KR101316642B1 - A Composition for Overcoming Resistance to Anticancer Drugs comprising Methyl pheophorbide - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for overcoming cancer drug resistance, a composition for inhibiting heme oxidase activity and a method thereof, and a composition for promoting apoptosis, including methyl pieophorbide as an active ingredient.

Description

A composition for overcoming resistance to anticancer drugs comprising Methyl pheophorbide}

The present invention relates to an anticancer drug overcoming pharmaceutical composition comprising methyl pheophorbide as an active ingredient, a composition for inhibiting heme oxidase activity and a method thereof, and a composition for promoting apoptosis and a method thereof.

In the last 30 years, the use of chemical drugs as cytostatic agents for the treatment of cancer constitutes one of the choices as the first-line treatment for some solid tumors and hematopoietic tumors. The most commonly used chemical drugs for the treatment of cancer are: cisplatin, taxol, alkaloids from Vinca, 5-fluorouracil, cyclophosphamide (Jackman AL, Kaye S., Workman P. (2004) The combination of cytotoxic and molecularly targeted therapies-can it be done? Drug Discovery Today 1: 445-454). However, results from clinical trials show a low therapeutic index for this type of drug in cancer treatment, as evidenced by the therapeutic marginal benefit with the high toxicity observed in patients (Schrader C., et al. M. (2005) Symptoms and signs of an acute myocardial ischemia caused by chemotherapy with paclitaxel (taxol) in a patient with metastatic ovarian carcinoma.Eur J Med Res 10: 498-501).

For example, although many authors agree that cisplatin constitutes the primary treatment of lung cancer, unchanged efficacy has been observed with a six-week increase in survival and little improvement in clinical symptoms (Grillo R., Oxman A., Julian J.). (1993) Chemotherapy for advanced non-small cell lung cancer.J Clin Oncol 11: 1866-1871; Bouquet PJ, Chauvin F., et al (1993) Polychemotherapy in advanced non-small cell lung cancer: a meta-analysis.Lancet 342: 19-21).

Therefore, current strategies for achieving optimal therapeutic benefit focus on pharmaceutical combinations based on cytostatic drugs with molecular targeted therapies.

Some of the current anticancer drugs, for example, target Glevex (Imitinib) targeting Abl kinase, which in turn plays a fundamental role in the development of chronic myeloid leukemia (Giles JF, Cortes JE, Kantarjian HM (2005) Targeting the Kinase Activity of the BCR-ABL Fusion Protein in Patients with Chronic Myeloid Leukemia.Current Mol Med 5: 615-623), and also Isar (Onn A., Herbst RS (targeting a tyrosine kinase that binds to epidermal growth factor (EGF) receptor 2005) Molecular targeted therapy for lung cancer.Lancet 366: 1507-1508) and Velcade (Vortezomib) which blocks the protein degradation by targeting the proteasome machinery (Spano JP, et al. (2005) Proteasome inhibition: a new approach for the treatment of malignancies.Bull Cancer 92: E61-66).

Given that the non-specific mechanisms of conventional chemotherapy converge on the abolition of cellular mitosis, the use of novel cancer targeted therapies provides an excellent prospect for achieving pharmaceutical combinations that produce synergistic effects of antitumor effects. do.

On the other hand, drug resistance was recognized as the primary cause of failure in cancer treatment when chemotherapeutic agents were used. Although suboptimal drug concentration in the tumor environment affects drug resistance, other factors such as cellular origin play an idiopathic role in chemical resistance to many tumors. Drug resistance is a multifactorial phenomenon with multiple independent mechanisms involved in intracellular detoxification, changes in cellular responses, tolerance to stress and deficiencies in apoptosis signaling pathways (Luqmani A. (2005) Mechanisms of drug resistance in cancer chemotherapy.Med Princ.Pract 14: 35-48).

