TWI648051B - Use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells - Google Patents

Use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells Download PDF

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TWI648051B
TWI648051B TW106125799A TW106125799A TWI648051B TW I648051 B TWI648051 B TW I648051B TW 106125799 A TW106125799 A TW 106125799A TW 106125799 A TW106125799 A TW 106125799A TW I648051 B TWI648051 B TW I648051B
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carnosine
hct
cells
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cell line
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TW201909893A (en
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謝淑玲
賴柏羽
吳志忠
謝淑貞
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謝淑玲
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Abstract

一種化合物用於製備調控腸癌細胞轉移之藥物的用途,以人類結腸癌(HCT-116)細胞株培養為實驗模式,探討肌肽(carnosine)對人類結腸癌細胞株轉移作用之影響。由結果可知,肌肽可藉由調節細胞遷移(migration)、侵犯(invasion)、內滲(intravasation)、外滲(extravasation)及黏附(adhesion)作用而降低結腸癌細胞之轉移能力。 The use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, and the culture of human colon cancer (HCT-116) cell line as an experimental model to investigate the effect of carnosine on the metastasis of human colon cancer cell lines. From the results, carnosine can reduce the metastatic ability of colon cancer cells by regulating cell migration, invasion, intravasation, extravasation, and adhesion.

Description

化合物用於製備調控腸癌細胞轉移之藥物的用途 Use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells

本發明係有關於一種化合物用於製備調控腸癌細胞轉移之藥物的用途,尤指涉及一種肌肽(carnosine)可藉由調節細胞遷移(migration)、侵犯(invasion)、內滲(intravasation)、外滲(extravasation)及黏附(adhesion)作用而降低結腸癌(HCT-116)細胞之轉移能力者。 The present invention relates to the use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, in particular to a carnosine which can regulate cell migration, invasion, intravasation, and external Extravasation and adhesion to reduce the metastatic ability of colon cancer (HCT-116) cells.

隨著社會經濟發展,國人之生活型態跟著改變,又因飲食精緻化與西化之影響,導致國人癌症發生率逐年增加。根據國際癌症研究署(International Agency for Research on Cancer,IARC)之報告指出,2012年全球約有140萬人罹患結腸癌(IARC,2016);而世界衛生組織(world health organization,WHO)亦曾指出2015年有69.4萬人因結腸癌而死亡(WHO,2016)。另外,在中華民國衛生福利部於2017年所公告之2016年台灣十大死亡原因之中,惡性腫瘤再度高居國人十大死因第一位;而且,在十大癌症死因中,結腸癌不僅高居第三位,因結腸癌死亡之人數每十萬人口就高達24.3人(衛生福利部,2017)。由此可知,結腸癌對我國人民之健康具有重大之威脅。因此,如何預防結腸癌之發生乃成為許多學者研究之重要課題之一。 With the development of social economy, the lifestyle of Chinese people has changed, and the incidence of cancer has increased year by year due to the influence of dietary refinement and westernization. According to the International Agency for Research on Cancer (IARC) report, about 1.2 million people worldwide suffered from colon cancer in 2012 (IARC, 2016); the World Health Organization (WHO) also pointed out In 2015, 694,000 people died of colon cancer (WHO, 2016). In addition, among the top ten causes of death in Taiwan announced by the Ministry of Health and Welfare of the Republic of China in 2017, malignant tumors once again ranked first among the top ten causes of death among Chinese people. Moreover, among the top ten causes of cancer, colon cancer is not only high. The number of deaths due to colon cancer is as high as 24.3 per 100,000 population (Ministry of Health and Welfare, 2017). It can be seen that colon cancer poses a major threat to the health of our people. Therefore, how to prevent the occurrence of colon cancer has become one of the important topics for many scholars to study.

已知癌細胞之轉移(metastasis)係造成癌症治療成效不佳之主要原因之一,一旦癌症發生轉移現象,其預後狀況皆相當不良,而且90%以上癌症病患之死因多是因癌細胞轉移至遠端器官所造成(Leber and Efferth,2009)。值得注意的是,在結腸癌之晚期階段,癌細胞常經 由淋巴或血液進行遠端轉移至肝臟、肺臟、骨骼及大腦等部位(Roth et al.,2009),對人類之健康造成危害。 It is known that metastasis of cancer cells is one of the main causes of poor treatment of cancer. Once the cancer metastasizes, the prognosis is quite poor, and more than 90% of cancer patients die because cancer cells are transferred to Caused by distal organs (Leber and Efferth, 2009). It is worth noting that in the advanced stage of colon cancer, cancer cells often The distant metastasis from the lymph or blood to the liver, lungs, bones and brain (Roth et al., 2009) poses a hazard to human health.

目前在臨床醫學上對於結腸癌之治療方法有手術切除、化學治療、標靶藥物合併放射治療等,但此等治療方式經常有不同之副作用(Li et al.,2010)。因此,近年來已有許多具有預防及改善癌症治療效果之天然活性成分逐漸受到重視。如何應用天然食品及其活性成分達到降低癌細胞轉移之現象已成為重要之研究議題。 At present, there are surgical resection, chemotherapy, target drugs combined with radiotherapy for clinical treatment of colon cancer, but these treatments often have different side effects (Li et al., 2010). Therefore, in recent years, many natural active ingredients that have the effect of preventing and improving the treatment of cancer have received increasing attention. How to apply natural foods and their active ingredients to reduce the metastasis of cancer cells has become an important research topic.

肌肽(carnosine)為存在於脊椎動物骨骼肌中之內源性雙勝肽,目前已知其具有抗疲勞、抗氧化、改善高血壓、糖尿病及抑制癌症細胞生長等作用。然而,肌肽對於人類結腸癌細胞(human colorectal cancer cells)轉移作用(metastasis)之影響則少有研究。 Carnosine is an endogenous double peptide existing in skeletal muscle of vertebrate. It is known to have anti-fatigue, anti-oxidation, high blood pressure, diabetes and inhibition of cancer cell growth. However, the effects of carnosine on human colorectal cancer cell metastasis have been rarely studied.

鑑於肌肽抑制癌細胞轉移之調控機制目前仍不完整,故,一般習用者係無法符合使用者於實際使用時之所需。 In view of the fact that the regulation mechanism of carnosine inhibiting cancer cell metastasis is still incomplete, the general practitioners cannot meet the needs of users in actual use.

本發明之主要目的係在於,克服習知技藝所遭遇之上述問題並提供一種肌肽(carnosine)可藉由調節細胞遷移(migration)、侵犯(invasion)、內滲(intravasation)、外滲(extravasation)及黏附(adhesion)作用而降低結腸癌(HCT-116)細胞之轉移能力之化合物用於製備調控腸癌細胞轉移之藥物的用途。 The main object of the present invention is to overcome the above problems encountered in the prior art and to provide a carnosine which can regulate cell migration, invasion, intravasation, extravasation by extravasation. And the use of a compound to reduce the metastatic ability of colon cancer (HCT-116) cells for the preparation of a medicament for regulating metastasis of intestinal cancer cells.

為達以上之目的,本發明係一種化合物用於製備調控腸癌細胞轉移之藥物的用途,包含一有效量之式I化合物-肌肽(2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid,carnosine) For the purposes of the above, the present invention is a use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, comprising an effective amount of a compound of the formula I - carnosine (2S)-2-[(3-Amino-1-oxopropyl) Amino]-3-(3H-imidazol-4-yl)propanoic acid,carnosine)

或其醫藥上可接受鹽,及醫藥上可接受載體(carrier)。 Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier.

於本發明上述實施例中,該化合物可治療癌症。 In the above embodiments of the invention, the compound treats cancer.

於本發明上述實施例中,該癌症包括結腸癌。 In the above embodiments of the invention, the cancer comprises colon cancer.

於本發明上述實施例中,該腸癌細胞為人類結腸癌細胞株HCT-116。 In the above embodiment of the invention, the intestinal cancer cell is human colon cancer cell line HCT-116.

