMSO, medicine Promethazine to be measured is diluted to 20mM stand-by.
B) getting the 20mM medicine 79 to be measured μ L configured in a) step adds in dilution plate the first row first hole, add the DMSO solvent of 54 μ L subsequently to the first row second hole in the 9th hole, 25 μ L solution are got in the second hole from the first hole, from the second hole, 25 μ L solution are got in the 3rd hole after mixing, the like to the 9th hole, ensure medicine successively carry out 3.16 times of multiple proportions gradient dilutions.
2) positive drug Doxorubicin (MCE, Cat.No.HY-15142) motherboard preparation
A) with DMSO, positive drug Doxorubicin is diluted to 6mM stand-by.
B) 6mM positive drug Doxorubicin solution is added in dilution plate, this medicine to be measured of DMSO solution 1:3.16 times of multiple proportions gradient dilution.
3, the preparation of medicine working plate and dosing
A) medicine to be measured and positive drug application of sample template are illustrated in fig. 1 shown below, wherein, S1208: positive drug Doxorubicin, DMSO: Positive control wells, Cpd1,2,3: medicine to be measured, DMSO final concentration is 0.5% (DMSO is compatible).
B) in working plate, add the specific complete medium of 95 μ l cell, corresponding 9 holes of each medicine, with multiple tracks volley of rifle fire difference transferase 45 μ l a series of (9 holes) solution (10X final concentration) that doubling dilution is good successively from medicine to be measured and positive drug doxorubicin motherboard, obtain the cell culture medium containing variable concentrations medicine.
C) from incubator, take out step 1 prepare HUH-796 porocyte culture plate and LOVO96 porocyte culture plate, by Fig. 1 dosing template arrangement mode (Fig. 1) respectively to adding the above-mentioned cell culture medium (10X final concentration) containing variable concentrations medicine b) prepared of 10 μ l in HUH-796 porocyte culture plate and LOVO96 porocyte culture plate, put into CO
2incubator 37 DEG C cultivates 72h, obtains the 96 hole medicine sieve plates of HUH-7 and the 96 hole medicine sieve plates of LOVO.
Not add the hole in contrast of any medicine.
Final concentration in 96 orifice plates of above-mentioned medicine to be measured, positive drug Doxorubicin and control wells and dosing situation as follows:
The final concentration (μM) of medicine to be measured in the 2-10 hole of Fig. 1 is followed successively by: 100,31.64557,10.01442,3.16912,1.002886,0.317369,0.100433,0.031783,0.010058;
The final concentration (μM) of positive drug Doxorubicin in the 2-10 hole of Fig. 1 is followed successively by: 30,9.493671,3.004326,0.950736,0.300866,0.095211,0.03013,0.009535,0.003017;
In addition, in 96 orifice plates S1208 hole (E1-H1 and A12-D12): 10 μ l final concentrations, 100 μMs of Doxorubicin solution (solvent is the complete medium solution comprising 0.5%DMSO), DMSO hole (A1-D1, E12-H12 and A11-H11): 10 μ l comprise the complete medium solution of 0.5%DMSO.
Two, CELLTITER-GLO fluorecyte activity monitor system detects
1, CellTiter-Glo preparation of reagents
A) before using, CellTiter-Glo reagent Buffer is melted, equilibrate to room temperature and use.
B) before using, CellTiter-Glo reagent agar substrate is melted, equilibrate to room temperature and use.
C) get the CellTiter-GloBuffer that 100ml balances to join in bottled CellTiter-Glo reagent agar substrate, make its abundant resuspended formation enzyme/substrate mixture, namely so-called CellTiter-Glo detectable.
D) mix vortex gently, and be repeatedly inverted the uniform solution of acquisition.In general, in 1 minute, CellTiter-Glo substrate reagent will fully dissolve, subpackage, keeps in Dark Place for subsequent use at-20 DEG C, avoids multigelation.
2, detect
A), before detecting, the 96 hole medicine sieve plates of above-mentioned one 3 HUH-7 obtained and the 96 hole medicine sieve plates of LOVO are balanced 20-30 minute at room temperature.
B) cellular morphology and the death condition of every block culture plate observed by inverted microscope, mark abnormal conditions and repetition measurement once.
