CN111297849B - Pharmaceutical composition for treating laryngeal cancer, preparation method and application thereof - Google Patents
Pharmaceutical composition for treating laryngeal cancer, preparation method and application thereof Download PDFInfo
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Abstract
The invention relates to the technical field of anti-cancer drugs, in particular to a pharmaceutical composition for treating laryngeal cancer, a preparation method and application thereof. The active ingredients of the pharmaceutical composition comprise amentoflavone, ginkgetin, hinokiflavone, bamboo cedarwood biflavone A, 7-demethyl ginkgetin and 7,4',7', 4' -tetramethoxy amentoflavone. The pharmaceutical composition has good effect on treating laryngeal cancer.
Description
Technical Field
The invention relates to the technical field of anti-cancer drugs, in particular to a pharmaceutical composition for treating laryngeal cancer, a preparation method and application thereof.
Background
The traditional Chinese medicine is a valuable cultural heritage in China, and makes a great contribution to the human health career. China has rich Chinese herbal medicine resources and clinical experience of preventing and treating diseases by using natural medicines for thousands of years, but has an explorable space for pharmacological actions or application of some natural medicines. For example, Selaginella tamariscina is also known as "Selaginella tamariscina", "Selaginella icteris", etc., and is derived from the whole plant of Selaginella tamariscina (Selaginella moellendorffii Hieron) belonging to Selaginellaceae. The plant form is perennial evergreen herbaceous and has strong biological activity. Selaginella tamariscina is mainly distributed in the south of the Yangtze river, from the north to the south of Shaanxi, and is grown under the forest or on the stream side. Selaginella tamariscina is recorded as folk herb and can clear heat, promote urination, promote blood circulation and reduce swelling. Can be used for treating acute infectious hepatitis, chest and hypochondrium contusion, general edema, and thrombocytopenia. However, the pharmacological actions of selaginella tamariscina in the prior art are not reported much, and particularly, related researches on the biflavone enrichment are few.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a pharmaceutical composition for treating laryngeal cancer, a preparation method and application thereof. The invention provides a pharmaceutical composition which can be used for treating laryngeal cancer.
The invention is realized by the following steps:
in a first aspect, embodiments provide a pharmaceutical composition for the treatment of laryngeal cancer, the active ingredients of which include amentoflavone, ginkgetin, hinokiflavone, thuja biflavone a, 7-demethylginkgetin and 7,4',7 ", 4'" -tetramethoxyamentoflavone.
In alternative embodiments, the active ingredient comprises a biflavone concentrate;
preferably, the active ingredient comprises hinokiflavone enrichment, and the hinokiflavone enrichment comprises amentoflavone, ginkgetin, hinokiflavone, tabebuifolin A, 7-demethylginkgetin and 7,4',7', 4' -tetramethoxyamentoflavone.
In an alternative embodiment, the amentoflavone, the ginkgetin, hinokiflavone, thujaplicin a, 7-demethylginkgetin and the 7,4',7 ", 4'" -tetramethoxyamentoflavone are present in a mass ratio of 42 to 76: 5-15: 3-9: 1-7: 6-17: 1-7; preferably 52.7:7.8:4.7: 2.3: 8.2:2.5.
In a second aspect, embodiments provide a method of preparing a pharmaceutical composition for treating laryngeal cancer according to any one of the preceding embodiments, comprising extracting a starting material to obtain the pharmaceutical composition comprising amentoflavone, ginkgetin, hinokiflavone, tabebuione a, 7-demethylginkgetin and 7,4',7 ", 4'" -tetramethoxyamentoflavone;
preferably, the medicinal composition containing the active ingredients is obtained by extracting selaginella tamariscina;
preferably, the extracting comprises: mixing and soaking the selaginella tamariscina with an alcohol solvent to form a crude extract; then, sequentially utilizing petroleum ether, ethyl acetate and n-butanol to extract the crude extract, and respectively collecting corresponding extraction liquid;
then, purifying the ethyl acetate extract to form a Jiangnan Chinese arborvitae biflavone enrichment substance, wherein the Jiangnan Chinese arborvitae biflavone enrichment substance comprises amentoflavone, ginkgetin, hinokiflavone, tabebuifolin A, 7-demethyl ginkgetin and 7,4',7', 4' -tetramethoxyamentoflavone;
preferably, the alcoholic solvent is a monohydric alcohol, preferably ethanol;
preferably, the step of forming the Jiangnan Chinese arborvitae twig and leaf flavonoid enrichment comprises the steps of adsorbing components in the ethyl acetate extraction liquid by using resin, then carrying out gradient elution, and collecting eluent to form the Jiangnan Chinese arborvitae twig and leaf flavonoid enrichment;
preferably, the gradient elution comprises elution with water-alcohol solvents of different mass concentrations.
In a third aspect, embodiments provide the use of a pharmaceutical composition for the treatment of laryngeal cancer according to any one of the preceding embodiments in the manufacture of a medicament for the treatment of laryngeal cancer;
preferably, the pharmaceutical composition is a medicament for inducing apoptosis of laryngeal cancer cells;
preferably, the pharmaceutical composition is a drug that inhibits the ability of cells to migrate.
In an alternative embodiment, the pharmaceutical composition is a drug that induces apoptosis of laryngeal cancer cells by affecting mitochondrial apoptosis signaling pathways;
preferably, the pharmaceutical composition is a drug which affects the mitochondrial apoptosis signal transduction pathway by down-regulating the expression level of Bcl-2 protein and/or up-regulating the expression level of Bax protein;
alternatively, the pharmaceutical composition is a drug that affects the mitochondrial apoptosis signaling pathway by up-regulating the expression levels of Caspase-3 and Caspase-9.
In an alternative embodiment, the pharmaceutical composition is a drug that induces apoptosis of the laryngeal cancer cell by affecting the JAK-STAT signaling pathway;
preferably, the pharmaceutical composition is a drug that in turn affects the JAK-STAT signaling pathway by downregulating the phosphorylation level of STAT3 protein.
In alternative embodiments, the pharmaceutical composition is an agent that induces apoptosis of the laryngeal cancer cell by affecting the Akt/NF- κ B signaling pathway;
preferably, the pharmaceutical composition is a drug that affects the Akt/NF- κ B signaling pathway by downregulating the phosphorylation level of Akt protein and NF- κ B protein.
