KR101638567B1 - Compositions for prevention and treatment of Cancer - Google Patents
Compositions for prevention and treatment of Cancer Download PDFInfo
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- KR101638567B1 KR101638567B1 KR1020150105877A KR20150105877A KR101638567B1 KR 101638567 B1 KR101638567 B1 KR 101638567B1 KR 1020150105877 A KR1020150105877 A KR 1020150105877A KR 20150105877 A KR20150105877 A KR 20150105877A KR 101638567 B1 KR101638567 B1 KR 101638567B1
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/34—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having five-membered rings with one oxygen as the only ring hetero atom, e.g. isosorbide
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/045—Hydroxy compounds, e.g. alcohols; Salts thereof, e.g. alcoholates
- A61K31/05—Phenols
- A61K31/055—Phenols the aromatic ring being substituted by halogen
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/16—Amides, e.g. hydroxamic acids
- A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
- A61K31/167—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the nitrogen of a carboxamide group directly attached to the aromatic ring, e.g. lidocaine, paracetamol
Abstract
The present invention relates to a pharmaceutical composition comprising 2-furanopropanenitrile, α- [2 [3,5-bis (trifluoromethyl) phenyl] hydradinylidene] - β-oxo- or a pharmaceutically acceptable salt thereof, And to a pharmaceutical composition for preventing or treating cancer.
According to the present invention, 2-furanopropanenitrile, α- [2 [3,5-bis (trifluoromethyl) phenyl] hydradinylidene] - β-oxo- or a pharmaceutically acceptable salt thereof, Induced mitochondrial autophagy, resulting in cancer cell death.
Description
The present invention relates to a pharmaceutical composition for preventing and treating cancer. Specifically
2-furanopropanenitrile, a- [2- [3, 5-bis (trifluoromethyl) phenyl] hydrazinylidene] 5-bis (trifluoromethyl) phenyl] hydrazinylidene] -β-oxo-) is administered to cancer cells to activate cancer cells and mitophagy, thereby killing cancer cells.
Autophagy is an action that causes cells to decompose their own proteins in response to nutrient deficiency, or to remove unnecessary cellular components during cell reconstruction. Physiologically, the self-predation process is a recycling system that builds on the basis of new organelles, supplying energy to improve cells or maintain homeostasis, rather than simply removing it. It plays an important role.
Eukaryotic cell death is largely dependent on three mechanisms, namely, apoptosis, autophagy and necrosis, depending on morphological and biochemical characteristics. Autophagy is defined as the process of autophagosomic-lysosomal proteolysis, which is activated in certain situations. Such autophagy is generally triggered under malnutrition conditions, but development, differentiation, It is also known to be associated with physiological processes such as neurodegenerative diseases and infections (Reggiori, F. et al . (2002) Eukaryot.
Mitophagy is a selective degradation process of mitochondria by autophagy, and is caused by mitochondria damaged by stress. The mitochondria prevent the accumulation of mitochondria that do not function to stimulate the circulation of mitochondria and alter the cells, thus making the cells the main factor for maintaining health.
Recently, the importance of active research and autopoiesis based on the control mechanism of cancer cell self-regulation has been established in various diseases, and it has been found that autopatch is not only a part of cell death, but also contributes to cancer cell survival have. However, it is noteworthy that cell organelle-specific autophagy exists, and this detailed autophagy is being studied as a therapeutic agent targeting specific cell death.
Accordingly, the present inventors have completed the present invention as a result of intensive studies to develop a method for treating various cancers by inducing cell organelle-specific autophagy of cancer cells.
Accordingly, it is an object of the present invention to provide a pharmaceutical composition for preventing and treating cancer, which can induce cancer cell death by activating mitochondrial-specific autophagy of cancer cells.
In order to accomplish the above object, the present invention provides a pharmaceutical composition for preventing or treating cancer, comprising a compound represented by the following
In the present invention, the compound represented by the formula (1) is preferably selected from the group consisting of 2-furanopropanenitrile, α- [2- [3,5-bis (trifluoromethyl) phenyl] hydrazinylidene] Acceptable salts include, unless otherwise indicated, salts of acidic or basic groups that may be present in the compounds of the present invention and may be prepared by methods known to those skilled in the art or by preparation of the salts.
In the present invention, since the mitochondrial selective degradation process induced by autophagic action, which is one of eukaryotic cell apoptosis mechanisms, induces mitochondria to induce apoptosis of cancer cells, Can be applied to all cancers, and preferably lung cancer, as long as the mitotic page of the subject can be induced.
