TW201204369A - Pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer and metrocarcinoma - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer and metrocarcinoma, which comprises at least one kind of STIM1 inhibitor. The present invention also relates to another pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer and metrocarcinoma, which comprises at least one kind of store-operated calcium channel inhibitor.

Description

201204369 VI. Description of the Invention: [Technical Field to Which the Invention Is Applicable] 〆 The present invention relates to a pharmaceutical composition for inhibiting colorectal cancer cells and uterine epithelial cancer = growth and metastasis. In particular, the present invention relates to the inhibition of colorectal cancer cells by inhibitors of matrix action molecule i (stromal interaction i〇le〇lecule [, stimi] inhibition or calcium pool regulation (store_operated calcium channds, s〇c). Growth and transfer. In addition, there was a significant positive correlation between the colony and the tumor sputum cancer embryo antigen (CEA) in the colonic f </ s> STIM1 table _ colorectal cancer. Therefore, STIM1 f intestinal rectal _ performance can be used as the power of the _ after the recovery of the molecular classification. [Prior Art] The most terrible place in the disease is the metastasis, and the two cancer cells will be added to the body (4) official organizations. According to statistics, most of the culprit is cancer cells, liver, i脎: ΐ ΐ and other important organs, only less than 10% is the first to grow from the original two H # into two cancer cells is a The continuous process: the cancer cells are peeled off from the original 'reported by the blood vessels to the position of the appropriate roots. It will build: ii, the mosquitoes hold their own growth, and grow up to the battle of the battle. Doing &amp; chemotherapeutic methods can prolong the metastasis of cancer cells. Because the medical community has always been aware of the process of cancer cell metastasis, &lt;music or therapeutic can really transfer the process, lie to cure the disease, no one else A major cause of cancerous discoloration. The car is fine, the ionic ion is thick _ change can scale various physiological functions. 2005 1 (stromal interaction molecule 201204369 1, referred to as STIM1) is an important calcium ion sensing molecule in the cell, which can detect changes in calcium ion concentration in the endoplasmic reticulum matrix and open the calcium pool to regulate calcium channel. The EF-hand structure of STIM1 binds to calcium ions. When the calcium concentration of the endoplasmic reticulum matrix decreases, STIM1 begins to accumulate on the endoplasmic reticulum and regulates the ion channel with the cell on the cell membrane (Store-Operated Calcium). The secondary unit Channelraii of Channels 'S0C for short) interacts to open the calcium pool to regulate calcium channels, allowing extracellular calcium ions to enter the cell (James W· P) J Cell Biol. 2005.9 169: 381-382 and Roos J . et al., J. Cell

Biol· 2005 169: 435-445). However, what is the role of calcium pools in regulating calcium channels in cancer cells? It is still unclear. In 2009, Yang et al. found that 0ran and STIMi have a significant relationship with the metastasis of cancer cells. For example, the inhibition of 〇rail and STIM1 can reduce the metastasis of breast cancer cells in experimental mice. However, the mechanism by which 〇rail and STIM1 cause cancer cell metastasis is unclear (Yang s et al, Cancer