Glycoprotein-P and Gluthathion S-transferase are major proteins that mediate intracellular detoxification associated with drug resistance in cancer (Saeki T., Tsuruo T., Sato W., Nishikawsa K. (2005) Cancer resistance in chemotherapy for breast cancer.Cancer Chemother Pharmacol 56: 84-89) (Hara T., et al. (2004) Gluthathione S-transferase P1 has protective effects on cell viability against camptothecin. Cancer Letters 203: 199-207 ). It has been reported that other proteins, such as beta-tubulin, are involved in drug resistance, a level directly related to tumor resistance to paclitaxel (Orr GA, et al. (2003) Mechanisms of Taxol resistance related to microtubules.Oncogene 22 : 7280-7295). Alternatively, Hisano T., et al. (1996) Increased expression of T-plastin gene in cisplatin-resistant human cancer cells: identification by mRNA differential display.FEBS Letters 397: 101-107 ), the Heat Shock Protein (HSP70) and (HSP90) (Jaattela M. (1999) Escaping cell death: survival proteins in cancer.Exp Cell Res 248: 30-43) and the transcription factor YB1 (Fujita T., et al. (2005) Increased nuclear localization of transcription factor Y-box binding protein accompanied by up-regulation of P-glycoprotein in breast cancer pretreated with paclitaxel.Clin Cancer Res 11: 8837-8844). The reception was reported.

In addition, glycolysis and the pyruvic acid pathway have been reported to play an idiopathic role in the chemical resistance phenomena observed in tumor cells (Boros LG, et al. (2004) Use of metabolic pathway flux information in targeted cancer drug design. Disc.Today 1: 435-443).

Reports from other groups indicated the presence of a set of proteins that inhibit apoptosis or increase cell viability in tumor cells, contributing to the chemical resistance phenomenon of cellular tumors. One example is the Nucleophosmin protein, which plays a pivotal role in cell cycle promotion, inhibition of apoptosis, which is considered a poor prognostic marker in cancer (Ye K. (2005) Nucleophosmin / B23, a multifunctional protein that can regulate apoptosis.Cancer Biol Ther 4: 918-923). Likewise, CK2 enzyme plays an important role in tumor cell resistance and cell growth towards apoptosis (Tawfic S., Yu S., Wang H., Faust R., Davis A., Ahmed K. (2001) Protein kinase CK2 signal in neoplasia.Histol.Histopathol. 16: 573-582). Previous findings have shown a 3- to 7-fold increase in CK2 activity in normal tissue epithelial tumors (Tawfic S., Yu S., et al. (2001) Protein kinase CK2 signal in neoplasia. Histol Histopatol 16: 573-582; Faust RA, Gapany M., et al (1996) Elevated protein kinase CK2 activity in chromatin of head and neck tumors: association with malignant transformation. Cancer Letters 101: 31-35). In addition, CK2 activity is an important cellular event in malignant transformation and constitutes a tumor progression marker (Seldin DC, Leder P. (1995) Casein Kinase IIa transgene-induced murine lymphoma: relation to theileroiosis in cattle.Science 267: 894. -897). CK2 phosphorylation represents a strong signal for protecting tumor cells from apoptosis, which led to the consideration of this enzyme as an apoptosis mediator in cell physiology (Ahmed K., Gerber DA, Cochet C. (2002) Joining the cell survival squad: an emerging role for protein kinase CK2.Trends Cell Biol, 12: 226-229; Torres J., Rodriguez J., et al (2003) Phosphorylation-regulated cleavage of the tumor suppressor PTEN by caspase-3: implications for the control of protein stability and PTEN-protein interactions.J Biol Chem, 278: 30652-60).

Competitive proteomics studies, along with molecular biology, allow some understanding of the molecular mechanisms involved in both cellular malignant transformation and tumor chemoresistance. Therefore, cancer therapy cures considerably to achieve an effective drug combination that significantly reduces cytotoxicity and also reduces the likelihood of increasing chemical resistance.

Therefore, a major goal in the treatment of cancer today is to increase the therapeutic index of current cytostatic drugs by reducing the effective amount and the intrinsic toxicity exhibited by this type of medication. Another current strategy is to bypass tumor chemoresistance to conventional cell proliferation inhibitory drugs.

In general, reactive oxygen species (ROS) are mainly produced as by-products of aerobic breathing, including superoxide anion (O2 ^— ), hydrogen peroxide (H ₂ O ₂ ), and hydroxyl anion (OH).

In order for reactive oxygen species to act as a signaling material for intracellular physiological activity and stress response, it must have mild activity that reversibly oxidizes target molecules and precisely regulates intracellular concentrations.

In order for active oxygen species to function as intracellular signaling agents, the role of antioxidant defense systems that can properly control their concentration and reversibly reduce oxidative modification of target proteins is important. Peroxidase, catalase and superoxide dismutase are well known antioxidant proteins that regulate the level of reactive oxygen species and thioredoxin, glutaredoxin and nitrosoglutathione reductase are examples of proteins that repair oxidative modification of target proteins.