In the present invention, the lung cancer is a malignant tumor of the lung, and includes primary lung cancer occurring in the lung itself and metastatic lung cancer in which cancer arising from other organs is transferred to the lung. Small cell lung carcinoma (NSCLC) and non-small cell lung carcinoma (NSCLC) are classified into squamous cell carcinoma, adenocarcinoma, and large cell carcinoma. Squamous cell carcinoma is a lung cancer caused by denaturation of squamous epithelium that constitutes the bronchial mucosa of the lung. It is mainly found in the lungs. Adenocarcinoma is a cancer that occurs in the cytoskeleton, which mainly functions as a secretion of specific substances in the peripheral part of the lungs. Large cell carcinomas mainly occur near the lung surface, and cancer cells are generally large. Small cell carcinoma It is characterized by the first onset of the airway and the strong malignancy.
In one embodiment of the present invention, the compound may have an anticancer effect through autophagic activity of cancer cells.
In the present invention, the self-predation is an action for the cell to remove unnecessary cellular components in the process of decomposing its own protein in response to a nutrient deficiency or rebuilding the cells. Physiologically, the self-predation process is a recycling system that supplies energy to improve cells or maintain homeostasis rather than merely the removal, and build a foundation for new organelles. If the self-predation is above a certain level, it causes apoptosis, which can lead to the death of cancer cells by autophagy. Specifically, when a pharmaceutical composition containing a compound according to the present invention is treated in a cancer cell in which autophagy is inhibited, it is possible to induce an autopoietic process and thereby cause self-destruction of cancer cells. It is known as the phenomenon. In particular, there are patients whose patients have anticancer drug resistance and whose cellular autoregulation is inhibited. When the pharmaceutical composition according to the present invention is treated with these, it can induce auto-apoptosis of cancer cells and can be used as a target drug development.
In one embodiment of the present invention, the self-feeding may be through the activity of LC3 or NIX.
In the present invention, the above-mentioned LC3 is a protein involved in the formation of an autogranular body and is caused by the following mechanism. In mammalian cells, when nutrients decrease, they form autogranular bodies by proteins such as atg and FIP200. In this case, beclin-1-class Ⅲ PI3K complex is required for self-repopulation, LC3 (light chain3) -II should be inserted into the membrane of autoprolide, LC3-Ⅱ is a protein similar to ubiquitin, .
In the present invention, the NIX is one of the proteins associated with mitochondrial activity, and is involved in a mechanism for preventing the accumulation of mitochondria over functions capable of promoting the recovery of mitochondria and inhibiting the cell environment. Furthermore, one of the active pathways in the mitochondrion can be made by the mitochondrion receptor on the surface of the mitochondrial outer membrane, including NIX1, BNIP3 and FUNDCI. Among these, BNIP3L forms a complex with NIX and is involved in the migration of lysosomal proteins from the mitochondrial cytoplasm to the mitochondrial matrix. In addition, all of the above receptors contain a LIR common sequence capable of binding LC3 / GABARAP and resulting in degradation of the mitochondria. One of the receptors, BNIP3, is up-regulated by HIF1α in a hypoxic environment, and BNIP3 then phosphorylates at the serine residue near the LIR sequence promoting LC3 binding. FUNDCI is also susceptible to hypoxic conditions despite being composed in the mitochondrial outer membrane under normal conditions.
Thus, treatment of cancer cells with the composition according to the present invention increases the levels of LC3 and NIX mRNA expression (see Examples 3.4 and 3.5), promotes autophagy formation and induces autophagy, resulting in cell death Can cause.
According to the present invention, 2-furanopropanenitrile, a- [2- [3,5-bis (trifluoromethyl) phenyl] hydrazinylidene] -? - oxo- or a pharmaceutically acceptable salt thereof, Induced mitochondrial-specific autophagy, resulting in the death of cancer cells.
BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a diagram showing information including the structure of a compound according to the present invention.
FIG. 2 shows the results of MTT analysis performed to compare the killing effect of the composition of the present invention on the cancer cell line, the control group and the positive control group.
FIG. 3 is a graph showing changes in expression levels of the LC3 mRNA, which is an autopoietic active gene of the control and the positive control when the composition of the present invention is treated.