In a fourth aspect, embodiments provide the use of a pharmaceutical composition for the treatment of laryngeal cancer according to any one of the preceding embodiments in the preparation of a medicament for the modulation of at least one pathway from (1) to (3) below;
(1) activating a mitochondrial apoptosis signaling pathway;
(2) inhibiting the JAK-STAT signaling pathway;
(3) inhibiting Akt/NF-kB signal channel.
In a fifth aspect, embodiments provide the use of a pharmaceutical composition for the treatment of laryngeal cancer according to any one of the preceding embodiments in the manufacture of a medicament for the modulation of at least one of (a) - (c);
(a) up-regulating the expression level of at least one of (4) - (6), wherein (4) is a Bax protein, (5) is Caspase-3 and (6) is Caspase-9 protein;
(b) down-regulating the expression level of at least one of (7) - (8), wherein (7) is an NF- κ B protein and (8) is a Bcl-2 protein;
(c) down-regulating the phosphorylation level of at least one protein from (9) to (10), wherein (9) is a STAT3 protein and (10) is an Akt protein.
The invention has the following beneficial effects: the invention can induce cancer cell apoptosis by adopting active ingredients containing amentoflavone, ginkgetin, hinokiflavone, bamboo cedarwood biflavone A, 7-demethyl ginkgetin and 7,4',7', 4' -tetramethoxy amentoflavone, thus having good treatment effect on laryngeal cancer and being used for preparing anti-laryngeal cancer drugs.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings needed to be used in the embodiments will be briefly described below, it should be understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and for those skilled in the art, other related drawings can be obtained according to the drawings without inventive efforts.
FIG. 1 is a schematic flow chart of the extraction process of the Jiangnan Selaginella moellendorfii Bifida enriched substance provided by the embodiment of the invention.
FIG. 2 is a graph showing the results of the test provided in Experimental example 1 of the present invention.
FIG. 3 is a graph showing the results of the test provided in Experimental example 2 of the present invention; wherein, A and C represent micrographs of two laryngeal cancer cell lines at 0, 12 and 24 hours after treatment with SM-BFRE (0, 5, 10 and 20. mu.g/mL), respectively, and the relative wound surface area in the wound healing test was calculated using Image J software, and B and D represent the expression of MMP-9 protein measured by western blot method and the relative expression amount of protein calculated by Image Lab software.
FIG. 4 is a graph showing the results of the test provided in Experimental example 3 of the present invention; wherein, A and C represent morphological changes after treatment of the two laryngeal cancer cell lines for 24h with SM-BFRE (0, 10, 30 and 50. mu.g/mL), and B and D represent changes of the two laryngeal cancer cell lines treated with SM-BFRE (0, 10, 30 and 50. mu.g/mL) for 24h and observed under a fluorescence microscope with Hoechst 3328 staining.
FIG. 5 is a graph showing the results of the test provided in Experimental example 4 of the present invention; wherein, A and C represent that Hep-2 and TU212 cells were treated with SM-BFRE (0, 10, 30 and 50. mu.g/mL) for 24 hours, cells were stained with Annexin V-FITC and PI, and the proportion of apoptosis was examined by flow cytometry, and B and D represent that the apoptosis of laryngeal cancer cells treated with SM-BFRE (0 and 50. mu.g/mL) was observed under a fluorescence microscope after examination by flow cytometry.
FIG. 6 is a graph showing the results of the test provided in Experimental example 5 of the present invention; wherein, A and B represent the relative protein expression of Bcl-2/Bax, clear caspase-9, caspase-3 and PARP after the two laryngeal cancer cell lines are treated by SM-BFRE (0, 10, 30 and 50 mug/mL) for 24h by a Western blot method.
FIG. 7 is a graph showing the results of the test provided in Experimental example 6 of the present invention; wherein, A and B represent tumor removal and photography after 4 weeks of Hep-2 tumor-bearing mice treated with SM-BFRE (0, 45 and 90mg/kg/day), C represents the volume of transplanted tumor measured with vernier calipers every 4 days during the experiment, and when the experiment was finished, tumor was removed and weighed, D and E represent tumor sections prepared, TUNEL and IHC staining was performed, and the proportion of apoptotic cells was calculated, F represents protein extraction from transplanted tumor tissue, and relative expression of Bcl-2/Bax, clear caspase-9, caspase-3 and PARP was detected by Western blot.
FIG. 8 is a graph showing the results of the test provided in Experimental example 7 of the present invention; wherein, A and B represent the relative expression of p-STAT3(Tyr705) in the two laryngeal cancer cell lines after treatment with SM-BFRE (0, 10, 30 and 50. mu.g/mL) for 24h, and C represents the extraction of protein from the transplanted tumor tissue and the relative expression of p-STAT3(Tyr705) by Western blot.
FIG. 9 is a graph showing the results of the test provided in Experimental example 8 of the present invention; wherein, A and B represent the relative expression of p-Akt (Ser473) and NF-kB in two laryngeal cancer cell lines after treatment with SM-BFRE (0, 10, 30 and 50. mu.g/mL) for 24h, and C represents the extraction of protein from transplanted tumor tissue and the detection of the relative expression of p-Akt (Ser473) and NF-kB by Western blot.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The features and properties of the present invention are described in further detail below with reference to examples.
First, the embodiment of the present invention provides a pharmaceutical composition for treating laryngeal cancer, active ingredients of the pharmaceutical composition include amentoflavone, ginkgetin, hinokiflavone, thuja biflavone a, 7-demethylginkgetin and 7,4',7 ", 4'" -tetramethoxyamentoflavone. The laryngeal cancer cell apoptosis can be effectively induced through the actions of the substances, and then laryngeal cancer is treated.
Specifically, the structural formula of amentoflavone (compound 1), ginkgetin (compound 2), tabebuia avellanedae A (compound 4), 7-demethylginkgetin (compound 5) and 7,4',7', 4' -tetramethoxyamentoflavone (compound 6) is as follows:
wherein the structural formula of hinokiflavone (compound 3) is as follows:
specifically, the mass ratio of amentoflavone, ginkgetin, hinokiflavone, tabebuia arborvitae A, 7-demethylated ginkgetin and 7,4',7', 4' -tetramethoxyamentoflavone is 42-76: 5-15: 3-9: 1-7: 6-17: 1-7; preferably 52.7:7.8:4.7: 2.3: 8.2:2.5.