於本發明上述實施例中,該式I化合物-肌肽之濃度為0.5mM至5mM。 In the above examples of the invention, the concentration of the compound of formula I - carnosine is from 0.5 mM to 5 mM.

第1圖,係本發明的肌肽對人類結腸癌細胞轉移之調控流程示意圖。 Fig. 1 is a schematic diagram showing the regulation process of carnosine of the present invention for human colon cancer cell metastasis.

第2圖,係本發明的肌肽對HCT-116細胞株的細胞生存力之影響示意圖。 Fig. 2 is a graph showing the effect of carnosine of the present invention on cell viability of HCT-116 cell line.

第3圖,係本發明的肌肽對HCT-116細胞株的遷移能力之影響示意圖。 Fig. 3 is a graph showing the effect of the carnosine of the present invention on the migration ability of the HCT-116 cell line.

第4圖,係本發明的肌肽對HCT-116細胞株的侵犯能力之影響示意圖。 Fig. 4 is a graph showing the effect of the carnosine of the present invention on the invasive ability of the HCT-116 cell line.

第5圖,係本發明的肌肽對HCT-116細胞株的MMP-2基因表現影響之長條示意圖。 Fig. 5 is a schematic view showing the effect of the carnosine of the present invention on the MMP-2 gene expression of the HCT-116 cell line.

第6圖,係本發明的肌肽對HCT-116細胞株的MMP-9基因表現之影響示意圖。 Fig. 6 is a graph showing the effect of the carnosine of the present invention on the MMP-9 gene expression of the HCT-116 cell line.

第7圖,係本發明的肌肽對EA.hy 926細胞株的TEER之影響示意圖。 Figure 7 is a graph showing the effect of carnosine of the present invention on TEER of EA.hy 926 cell line.

第8圖,係本發明的肌肽對HCT-116細胞株黏附至EA.hy 926細胞株之影響示意圖。 Fig. 8 is a schematic view showing the effect of carnosine of the present invention on adhesion of HCT-116 cell line to EA.hy 926 cell line.

第9圖,係本發明轉移調節蛋白表現後肌肽之治療效果示意圖。 Fig. 9 is a schematic view showing the therapeutic effect of carnosine after expression of the transfer regulatory protein of the present invention.

第10圖,係本發明的肌肽對HCT-116細胞株的細胞型態影響之顯微照片。 Fig. 10 is a photomicrograph showing the effect of the carnosine of the present invention on the cell type of the HCT-116 cell line.

請參閱『第1圖~第10圖』所示,係分別為本發明的肌肽對人類結腸癌細胞轉移之調控流程示意圖、本發明的肌肽對HCT-116細胞株的細胞生存力之影響示意圖、本發明的肌肽對HCT-116細胞株的遷移能力之影響示意圖、本發明的肌肽對HCT-116細胞株的侵犯能力之影響示意圖、本發明的肌肽對HCT-116細胞株的MMP-2基因表現影響之長條示意圖、本發明的肌肽對HCT-116細胞株的MMP-9基因表現之影響示意圖、本發明的肌肽對EA.hy 926細胞株的TEER之影響示意圖、本發明的肌肽對HCT-116細胞株黏附至EA.hy 926細胞株之影響示意圖、本發明轉移調節蛋白表現後肌肽之治療效果示意圖、及本發明的肌肽對HCT-116細胞株的細胞型態影響之顯微照片。如圖所示:本發明係一種化合物用於製備調控腸癌細胞轉移之藥物的用途,係利用肌肽(carnosine)為實驗材料,並利用人類結腸癌HCT-116細胞株之培養以不同濃度之肌肽處理不同時間後,以細胞生存率(MTT assay)試驗、細胞形態(morphology examination)觀察,瞭解肌肽對細胞生存能力(cell viability)之影響。配合細胞損傷試驗(wound healing assay)及分析上皮鈣黏蛋白(E-cadherin)表現探討肌肽對癌細胞遷移之能力;以細胞侵犯試驗(cell invasion assay)及基質金屬蛋白酶(matrix metalloproteinases,MMPs)之MMP-2與MMP-9表現,探討肌肽對癌細 胞侵犯能力之影響。利用經螢光標示之HCT-116細胞與人類血管內皮(EA.hy 926)細胞共同培養,進行細胞黏附試驗(cell adhesion assay)以及分析整合素β1(Integrin β1)、E-選擇素(E-selectin)及細胞黏附分子-1(ICAM-1)表現,探討肌肽對癌細胞黏附於內皮細胞能力之影響。並利用內皮細胞之培養模式,進行內皮細胞通透性分析與血管內皮鈣離子依賴性黏著分子(vascular endothelial cadherin,VE-cadherin)磷酸化之表現。如第1圖之實驗架構所示,期望本發明之進行可以瞭解肌肽對於人類結腸癌HCT-116細胞株遷移(migration)、侵犯(invasion)、內滲(intravasation)、外滲(extravasation)及黏附(adhesion)之作用。 Please refer to FIG. 1 to FIG. 10 , which are schematic diagrams showing the regulation process of carnosine-to-human colon cancer cell metastasis of the present invention, and the effect of carnosine of the present invention on cell viability of HCT-116 cell line, The effect of the carnosine of the present invention on the migration ability of the HCT-116 cell line, the effect of the carnosine of the present invention on the invasion ability of the HCT-116 cell line, and the MMP-2 gene expression of the carnosine of the present invention on the HCT-116 cell line Schematic diagram of the influence of the long strip, the effect of the carnosine of the present invention on the MMP-9 gene expression of the HCT-116 cell line, the effect of the carnosine of the present invention on the TEER of the EA.hy 926 cell line, and the carnosine pair HCT- of the present invention Schematic diagram of the effect of the 116 cell line adhering to the EA.hy 926 cell line, the therapeutic effect of the carnosine after the expression of the transfer regulatory protein of the present invention, and the micrograph of the effect of the carnosine of the present invention on the cell type of the HCT-116 cell line. As shown in the figure: The present invention is a use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, using carnosine as an experimental material, and using human colon cancer HCT-116 cell strain to culture different concentrations of carnosine. After treatment for different time, the effects of carnosine on cell viability were investigated by MTT assay and morphology examination. The ability of carnosine to migrate to cancer cells was explored in conjunction with the wound healing assay and the analysis of E-cadherin; cell invasion assay and matrix metalloproteinases (MMPs) were used. MMP-2 and MMP-9 performance, explore carnosine to cancer The impact of cell invasion. Fluorescently labeled HCT-116 cells were co-cultured with human vascular endothelium (EA.hy 926) cells for cell adhesion assay and analysis of integrin β1 and E-selectin (E- Selectin) and cell adhesion molecule-1 (ICAM-1) showed the effect of carnosine on the ability of cancer cells to adhere to endothelial cells. The endothelial cell permeability model was used to analyze endothelial cell permeability and phosphorylation of vascular endothelial cadherin (VE-cadherin). As shown in the experimental scheme of Figure 1, it is expected that the present invention can be understood to understand the migration, invasion, invasion, extravasation, extravasation and adhesion of carnosine to human colon cancer HCT-116 cell line. The role of (adhesion).

由於E-cadherin、MMP-2、MMP-9、Integrin β1、E-selectin、ICAM-1及VE-cadherin等基因之啟動區中都包含有轉錄因數NF-κB之結合調控區。因此,實驗模式也將探討肌肽是否經由調控NF-κB之活化而影響癌細胞轉移之作用。 The promoter region of the transcription factor NF-κB is contained in the promoter regions of E-cadherin, MMP-2, MMP-9, Integrin β1, E-selectin, ICAM-1 and VE-cadherin. Therefore, the experimental model will also explore whether carnosine affects cancer cell metastasis via regulation of NF-κB activation.

以下實施例僅用於解釋本發明,但本發明的保護範圍並不僅限以下實施例。為了讓本發明之上述和其他目的、特徵、和優點能更明顯易懂,下文特舉較佳實施例,作詳細說明如下: The following examples are merely illustrative of the invention, but the scope of protection of the invention is not limited to the following examples. The above and other objects, features and advantages of the present invention will become more apparent and understood.