Free radicals, on the other hand, contribute to the tumorigenization process in various ways; ① Gene damage and genetic variation ② Function as mediator of cell proliferation and cell survival signaling induced by growth factor and adhesion factor ③ Promotes cell mobility and invasiveness ④ Induces inflammatory response and neovascularization tumor microenvironment formation

Most scholars in the field of tumorigenization research have understood the importance of free radicals in terms of their role as important mediators of mitogenic signals (EGF, PDGF, Ras, and PI3K-Akt).

However, it is very important that cancer cells themselves also protect themselves from oxidative damage and gain survival and metastasis. That is, the regulation function of the intracellular system that maintains the level of reactive oxygen species appropriate as a signaling material may be very important. This view suggests a new direction that antioxidant systems, which have been regarded as negative signals of tumorigenesis, may be very important as positive signals of tumorigenization. However, many researchers in the field have overlooked the research from this point of view and there is almost no relevant research to date.

No key antioxidant system has been found that substantially tunes the oxidative stress environment of cancer cells, ie the levels of free radicals.

In addition, the mechanism of induction of antioxidant system activation due to the increase of reactive oxygen species has not been well-established.In addition to the regulation function of reactive oxygen species in the tumor environment, the role and mechanism of action of the antioxidant system inducing cancer progression from various directions are revealed. You must lose. There is a lack of research information on the discovery of key factors and the development of a strategic system that can convert the role of reactive oxygen species from tumor promoter to apoptosis inducer.

On the other hand, the expression of the heme oxygenase-1 gene (hereinafter, referred to as 'HO-1') is an inducible gene which is very low under normal conditions but increases rapidly in an environment where oxidative stress is induced. Thus, expression is maintained at a very low level in normal cells, but the expression may be continuously increased in conditions where oxidative stress such as cancer cells are formed.

In related prior patents, Korean Patent Publication No. 1020090030251 relates to the use of CB2 inhibitors for the treatment and chemosensitivity of non-responsive tumors to anti-cancer drugs. Casein kinase is combined with standard chemotherapeutic drugs used for cancer treatment. A pharmaceutical combination comprising a 2 (CK2) peptide deterrent and administered separately, or sequentially, wherein the chemotherapeutic drug is cisplatin, taxol, vinca-derived alkaloid, 5-fluorouracil, doxorubicin, cyclophosphamide , Etoposide, mitomycin C, imatinib, iresa and melcade (bortesomib). The synergistic effect between the P15 peptide and the anticancer drug ensures that the effective concentration of each cytostatic drug in the combination is 10- to 100-fold lower than when each cytostatic drug alone is used, and the pharmaceutical combinations described are reported by the anticancer drug. It shows lower toxicity than that, which represents a decisive advantage for its use in cancer treatment. In addition, sequential administration of this pharmaceutical combination via pretreatment with P15 peptides has been described to elicit chemosensitivity of tumors resistant to chemotherapy.

In another related prior art, Korean Patent Publication No. 1020070050918 relates to 'treatment of cancer', in the method of preparing a medicament for the treatment of cancer, comprising: (i) an antibody or antigen thereof specific for at least one epitope of Hsp90; Binding fragments; And (ii) at least one anticancer agent selected from the group consisting of doxorubicin, daunorubicin, epirubicin, herceptin, docetaxel and cisplatin.

The present invention has been made by the above necessity, an object of the present invention to provide a composition for preventing and treating cancer-resistant cancer.

Another object of the present invention is to provide a composition for inhibiting heme oxidase 1.

Still another object of the present invention is to provide a composition for promoting caspase activity.

Still another object of the present invention is to provide a composition for promoting apoptosis.

In order to achieve the above object, the present invention provides a composition for the prevention and treatment of cancer-resistant cancer, including methyl pheophorbide as an active ingredient.