The active substance comprises biflavone enrichment, further, the active ingredient comprises Jiangnan Chinese arborvitae biflavone enrichment, and the Jiangnan Chinese arborvitae biflavone enrichment comprises amentoflavone, ginkgetin, hinokiflavone, bamboo cedarwood biflavone A, 7-demethyl ginkgetin and 7,4',7', 4' -tetramethoxy amentoflavone.
That is, the pharmaceutical composition for treating laryngeal cancer of the present application includes the hinokiflavone concentrate including amentoflavone, ginkgetin, hinokiflavone, tabebuiflavone a, 7-demethylginkgetin and 7,4',7 ", 4'" -tetramethoxyamentoflavone. Certainly, the pharmaceutical composition can also comprise other components with treatment effect on laryngeal cancer so as to further improve the treatment effect of the pharmaceutical composition, or can also comprise other active components so as to reduce the toxic and side effects of the pharmaceutical composition or improve the treatment effect of the pharmaceutical composition, or the pharmaceutical composition can only comprise the Jiangnan Chinese arborvitae biflavone enrichment substance and can also ensure the treatment effect of the pharmaceutical composition, or the pharmaceutical composition can also comprise pharmaceutically acceptable carriers or auxiliary materials and can be prepared into a medicament with a certain dosage form.
It should be noted that, in the present embodiment, the term "pharmaceutically acceptable" refers to a compound which is physiologically acceptable when the compound is administered to a human, and does not cause allergic reactions such as gastrointestinal disorders, dizziness, and the like, or systemic allergic reactions similar to these allergic reactions.
In embodiments of the invention, a "pharmaceutically acceptable carrier or excipient" includes, but is not limited to: binders (such as microcrystalline cellulose, alginates, gelatin, and polyvinylpyrrolidone), fillers (such as starch, sucrose, glucose, and anhydrous lactic acid), disintegrants (such as crosslinked PVP, sodium crosslinked carboxymethyl starch, sodium crosslinked carboxymethyl cellulose, and low-substituted hydroxypropyl cellulose), lubricants (magnesium stearate, aluminum stearate, talc, polyethylene glycol, sodium benzoate), wetting agents (such as glycerin), surfactants (such as cetyl alcohol), and absorption enhancers, flavors, sweeteners, diluents, coating agents, and the like.
Meanwhile, in order to allow the drug to release the active ingredient rapidly, continuously and over a long period of time, the pharmaceutical composition of the present invention may be manufactured according to conventional methods disclosed in those technical fields. The route of administration of the drug of this embodiment may be oral, nasal inhalation, or parenteral administration. The preparation containing the composition can be tablet, soft capsule, hard capsule, oral liquid, pill, suppository, powder, granule, emulsion, syrup, aerosol, sterile injection, sterile powder, etc.
The embodiment of the invention also provides a preparation method of the pharmaceutical composition for treating laryngeal cancer, which comprises the step of extracting selaginella tamariscina to obtain the pharmaceutical composition containing the active ingredients.
Further, mixing and soaking the selaginella tamariscina with an alcohol solvent to form a crude extract; then, petroleum ether, ethyl acetate and n-butanol are sequentially used for extracting the crude extract, and corresponding extraction liquid is respectively collected. The alcohol solvent used is a monohydric alcohol, preferably ethanol, most preferably ethanol at a concentration of 95% (volume fraction). The embodiment of the invention adopts an immersion method for extraction, 95% ethanol is used for leaching for 4 times, the dosage of the 95% ethanol is 2-3 liters each time, the extraction effect is ensured, and meanwhile, the method is simple to operate.
And then, purifying the ethyl acetate extract to form a Jiangnan Chinese arborvitae biflavone enrichment, wherein the Jiangnan Chinese arborvitae biflavone enrichment comprises amentoflavone, ginkgetin, hinokiflavone, tabebuia bambusicola biflavone A, 7-demethyl ginkgetin and 7,4',7', 4' -tetramethoxyamentoflavone.
Further, the step of forming the Jiangnan Chinese arborvitae twig and leaf biflavone enrichment substance comprises the steps of adsorbing components in the ethyl acetate extraction liquid by using resin, such as a D101 macroporous resin column, then carrying out gradient elution, and collecting eluent to form the Jiangnan Chinese arborvitae twig and leaf biflavone enrichment substance; preferably, the gradient elution comprises elution with water-alcohol solvents of different mass concentrations; preferably, the gradient elution includes gradient elution using 10%, 30%, 50%, 70%, 80%, and 95% water-ethanol mixed solutions in sequence.
And performing high performance liquid (hereinafter referred to as high performance liquid) analysis on the eluents, combining the eluents having the same high performance liquid analysis result according to the HPLC analysis result, meanwhile, the high-liquid analysis shows that the eluents with consistent chromatographic peaks are combined and mainly contain 6 compounds of amentoflavone (compound 1, retention time 4.75 minutes), ginkgetin (compound 2, retention time 8.83 minutes), hinokiflavone (compound 3, retention time 10.08 minutes), hinokiflavone A (compound 4, retention time 13.81 minutes), 7-demethylginkgetin (compound 5, retention time 13.81 minutes) and 7,4',7', 4' -tetramethoxyamentoflavone (compound 6, retention time 17.79 minutes), the purified Jiangnan Chinese arborvitae biflavone enrichment substance is obtained, and the biflavone content is more than 85 percent calculated by the peak area of 6 compounds.
And separating the Jiangnan Chinese arborvitae biflavone enriched substance by using a chromatographic column, such as a Phenomenex Gemini C18 HPLC chromatographic column, to obtain 6 fractions with different peaks. Simultaneously, the above 6 fractions were dissolved in DMSO-d, respectively6The NMR test is carried out, so that the test not only confirms that the compounds 1-6 are actually present in the Jiangnan Cedar biflavone enrichment, but also confirms the respective contents of the compounds 1-6, and each gram of the Jiangnan Cedar biflavone enrichment contains 400-620 mg of the compound 1, 47-93 mg of the compound 2, 34-62 mg of the compound 3, 14-33 mg of the compound 4, 63-104 mg of the compound 5 and 14-37 mg of the compound 6.
Furthermore, the embodiment of the invention also provides an application of the pharmaceutical composition for treating laryngeal cancer in preparing a medicament for treating laryngeal cancer; preferably, the pharmaceutical composition is a medicament for inducing apoptosis of laryngeal cancer cells; preferably, the pharmaceutical composition is a drug that inhibits the ability of cells to migrate.