[實施例一]實驗材料 [Example 1] Experimental materials

肌肽來源 Carnosine source

本實驗所使用之化合物-肌肽(2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid,carnosine),乃購置於Sigma公司(Sigma-Aldrich Co.USA)。 The compound used in this experiment, carnosine (2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid, carnosine), was purchased at Sigma ( Sigma-Aldrich Co. USA).

細胞培養 Cell culture

1. 人類結腸癌HCT-116細胞株 Human colon cancer HCT-116 cell line

本實驗所使用之細胞株為人類結腸癌HCT-116細胞株,乃購自食品工業發展研究所生物資源保存及研究中心。HCT-116細胞株培養於包含2.2g/L重碳酸鈉(sodium bicarbonate)、1%青黴素/鏈黴素(penicillin/streptomycin)100U/mL與10%胎牛血清(fetal bovine serum)之McCoy’s 5A培養基中,置於37℃含有5%二氧化碳(CO2)之恆溫培養箱中培養。 The cell line used in this experiment was human colon cancer HCT-116 cell line, which was purchased from the Center for Biological Resource Conservation and Research of the Food Industry Development Research Institute. The HCT-116 cell line was cultured in McCoy's 5A medium containing 2.2 g/L sodium bicarbonate, 1% penicillin/streptomycin 100 U/mL and 10% fetal bovine serum. The medium was cultured in a constant temperature incubator containing 5% carbon dioxide (CO 2 ) at 37 °C.

液態桶中取出細胞冷凍管,於37℃水浴槽中使其完全溶解,消毒後於無菌操作臺中將解凍之細胞液取出置於含有McCoy’s 5A培養基之離心管中,以1000rpm離心2分鐘,去除上清液後,加入新鮮之McCoy’s 5A培養基並將細胞充分混勻,再將細胞液種入10公分培養皿中並左右輕微搖晃使細胞均勻分散,最後將培養皿置於37℃含有5% CO2之培養箱中培養。於隔日確定細胞是否有貼附於培養皿中,並更換新鮮之McCoy’s 5A培養基進行培養。 The cell cryotube was taken out in a liquid barrel and completely dissolved in a 37 ° C water bath. After disinfection, the thawed cell liquid was taken out in a sterile tube and placed in a centrifuge tube containing McCoy's 5A medium, centrifuged at 1000 rpm for 2 minutes, and removed. After clearing the solution, add fresh McCoy's 5A medium and mix the cells thoroughly. Then, mix the cell liquid into a 10 cm culture dish and shake it gently to make the cells evenly dispersed. Finally, place the culture dish at 37 ° C with 5% CO 2 . Cultivate in the incubator. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh McCoy's 5A medium for culture.

細胞於培養皿中生長至九分滿時,以包含9Mm磷酸氫二鈉(Na2HPO4)、140mM氯化鈉(NaCl)、及1mM磷酸二氫鈉(NaH2PO4)組成pH 7.4磷酸緩衝液(phosphate buffered saline,PBS)清洗兩次後,加入0.25%胰蛋白酶(trypsin/EDTA)置於37℃含有5% CO2之培養箱中作用3分鐘後拍盤,以McCoy’s 5A培養基沖下細胞,並將細胞液收集至離心管中,以1000rpm離心2分鐘,去除上清液,加入新鮮之McCoy’s 5A培養基將細胞充分混勻,再取出所需細胞液種入10公分培養皿中並左右輕微搖晃使細胞均勻分散,最後將培養皿置於37℃含有5% CO2之培養箱中培養。於隔日確定細胞是否有貼附於培養皿中,並更換新鮮之McCoy’s 5A培養基進行培養。 The cells were grown in petri dishes until they were nine minutes full, and consisted of 9 Mm disodium hydrogen phosphate (Na 2 HPO 4 ), 140 mM sodium chloride (NaCl), and 1 mM sodium dihydrogen phosphate (NaH 2 PO 4 ) to form pH 7.4 phosphoric acid. After washing twice with phosphate buffered saline (PBS), add 0.25% trypsin (trypsin/EDTA) and place in a 37 ° C incubator with 5% CO 2 for 3 minutes, then take the plate and wash it down with McCoy's 5A medium. The cells were collected, and the cell liquid was collected into a centrifuge tube, centrifuged at 1000 rpm for 2 minutes, the supernatant was removed, the cells were thoroughly mixed by adding fresh McCoy's 5A medium, and the desired cell liquid was taken out into a 10 cm culture dish and left and right. The cells were evenly dispersed by gentle shaking, and finally the dishes were incubated in an incubator containing 5% CO 2 at 37 °C. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh McCoy's 5A medium for culture.

利用血球計數器計算細胞數為每毫升1×106個細胞,加入1mL含有10%二甲亞碸(dimethyl sulfoxide,DMSO)之McCoy’s 5A培養基並使細胞充分混勻後,移至冷凍管中,放置於-80℃冷凍庫,於24小時後再移至液態氮桶中凍存。 Count the number of cells to 1 × 10 6 cells per ml using a hemocytometer, add 1 mL of McCoy's 5A medium containing 10% dimethyl sulfoxide (DMSO), and mix the cells thoroughly, then transfer to a cryotube and place. The library was frozen at -80 ° C and then transferred to a liquid nitrogen drum for 24 hours.

2. 人類血管內皮EA.hy 926細胞株 2. Human vascular endothelial EA.hy 926 cell line

本實驗所使用之人類血管內皮EA.hy 926細胞株乃由中國醫藥大學李宗貴老師實驗室所提供。EA.hy 926細胞株培養於包含3.7g/L重碳酸鈉、1%非必需胺基酸(Non-Essential Amino Acid,NEAA)、1%丙酮酸鈉(sodium pyruvate)、1%青黴素/鏈黴素100U/mL與10%胎牛血清之DMEM(Dulbecco’s Modified Eagle Medium)培養基中,置於37℃含有5% CO2之培養箱中培養。 The human vascular endothelial EA.hy 926 cell strain used in this experiment was provided by the laboratory of Teacher Li Zonggui of China Medical University. The EA.hy 926 cell line is cultured to contain 3.7 g/L sodium bicarbonate, 1% Non-Essential Amino Acid (NEAA), 1% sodium pyruvate, 1% penicillin/streptococcus In a DMEM (Dulbecco's Modified Eagle Medium) medium containing 100 U/mL and 10% fetal calf serum, it was cultured in an incubator containing 5% CO 2 at 37 °C.

液態桶中取出細胞冷凍管,立即放入37℃水浴槽中使其完全溶解,消毒後於無菌操作臺中將解凍之細胞液取出置於含有DMEM培養基之離心管中,以離心機1000rpm離心2分鐘,去除上清液後,加入新鮮之DMEM培養基並將細胞充分混勻,再將細胞液種入10公分培養皿中並左右輕微搖晃使細胞均勻分散,最後將培養皿置於37℃含有5% CO2之培養箱中培養。於隔日確定細胞是否有貼附於培養皿中,並更換新鮮之DMEM培養基進行培養。 The cell cryotube was taken out in a liquid barrel and immediately placed in a 37 ° C water bath to completely dissolve it. After disinfection, the thawed cell liquid was taken out in a sterile tube and placed in a centrifuge tube containing DMEM medium, and centrifuged at 1000 rpm for 2 minutes in a centrifuge. After removing the supernatant, add fresh DMEM medium and mix the cells thoroughly. Then, mix the cell solution into a 10 cm culture dish and shake it gently to make the cells evenly dispersed. Finally, the culture dish is placed at 37 ° C and contains 5%. Culture in a CO 2 incubator. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh DMEM medium for culture.

