KR101637640B1 - A peptide derived from fermented marine microalgae pavlova lutheri and a pharmaceutical composition for preventing and treating cancer comprising the same - Google Patents

Abstract

The present invention relates to an anticancer composition of a peptide purified and isolated from a fermentation product of a microalgae pavlova luteri, comprising the steps of: determining the amino acid sequence of a peptide isolated and purified from a fermentation product of microalgae Pavlovireri; A pharmaceutical composition for prevention and treatment of cancer comprising as an active ingredient a novel peptide of pablovireri obtained through a step of comparing and evaluating cytotoxicity, MMP activity and cell migration inhibitory activity using the peptide obtained in the above step is provided There is an excellent effect.

Description

FIELD OF THE INVENTION The present invention relates to peptides derived from a fermentation product of marine microalgae pavlova luteri and a pharmaceutical composition for preventing and treating cancer containing the same,

The present invention relates to a peptide purified and purified from a fermentation product of a microalgae pavlova luteri, and a pharmaceutical composition for preventing and treating cancer containing the same.

Matrix metalloproteinases (MMPs) play an important role in the degradation of extracellular matrix by proteolytic enzymes, which are complex mixtures of structural and functional proteins. MMPs affect a variety of physiological processes, including tissue morphogenesis, growth, and wound healing. Recently, a number of papers have reported that MMPs are involved in diseases and pathological processes involved in connective tissue degradation such as inflammatory reactions, arthritis, tumor invasion and metastasis as well as physiological processes.

In the tumor progression, the substrate barrier surrounding the cancer composed mainly of collagen type IV is destroyed by the MMPs produced by the cancer cells. Through the destroyed substrate barrier, the cancer cells invade the peripheral connective tissue, . Barriers to cancer cell migration are degraded by gelatinase and collagenases type IV, MMP-9 and MMP-2. Furthermore, since MMPs are more expressed and activated in cancer cells than normal tissues, MMPs are thought to play a role in transferring cancer cells.

In human fibrosarcoma cells (HT1080 cells), fibroblasts become malignant tumors. Fibroblasts produce collagen and are considered to be the most important source of extracellular quality. It also plays a central role in tissue remodeling and wound healing processes by producing MMP-2 and MMP-9. Therefore, HT1080 cells have been used in a number of studies.

Marine microalgae are the primary primary producer in the aquatic environment and have been considered to have significant nutritional value due to high protein, fat and vitamin content. The use of microalgae and the removal of carbon dioxide in biodiesel and biohydrogen production have received special attention. Recently, a number of reports have been reported on microalgae useful as a new source of pharmacologically active substances. Marine microalgae Pavlova lutheri is one of the largest producers of biomass in the marine environment and is a novel material containing high protein and also producing chemically diverse active metabolites. Although the peptide has been reported to have antioxidant properties, the inhibitory effect of HT1080 on cancer cell metastasis under cell migration conditions has not been elucidated.

Accordingly, an object of the present invention is to provide a peptide for inhibiting cancer cell migration.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting cancer metastasis comprising the peptide as an active ingredient.

The above object of the present invention can be accomplished by a method for determining the amino acid sequence of a peptide isolated and purified from a fermentation product of microalgae pavloviruleri; The peptide obtained in the above step was used to compare and evaluate cytotoxicity, MMP inhibitory activity and cell migration inhibitory activity.

The present invention has an excellent effect of providing a pharmaceutical composition for preventing and treating cancer, which contains a novel peptide of Pavlovir lutein as an active ingredient.

1 is a graph showing the amino acid sequence of a peptide (PFPL) according to the present invention.
2 is a graph showing cell viability of a peptide (PFPL) according to the present invention.
FIG. 3 is a photograph showing the activity of MMP-2 and MMP-9 of the peptide (PFPL) according to the present invention.
4 is a photograph showing the cell migration inhibitory activity of the peptide (PFPL) according to the present invention.
FIG. 5 is a photograph showing the MMP expression of a peptide (PFPL) according to the present invention at a protein level. FIG.
FIG. 6 is a photograph showing the MMP expression of the peptide (PFPL) according to the present invention at the mRNA level.

The present invention is described in more detail by the following examples. However, the scope of the present invention is not limited thereto.