Specifically, the pharmaceutical composition induces laryngeal cancer cell apoptosis by influencing a mitochondrial apoptosis signal transduction pathway, wherein the expression and regulation of bcl-2 family are one of key factors influencing mitochondrial apoptosis signal transduction, and the main action site of the pharmaceutical composition is on a mitochondrial membrane. After the cell is stimulated by death signals, the pro-apoptotic protein in the bcl-2 family generates conformation change under the action of protease, is displaced from cytoplasm to the membrane of an organelle, particularly to the outer membrane of mitochondria, and interacts with the anti-apoptotic protein on the membrane of the organelle and in the membrane, so that the anti-apoptotic protein loses the inhibiting effect on apoptosis, the function of the organelle is lost, various pro-apoptotic factors are released, and finally, the laryngeal cancer cell is apoptotic.
Bcl-2 and Bax are the most representative apoptosis inhibiting and apoptosis promoting genes, respectively, in the Bcl-2 family. In normal cells, Bcl-2 is usually expressed negatively, while Bax is expressed positively. However, in malignant tumors such as laryngeal cancer, liver cancer, lung cancer, stomach cancer, breast cancer, neuroblastoma, nasopharyngeal cancer, prostate cancer, bladder cancer, etc., the up-regulation and Bax down-regulation of Bcl-2 are observed, and the lower the degree of cell differentiation, the more remarkable the trend.
The inventor finds that the pharmaceutical composition can influence the mitochondrial apoptosis signal transduction pathway and then induce laryngeal cancer cell apoptosis by down-regulating the expression level of Bcl-2 protein and/or up-regulating the expression level of Bax protein.
Meanwhile, the change of the Bcl-2/Bax ratio finally causes the apoptosis caused by the caspase family. The caspase family is a class of cysteine proteases that specifically recognize aspartate sites and are involved in multiple processes of apoptotic signal transduction. In the mitochondrial cell-mediated apoptosis pathway, Caspase family participates in the amplification and apoptosis effect of apoptosis signals, amplifies the apoptosis signals in an enzymatic cascade reaction mode, catalyzes apoptosis-related effect proteins and finally leads to the apoptosis of cells. For example, under the regulation of Bcl-2 family, mitochondria release apoptosis activating factor 1(Apaf1), Apaf1 combines with cytochrome C (cytoC) and ATP, and through self conformation change and self activation, Apaf 1N end forms a surface structure which can activate Caspase 8/9, further activate Caspase 3/6/7, and finally Caspase 3/6/7 catalyzes a series of apoptosis related substrates to cause apoptosis.
The inventor also finds that the pharmaceutical composition influences the mitochondrial apoptosis signal transduction pathway by up-regulating the expression levels of Caspase-3 and Caspase-9, and then induces laryngeal cancer cell apoptosis.
Meanwhile, the inventor also finds that the pharmaceutical composition induces the laryngeal cancer cell apoptosis by influencing JAK-STAT signal path.
The JAK-STAT signaling pathway is a cytokine-stimulated signaling pathway discovered in recent years, and is involved in many important biological processes such as proliferation, differentiation, apoptosis, and immunoregulation of cells. Among them, STAT3 activity plays a critical role in the process of cell malignancy. STAT proteins (particularly STAT3) are required for the maintenance of the post-translational phenotype in many tumor-derived cell lines, including laryngeal, liver, breast, lung, stomach, neuroblastoma, nasopharyngeal, prostate, or bladder cancer.
The following discussion will be made in detail, taking laryngeal carcinoma as an example:
many risk factors for laryngeal cancer may contribute to activation of STAT 3. The delivery process of the JAK-STAT signaling pathway is relatively simple. The signal transmission process is as follows: the binding of cytokines to the corresponding receptors causes dimerization of the receptor molecules, which brings the JAK kinases coupled to the receptors into proximity and activation by interactive tyrosine phosphorylation. Following JAK activation, tyrosine residues on the catalytic receptor undergo phosphorylation modification, and these phosphorylated tyrosine sites form a "docking site" with surrounding amino acid sequences, and STAT proteins containing the SH2 domain are recruited to this "docking site". Finally, kinase JAK catalyzes STAT protein combined on a receptor to generate phosphorylation modification, and the activated STAT protein enters a cell nucleus in a dimer form to be combined with a target gene so as to regulate and control the transcription of the gene. STAT3 is highly phosphorylated in almost all head and neck tumors. Therefore, STAT3 is used as a very important molecular target for hepatocellular carcinoma treatment.
The inventor finds that the pharmaceutical composition can influence the JAK-STAT signal pathway by down-regulating the phosphorylation level of STAT3 protein, so as to achieve the aim of inducing laryngeal cancer cell apoptosis.
Further, the pharmaceutical composition induces apoptosis in said laryngeal cancer cells by affecting the Akt/NF- κ B signaling pathway, the nuclear factor κ B (NF- κ B) is a family of closely related transcription factors, an important nuclear transcription factor in the cells. It is mainly involved in the inflammatory and immune reactions of the human body. However, there are increasing reports indicating that NF-. kappa.B regulates the expression of genes that are critical in the development and progression of tumors, including cell proliferation, migration and apoptosis. NF-. kappa.B is generally thought to exist in the cytoplasm as a dimer in the form of an inhibitor of its inactive state (I.kappa.Bs). Upon stimulation, I κ B proteins are degraded by phosphorylation and ubiquitination, and NF- κ B dimers are released and eventually transferred to the nucleus, which binds to the target genes to facilitate their transcription. Studies have shown that activation of the PI3K/Akt signaling pathway is also associated with activation of NF-. kappa.B. Akt has two phosphorylation sites (Thr308 and Ser473) and can be activated by dual phosphorylation. Phosphorylation of the Thr308 site activates the Akt moiety, but phosphorylation of Ser473 is required for overall Akt functional activity. A decrease in expression of phosphorylated Akt (p-Akt Ser473) is a key indicator of PI3K/Akt pathway activation. As mentioned previously, NF-. kappa.B has a regulatory effect on apoptosis, and it is generally believed that NF-. kappa.B inhibits apoptosis.
The inventor finds that the pharmaceutical composition can reduce the phosphorylation level of Akt protein and the expression of NF-kB protein, then regulates an Akt/NF-kB signal channel, and further induces the apoptosis of laryngeal cancer cells.