C) in all medicine sieve apertures, the CellTiter-Glo reagent of above-mentioned 1 preparation of 100 μ l is added, mixing.
D) on 96 hole microwell plate agitators, fully shake mixing 2 minutes, make the complete cracking of cell.
E) lucifuge room temperature is placed after 15 minutes and is carried out luminescence signal detection, ensures the stability of signal.
F) use during the multi-functional plate reading machine 570nm of EnSpire and read luminescence signal.
G) analyzing and processing data.
The 96 hole medicine sieve plate results of HUH-7 as shown in Figure 2.
The 96 hole medicine sieve plate results of LOVO as shown in Figure 3.
Calculate IC50 value, result is as shown in table 1.
Adopting uses the same method detects Promethazine effect Proliferation of Human Ovarian Cell SKOV-3 and maxicell lung carcinoma cell NCI-H661, and the IC50 value of cell, result is as table 1.
Adopting uses the same method detects Promethazine effect lung carcinoma cell, and the IC50 value of cell, result is as table 1 and Fig. 4.
NCI-H2141 cell, complete medium is HITES (DMEM:F12Medium), is life product, 11330057, fishplate bar density (cells/well) is 16000.
Can find out, Promethazine has Specific Inhibitory Effect to hepatocarcinoma and/or colon cancer and/or proliferation of lung cancer cells, can be used as the medicine of Hepatoma therapy and/or colon cancer and/or pulmonary carcinoma.
Table 1 is the IC50 value of different cell under Promethazine drug effect
Claims (7)
1.Promethazine is preparing the application in Hepatoma therapy and/or colon cancer and/or pulmonary carcinoma product.
The application of 2.Promethazine in preparation suppression hepatocarcinoma and/or colon cancer cell and/or proliferation of lung cancer cells product.
3. application according to claim 1 and 2, is characterized in that: described hepatoma carcinoma cell is HUH-7; Described colon cancer cell is LOVO, and described lung carcinoma cell is NCI-H2141.
4., according to described application arbitrary in claim 1-3, it is characterized in that: described product is medicine or test kit.
5. a product, its active component is Promethazine; Described product has following 1) and/or 2) function:
1) Hepatoma therapy and/or colon cancer and/or pulmonary carcinoma;
2) hepatocarcinoma and/or colon cancer and/or proliferation of lung cancer cells is suppressed.
6. product according to claim 5, is characterized in that: described hepatoma carcinoma cell is HUH-7; Described colon cancer cell is LOVO, and described lung carcinoma cell is NCI-H2141.
7. the product according to claim 5 or 6, is characterized in that: described product is medicine or test kit.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510386589.8A CN105030785B (en) | 2015-06-30 | 2015-06-30 | Applications of the Promethazine in anti-liver cancer and anti-and/or colon cancer and/or lung cancer product is prepared |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510386589.8A CN105030785B (en) | 2015-06-30 | 2015-06-30 | Applications of the Promethazine in anti-liver cancer and anti-and/or colon cancer and/or lung cancer product is prepared |
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|---|---|
| CN105030785A true CN105030785A (en) | 2015-11-11 |
| CN105030785B CN105030785B (en) | 2017-11-10 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040072824A1 (en) * | 2001-06-01 | 2004-04-15 | Adam Telerman | Methods and compositions for the treatment of cancer |
| CN1681511A (en) * | 2002-07-11 | 2005-10-12 | 康宾纳特克斯公司 | Combinations of drugs for the treatment of neoplasms |
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- 2015-06-30 CN CN201510386589.8A patent/CN105030785B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20040072824A1 (en) * | 2001-06-01 | 2004-04-15 | Adam Telerman | Methods and compositions for the treatment of cancer |
| CN1681511A (en) * | 2002-07-11 | 2005-10-12 | 康宾纳特克斯公司 | Combinations of drugs for the treatment of neoplasms |
Non-Patent Citations (2)
| Title |
|---|
| G. R. N. JONES: "Successful Cancer Therapy with Promethazine:The Rationale", 《MEDICAL HYPOTHESES》 * |
| YAPRAK DO¨NMEZ等: "Effect of MDR modulators verapamil and promethazine on gene expression levels of MDR1 and MRP1 in doxorubicin-resistant MCF-7 cells", 《CANCER CHEMOTHER PHARMACOL》 * |
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