細胞於培養皿中生長至九分滿時,以PBS清洗細胞兩次後,加入胰蛋白酶置於37℃含有5% CO2之培養箱中作用3分鐘後拍盤,利用DMEM培養基沖下細胞,並將細胞液收集至離心管中,以1000rpm離心2分鐘,去除上清液,加入新鮮之DMEM培養基並將細胞充分混勻,再取出所需細胞液種入10公分培養皿中並左右輕微搖晃使細胞均勻分散, 最後將培養皿置於37℃含有5% CO2之培養箱中培養。於隔日確定細胞是否有貼附於培養皿中,並更換新鮮之DMEM培養基進行培養。 When the cells were grown in a petri dish until the end of nine minutes, the cells were washed twice with PBS, and trypsin was added to an incubator containing 5% CO 2 at 37 ° C for 3 minutes, and the cells were plated, and the cells were washed down with DMEM medium. The cell fluid was collected into a centrifuge tube, centrifuged at 1000 rpm for 2 minutes, the supernatant was removed, fresh DMEM medium was added and the cells were thoroughly mixed, and the desired cell solution was taken out into a 10 cm culture dish and gently shaken left and right. The cells were uniformly dispersed, and finally the dishes were incubated in an incubator containing 5% CO 2 at 37 °C. On the next day, it was determined whether the cells were attached to the culture dish and replaced with fresh DMEM medium for culture.

利用血球計數器計算細胞數為每毫升5×105個細胞,加入1mL含有10% DMSO之DMEM培養基並使細胞充分混勻後,移至冷凍管中,放置於-80℃冷凍庫,於24小時後再移至液態氮桶中凍存。 Count the number of cells to 5 × 10 5 cells per ml using a hemocytometer, add 1 mL of DMEM medium containing 10% DMSO and mix the cells well, transfer to a cryotube, and place in a freezer at -80 ° C, after 24 hours. Move to a liquid nitrogen drum for cryopreservation.

[實施例二]實驗方法 [Example 2] Experimental method

樣品處理 Sample processing

HCT-116細胞株及EA.hy 926細胞株分別以每毫升1×106及5×105個細胞培養於直徑3.5公分培養皿中。培養24小時後,分別加入0.5、1、或5mM肌肽,培養24小時後,進行後續分析。 The HCT-116 cell line and the EA.hy 926 cell line were cultured in a 3.5 cm diameter culture dish at 1 × 10 6 and 5 × 10 5 cells per ml, respectively. After 24 hours of culture, 0.5, 1, or 5 mM carnosine was added, respectively, and after 24 hours of culture, subsequent analysis was performed.

細胞生存力 Cell viability

1. MTT試驗(MTT assay) 1. MTT assay (MTT assay)

本實驗採用細胞生存率(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide,MTT)試驗,作為測定HCT-116細胞株存活率之方法。位於活細胞中粒線體之琥珀酸脫氫酶(succinate dehydrogenase,SDH)與細胞色素C(cytochrome C)之作用下會使MTT之四唑(tetrazolium)環裂開,生成藍色結晶物質甲臢(formazan),此結晶物質之生成量與活細胞數目成正比,利用異丙醇(isopropanol)將formazan結晶溶解,再以酵素免疫分析儀(Enzyme-linked Immuno-sorbent Assay reader,ELISA reader)測定其在波長570nm之吸光值。此吸光值代表粒腺體之活性,與活細胞數目成正比,故MTT試驗可用作細胞存活率之指標。 In this experiment, the cell survival rate (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, MTT) was used as a method for determining the survival rate of HCT-116 cell line. The succinate dehydrogenase (SDH) and cytochrome C (Cytochrome C) in the mitochondria of living cells will cleave the tetrazolium ring of MTT to form a blue crystalline substance. (formazan), the amount of the crystalline substance produced is proportional to the number of living cells, and the formazan crystal is dissolved by isopropanol, and then determined by an enzyme-linked immuno-sorbent Assay reader (ELISA reader). Absorbance at a wavelength of 570 nm. This absorbance value represents the activity of the granulosa, which is proportional to the number of viable cells, so the MTT assay can be used as an indicator of cell viability.

本實驗將每毫升1×106個HCT-116細胞種植於3.5公分培養皿中,培養於37℃含有5% CO2之細胞恆溫培養箱中培養至8分滿。分別給予0.5、1、或5mM肌肽,並以僅含無菌水之培養基為控制組。培養24及48小時後,去除培養基,以PBS清洗二次,並加入含有0.5mg/mL MTT之培養基置於37℃與5% CO2之細胞恆溫培養箱中培養3小時,去除培養液,加入1mL異丙醇將formazan溶解,經10分鐘震盪搖勻後移入微量離心管中,以7,500rpm離心5分鐘,取200μL上清液於96孔盤中,以ELISA reader測定其在波長570nm之吸光值。 In this experiment, 1×10 6 HCT-116 cells per ml were planted in a 3.5 cm culture dish, and cultured in a constant temperature incubator containing 5% CO 2 at 37 ° C until 8 minutes. 0.5, 1, or 5 mM carnosine was administered separately, and the medium containing only sterile water was used as the control group. After 24 and 48 hours of culture, the medium was removed, washed twice with PBS, and added to a medium containing 0.5 mg/mL MTT, and cultured in a constant temperature incubator at 37 ° C and 5% CO 2 for 3 hours to remove the culture solution and added. 1 mL of isopropanol was dissolved in formazan, shaken for 10 minutes, transferred to a microcentrifuge tube, centrifuged at 7,500 rpm for 5 minutes, and 200 μL of the supernatant was taken in a 96-well plate. The absorbance at 570 nm was measured by ELISA reader. .

2. 細胞形態觀察與紀錄 2. Cell morphology observation and record

本實驗將每毫升1×106個HCT-116細胞種植於3.5公分培養皿中,培養於37℃含有5% CO2之細胞恆溫培養箱中培養至8分滿。分別給予0.5、1、或5mM肌肽,並以僅含無菌水之培養基為控制組,培養24及48小時後,採用倒立式相位差顯微鏡觀察並記錄其細胞數量及形態之變化。 In this experiment, 1×10 6 HCT-116 cells per ml were planted in a 3.5 cm culture dish, and cultured in a constant temperature incubator containing 5% CO 2 at 37 ° C until 8 minutes. 0.5, 1, or 5 mM carnosine was administered separately, and the medium containing only sterile water was used as the control group. After 24 and 48 hours of culture, the number and morphology of the cells were observed and recorded by an inverted phase contrast microscope.

肌肽對HCT-116細胞株傷口癒合能力之分析 Analysis of wound healing ability of carnosine on HCT-116 cell line

本實驗採用Ang等人(2010)實驗方法使細胞培養盤產生損傷區域,利用細胞移動過程使損傷面積改變,藉以表示細胞遷移之能力。 In this experiment, the experimental method of Ang et al. (2010) was used to make the cell culture plate produce a damaged area, and the cell movement process was used to change the damage area, thereby indicating the ability of cell migration.

本實驗以3.5公分培養皿培養每毫升1×106個HCT-116細胞,待細胞長至滿盤後,以10μL無菌微量吸管(tip)輕劃培養皿使產生損傷區域後,利用PBS清洗兩次,以0.5、1、或5mM肌肽處理0、12、24及48小時後,採用倒立式相位差顯微鏡進行細胞遷移之觀察及記錄;進一步利用Image J軟體進行損傷面積之檢測(Image J 1.47t,National Institutes of Health,USA)。 In this experiment, 1×10 6 HCT-116 cells per ml were cultured in a 3.5 cm culture dish. After the cells were grown to full volume, the culture dish was lightly drawn with a 10 μL sterile micropipette tip to create a damaged area, and then washed with PBS. After treatment with 0.5, 1, or 5 mM carnosine for 0, 12, 24, and 48 hours, the cell migration was observed and recorded using an inverted phase contrast microscope; the damage area was further examined using Image J software (Image J 1.47t) , National Institutes of Health, USA).