In one embodiment of the present invention, the anticancer agent is busulfan, carboquone, cyclophosphamide, cyclophosphamide, ifosfamide, melfaran, nitrogen mustard, nitrogen mustard. ), Alkylating agents such as thiotepa, uracil mustard, carmustine (BCUN), nimustine hydrochloride (ACNU), and estramustine phosphate; Azathiorpine, ancitabine, carmofur, doxifluridine, fluorouracil (5-FU), mercaptopurine (6-MP), thio Metabolic antagonists such as inosine, tegafur, cytarabine ocfosfate, pentostatin, etc .; Dactinomycin, mitomycin C, bleomycin (BLM), daunorubicin, adriamycin (doxorubicin) (adriamycin (doxorubicin), neocarzinostatin (NCS), hydrochloric acid Antitumor antibiotics such as idarubicin hydrochloride; etoposide (VP-16), teniposide, vindesine, vincristine, vinblastine Plant-derived anti-tumor substances such as taxol (paclitaxel), irinotecan hydrochloride; platinum complexes such as cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (NDP) Predonisone, prednisolone, testosterone, estramustine, norethisterone, goserelin acetate, leuprotelin acetate ), Citric acid toremfene (toremifene citrate), hydrochloric acid Hormonal agents such as fadrozole hydrochloride, tamoxifen, anthracycline compounds such as mitoxantrone (MXT), and the like, but are not limited to these.A preferred anticancer agent is cisplatin. Platinum complexes such as carboplatin and nedaplatin; dactinomycin, mitomycin C; bleomycin, daunorubicin, adriamycin antitumor antibiotics such as adriamycin), neocarzinostatin, and idarubicin hydrochloride; Plant-derived antitumor agents such as etoposide, teniposide, vindesine, vincristine, vinblastine, taxol and irinotecan hydrochloride Substance and the like. More preferred are cisplatin, carboplatin, nedaplatin, adriamycin and taxol, particularly preferably cisplatin.

Methyl pheophorbide (MPP) used in the present invention can be purchased from Shanghai Xianhui Pharmaceutical Co., Ltd. and the like.

The MPP compound according to the present invention has excellent anticancer resistance coping action and anticancer effect enhancing action. That is, the anticancer drug resistance overcomer or anticancer effect enhancing composition of the present invention exhibits anticancer activity of the anticancer agent against cancers resistant by coadministration with the anticancer agent.

 When the compound of the present invention is used as an anticancer drug resistance or anticancer effect enhancing composition, it is usually administered orally or parenterally or systemically. Parenteral administration includes intravenous administration (including drip infusion), intraarterial administration, intramuscular administration, subcutaneous administration, intraperitoneal administration, intrathoracic administration, bladder administration, intramuscular administration, Transdermal administration, transmucosal administration, rectal administration, intratumoral administration, etc. are mentioned.

The anticancer drug resistance survivor or anticancer drug enhancing composition of the present invention may be administered simultaneously with the anticancer agent, may be administered before or after the anticancer agent, or may be administered during the drug holiday. The route of administration of the anticancer drug resistant drug or the anticancer effect enhancer and the anticancer agent may be the same or different.

The anticancer drug resistance overcoming pharmaceutical composition of the present invention is administered in combination with an anticancer agent, thereby preventing lung cancer (non-small cell lung cancer, small cell lung cancer), colon cancer (rectal cancer, colon cancer), small intestine cancer, gastric cancer, esophageal cancer, and liver cancer in mammals including humans. , Pancreatic cancer, malignant melanoma, kidney cancer, bladder cancer, uterine cancer (uterine cancer, uterine carcinoma), ovarian cancer, breast cancer, osteosarcoma, malignant lymphoma, prostate cancer, leukemia (acute leukemia, chronic leukemia), myeloma, neuroblastoma, head and neck cancer It can be used for the treatment of cancer (malignant tumor) such as skin cancer, testicular tumor, and the like.

In the present invention, "concomitant administration" means administration of two kinds of drugs at the same time, continuously or at a time interval. Two kinds of drugs may be administered as a mixture, or may be administered as respective agents. When administering as each agent, each administration route may be same or different.

The dosage of the MPP compound, salt, or prodrug thereof varies depending on age, weight, symptoms, therapeutic effect, method of administration, treatment time, etc., but is usually oral once or several times a day in the range of 0.01 mg to 1 g per adult. Parenteral administration.

The dose of anticancer agent administered in combination may be a dose used for the treatment of ordinary cancer, or a lower dose. In addition, the administration route of the anticancer agent administered in combination may be the same as the administration route used in the treatment of normal cancer.

Moreover, it can also administer as a pharmaceutical composition containing an MPP compound, its salt, its prodrug, and an anticancer agent. Although the weight ratio of the MPP compound, its salt, or its prodrug and anticancer agent in a composition may be 1: 100-100: 1, it is not limited to this range.