Published sample

The Pavlova luteri ( KMCC H-006) used in the present invention was supplied from the Korea Marine Microalgae Culture Center. Hansenula polymorpha KCTC 7538, obtained from the Korea Biological Resource Center, was stored on standard F / 2 (Guillard) medium. HT1080 cells were purchased from the American Type of Culture Collection, and Dulbecco's Modified Eagle Medium (DMEM), trypsin-ethylenediamine tetraacetate (EDTA), phenyllysine / streptomycin and fetal bovine serum (FBS) Gibco BRL, Life Technologies (USA). (MTT), gelatin (type A) and povol 12-myristate 13-acetate (PMA) were purchased from Sigma Chemical ( USA). The primary and secondary antibodies for Western blot were MMP-2 (sc-13595), MMP-9 (sc-10737), NF-κB p65 (sc-8008), β- rabbit IgG-HRP (sc-2004) and goat anti-mouse IgG1-HRP (sc-2060) were purchased from Santa Cruz Biotechnology (USA).

Example  1: Microalgae Fermented  Produce

The microalgae powder was added to sodium phosphate buffer (pH 7.0) at a ratio of 1: 15 (w / v) and a Hansenula polyMorpha solution (500: 1) based on the amount of microalgae was added to the mixture. At 121 ℃ after pressure sterilization for 30 minutes to give the algae solution and the solution Lactobacillus brevis (Lactobacillus were fully mixed together by the addition of 1% of KCTC 11377BP) culture solution (v / v) concentration, and cultured at 37 ℃ 3 days, 6 days and 12 days: brevis) BJ20 (Accession No. The microalgae fermented product was recovered, lyophilized and hydrolyzed in 6N HCl containing 0.1% thioglycolic acid for 24 hours at 110 ° C under vacuum. High performance liquid chromatography (HPLC) was used to purify the peptides induced by phenylisothiocyanate.

Example  2: determination of amino acid sequence

The exact molecular weight and amino acid sequence of the purified peptide was determined using a Q-TOF mass spectrometer (Micromass, UK) equipped with an electrostatic spray ionization (ESI) source. The purified peptide was dissolved in methanol / water (1: 1, v / v) and then injected into the electrostatic injection source and determined by doubly charged condition in the mass spectrum. Following molecular weight determination, the peptide to be fragmented was automatically selected and sequence information was obtained by tandem mass spectrometry.

As a result of the experiment, the amino acid sequence of the peptide (PFPL; peptide from fermented P. lutheri ) derived from the pablovireri fermentation product was determined as shown in Fig. 1, which is identical to SEQ ID NO: 1 (Val-Ser-Pro-Gln-Ile).

Cell culture

Human fibrosarcoma cells (HT1080) were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin at 37 ° C and humidified conditions containing 5% CO ₂ . For experiments, the cells were trypsin-EDTA treated and transferred to a 6-well plate at a density of 1 x 10 ⁶ per well, at a density of 2 x 10 ⁵ per well and at a density of 5 x 10 ³ in a 96-well plate .

Experimental Example  1: Assessment of cell viability

The cell viability of PFPL isolated from pablovireri was evaluated by MTT analysis. HT1080 cells were transferred to 96-well plates at a density of 5 x 10 < ³ &gt; per well and cultured for 24 hours with various concentrations of PFPL separated from pavlova lutein in the presence and absence of animal serum. 100 占 퐇 of MTT was then added to each well and cultured at 37 占 폚 for 4 hours. During incubation, MTT was converted to blue formazan crystals by the dehydrogenase of living cells. Blue formazan crystals were dissolved in DMSO and absorbance was measured at 540 nm using a UV microplate reader. Relative cell viability was calculated relative to the untreated group. Data were presented as mean values of at least 4 experiments and P <0.05 was considered valid.

As shown in FIG. 2, when the cells were treated with PFPL to a concentration of 200 μg / mL, no significant toxic effect was observed. Based on the results, PFPL ranging from 10-100 μg / mL was selected for MMP activity analysis.

Experimental Example  2: MMP  Active crystal

The expression and activity of MMP-9 and MMP-2 were determined in HT1080 cells treated with PFPL using gelatin zymography. First, HT1080 cells were seeded in a 24-well plate using serum-free medium. Cells were treated with various concentrations of PFPL, and then stimulated with MMP expression for 24 hours under serum-free conditions after 1 hour. ). The medium supernatant, as measured by Bradford and containing the average protein content, was electrophoresed on a 10% polyacrylamide gel containing 0.15% gelatin. After electrophoresis, the gel was washed with 2.5% Triton X-100 solution for 1 hour, rinsed with water, and then eluted with 50 mM TRIS-HCl, pH 7.50, 200 nM NaCl, 5 mM CaCl ₂ .2H ₂ O and 0.02% Brif- And incubated in a developing buffer at 37 ° C for 24 hours. The gel was stained with 45% methanol and 10% glacial acetic acid in 1% Coomassie Brilliant Blue and destained with the same solution except Coomassie Blue dye. Enzymatic active regions are represented by negative staining: the proteolytic region appears as a bright band versus a blue background.