肌肽對HCT-116細胞株轉移相關基因表現之分析 Analysis of carnosine on the expression of metastasis-associated genes in HCT-116 cell line

1. 肌肽對之前製備 1. Preparation of carnosine pair

本實驗將每毫升1×106個HCT-116細胞種植於3.5公分培養皿中,培養於37℃含有5% CO2之細胞恆溫培養箱中培養至8分滿。分別給予0.5、1、或5mM肌肽,並以僅含無菌水之培養基為控制組,培養24小時。 In this experiment, 1×10 6 HCT-116 cells per ml were planted in a 3.5 cm culture dish, and cultured in a constant temperature incubator containing 5% CO 2 at 37 ° C until 8 minutes. 0.5, 1, or 5 mM carnosine was administered separately, and cultured in a medium containing only sterile water for 24 hours.

2. HCT-116細胞株全量核糖核酸(total RNA)之萃取 2. Extraction of total RNA from HCT-116 cell line

在進行實驗以前,所有物品必須經過高溫高壓滅菌,以除去殘存之核糖核酸醇(RNase)。取上述之細胞,用冷的PBS洗兩次,加入1mL核酸蛋白質三相萃取試劑(TRI reagent)並充分混合震盪,加入200μL氯仿(chloroform)輕搖混合均勻,冰浴5分鐘,於4℃、13000rpm離心15分鐘,取上清液到新的tube裡加入等體積之異丙醇(即1:1)震盪均勻並離心,之後冰浴5分鐘,在4℃、13000rpm離心15分鐘,吸除上清液,留下底部RNA,加入75%酒精1m,以4℃、13000rpm離心10分鐘,吸除上清液,留下沉澱物,再以4℃、13000rpm空離1分鐘,將殘存之酒精去除,之後RNA沉澱物自然風乾15分鐘,最後溶解於DEPC-H2O溶液中,定量後保存於-20℃備用。 Prior to the experiment, all items must be autoclaved to remove residual RNase. Take the above cells, wash twice with cold PBS, add 1 mL of nucleic acid protein three-phase extraction reagent (TRI reagent) and mix well with shaking, add 200 μL chloroform, gently shake and mix, ice bath for 5 minutes, at 4 ° C, Centrifuge at 13000 rpm for 15 minutes, take the supernatant into a new tube, add an equal volume of isopropanol (ie 1:1), shake well and centrifuge, then ice bath for 5 minutes, centrifuge at 13000 rpm for 15 minutes at 4 ° C, aspirate Clear the supernatant, leave the bottom RNA, add 7% alcohol 1m, centrifuge at 4 ° C, 13000 rpm for 10 minutes, aspirate the supernatant, leave a precipitate, and then detach at 4 ° C, 13000 rpm for 1 minute, remove the residual alcohol After that, the RNA pellet was naturally air-dried for 15 minutes, finally dissolved in DEPC-H 2 O solution, quantified and stored at -20 ° C until use.

3. total RNA之定量 3. Quantification of total RNA

total RNA之定量係利用核酸蛋白分析系統測定,測定前先取2μL DEPC-H2O(作為對照)於樣品放置槽內測定,測定完成後將置於樣品放置槽內以DEPC-H2O擦拭乾淨,接著從全量核糖核酸溶液中取2μL加於樣品放置槽測定,測定完成後一樣將樣品放置槽內之total RNA溶液擦拭乾淨,依序重複上述動作此核酸蛋白定量分析系統與電腦連接,藉由電腦軟體來運算,當每測定完成一個樣品後,就會把樣品濃度、比值OD260/OD280算出。 The total RNA was determined by the nucleic acid protein analysis system. Before the measurement, 2 μL of DEPC-H 2 O (as a control) was taken in the sample placement tank. After the measurement was completed, it was placed in the sample placement tank and wiped with DEPC-H 2 O. Then, 2 μL of the whole amount of ribonucleic acid solution is added to the sample placement tank, and after the measurement is completed, the total RNA solution in the sample placement tank is wiped clean, and the above-mentioned action is repeated in sequence to connect the nucleic acid protein quantitative analysis system with the computer. The computer software calculates the sample concentration and the ratio OD260/OD280 after each measurement is completed.

4. total RNA反轉錄成互補去氧核醣核酸(complementary DNA,cDNA) 4. Total RNA is reverse transcribed into complementary DNA (cDNA)

本實驗參考Promega廠商所建議之方法進行。單股cDNA(first cDNA)之合成係於1.5mL微量試管中先加入10mM去氧核苷三磷酸(dNTP)1μL、寡脫氧胸苷酸(oligo(Dt))0.5μL、及RNA 3μg,再添加滅菌水補足到總體積20μL,接著將微量試管置放於加熱器上65℃作用5分鐘,之後再置於冰上5分鐘,接著添加下列試劑:1μL RNA酶抑制劑(RNasin)、1μL M-MLV反轉錄酶(M-MLV Reverse transcriptase)、及5μL M-MLV RT 5X buffer,最後總體積為27μL,將所有試劑混合均勻後,置放於37℃加熱板下作用1小時,即得單股cDNA溶液。最後置放於-20℃保存備用。 This experiment was carried out in accordance with the method recommended by the Promega manufacturer. The synthesis of single-strand cDNA (first cDNA) was first added to 1 μL of 10 mM deoxynucleotide triphosphate (dNTP), 0.5 μL of oligo (Dt), and 3 μg of RNA in a 1.5 mL microtube. The sterilized water was made up to a total volume of 20 μL, and then the microtubes were placed on a heater at 65 ° C for 5 minutes, and then placed on ice for 5 minutes, followed by the addition of the following reagents: 1 μL of RNasein (RNasin), 1 μL of M- MLV reverse transcriptase (M-MLV Reverse transcriptase), and 5 μL M-MLV RT 5X buffer, the final total volume is 27 μL, all the reagents are evenly mixed, placed in a 37 ° C heating plate for 1 hour, that is, a single strand cDNA solution. Finally placed at -20 ° C for storage.

5. 即時聚合酶連鎖反應(Real-time reverse transcription-polymerase chin reaction,Real-time PCR) 5. Real-time reverse transcription-polymerase chin reaction (Real-time PCR)

Real-time PCR與PCR之不同,在於Real-time PCR會在PCR反應中加入一個換和雙股DNA鍵結,而且在鹵素燈激發下會產生螢光之染劑SYBR green I,每一次之PCR循環反應增加目標基因(target gene)合成數量之同時,SYBR green I與雙股DNA結合,因此SYBR green I螢光量愈高顯示其基因表現量就越多。Real-time PCR之原理就是利用不同濃度之範本在相同PCR反應條件,含有高濃度範本之反應會較快達到幾何相位(geometric phase),此時DNA之合成係以兩倍來倍增,定義中達到geometric phase中點之臨界PCR循環數目稱為閥值循環(threshold cycle,CT),亦即CT值有隨範本濃度降低而升高。且在整個反應過程中為了確認在操作上有無汙染,故必須加入一組不含範本之陰性控制組(negative control),或稱為沒有範本的控制組(non-template control,NTC),另外為了可將每一次定量進行相互比較,並避免PCR反應效力差異導致定量分析偏差,因此每次定量分析 中皆會加入一組以上之內部控制組(Internal control(quality control,QC)),因為專一性之引子反應Internal control所換算之濃度可用於確認每次定量之準確度。而實際操作定量分析試劑添加如下:2.5μL雙蒸水(ddH2O)、5μL 2X SYBR Fast MM、0.5μL 20X目標引子集(Target Primer set)、及2μL cDNA(5ng),最後總體積為10μL,當反應試劑添加完畢後,加PCR專用之8連盤置入即時聚合酶連鎖反應儀(LightCyvler 96 Real-Time PCR System,Roche Life Science,Basel,Switzerland)進行反應,設定Real-Time PCR條件如表一所示: Real-time PCR differs from PCR in that Real-time PCR adds a double-stranded DNA bond to the PCR reaction and generates a fluorescent dye SYBR green I under the excitation of a halogen lamp. While the circulating reaction increases the amount of target gene synthesis, SYBR green I binds to the double-stranded DNA, so the higher the amount of SYBR green I fluorescence, the more the gene expression is. The principle of Real-time PCR is to use different concentrations of the template in the same PCR reaction conditions, the reaction containing high concentration of the template will reach the geometric phase faster, at this time the synthesis of DNA is doubled in the definition, reached in the definition The number of critical PCR cycles at the midpoint of the geometric phase is called the threshold cycle (CT), that is, the CT value increases as the concentration of the template decreases. In order to confirm the operation or not pollution during the whole reaction process, it is necessary to add a negative control group without a template, or a non-template control (NTC), and Each quantification can be compared with each other, and the difference in the efficiency of the PCR reaction can be avoided to lead to quantitative analysis bias. Therefore, more than one internal control (quality control (QC)) is added to each quantitative analysis because of the specificity. The concentration converted by the internal control can be used to confirm the accuracy of each quantification. The actual quantitative reagents were added as follows: 2.5 μL double distilled water (ddH 2 O), 5 μL 2X SYBR Fast MM, 0.5 μL 20X Target Primer set, and 2 μL cDNA (5 ng), and the final total volume was 10 μL. After the addition of the reaction reagent, the PCR-specific 8-plate was placed in a real-time polymerase chain reaction reactor (LightCyvler 96 Real-Time PCR System, Roche Life Science, Basel, Switzerland) to perform the reaction, and the Real-Time PCR conditions were set as follows. Table 1 shows:

由於相對定量時需選用結腸癌HCT-116本身具有之恆定表現特性基因來作為正常 之必要之計算值,因此本發明之試驗方法選用結腸癌HCT-116 GAPDH來當對照組,Real-Time PCR使用之各基因特異性引子於表二。 Since the relative quantification requires the constant performance characteristic gene of colon cancer HCT-116 as a necessary calculation value for normal, the test method of the present invention uses colon cancer HCT-116 GAPDH as a control group, and Real-Time PCR is used. Each gene-specific primer is shown in Table 2.

肌肽對HCT-116細胞株侵犯能力之分析 Analysis of the invasive ability of carnosine on HCT-116 cell line

本實驗使用24孔(transwell)培養皿之細胞培養模式,事先於8.0μm細胞培養皿內槽(transwell insert)注入70μL含有基底膜基質(matrigel)之培養基(以不含血清之McCoy’s 5A培養基1:6稀釋)於37℃培養箱培養60分鐘。計數每毫升5×104個細胞分別加入含有0.5、1、或5mM肌肽且不含FBS之200μL McCoy’s 5A培養基注入transwell insert中,於24孔培養皿中加入含FBS之McCoy’s 5A培養基500μL,置於37℃含有5% CO2之培養箱中培養48小時。取出transwell insert,以PBS清洗2 次後加入4%甲醛溶液進行細胞固定30分鐘,加入0.5%結晶紫染色15分鐘,以侵犯表現,最後加入1mL甲醇於室溫下搖晃10分鐘使結晶紫褪色,將甲醇移至微量離心管中混勻,取200μL細胞液於96孔盤中,以ELISA reader測定其在波長540nm之吸光值。 In this experiment, a cell culture mode of a 24-well (transwell) culture dish was used, and 70 μL of a medium containing a matrigel medium (in serum-free McCoy's 5A medium 1) was previously injected into a 8.0 μm cell culture dish. 6 dilution) Incubate in a 37 ° C incubator for 60 minutes. Count 5×10 4 cells per ml, add 200 μL McCoy's 5A medium containing 0.5, 1, or 5 mM carnosine and no FBS to the transwell insert, and add 500 μL of FCo-containing McCoy's 5A medium to the 24-well culture dish. Incubate for 48 hours in an incubator containing 5% CO 2 at 37 °C. The transwell insert was taken out, washed twice with PBS, and then fixed with 4% formaldehyde solution for 30 minutes, and stained with 0.5% crystal violet for 15 minutes to invade the performance. Finally, 1 mL of methanol was added and shaken at room temperature for 10 minutes to discolor the crystal violet. The methanol was transferred to a microcentrifuge tube and mixed, and 200 μL of the cell solution was taken in a 96-well plate, and its absorbance at a wavelength of 540 nm was measured by an ELISA reader.

[實施例三]實驗結果 [Example 3] Experimental results

統計分析 Statistical Analysis

實驗數據利用SPSS統計套裝軟體中之變異分析(analysis of variance,ANOVA)配合Duncan’s test進行統計分析,以判斷實驗數據間是否具有顯著性差異。 The experimental data were statistically analyzed using the analysis of variance (ANOVA) in the SPSS statistical software package with Duncan's test to determine whether there was a significant difference between the experimental data.

1. 以肌肽處理HCT-116細胞株24、48、72小時後,對其細胞生存力之影響 1. Effect of cellulite on HCT-116 cell line after 24, 48 and 72 hours of cell viability

HCT-116細胞株在含有0.5、1及5mM肌肽之McCoy's 5A培養基中培養,並以無菌水處理HCT-116細胞株作為控制組,在處理24、48及72小時後,其細胞生存力之變化如第2圖所示,其中數據來自四個獨立之實驗,以平均值±SD表示,並由Duncan’s test進行統計分析評估細胞存活力之顯著差異,圖中不同字母之間表示細胞存活力具有顯著差異(P<0.05)。 HCT-116 cell line was cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cell line was treated with sterile water as a control group, and cell viability was changed after 24, 48 and 72 hours of treatment. As shown in Figure 2, the data were from four independent experiments, expressed as mean ± SD, and statistically analyzed by Duncan's test to assess significant differences in cell viability, with significant cell viability between the different letters in the figure. Difference (P<0.05).

HCT-116細胞株分別以0.5、1及5mM肌肽處理24小時後,其生存能力分別為101.3%、98.9%及99.63%,與控制組(100%)相比,在1mM與5mM組顯著低於控制組,在48小時後,其生存力分別為96.2%、91.2%及86.9%都顯著低於控制組,且在72小時後,其生存力分別為82.5%、78.9%及80.2%也都顯著低於控制組(P<0.05)。結果顯示, 當HCT-116細胞株以1或5mM肌肽處理24、48及72小時皆可發現,肌肽具有抑制癌細胞生存力之能力。 After treatment with 0.5, 1 and 5 mM carnosine for 24 hours, the viability of HCT-116 cells was 101.3%, 98.9% and 99.63%, respectively, which was significantly lower than that of the control group (100%) in the 1 mM and 5 mM groups. In the control group, after 48 hours, their viability was 96.2%, 91.2% and 86.9%, respectively, which were significantly lower than the control group, and after 72 hours, their viability was 82.5%, 78.9% and 80.2%, respectively. Below the control group (P <0.05). The results show, When the HCT-116 cell line was treated with 1 or 5 mM carnosine for 24, 48 and 72 hours, carnosine has the ability to inhibit the viability of cancer cells.

2. 肌肽對HCT-116細胞株遷移能力之影響 2. Effect of carnosine on migration ability of HCT-116 cell line

利用倒立式顯微鏡觀察發現,HCT-116細胞株在含有0.5、1及5mM肌肽之McCoy's 5A培養基中培養,並以無菌水處理HCT-116細胞株作為控制組,在處理0、12、24及48小時後,癌細胞傷口癒合能力之變化分別如第3圖所示,其中數據來自四個獨立之實驗,以平均值±SD表示,並由Duncan’s test進行統計分析評估抑制細胞遷移之顯著差異,圖中不同字母之間表示細胞遷移具有顯著差異(P<0.05)。 Using inverted microscope, HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group at treatments 0, 12, 24 and 48. After hours, changes in cancer cell wound healing were shown in Figure 3, with data from four independent experiments, expressed as mean ± SD, and statistical analysis by Duncan's test to assess significant differences in inhibition of cell migration, There was a significant difference in cell migration between the different letters (P < 0.05).