According to the present invention, there is provided a pharmaceutical kit for cancer treatment comprising two kinds of drugs. In the pharmaceutical kit for cancer treatment of the present invention, the first agent is an anticancer drug-resistant drug or an anticancer drug-enhancing agent containing an MPP compound, a salt thereof, or a prodrug thereof, and the second agent is an anticancer agent. By using these two kinds of drugs in combination, it can be used for the treatment of cancer, in particular for the treatment of cancer resistant to anticancer agents. The anticancer drug resistance surviving pharmaceutical composition may be administered simultaneously with the anticancer agent, or may be administered before or after the administration of the anticancer agent, and in some cases, may be administered during the drug holiday. The route of administration of these anticancer drug resistance pharmaceutical compositions may be the same as or different from that of the anticancer agent.

When the compound of the present invention is used as a solid composition for oral administration, it is possible to formulate tablets, pills, powders, granules and the like. In such solid compositions, one or more active substances may be at least one inert diluent, dispersant, or adsorbent, such as lactose, mannitol, glucose, hydroxypropyl cellulose, microcrystalline cellulose, starch. , Polyvinylpyrrolidone, magnesium metasilicate aluminate or silicic anhydride powder and the like. In addition, the composition may mix additives other than a diluent according to a conventional method.

When prepared by tablets or pills, it may be coated with a film of gastric or enteric material such as white sugar, gelatin, hydroxypropyl cellulose or hydroxymethyl cellulose phthalate, or may be coated with two or more layers as necessary. In addition, a capsule of a substance such as celatin or ethyl cellulose may be used.

In the case of a liquid composition for oral administration, formulations such as pharmaceutically acceptable emulsions, solubilizers, suspensions, syrups or elixirs may be used. Examples of the abrasives to be used include purified water, ethanol, vegetable oil or emulsifier. In addition to the diluent, the composition may be mixed with auxiliaries such as wetting agents, suspending agents, sweetening agents, flavoring agents, fragrances or preservatives.

When preparing a parenteral injection, a sterile aqueous or nonaqueous solution, solubilizer, suspending agent or emulsifying agent is used. As aqueous solutions, solubilizers, and suspending agents, for example, organic amines such as water for injection, distilled water for injection, saline, cyclotextrin and derivatives thereof, triethanolamine, diethanolamine, monoethanolamine and triethylamine Or an inorganic alkali solution.

In the case of using a water-soluble solution, a vegetable oil such as propylene glycol, polyethylene glycol or olive oil, or an alcohol such as ethanol may be used. As the solubilizer, for example, a surfactant (mixed micelle formation) such as polyoxyethylene hardened castor oil, sucrose fatty acid ester, or lecithin or hydrogenated lecithin (liposome formation) may be used. Moreover, it can also be set as the emulsion agent which consists of water-insoluble dissolving agents, such as vegetable oil, lecithin, polyoxyethylene hardened castor oil, polyoxyethylene polyoxypropylene glycol, etc.

Other compositions for parenteral administration include one or more active substances, and may be external preparations, coating agents such as ointments, suppositories or pessaries, etc., which are prescribed by methods known per se.

In another embodiment of the present invention, the effective concentration of the methyl pheophorbide (Methyl pheophorbide) is preferably 10μM, but is not limited thereto.

In another aspect, the present invention provides a composition for inhibiting heme oxidase 1 containing methyl pheophorbide as an active ingredient.

In another aspect, the present invention provides a method for inhibiting heme oxidase 1 activity by treating the methyl phenophorbide enzyme with heme oxidase 1 enzyme.

In another aspect, the present invention provides a composition for promoting caspase activity comprising methyl pheophorbide and cisplatin as an active ingredient.

In one embodiment of the present invention, the effective concentration of the methyl pheophorbide (Methyl pheophorbide) is preferably 5 to 10μM, but is not limited thereto.

The present invention also provides a method of promoting caspase activity by treating caspase enzyme with methyl pheophorbide and cisplatin.

The present invention also provides a composition for promoting apoptosis (apoptosis) comprising methyl pheophorbide and cisplatin as an active ingredient.

In one embodiment of the present invention, the effective concentration of the methyl pheophorbide (Methyl pheophorbide) is preferably 5 to 10μM, but is not limited thereto.