It is further explained that the above pharmaceutical composition for the treatment of laryngeal cancer can be applied to the preparation of a modulating drug for inhibiting at least one pathway of the following (1) to (3); (1) activating a mitochondrial apoptosis signaling pathway; (2) inhibiting the JAK-STAT signaling pathway; (3) inhibiting Akt/NF-kB signal channel.
It is further illustrated that the above pharmaceutical composition for the treatment of laryngeal cancer can be applied for the preparation of a medicament for the modulation of at least one of the conditions of (a) - (c); (a) up-regulating the expression level of at least one of (4) - (6), wherein (4) is a Bax protein, (5) is a Caspase-3 protein and (6) is a Caspase-9 protein; (b) down-regulating the expression level of at least one of (7) - (8), wherein (7) is an NF- κ B protein and (8) is a Bcl-2 protein; (c) down-regulating the phosphorylation level of at least one protein from (9) to (10), wherein (9) is a STAT3 protein and (10) is an Akt protein. Or the pharmaceutical composition for treating laryngeal cancer can be applied to the preparation of drugs for inhibiting the cell migration ability of laryngeal cancer.
Example 1
The embodiment provides a pharmaceutical composition, the active ingredients of which comprise amentoflavone, ginkgetin, hinokiflavone, sabdariffaein A, 7-demethylginkgetin and 7,4',7 ", 4'" -tetramethoxyamentoflavone, and the mass ratio of the amentoflavone to the ginkgetin A is 52.7:7.8:4.7: 2.3: 8.2:2.5.
The embodiment of the invention also provides a preparation method of the pharmaceutical composition, which comprises the following steps:
with reference to the process flow shown in fig. 1, selaginella tamariscina is extracted to obtain the pharmaceutical composition containing the active ingredients.
Specifically, the dried whole herb of Selaginella moellendorfii hieron (500g) was ground and then immersed in 95% EtOH four times (2.5L each for 3 days) in sequence at room temperature for leaching. The solvent was evaporated under reduced pressure to give a crude extract (61.5 g). Suspending the crude extract in water at a ratio of 1:10, sequentially extracting with equal volumes of Petroleum Ether (PE), ethyl acetate (EtOAc) and n-butanol (n-BuOH) for 4 times, mixing the corresponding extracts, concentrating and drying to obtain PE fraction (4.3g), EtOAc fraction (9.4g), n-BuOH fraction (23.7g) and water fraction.
Subjecting the EtOAc part (8g) to D101 macroporous resin column chromatography, and gradient eluting with water-ethanol with mass concentration of 10%, 30%, 50%, 70%, 80% and 95% to obtain 142 kinds of eluates. The different eluates were combined into seven fractions (frs.a-G) according to HPLC analysis. Wherein the eluates with retention time of 4.75 min, 8.83 min, 10.08 min, 13.81 min and 17.79 min chromatographic peaks were combined and concentrated as purified Asparagus ferox biflavone enrichment (accession number: SM-BFRE, 3.5 g).
And (3) carrying out chemical analysis on the Jiangnan selaginella biflavone enrichment by an HPLC-PDA method. Gradient elution was optimized for analysis using a Phenomenex Gemini-NX C18 HPLC column (5 μm, 4.6 x 250mm) (acetonitrile in water with 0.1% formic acid). The gradient program is as follows: 0-13 minutes, 40-70% acetonitrile; 13-13.05 minutes, and 70-90% of acetonitrile; 13.05-20 minutes, and acetonitrile 90-95%; 20-25 min, 95% acetonitrile. The flow rate for the assay was 1.0 mL/min. 100mg of SM-BFRE was dissolved in 2.0mL of methanol and the solution was filtered. Semi-preparative HPLC was performed with Phenomenex Gemini C18 HPLC column (5 μm, 10 × 250mm) (acetonitrile containing 0.1% formic acid in 40% to 60% water from 100% SM-BFRE for 16 min from 60%) reduced to 90% in the next 0.05 min, to 95% in the next 8.95 min and held at 95% acetonitrile for 5 min) at a flow rate of 4.0 mL/min. Fractions of 6 chromatographic peaks were collected manually and 20 injections were repeated, after which 6 fractions were taken in DMSO-d6 for test NMR, to confirm that the 6 fractions were Compound 1(52.7mg), 2(7.8mg), 3(4.7mg), 4(2.3mg), 5(8.2mg) and 6(2.5mg), respectively.
Examples of the experiments
The pharmacodynamic test is combined to evaluate the pharmacodynamic effect of the Jiangnan Chinese arborvitae twig and leaf flavonoid enrichment provided in the embodiment 1 of the invention.
Sample preparation:
and (3) a to-be-detected product: the hinokiflavone concentrate provided in example 1;
comparative example 1: the crude extract provided in example 1;
comparative example 2: the ethyl acetate extract provided in example 1;
experimental example I, test of the Effect of the test substance and comparative examples 1 and 2 on the cell viability of human laryngeal carcinoma cells Hep-2 and TU212 and normal laryngeal epithelial cells HBE and IC50Value of
Taking Hep-2 and TU212 cell strains in logarithmic growth phaseRegulating cell concentration to 5X 104Inoculating 0.1mL of the culture solution into a 96-well culture plate per well, continuously culturing for 24h, and then respectively adding 0.2mL of a sample to be tested prepared by DMEM solution containing 10% calf serum into each well to ensure that the sample concentration is respectively 5 mu g/mL, 10 mu g/mL, 20 mu g/mL, 50 mu g/mL and 100 mu g/mL, and continuously culturing for 12h, 24h and 48 h; each concentration was done in 3 wells, with experimental control wells without test sample and blank control wells without sample and cells. And adding 0.1mL of MTT into each hole after the administration time is reached, continuously incubating for 30min, sucking the culture solution after the incubation time is reached, adding 0.15mL of DMSO into each hole, fully dissolving purple formazan crystals generated in the cells, and measuring the absorbance A value of each hole at 562 nm. HBE cells were cultured and administered for 24 hours in the same manner as described above.