由第3圖上圖可見HCT-116細胞株以0.5、1及5mM肌肽處理48小時後,細胞損傷區域與控制組相比有顯著減少之差異。由第3圖下圖可見以0.5、1及5mM肌肽處理12及24小時後,癌細胞損傷區域與控制組相比無明顯差異,但在48小時後其細胞遷移能力分別為84.9%(控制組)、79.2%、74.1%及68.8%,皆有顯著低於控制組之現象(P<0.05)。由上述結果可得知,當HCT-116細胞株分別以0.5、1或5mM肌肽處理48小時後,可顯著抑制HCT-116細胞株之遷移能力。 It can be seen from the upper panel of Fig. 3 that the HCT-116 cell line was treated with 0.5, 1 and 5 mM carnosine for 48 hours, and the cell damage area was significantly reduced compared with the control group. It can be seen from the lower panel of Figure 3 that after treatment with 0.5, 1 and 5 mM carnosine for 12 and 24 hours, there was no significant difference between the cancer cell injury area and the control group, but the cell migration ability was 84.9% after 48 hours (control group). ), 79.2%, 74.1%, and 68.8%, all significantly lower than the control group (P <0.05). From the above results, it was found that when the HCT-116 cell line was treated with 0.5, 1 or 5 mM carnosine for 48 hours, the migration ability of the HCT-116 cell line was significantly inhibited.

3. 肌肽對HCT-116細胞株侵犯能力之影響 3. Effect of carnosine on the invasion ability of HCT-116 cell line

第4圖為HCT-116細胞株分別在含有0.5、1、及5mM肌肽之McCoy's 5A培養基中培養,並以無菌水處理HCT-116細胞株作為控制組,在電鍍處理24小時後,細胞從過濾器頂部取出,將侵入膜之細胞固定並計數,對其侵犯能力之變化,其中數據來自四個獨立之實驗,以平均值 ±SD表示,並由Duncan’s test進行統計分析評估細胞侵犯之顯著差異,圖中不同字母之間表示細胞侵犯具有顯著差異(P<0.05)。 Figure 4 shows that HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1, and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After 24 hours of electroplating, cells were filtered. The top of the device is removed, the cells invading the membrane are fixed and counted, and the invasive ability is changed. The data is from four independent experiments, with an average value. ±SD indicates that statistical analysis of Duncan’s test was performed to assess significant differences in cell invasion, and there was a significant difference in cell invasion between the different letters in the figure (P<0.05).

由第4圖上圖可見HCT-116細胞株以0.5、1及5mM肌肽處理24小時後,其紫藍色區塊隨肌肽濃度越高,則顏色越少,與控制組相比有明顯差異,顯示HCT-116細胞株侵犯能力下降。由第4圖下圖可見以0.5、1及5mM肌肽處理24小時後,其侵犯能力分別為94.3%、65.5%及52.3%有顯著低於控制組(P<0.05)。由上述結果可得知,HCT-116細胞株以1或5mM肌肽處理24小時下,可顯著抑制HCT-116之侵犯作用,及顯著增加E-cadherin蛋白質之表現(P<0.05)。 It can be seen from the above figure in Fig. 4 that the HCT-116 cell line treated with 0.5, 1 and 5 mM carnosine for 24 hours, the higher the concentration of carnosine in the purple-blue block, the less the color, and the difference compared with the control group. It shows that the invasion ability of HCT-116 cell line is decreased. From the lower panel of Fig. 4, the invasive ability after treatment with 0.5, 1 and 5 mM carnosine for 24 hours was 94.3%, 65.5% and 52.3%, respectively, which was significantly lower than that of the control group (P<0.05). From the above results, it was found that HCT-116 cell line treated with 1 or 5 mM carnosine for 24 hours significantly inhibited the invasion of HCT-116 and significantly increased the expression of E-cadherin protein (P<0.05).

4. 肌肽對HCT-116細胞株MMP-2基因表現之影響 4. Effect of carnosine on the expression of MMP-2 gene in HCT-116 cell line

第5圖為HCT-116細胞株分別在含有0.5、1及5mM肌肽之McCoy's 5A培養基中培養,並以無菌水處理HCT-116細胞株作為控制組,在處理24小時後,對其MMP-2基因表現之影響,其中數據來自四個獨立之實驗,以平均值±SD表示,並由Duncan’s test進行統計分析評估MMP-2基因表現之顯著差異,圖中不同字母之間表示MMP-2基因表現具有顯著差異(P<0.05)。 Figure 5 shows that HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After treatment for 24 hours, MMP-2 was treated. The effects of gene expression, including data from four independent experiments, expressed as mean ± SD, and statistical analysis by Duncan's test to assess significant differences in MMP-2 gene expression, showing MMP-2 gene expression between different letters in the figure There was a significant difference (P < 0.05).

由圖中顯示HCT-116細胞株分別以0.5、1及5mM肌肽處理24小時後,其MMP-2基因分別為5.2、9.6及8.4,皆有顯著高於控制組之現象(P<0.05)。 The HMP-116 cell line was treated with 0.5, 1 and 5 mM carnosine for 24 hours, and the MMP-2 genes were 5.2, 9.6 and 8.4, respectively, which were significantly higher than those of the control group (P<0.05).

5. 肌肽對HCT-116細胞株MMP-9基因表現之影響 5. Effect of carnosine on the expression of MMP-9 gene in HCT-116 cell line

第6圖為HCT-116細胞株分別在含有0.5、1及5mM肌肽之McCoy's 5A培養基中培養,並以無菌水處理HCT-116細胞株作為控制組,在處理 24小時後,對其MMP-9基因表現之影響,其中數據來自四個獨立之實驗,以平均值±SD表示,並由Duncan’s test進行統計分析評估MMP-9基因表現之顯著差異,圖中不同字母之間表示MMP-9基因表現具有顯著差異(P<0.05)。 Figure 6 shows that HCT-116 cells were cultured in McCoy's 5A medium containing 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After 24 hours, the effect of MMP-9 gene expression, data from four independent experiments, expressed as mean ± SD, and statistical analysis by Duncan's test to assess the significant difference in MMP-9 gene performance, different in the figure There was a significant difference in the expression of MMP-9 gene between the letters (P<0.05).

由圖中顯示HCT-116細胞株分別以0.5及1mM以肌肽處理24小時後,其MMP-9基因分別為0.9及0.7,皆顯著低於控制組(P<0.05)。 The HMP-116 cells were treated with carnosine at 0.5 and 1 mM for 24 hours, respectively, and the MMP-9 genes were 0.9 and 0.7, respectively, which were significantly lower than the control group (P < 0.05).

6. 肌肽對EA.hy 926細胞株上皮電阻(trans epithelial electric resistance,TEER)之影響 6. Effect of carnosine on trans epithelial electric resistance (TEER) of EA.hy 926 cell line

第7圖為EA.hy 926細胞株以0.5、1及5mM肌肽處理,並以無菌水處理EA.hy 926細胞株作為控制組,在處理24小時後,對其TEER之影響。由圖中顯示EA.hy 926細胞株分別以0.5、1及5mM以肌肽處理24小時後,其TEER皆有顯著低於控制組之現象(P<0.05)。 Figure 7 shows the effect of EA.hy 926 cell line treated with 0.5, 1 and 5 mM carnosine and treated with sterile water as EA.hy 926 cell line as control group for 24 hours after treatment. The TEER of the EA.hy 926 cell line treated with carnosine at 0.5, 1 and 5 mM for 24 hours was significantly lower than that of the control group (P < 0.05).

7. 肌肽對HCT-116細胞株黏附至EA.hy 926細胞株之影響 7. Effect of carnosine on adhesion of HCT-116 cell line to EA.hy 926 cell line

由第8圖中顯示細胞黏附能力分析結果可知,以0.5、1或5mM肌肽處理EA.hy 926細胞株24小時後,可顯著降低HCT-116對EA.hy926細胞株之黏附作用(P<0.05)。而第9圖為遷移調節蛋白表現後肌肽之治療效果,以1或5mM肌肽處理HCT-116細胞株後,皆可顯著抑制HCT-116細胞株中Integrin β1表現(P<0.05);而且可顯著降低EA.hy926內皮細胞內黏附因數E-selectin與ICAM-1之表現(P<0.05)。 From the results of cell adhesion analysis shown in Fig. 8, it can be seen that the treatment of EA.hy 926 cell line with 0.5, 1 or 5 mM carnosine for 24 hours significantly reduced the adhesion of HCT-116 to EA.hy926 cell line (P<0.05). ). Figure 9 shows the therapeutic effect of carnosine after migration of regulatory proteins. After treatment with HCT-116 cells with 1 or 5 mM carnosine, the expression of Integrin β1 in HCT-116 cells was significantly inhibited (P<0.05). The expression of E-selectin and ICAM-1 in EA.hy926 endothelial cells was decreased (P<0.05).