The present invention also provides a method of promoting apoptosis by treating methyl pheophorbide and cisplatin in cells.

In one embodiment of the present invention, the method is preferably treated with 5-10 μM of methyl pheophorbide (Methyl pheophorbide), but is not limited thereto.

Hereinafter, the present invention will be described.

Methyl pheophorbide  ( MPP Cytotoxic effect of

The toxicity range of MPP was analyzed in the metastatic breast cancer cell line MDA-MB231. After treatment with various concentrations of MPP cells for 24 hours under UV-blocked conditions, cells were harvested and MTT assay was performed. Cytotoxic effect was not significantly different compared to control at 50 uM concentration and 10% toxic effect was observed at 100 uM (Fig. 1). In the present invention, since the MPP itself is less toxic and expects a therapeutic effect that can increase cancer cell death when combined with an anticancer agent, 10 uM MPP was used in the present invention.

MPP On by Heme oxygenase  ( HO Inhibition of activity

HO is increased in cancer cells and acts as a factor in conferring resistance to anticancer drugs. It was investigated whether MPP could inhibit the activity of HO.

First, HO-1 expression levels were examined in normal human mammary cell lines and cancer cell lines. E-cadherin expression was used as a marker to distinguish between normal and cancer cells. The expression level of HO-1 was significantly higher in metastatic cancer cell lines Hs578T and MDA-MB231, which is opposite to that of E-cadherin (Fig. 2A).

The effect of MPP on the activity of HO was also analyzed. MPP was treated to the cell line extracts under UV-blocked conditions, and then the activity of HO was identified. Zinc protophorphyrin IX (ZnPP) was used as a positive control as a well known inhibitor of HO. The inhibitory effect was 10-fold lower than that of ZnPP, but significantly lowered the activity of HO at 10 uM (Fig. 2B). However, the effectiveness of MPP is significant given ZnPP's strong cytotoxicity. These results demonstrate the new effect of MPP used as a phorosensitizer.

MPP Wow Cisplatin  Cancer cell death effect by combined treatment

MPP was treated with MDA-MB231 cell line for 30 minutes in the absence of UV light and cisplatin (50 uM) for 24 hours and analyzed for apoptosis effect. 10 uM MPP or cisplatin itself did not show apoptosis effect. An increase in apoptosis was significantly observed under the conditions of MPP and Cisplatin treatment (Fig. 3A). This effect was also observed in flow cytometric analysis (FACS) experiments in which subG1 was stained with propidium iodide (PI) (Fig. 3B).

MPP Wow Cisplatin  By compound processing Caspase  Active increase effect

The activity of caspase, a marker factor of apoptosis, was analyzed under the same conditions as shown in FIG. In combination with MPP and cisplatin, caspase-3 in activated cleaved form increased significantly with increasing MPP concentration (Fig. 4A). This increase in cleaved caspase-3 was also confirmed in experiments identifying caspase activity (Fig. 4B).

As can be seen through the present invention, the present invention shows that the MPP has a new efficacy irrelevant to the photosensitizer. In other words, MPP has an effect of significantly increasing the apoptosis sensitivity of cancer cells exhibiting resistance to anticancer drugs by inhibiting the increased HO activity in cancer cells.

1 is a diagram showing the cytotoxic effect of Methyl pheophorbide (MPP).
Figure 2 is a diagram showing the HO inhibitory effect by Methyl pheophorbide (MPP).
Figure 3 is a diagram showing the effect of cancer cell death by the combination of Methyl pheophorbide (MPP) and cisplatin.
Figure 4 is a diagram showing the effect of Caspase activation by the combination of Methyl pheophorbide (MPP) and cisplatin.

The present invention will now be described in more detail by way of non-limiting examples. However, the following examples are intended to illustrate the present invention and the scope of the present invention is not to be construed as limited by the following examples.