FIG. 4 is a Western blotting result for confirming the expression of LC3, an autopoietic active protein of the control and the positive control, when the composition of the present invention was treated.
FIG. 5 is a graph showing changes in NIX mRNA expression level when the composition of the present invention was treated and when the control and positive control mice were treated with the composition of the present invention.
Hereinafter, the present invention will be described in detail with reference to examples. However, these examples are intended to further illustrate the present invention, and the scope of the present invention is not limited to these examples.
≪ Example 1 >
1.1 Culture of Lung Cancer Cell Lines
Human non-RPMI 1640 medium to large cell carcinoma cell line, H460 cell line of the small cell carcinoma (Gibco BRL Life technologies) using
1.2 Statistical analysis
All experiments were repeated three independent experiments and then expressed as mean ± standard deviation, and were recorded as p <0.05 (*), p <0.01 (**).
≪ Embodiment 2 > MTT analysis
The H460 cell line cultured in accordance with the above Example 1.1 was dispensed into 1x10 3 cells in a 96-well plate. The next day, 10 ul of Ez-Cyto (MTT) reagent was added to 90 ul of fresh medium and allowed to react for 30 minutes. Ez-Cytox is a product that measures the amount of living cells using WST and can be used for cell viability, proliferation and cytotoxicity analysis. The substrate, WST, is a highly-sensitive, water-soluble tetrazolium salt that reacts with the dehydrogenase of living cells to produce an orange-colored aqueous formazan. Dehydrogenase, which reacts with WST, is metabolically active It is an enzyme present in the mitochondrial electron transport system of cells and is only effective for living cells. Therefore, the formation of formazan has a linear correlation with the number of living cells, which can be confirmed by measuring absorbance at 450 nm (reference 650 nm) of the plate after the reaction.
As a result, it was confirmed that the cell survival rate was decreased in the group treated with the composition of the present invention (ID-45681) as compared with the control group (CON) in which nothing was treated as in FIG. The results are similar to those of positive control (CCCP), and thus it was confirmed that the composition of the present invention has a cancer cell killing effect.
≪ Example 3 > LC3 and NIX expression analysis
3.1 mRNA extraction and cDNA synthesis
The H460 cell line cultured in accordance with the above Example 1.1 was dispensed into 6 well plates at 3 × 10 5 per 3 wells and replaced with 2 ml of fresh media in 3 wells the following day. CCCP was then treated to a final concentration of 30 uM in the positive control wells and 2-furanopropanenitrile, a- [2- [3,5-bis (trifluoromethyl) phenyl] hydrazinylidene] -β-oxo- (ID-45681 compound) was treated to a final concentration of 3 μM and cultured for 8 hours.
After the incubation, the media was removed, washed with 1X PBS, and then mRNA was extracted using the PureHelix RNA Extraction Solution (Nanohelix Co., South Korea) according to the protocol provided. Then, the mixture was quantitated with GE Nano vue and synthesized with 1 μg of cDNA synthesis Kit- 5x RT premix (reverse transcription-elips biotech).
3.2 Real-Time Quantitative-PCR (RT Q-PCR)
PCR was performed using the primers below (Table 1 below).
SYBR Green Kit-SYBR green with low ROX (enzynomics) 10 μl and the rest 20 μl with H 2 O, using a StepOne Plus real-time PCR system (AB Applied biosystems ) Machine. The PCR cycle is shown in Table 2 below.
The statistical analysis of the results was performed with one-way ANOVA and the significance was confirmed at the level of p <0.05 after calculating the mean ΔCT (normalized cycle threshold) value and the standard deviation value. House-keeping gene is calibrated against GAPDH analysis value.
3.3 Western blotting analysis
The H460 cell line cultured according to Example 1.1 was dispensed into 3 wells of 3 × 10 5 cells in 6 well plates, and the medium was changed to the control group the next day. CCCP (Carbonyl cyanide m-chlorophenyl hydrazone) was added to the positive control group The final concentration was adjusted to 30 uM, and the other plate was treated with 2 ml of the medium so that the composition of the present invention became 10 uM and cultured for 8 hours.