As shown in FIG. 3, PFPL treatment inhibited MMP-2 and MMP-9 expression in a concentration-dependent manner.

Experimental Example  3: Cell migration analysis

Cells were seeded in 6-well plates, grown in 10% serum-containing medium for 24 hours, transferred to serum-free medium and further cultured for 12 hours. The plate was scratched with the tip of the plate to make an empty space, and the cells were treated with PFPL and incubated at 37 ° C. Photographs were taken after 0, 12 and 24 hours. Cell migration was determined by measuring the distance the cells migrated into the empty space in the plate and was performed by measuring the distance between the two boundaries of the cells in the same magnification photograph. The measurements were performed independently by two persons without prior knowledge to eliminate subjectivity, and the error between the two measurement data was within 5%.

As shown in FIG. 4, various concentrations of PFPL reduced the migration of HT1080 cells.

Experimental Example  4: Western Blot  analysis

Expression of MMP-9 and MMP-2 in HT1080 cells treated with PFPL was determined using Western blot analysis. First, HT1080 cells were seeded in a 6-well plate containing 10% serum-containing medium. Cells were treated with various concentrations of PFPL and treated with PMA (4 ng / mL) for 24 h in serum-containing conditions 1 hour later to stimulate MMP expression. The cells were dissolved in RIPA buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 2 mM EDTA and 0.1% SDS). The average protein content as measured by the BCA method was electrophoresed on a 10% polyacrylamide gel and transferred to a nitrocellulose membrane. The membrane was blocked with 5% skim milk in TBS-T buffer and incubated overnight at 4 ° C with primary antibody (1: 1,000) in 2.5% skim milk. After washing with TBS-T buffer, the cells were incubated with secondary antibody (1: 5,000) in TBS-T buffer for 1 hour at room temperature. The band was confirmed by the enhanced chemiluminescence method and the LAS-mini imaging system.

Experimental Example  5: RT - PCR

Cells were treated with various concentrations of PFPL and then treated with PMA (4 ng / mL) for 24 hours in serum-containing conditions for 1 hour to stimulate MMP expression. RNA was extracted with trisol and reverse transcriptase was used in the presence of oligo dT And the cDNA was reverse transcribed. (MMP-2 primer: 5'-GGGGCCTCTCCTGACATT-2) was performed in 27 cycles (GAPDH) and 30 cycles (MMP-9, MMP-2, TIMP-1 and TIMP- 3 '5'-TCACAGTCCATCACTTGGTTAT-3' TIMP-1 primer: 5'-CCCTATGCCTACATCCGTGA-3 '5'-TCCATCCATCACTTGGTTAT-3'; TIMP-2 primer: 5'-GGTCTCGCTGGACGTTGGAG-3 '5'-GGAGCCGTCACTTCTCTTG-3'; MMP-9 primer: 5'-TCCAACCACCACCACACCGC-3 '5'-CAGAATCGCCAGTACTT-3'; GAPDH primer: 5'-ACGGATTTGGTCGTATTGGG-3 '5'-TGATTTTGGAGGGATCTCGC-3'). The amplified cDNA was separated by electrophoresis on 1% and 1.2% agarose gel, and the gel was stained with 0.01% etidium bromide and quantified using a Gel Doc image analysis system.

As shown in FIG. 5 and FIG. 6, expression of MMP-2 and MMP-9 was inhibited by PFPL in a dose-dependent manner. While TIMP-2 was increased in the presence of PFPL, TIMP-1 was dose-dependently increased by PFPL treatment. PFPL inhibition was due to down-regulation of JNK and ERK and p38 levels were dose-dependently increased by PFPL treatment.

<110> Pukyong National University Industry-University Cooperation Foundation <120> A peptide derived from fermented marine microalgae pavlova          Lutheran and a composition for anticancer comprising the same <130> 2 <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 5 <212> PRT <213> Artificial Sequence <220> <223> peptide derived from fermented pavlova lutheri <400> 1 Val Ser Pro Gln Ile   1 5

Claims (2)

A peptide for suppressing cancer metastasis consisting of the amino acid sequence of SEQ ID NO: 1 derived from a Pavlova lutheri fermentation product. A pharmaceutical composition for prevention and treatment of cancer containing the peptide of claim 1.

Images