Growth inhibition rates of Hep-2, TU212 and HBE cells ═ 1- (experimental a value-blank a value)/(control a value-blank a value)]X 100%. Half maximal Inhibitory Concentration (IC) was calculated using SPSS 19.050)。
Referring to fig. 2, the hinokiflavone concentrate provided in example 1 of the present invention shows a good activity inhibition on laryngeal cancer cells within a concentration range of 5-100 μ g/mL, and also shows an obvious dose-effect and aging relationship, but has little effect on normal laryngeal epithelial cells HBE. Compared with the above, the crude ethanol extract (SME) and ethyl acetate fraction (SMEA) of Selaginella moellendorfii hieron have significantly weaker inhibitory effect on two laryngeal cancer cells.
Experimental example II, the influence of the test substance on the migration ability of Hep-2 and TU212 cells was examined
Hep-2 and TU212 cells were collected at 5X 10 in logarithmic growth phase4Inoculating the cells/well into a 6-well plate, culturing for 24h, removing the culture solution by suction, adding the Jiangnan Chinese arborvitae flavonid concentrates with different concentrations (the sample concentrations are respectively 0 mu g/mL, 5 mu g/mL, 10 mu g/mL and 20 mu g/mL), and scraping the plate surface by using a 200 mu L sterile pipette tip to form a wound. The wound area was observed under an inverted microscope after 0h, 12h and 24h of administration. Measurements of wound surface area were calculated by Image J software.
Matrix Metalloproteinases (MMPs) play a major role in the metastasis and invasion of tumor cells. Among them, MMP-9 plays a key role in promoting cell migration. Therefore, we examined the expression of MMP-9 protein in two laryngeal cancer cells by western blot.
The results are shown in fig. 3, where the wound heals significantly after 12h and 24h, i.e. the wound heals more over time, compared to 0 h. Meanwhile, the inhibition effect of SM-BFRE on the migration of two laryngeal cancer cells is gradually increased along with the increase of the dosage. The results of the WB experiments showed that SM-BFRE dose-dependently inhibited MMP-9 expression. The result shows that the Jiangnan Chinese arborvitae twig concentrate provided by the embodiment 1 of the invention has a remarkable inhibiting effect on the migration of laryngeal cancer cells.
Experimental example III, the influence of the test substance on the morphology of Hep-2 and TU212 cells is detected
Hep-2 and TU212 cells were collected at 5X 10 in logarithmic growth phase4Inoculating the cells into a 6-well plate at each concentration, culturing for 24h, removing the culture solution by suction, adding the Jiangnan Chinese arborvitae twig-flavone concentrates with different concentrations (the sample concentrations are 0 mu g/mL, 10 mu g/mL, 30 mu g/mL and 50 mu g/mL respectively), continuing culturing for 24h, observing the cell morphology by a microscope in a bright field, removing the culture solution by suction, adding a fixing solution (methanol: glacial acetic acid ═ 3:1) into each well, removing the culture solution by suction after fixing, adding PBS (phosphate buffer solution) for cleaning twice, 3min each time, removing the liquid by suction, and air drying. Hoechst33258 staining solution was added to cover all cells. The cell morphology was observed using a fluorescence inverted microscope.
The results are shown in FIG. 4, the Hep-2 cells of the two blank groups showed good adherence, high total number, clear boundary, uniform cell distribution, normal nuclear morphology, circular or irregular oval shape, uniform nuclear mass distribution, and uniform fluorescence. After the Jiangnan Chinese arborvitae biflavone enrichment substance is treated, apoptosis of different degrees appears along with the increase of dosage, and the apoptosis characteristics of Hep-2 cells are gradually obvious. The cell morphology of the low-dose group (the drug concentration is 10 mug/mL) is not uniform, the number of adherent cells is reduced, and apoptotic bodies begin to appear; the number of adherent cells in the medium dose group (30 mu g/mL) is further reduced, a small amount of apoptotic bodies appear, and compact granular fluorescence can be seen in cell nuclei; the number of adherent cells in the high dose group (drug concentration 50. mu.g/mL) was significantly reduced, the nuclei were pyknomized, and more apoptotic bodies appeared. The experimental results for TU212 cells were consistent with that of Hep-2 cells.
Fourth experiment example, detecting the influence of the test substance on the apoptosis number of Hep-2 and TU212 cells
The cell culture and administration method is shown in experiment three. After 24h of cell administration and culture, cells are digested by trypsin without ethylenediaminetetraacetic acid (EDTA), 300g is centrifuged for 5min to collect suspended cells, the cells are washed twice by cold PBS, 400 mul of 1 Xannexin V is added to resuspend the cells, 5 mul of Annexin V-FITC staining solution is added firstly to incubate for 15min in the dark, 5 mul of PI staining solution is added to incubate for 5min in the dark, and the cells are immediately observed by a flow cytometer and a fluorescence microscope. The percentage of apoptotic cells was analyzed by FlowJo V10 software.
As a result, as shown in FIG. 5, in the flow cytometry results, the percentages of Q1, Q2, Q3 and Q4 in Hep-2 cells were 15.47%, 16.63%, 39.00% and 50.00% respectively, while those in TU212 cells were 10.92%, 17.56%, 28.20% and 51.60% respectively, at each dose of the spikenard and spikemoss enrichment groups (0. mu.g/mL, 10. mu.g/mL, 30. mu.g/mL and 50. mu.g/mL) provided in example 1 of the present invention, indicating that SM-BFRE dose-dependently induced apoptosis of laryngeal cancer cells. Under a fluorescence microscope, early apoptotic cells are stained with Annexin V-FITC to enable cell membranes to show green fluorescence, and on the basis, late apoptotic cells are stained with Propidium Iodide (PI) to enable cell nuclei to show. The results show that the number of apoptotic cells increases significantly at a concentration of SM-BFRE of 50. mu.g/mL compared to 0. mu.g/mL.
Experimental example five, detecting the influence of the test sample on the expression of Hep-2 and TU212 cell mitochondrial apoptosis signal transduction pathway-related proteins in vitro
Apoptosis is accompanied by a series of changes in the protease enzyme. The activity change of Bcl-2 family and Caspase family is involved in the initiation and execution process of mitochondrial apoptosis pathway. Bcl-2, Bax, Caspase-3 and Caspase-9 are key participatory molecules, and the detection of the Bcl-2, Bax, Caspase-3 and Caspase-9 can reflect whether the medicine induces the apoptosis of the liver cancer cells through the mitochondrial pathway.