After incubation, the media was removed, washed with 1X PBS, and then eluted with 20 mM Tris-HCl (pH 7.5), 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 0.5% NP40, 10 mM β-glycerophosphate ), 50 mM sodium fluoride, 0.2 mM sodium vanadate, 10 mM PNPP, 5 mM PMSF and 10 μg / ml leupeptin in a lysis buffer . Protein concentration was determined by Bradford protein assay (Bio-Rad, Hercules, Calif.), And 10 μg of the protein was dissolved. Laemmli sample buffer was mixed with the concentration, heated, loaded onto SDS-PAGE, And transferred to the membrane. The cells were blocked with skim milk, washed with TBS-T, and incubated with LC3 as a primary antibody for one day. The next day, the TBS-T-washed rabbit secondary antibody was incubated for 1 hour, washed with TBS-T 3 times for 20 minutes, and developed with a film.
3.4 Identification of LC3 mRNA expression levels
The cDNA synthesized according to Examples 3.1 and 3.2 above was corrected with LC3 / GAPDH. As a result, as compared with the control group (CON) in which nothing was treated as in FIG. 3, 2-furanopropanenitrile, a- [2- [3,5- bis (trifluoromethyl) phenyl] Nylidene] -β-oxo-treated group (ID-45681), the expression level of LC3 mRNA was increased. This was similar to the positive control group (CCCP) and showed a better effect when the composition of the present invention was treated than the positive control group.
As a result of Western blotting according to Example 3.3 above, 2-furanopropanenitrile of the present invention,? - [2 [3, It was confirmed that the expression of LC3 was increased in the group treated with 5-bis (trifluoromethyl) phenyl] hydradinylidene] -? - oxo - (ID-45681) similarly to the positive control group (CCCP).
Therefore, it was confirmed that the composition of the present invention promotes the expression of LC3, which is one of the genes necessary for the formation of autogranular bodies.
3.5 Determination of NIX expression level
The cDNA synthesized according to Examples 3.1 and 3.2 was corrected with NIX / GAPDH. As a result, as compared with the control group (CON) in which nothing was treated as in FIG. 5, 2-furanopropanenitrile of the present invention, a- [2- [3,5- bis (trifluoromethyl) phenyl] Nidene] -? - oxo-treated group (ID-45681), the expression level of NIX mRNA was increased. This was similar to positive control (CCCP). Therefore, it was confirmed that the composition of the present invention promotes the expression of NIX, which is one of the mitochondrial mitogen activated genes.
That is, the composition according to the present invention has an effect of killing cancer cells when treated directly with cancer cells (see Example 2), has an excellent effect of promoting the expression of LC3 involved in autophagic activity (see Example 3.4) , Mitochondrial function regulation and expression of NIX involved in mitotic activity (see Example 3.5). Therefore, by treating the composition of the present invention with cancer cells, autopatching of cancer cells can be induced and utilized for the treatment of cancer.
The present invention has been described with reference to the preferred embodiments. It will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention as defined by the appended claims. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the present invention is defined by the appended claims rather than by the foregoing description, and all differences within the scope of equivalents thereof should be construed as being included in the present invention.
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[Chemical Formula 1]
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20100022640A1 (en) * | 2006-12-20 | 2010-01-28 | Donald Wesley Stoutamire | Esters of 2-phenylalkanenitriles and antifungal compositions containing them |
| KR20100022640A (en) | 2008-08-20 | 2010-03-03 | 주식회사 컴퍼니원헌드레드 | Configuration method for mobile application program based on 3 dimensional graphics user interface modeling language |
| JP4719477B2 (en) * | 2005-02-04 | 2011-07-06 | 花王株式会社 | Hair dye composition |
| KR20110082159A (en) | 2008-10-03 | 2011-07-18 | 콸콤 인코포레이티드 | Double broken seal ring |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP4719477B2 (en) * | 2005-02-04 | 2011-07-06 | 花王株式会社 | Hair dye composition |
| US20100022640A1 (en) * | 2006-12-20 | 2010-01-28 | Donald Wesley Stoutamire | Esters of 2-phenylalkanenitriles and antifungal compositions containing them |
| KR20100022640A (en) | 2008-08-20 | 2010-03-03 | 주식회사 컴퍼니원헌드레드 | Configuration method for mobile application program based on 3 dimensional graphics user interface modeling language |
| KR20110082159A (en) | 2008-10-03 | 2011-07-18 | 콸콤 인코포레이티드 | Double broken seal ring |
Non-Patent Citations (2)
| Title |
|---|
| 논문(JOURNAL OF CHEMICAL RESEARCH, VOLUME 2004, NUMBER 12) * |
| 논문(PLOS PATHOGENS, VOLUME 8, ISSUE 4, 2012.04) * |
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