Example  One: Caspase Assay

For this material,

10 mM N-acetyl-Asp-Glu-Val-Asp-ρ-nitroanlide (Ac-DEVD-pNA), 100 mM DTT, HEPES Lysis buffer (1% NP-40, 25 mM Hepes, pH 7.4, 150 mM NaCl, 1 mM EDTA, 100 mM NaF, 10% glycerol, 2 mM Na2VO3, 50 mg / ml aprotinin, 5 mg / ml antipain, 2 mg / ml leupeptin, and 1 mM PMSF) and caspase assay buffer (1M HEPES pH7.5, 5 mM EDTA),

After seeding 1x10 ⁶ cells on a 60 mm cell culture plate, pretreat the MPP by concentration, and after 30 minutes, treat cisplatin 50 uM for 24 hours. After removing the media and washed with PBS, cells were lysed in 300 ul HEPES Lysis buffer. The 75 ul of cell lysate was added to 10 mM DEVD 2.1 ul, 100 mM DTT 10 ul, DMul 2.5 ul and 100 ul of caspase buffer and incubated at 37 ° C. for 4 hours and measured every 12 hours at 405 nm.

Example  2: HO -One Activity assay

Homogenization buffer has a composition of [30 mM Tris-HCl, 0.25 M sucrose, 0.15 M NaCl] and [10 ug / ml leupeptin, 10 ug / ml trypsin inhibitor, 2ug / ml aprotinin, 1 mM PMSF]

HO active buffer 2 mM MgCl ₂ 100 mM PBS buffer, 8.71 g KH ₂ PO _4, 8.71 g K ₂ HPO ₄ , MgCl _{2 in} 1 L of distilled water ^. 0.407 g of 6H ₂ O (Subtract MW. 6H ₂ O) was added and prepared by adjusting to pH 7.4 with KOH.

The reaction mixture was performed with 0.8 mM NADPH, 2 mM glucose-6 phosphate, 0.2 U glucose-6-phosphate dehydrogenase, 20 uM hemin, 100 mM potassium phosphate buffer (pH 7.4), and 1 mg rat liver cytosol.

Example  3: Microsome  Preparation of Extracts (Cells or Enough Tissue)

Cell (~ 5X10 ⁶ cells / 100 mm plates) were washed twice with cold PBS (pH 7.4), scraped with 1 ml of cold homogenization buffer, homogenized and centrifuged at 10,000 g for 15 minutes at 4 ° C.

The supernatant was collected and centrifuged at 100,000 g for 1 hour at 4 ° C. by ultracentrifugation, and the microsome pellets were then treated with 2 mM MgCl ₂ , 10 ug / ml leupeptin, 10 ug / ml trypsin inhibitor, 2 ug / ml aprotinin. And resuspended in 100 mM potassium phosphate buffer (pH 7.4) containing 1 mM PMSF.

Example  4: biliverdin reductase As a source of rat  liver Cytosolic  Produce

Mouse liver was washed several times with PBS until red blood was completely removed, liver was homogenized under ice in HO activity buffer (1 or 2 ml), then centrifuged at 10,000 g, 4 ° C. for 20 minutes and then the supernatant Was transferred to an ultracentrifuge tube, centrifuged at 100,000 g, 4 ° C. for 1 hour, and the supernatant was used as a source of BVR.

The reaction was carried out as follows.

Microsome fraction (200 ul) is added to the reaction mixture (200 ul),

The samples were incubated in a 37 ° C. water bath for 1 hour under dark conditions (cf. Blank = all materials except sample microsomes),

Place the sample on ice for at least 2 minutes,

Stop the reaction by adding 500-600 ul chloroform,

Vortexed for 30 seconds, centrifuged at 15,000 g for 20 minutes at room temperature,

Remove the brown film that appears between the water layer (top) and the chloroform layer (bottom layer),

The lower layers were collected to measure OD at 464 nm and 530 nm (using rock-made cubic for OD measurement).

bilirubin was determined by calculation of the absorbance difference between 464 nm and 530 nm (absorption coefficient, 40 mM ^-1 cm ^{-1 for} bilirubin)

Example  5: PI  dyeing

For flow cytometry, MDA-MB231 cells were grown in 6-well plates, incubated at 37 ° C. for 24 hours, pre-incubated with MPP and treated with cisplatin. After 24 hours the cells were pooled and washed twice with PBS (pH 7.4). After 30 min fixation with 80% ethanol, the cells were washed twice and resuspended in PBS (pH 7.4) containing 0.1% Triton X-100 and 5 μg / ml propidium iodide (PI), followed by FACScan cytometer (Program Cell-Quest, BD Biosciences).

Claims (12)

delete delete delete delete delete delete delete delete delete delete A method of promoting apoptosis of breast cancer cell lines in vitro by treating breast cancer cells with 5-10 μM methyl pheophorbide in vitro and 50 μM cisplatin. delete

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