Cell 2009 3; 15(2): 124-34) 'The clinically relevant research is also quite lacking. - The stimulation of soil growth factor (EGF) plays an important role in the regulation of cancer cell growth. Color, EGF receptor (EGFR) blockers have also been cancer, an important drug for treatment; The stimulation of the foot can be separated from the activation of the ion channel. However, in the process of epithelial growth factor-induced co-proliferation = shift, the role of the sub-channel is still not in the sound. SUMMARY OF THE INVENTION Summary of the invention: Cell and cancer: increased in 201204369, its pharmaceutically acceptable salts and a variety of medicines / · cell supply - observation of rectal cancer, at least - ί st itft transfer of pharmaceutical composition, Contains medicine: and sub-intestinal rectal cancer. Detailed Description of the Invention The STIM1 described herein refers to a matrix action molecule; the sac described herein refers to a calcium pool regulating calcium channel; the egfr described herein refers to an epithelial growth factor. Receptor; 〇 rai refers to the subunit of calcium pool regulation (SOC), including 〇rail, 0 rai2, and 〇 rai3; this article refers to epithelial growth factor, EGFR refers to epithelial growth factor receptor; 2-APB (2-aminoethoxydiphenyl borate) refers to the calcium channel regulating calcium channel blocker 2-aminoethylene diphenylboronic acid. The SW480 and HT29 colorectal cancer cell lines and the A431 uterine epithelial cancer cell line used in the present invention are from the Center for Bioresource Conservation and Research (BCRC) of the Food Industry Development Research Institute; the liposome transfection (iipofecti〇n) system of the present invention Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) was used; the siSTIM1 small fragment nucleic acid system used in the present invention is the product of invitr〇gen company number HSS186144 (SEQ ID N0.1), and is additionally attached at 3, Thymidine (tt). The present invention relates to a medical composition for inhibiting colorectal cancer cells and uterus 201204369: carrier or thin-cell cancer cell proliferation, and which can be used as a salt of a party and a medicine. Acceptable, Λ 好 好 红 赖 细胞 细胞 cell line is STIM1 sensitive cancer cells. In a preferred embodiment, the cell line is obtained from a tissue of a patient with a colorectal cancer; in the preferred embodiment of HT29SW4B0, the uterine epithelial cancer cell line is an A43i cell line exhibiting £gfr. The county combination age of the present invention is caused by the overexpression of the STIM1 gene, and the expression of the STIM1 gene is related to the molecular marker CEA of the blood production tumor. In a preferred embodiment, the STiMi 过度 has a positive phase with the _ molecular standard CEA. Therefore, the molecular markers of STIM1 in the prediction of the surface potential of colorectal cancer and uterine epithelial cancer tissues. The pharmaceutical composition towel of the present invention comprises a small fragment nucleic acid molecule (siRNA) or a chemical inhibitor in a STIM1 inhibitor; in a preferred embodiment, the small fragment nucleic acid molecule is SEQ ID NO: 1. The invention further relates to a pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer cells and uterine epithelial cancer cells, which comprises at least one inhibitor of Store-Operated Calcium Channels (S0C) activity. Or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent. In one embodiment, the rectal cancer cell and the uterine epithelial cancer cell line are STIM1-sensitive cancer cells, wherein the cancer cell proliferation and metastasis is caused by the overexpression of the STIM1 gene in 201204369, and the tumor molecular marker CEA and T 4 M The degree of expression of the STIM1 gene and the colorectal cancer cell line are taken from the colon. In a preferred embodiment, the colorectal cancer is % tissue-like tissue; real

In another preferred embodiment, the ^4itw480 or adipose 9 cell line; in the A431 cell line. U.S. epithelial cancer cell line is overexpressing EGFR

_ί二实二: 匕之: chest control in the drug composition = "better example one ^ (four) composition fairy machine dragon figure such as 22 t! In normal cells, the normal performance of stimi can be maintained

The normal cell concentration of the cells makes the cells proliferate normally; the TU1 and the silk of the 12 U cells are too high, causing the concentration of the cell ions to be too high, which promotes the fine growth and metastasis, and inhibits the STIM1 by siSTIM1. 'Or use SOC blocker 2-APB to block s〇c, which can inhibit abnormal proliferation and metastasis of cells. The use of the pharmaceutical compositions of the present invention will now be described by way of specific examples, which are intended to be merely representative of the present invention and are not to be construed as limited to the embodiments. Reveal the content. [Examples] Example 1: Correlation analysis between STIM1 expression and colorectal tumors Tumor tissue samples of 24 colorectal cancer patients were taken, and STIM1 was performed by using real-time PCR for 201204369 quantitative polymerase chain reaction (real-time PCR). Analysis of gene expression. Comparing the degree of STIM1 gene expression between the tumor tissue of the patient and the normal tissue and calculating the ratio of the quantitative number # (Figure 1), it was found that about 37% of the tumor tissues of colorectal cancer patients showed excessive expression of STIM1 gene. The situation. The case tissue sections of the STIM1 gene overexpressing were immunofluorescent stained. The STIM1 protein expression of colorectal cancer cells was found to be much higher than that of normal tissues (Fig. 2). According to the over-expression of STIM1 gene, the patients were further divided into two groups: high STIM1 performance and low STIM1 expression. Then, the clinical test values of these two groups were used for statistical analysis. The results showed that the level of STIM1 and tumor size, metastasis and invasion There was no significant correlation between sex and other properties, but the STIM1 gene expression was significantly correlated with the level of tumor molecules labeled CEA (Carcinoembronic Antigen, cancer embryo antigen, an important indicator of general gastrointestinal tumors) (Figure 3). Example 2: Correlation analysis between the expression level of STIM1 and the proliferation and metastasis ability of colorectal tumor and uterine epithelial cancer cells. The STIM1 gene was transfected into cells using the cancer cell line SW480 (the control group was emptied into the empty group). The vector was cultured for 24 hours, followed by administration of EGF stimulation at a concentration of 25 Ng/ml to the control group for control, and a 48-hour in vitro cell transfer assay for the migration assay or the wound-healing assay. Based on the group that did not receive EGF and leaned into empty vector, the relative metastasis ability and proliferation of each group were compared. It was found that the STIM1 expression increased in the group without EGF (Figure 4A). The cell transfer capacity (Fig. 4B) and the degree of hyperplasia (Fig. 4C) were also significantly improved, while there was no significant difference between the groups administered EGF (Fig. 4C).