Hep-2 and TU212 cells were harvested at 1.37X 10 in logarithmic growth phase6The concentration of each cell is inoculated in a cell culture dish of 100mm multiplied by 20mm for 24h,adding hinokiflavone enrichment substances with different concentrations (the sample concentrations are respectively 0 mug/mL, 10 mug/mL, 30 mug/mL and 50 mug/mL) for 24h, collecting cell sap, washing with PBS, adding cell lysate for cracking, performing ultrasonic crushing, performing boiling water bath for 10min, and determining the protein concentration by using a BCA protein concentration determination kit. Preparing concentrated gel and separating gel, separating total protein by adopting 12% SDS-PAGE gel electrophoresis, transferring the intracollagenic protein onto a PVDF membrane according to an immunoblotting method, sealing the membrane by using 5% skimmed milk powder solution overnight, respectively adding primary antibodies of anti-Bax, anti-Bcl-2, anti-cleared-Caspase-3, anti-cleared-Caspase-9, anti-PARP and anti-beta-actin, incubating the membrane overnight, washing the membrane by TBST for three times, adding a secondary antibody marked by horseradish peroxidase, incubating the membrane for 2 hours at room temperature, washing the membrane by TBST for 3 times, sequentially adding a luminescent substrate according to an ECL method, exposing, developing and fixing the membrane, and analyzing the strip by adopting Image software.
The results are shown in FIG. 6, the expression levels of some important proteins in Hep-2 and TU212 cells are obviously changed, the expression levels of Bax, clear-Caspase-3, clear-Caspase-9 and clear-PARP are increased along with the increase of the administration concentration of the Jiangnan selaginella biflavone enrichment, the expression level of Bcl-2 is reduced, and the ratio of Bcl-2/Bax is obviously reduced. The result shows that the Jiangnan selaginella biflavone enrichment provided by the embodiment 1 of the invention plays a role in resisting laryngeal cancer by influencing the expression of related proteins of mitochondrial apoptosis pathways.
Sixth Experimental example, detecting the influence of the in vitro test substance on the expression of mitochondrial apoptosis signal transduction pathway-related proteins in animals
The laryngeal carcinoma cell Hep-2 was inoculated into the right rear side of nude mice (BALB/c, SPF grade, female, 16-18g, 4-5 weeks old) at a concentration of 1X 107A/only. When the tumor grows to about 100mm3In time, mice were randomized into three groups (five mice per group) and injected intraperitoneally (ip) with the Jiangnan Tourette biflavone concentrate (0, 45 and 90mg/kg/day) for 28 days, with tumor volumes measured every four days. After 28 days, the transplanted tumors were removed for evaluation. All procedures involved in our animal experiments were in accordance with the requirements of the animal protection and use committee of the university of south-central ethnic group (wuhan, china).
As shown in FIG. 7, it can be seen from A-C in FIG. 7 that the enrichment of the oncidin according to example 1 of the present invention dose-dependently inhibited the proliferation of transplanted tumors in nude mice. From D-E in FIG. 7, the effect of the enriched Jiangnan Tourethrinkia biflavone on apoptosis in vivo was examined by TUNEL and immunohistochemical staining (IHC). TUNEL and IHC stained the nuclei of apoptotic cells brown, indicating that the number of apoptotic cells in the transplanted tumor gradually increased with increasing SM-BFRE dose. According to F in FIG. 7, WB experiment further proves the function of Jiangnan Chinese arborvitae flavonid enrichment in promoting laryngeal cancer cell apoptosis in vivo. It was found that with increasing SM-BFRE concentration, the expression ratio of Bcl-2/Bax decreased significantly, while the expression of clear-Caspase-9, -3 and PARP increased gradually, consistent with the results obtained in vitro.
Seventh experiment example, detecting the influence of the test substance on the expression of STAT3 pathway-related proteins in Hep-2 and TU212 cells
The JAK/STAT signal transduction pathway is widely involved in various physiological and pathological processes of proliferation, differentiation, apoptosis, immunoregulation, inflammation, tumor and the like of cells. Constitutive STAT3 is formed by dimerization of phosphorylated STAT3, an activated form of STAT3, whose aberrant activation is mainly caused by aberrant activation and expression of its upstream elements. STAT3 is directly or indirectly activated by a variety of tyrosine kinases, and STAT3 phosphorylation at the Tyr705 site is generally considered a prerequisite for activation of the STAT3 signaling pathway. The degree of phosphorylation of the STAT3Tyr705 site may reflect whether the drug induces apoptosis of laryngeal cancer cells by inhibiting the JAK2/STAT3 signaling pathway.
Cell manipulation and immunoblotting methods are shown in experiment five, wherein anti-STAT3, anti-pTyr705-STAT3 and anti-beta-actin are used as primary antibody.
The results are shown in fig. 8, and the phosphorylation levels of STAT3 in Hep-2 and TU212 cells are reduced along with the increase of the administration concentration of the hinokiflavone enrichment provided by the invention in example 1, which indicates that the hinokiflavone enrichment induces the apoptosis of laryngeal cancer cells to play a role by influencing the STAT3 signal transduction pathway. In fig. 8, a and B are both in vitro experiments, and C is the result of in vivo experiment in fig. 8.
Experimental example eight, detecting the influence of the test substance on the expression of Akt/NF-kB pathway related proteins of Hep-2 and TU212 cells
NF-kB is mainly involved in the inflammatory and immune responses of the human body. However, there are increasing reports indicating that NF-. kappa.B regulates the expression of genes that are critical in the development and progression of tumors, including cell proliferation, migration and apoptosis. NF-. kappa.B is usually present in the cytoplasm in the form of a dimer, which is released upon stimulation and eventually transferred to the nucleus, which binds to a target gene to promote its transcription. Research shows that the activation of a PI3K/Akt signal pathway is also related to the activation of NF-kappa B, and the reduction of the expression of phosphorylated Akt at a Ser473 site is a key index of the activation of a PI3K/Akt pathway. Therefore, the phosphorylation degree of Akt Ser473 site and the expression of NF-kB can reflect whether the drug induces the laryngeal cancer cell apoptosis by inhibiting Akt/NF-kB signal transduction pathway.
The results are shown in FIG. 9, the Akt phosphorylation level and NF-kB expression in Hep-2 and TU212 cells are reduced along with the increase of the administration concentration of the Jiangnan Chinese arborvitae biflavone enrichment, which indicates that the Jiangnan Chinese arborvitae biflavone enrichment induces the apoptosis of laryngeal cancer cells to play a role by influencing an Akt/NF-kB signal transduction pathway, and the inhibition effect of the Jiangnan Chinese arborvitae biflavone enrichment on NF-kB is probably caused by inhibiting the activation of the upstream Akt thereof. In fig. 9, a and B are both in vitro experiments, and C is the result of in vivo experiment in fig. 9.