8. 肌肽處理HCT-116細胞株後,對其細胞形態之影響 8. Effect of carnosine on HTD-116 cell line after cell morphology

利用電子顯微鏡觀察發現,HCT-116細胞株在以0.5、1及5mM肌肽處理,並以無菌水處理HCT-116細胞株作為控制組,在處理不同時間後,其細胞形態之變化如第10圖所示。HCT-116細胞株分別以不同劑量處理不同時間後,其細胞形態由電子顯微鏡觀察發現,細胞數量減少與控制組相比有顯著差異。由上述結果得知,當HCT-116細胞株以1或5mM肌肽處理不同時間都可以發現,肌肽具有抑制癌細胞增生之能力。 Observation by electron microscopy showed that HCT-116 cells were treated with 0.5, 1 and 5 mM carnosine, and HCT-116 cells were treated with sterile water as control group. After treatment for different time, the cell morphology changed as shown in Fig. 10. Shown. The HCT-116 cell line was treated with different doses for different time, and its cell morphology was observed by electron microscopy. The decrease in cell number was significantly different from that of the control group. From the above results, it was found that when the HCT-116 cell line was treated with 1 or 5 mM carnosine for various times, carnosine has the ability to inhibit the proliferation of cancer cells.

藉此,本發明以人類結腸癌(HCT-116)細胞株培養為實驗模式,探討肌肽對人類結腸癌細胞株轉移作用之影響。由結果得知,當HCT-116細胞株分別以0.5、1或5mM肌肽處理48小時後,可顯著抑制HCT-116細胞株之遷移能力(P<0.05);以0.5、1或5mM肌肽處理48小時後,可顯著降低MMP-9之表現(P<0.05)。另外,以1或5mM肌肽處理HCT-116細胞株24小時後,可顯著抑制HCT-116之侵犯作用(P<0.05),及顯著增加E-cadherin蛋白質之表現(P<0.05)。由細胞黏附能力分析結果可知,以0.5、1或5mM肌肽處理內皮(EA.hy926)細胞株24小時後,可顯著降低HCT-116對EA.hy926細胞株之黏附作用(P<0.05)。以1或5mM肌肽處理HCT-116細胞株後,皆可顯著抑制HCT-116細胞株中Integrin β1表現(P<0.05);而且可顯著降低EA.hy926內皮細胞內黏附因子E-selectin與ICAM-1之表現(P<0.05)。由掃描電子顯微鏡觀察得知,肌肽可以降低LPS誘發之細胞扁平化;並由增加單層細胞之跨上皮細胞電阻(TEER),而且顯著降低細胞中VE-cadherin磷酸化之表現(P<0.05),降低內皮細胞之通透性,顯示肌肽具有調控細胞侵犯作用之能力。由上述結果可知,肌肽可藉由調節細胞遷移、侵犯、侵犯與黏附作用而降低結腸癌細胞之轉移能力。 Therefore, the present invention uses human colon cancer (HCT-116) cell line culture as an experimental model to investigate the effect of carnosine on the metastasis of human colon cancer cell lines. It was found that when the HCT-116 cells were treated with 0.5, 1 or 5 mM carnosine for 48 hours, the migration ability of HCT-116 cells was significantly inhibited (P<0.05); treatment with 0.5, 1 or 5 mM carnosine 48 After an hour, the performance of MMP-9 was significantly reduced (P < 0.05). In addition, treatment of HCT-116 cells with 1 or 5 mM carnosine for 24 hours significantly inhibited the invasion of HCT-116 (P < 0.05) and significantly increased the performance of E-cadherin protein (P < 0.05). From the results of cell adhesion analysis, it was found that treatment of endothelial (EA.hy926) cell line with 0.5, 1 or 5 mM carnosine significantly reduced the adhesion of HCT-116 to EA.hy926 cell line (P<0.05). Treatment of HCT-116 cell line with 1 or 5 mM carnosine significantly inhibited the expression of Integrin β1 in HCT-116 cell line (P<0.05), and significantly decreased the adhesion factor E-selectin and ICAM- in EA.hy926 endothelial cells. 1 performance (P <0.05). Observed by scanning electron microscopy, carnosine can reduce LPS-induced cell flattening; increase the transepithelial resistance (TEER) of monolayer cells, and significantly reduce the expression of VE-cadherin phosphorylation in cells (P<0.05). It reduces the permeability of endothelial cells and shows that carnosine has the ability to regulate cell invasion. From the above results, carnosine can reduce the metastatic ability of colon cancer cells by regulating cell migration, invasion, invasion and adhesion.

綜上所述,本發明係一種化合物用於製備調控腸癌細胞轉移之藥物的用途,可有效改善習用之種種缺點,所提肌肽(carnosine)可藉由調節細胞遷移(migration)、侵犯(invasion)、內滲(intravasation)、外滲(extravasation)及黏附(adhesion)作用而降低結腸癌(HCT-116)細胞之轉移能力,進而使本發明之產生能更進步、更實用、更符合使用者之所須,確已符合發明專利申請之要件,爰依法提出專利申請。惟以上所述者,僅為本發明之較佳實施例而已,當不能以此限定本發明實施之範圍;故,凡依本發明申請專利範圍及發明說明書內容所作之簡單的等效變化與修飾,皆應仍屬本發明專利涵蓋之範圍內。 In summary, the present invention is a use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, which can effectively improve various disadvantages of the conventional use, and the carnosine can regulate cell migration and invasion (invasion). ), intravasation, extravasation, and adhesion to reduce the metastatic ability of colon cancer (HCT-116) cells, thereby making the invention more progressive, more practical, and more user-friendly. It must have met the requirements of the invention patent application and filed a patent application according to law. However, the above is only the preferred embodiment of the present invention, and the scope of the present invention is not limited thereto; therefore, the simple equivalent changes and modifications made in accordance with the scope of the present invention and the contents of the invention are modified. All should remain within the scope of the invention patent.

Claims (4)

一種化合物用於製備調控腸癌細胞轉移之藥物的用途,包含一有效量之式I化合物-肌肽(2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol-4-yl)propanoic acid,carnosine) 或其醫藥上可接受鹽,及醫藥上可接受載體(carrier);其中,該式I化合物-肌肽之濃度為0.5mM至5mM。 Use of a compound for the preparation of a medicament for regulating metastasis of intestinal cancer cells, comprising an effective amount of a compound of formula I - carnosine (2S)-2-[(3-Amino-1-oxopropyl)amino]-3-(3H-imidazol -4-yl)propanoic acid, carnosine) Or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier; wherein the concentration of the compound of formula I - carnosine is from 0.5 mM to 5 mM. 依申請專利範圍第1項所述之用途,其中,該化合物可治療癌症。 The use according to claim 1, wherein the compound treats cancer. 依申請專利範圍第2項所述之用途,其中,該癌症包括結腸癌。 The use according to claim 2, wherein the cancer comprises colon cancer. 依申請專利範圍第1項所述之用途,其中,該腸癌細胞為人類結腸癌細胞株HCT-116。 The use according to claim 1, wherein the intestinal cancer cell is human colon cancer cell line HCT-116.
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JP2012219080A (en) * 2011-04-12 2012-11-12 Anbas:Kk Composition for suppressing formation or proliferation of tumor by oral administration

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US20110064828A1 (en) * 2009-09-11 2011-03-17 Novelos Therapeutics, Incorporated Treatment of metastatic tumors and other conditions
JP2012219080A (en) * 2011-04-12 2012-11-12 Anbas:Kk Composition for suppressing formation or proliferation of tumor by oral administration

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