In conclusion, the Jiangnan selaginella biflavone enrichment substance provided by the embodiment 1 of the invention shows good effects in-vitro and in-vivo anticancer activity researches. The Jiangnan Chinese arborvitae flavonid enrichment can effectively induce laryngeal cancer cell strain apoptosis, and has small influence on normal laryngeal epithelial cells. The research on the mechanism of promoting apoptosis shows that the Jiangnan Chinese arborvitae biflavone enrichment can increase the expression level of Bax and reduce the level of Bcl-2, thereby reducing the ratio of Bcl-2/Bax and further up-regulating the levels of clear-Caspase-9, clear-Caspase-3 and clear-PARP to induce apoptosis. The pharmaceutical composition containing amentoflavone, ginkgetin, hinokiflavone, tabebuia avellanedae A, 7-demethylginkgetin and 7,4',7', 4' -tetramethoxyamentoflavone is proved to be used for treating laryngeal cancer, and the treatment principle is as above.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (16)
1. A pharmaceutical composition for treating laryngeal cancer, characterized in that the active ingredients of the pharmaceutical composition comprise amentoflavone, ginkgetin, hinokiflavone, tabebeflavone A, 7-demethylginkgetin and 7,4',7' ',4' '' -tetramethoxyamentoflavone;
the preparation method of the pharmaceutical composition comprises the following steps: extracting selaginella tamariscina to obtain a crude extract, then sequentially extracting the crude extract by using petroleum ether, ethyl acetate and n-butyl alcohol, and respectively collecting corresponding extract liquor; adsorbing the components in the ethyl acetate extract by using resin, then performing gradient elution, and collecting and combining eluates with retention time of 4.75 minutes, 8.83 minutes, 10.08 minutes, 13.81 minutes and 17.79 minutes to form the pharmaceutical composition; wherein, the gradient elution comprises gradient elution by using 10%, 30%, 50%, 70%, 80% and 95% of water-ethanol mixed solution in sequence.
2. The pharmaceutical composition for the treatment of laryngeal cancer according to claim 1, characterised in that the active ingredient comprises a biflavone concentrate.
3. The pharmaceutical composition for the treatment of laryngeal cancer according to claim 1, wherein the active ingredient comprises a hinokiflavone concentrate comprising amentoflavone, ginkgetin, hinokiflavone, tabebeflavone a, 7-demethylginkgetin and 7,4',7 ", 4"' -tetramethoxyamentoflavone.
4. The pharmaceutical composition for treating laryngeal cancer according to claim 1, wherein the weight ratio of amentoflavone, ginkgetin, hinokiflavone, tabebeflavone a, 7-demethylginkgetin and 7,4',7 ", 4"' -tetramethoxyamentoflavone is 42-76: 5-15: 3-9: 1-7: 6-17: 1 to 7.
5. The pharmaceutical composition for treating laryngeal cancer according to claim 1, wherein the mass ratio of amentoflavone, ginkgetin, hinokiflavone, tabebeflavone a, 7-demethylginkgetin and 7,4',7 ", 4"' -tetramethoxyamentoflavone is 52.7:7.8:4.7: 2.3: 8.2:2.5.
6. The pharmaceutical composition for the treatment of laryngeal cancer according to claim 1, characterized in that extraction comprises: mixing and soaking the selaginella tamariscina with an alcohol solvent to form a crude extract; the alcohol solvent is monohydric alcohol.
7. The pharmaceutical composition for the treatment of laryngeal cancer according to claim 6, wherein the alcoholic solvent is ethanol.
8. Use of the pharmaceutical composition for the treatment of laryngeal cancer according to any one of claims 1 to 7 in the manufacture of a medicament for inducing apoptosis or inhibiting the ability of laryngeal cancer cells to migrate.
9. The use of claim 8, wherein the pharmaceutical composition is a medicament for inducing apoptosis in laryngeal cancer cells by affecting mitochondrial apoptosis signaling pathways.
10. The use of claim 9, wherein the pharmaceutical composition is a medicament that affects the mitochondrial apoptotic signal transduction pathway by down-regulating the expression level of Bcl-2 protein and/or up-regulating the expression level of Bax protein;
alternatively, the pharmaceutical composition is a drug that affects the mitochondrial apoptosis signaling pathway by up-regulating the expression levels of Caspase-3 and Caspase-9.
11. The use according to claim 8, wherein the pharmaceutical composition is a medicament for inducing apoptosis of the laryngeal cancer cell by affecting the JAK-STAT signaling pathway.
12. The use according to claim 11, wherein the pharmaceutical composition is a medicament that in turn affects the JAK-STAT signaling pathway by downregulating the phosphorylation level of STAT3 protein.
13. The use of claim 8, wherein the pharmaceutical composition is a medicament that induces apoptosis of the laryngeal cancer cell by affecting the Akt/NF- κ B signaling pathway.
14. The use of claim 13, wherein the pharmaceutical composition is a medicament that affects the Akt/NF- κ B signaling pathway by downregulating the phosphorylation level of Akt protein and NF- κ B protein.
15. Use of the pharmaceutical composition for treating laryngeal cancer according to any one of claims 1 to 7, in the preparation of a medicament for modulating at least one of the following pathways (1) to (3) to treat laryngeal cancer;
(1) activating a mitochondrial apoptosis signaling pathway;
(2) inhibiting the JAK-STAT signaling pathway;
(3) inhibiting Akt/NF-kB signal channel.
16. Use of a pharmaceutical composition according to any one of claims 1 to 7 for the manufacture of a medicament for effecting modulation of at least one of (a) - (c) for the treatment of laryngeal cancer;
(a) up-regulating the expression level of at least one of (4) - (6), wherein (4) is a Bax protein, (5) is a Caspase-3 protein and (6) is a Caspase-9 protein;
(b) down-regulating the expression level of at least one of (7) - (8), wherein (7) is an NF- κ B protein and (8) is a Bcl-2 protein;
(c) down-regulating the phosphorylation level of at least one protein from (9) to (10), wherein (9) is a STAT3 protein and (10) is an Akt protein.
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