' I » Using the cancer cell line A431, the STIM1 gene was transfected into 201204369 (transfection) into cells (the control group was incubated into an empty vector), cultured for 24 hours, and then stimulated with EGF at a concentration of 25 Ng/ml. Control with the control group and perform an in vitro cell invasion experiment (9) vz &gt; 〇 〇 in invasion assay) for 12 to 48 hours. Based on the group that did not give EGF and colonized the empty vector, compared with the relative invasive ability and hyperplasia of the cells between the groups at 24 hours, it was found that the STIM1 expression increased with or without EGF (Figure 5A). ), the degree of hyperplasia of the fine ^ is also significantly improved (Figure 5B). Comparing the degree of cell invasion in each group for 12 hours and 48 hours, it was found that the group transfected with STIM1 gene had obvious invasion status, and the group with EGF had stronger invasive ability (Fig. 5 • Q°) It shows that the over-expression of STIM1 gene is closely related to the growth and metastasis of colorectal cancer, and EGF also promotes the growth and metastasis of colorectal cancer and uterine epithelial cancer. 实施 Example 3 · Small fragment nucleic acid siSTIM1 for cancer cell growth and metastasis Sexual analysis 0 lipofected (lipofection) 20 nmer ~ 1 〇〇 nemall small

Fragment nucleic acid sequence SEQ ID No. 1 to cancer cell line SW480 transfected with STIM1 gene, inhibiting the expression of STIM1 gene, and then treating the cell with 25 Ng/ml of EGF for 48 hours of in vitro cell transfer assay and measuring The amount of STIM1 protein expression. As shown in Figure 6A, the performance of STIM1 protein was significantly reduced in the group transfected with siSTIM1 compared to the group not transfected with siSTIM1. Based on the group that was not transfected with siSTIM1 and did not receive EGF, the cells in each group were compared, and the relative metastatic ability and proliferation were compared. It was found that the degree of cell proliferation was observed in the group transfected with siSTIM1 with or without EGF treatment. Both decreased significantly (Fig. 6 201204369 B), and transfection of siSTIM1 was effective in reducing the cell transfer ability caused by eGF (Fig. 6C, 6D). Example 4. Measuring calcium pool regulation of calcium ion channel (s〇c) activity Calcium imaging experiments recorded calcium channel signals, and the results were recorded on an Olympus Cell R fluorescence microscope system at room temperature (20-25 °C). All experiments were performed in the dark. The cells were first added with 1 micromolar Fluo-4, followed by a cerium ion solution. Fluo-4 is a fluorescent agent whose fluorescence intensity is directly proportional to the calcium ion concentration. The composition of the calcium ion solution is gasified sodium strontium 45 millimolar, potassium chloride 54 millimoles 'calcium chloride 2.0 millimoles' magnesium sulfate 2 millimolar, HEPES 20 millimolar, D-glucose 5.5 millimolar Ear, sodium aroxide, pH 7 4. The cancer cell line A431 transfected with the STIM1 gene was treated with EGF at 25 ng/ml, respectively, and the change in intracellular calcium ion concentration was observed by fluorescence intensity. Figure 7 shows that after administration of 2 millimoles of calcium ion solution, the intracellular calcium concentration of cells transfected with STIM1 gene increased significantly in a short period of time, and the concentration and concentration of intracellular calcium ions increased. It is much higher than the control group, showing a significant increase in the number or permeability of the fine-grained STIM1 gene-regulated calcium channel (SOC). Further, Examples 2 and 3 show the overexpression of the STIM1 gene and the development of colorectal cancer: this also implies an increase in intracellular calcium concentration and colonicity. "For the fresh pool, the calcium channel is regulated. Example 5. Small fragment nucleic acid siSTIM1 (SOC) activity impact analysis was performed. The experimental group was subjected to a 48-hour in vitro cell transfer experiment with a small fragment nucleic acid siSTIM1 by the cancer cell line A431 201204369 of the transgenic gene STIM1. The expression amount of the STIM1 protein was measured. As shown in Figure VIII, compared with the untransfected siSTIM1 group, the performance of STIM1 protein was significantly reduced in the group transfected with siSTIM1. Based on the group that was not transfected with siSTIM1 and did not receive EGF, the relative metastatic ability and proliferation of the cells were compared between the groups, and it was found that the degree of cell proliferation was detected by EGF-treated group with transfected siSTIM1 ( Figure 8 B) and cell transfer capacity (Figures 8 c, 8 d) showed a significant decrease. / • y The intracellular calcium ion concentration of EGF-treated A431 cells was observed using the experimental procedure as in Example 4, and it was found that after administration of 2 mmol of calcium ion, the cells transfected with siSTIM1 had intracellular calcium. The increase in ion concentration was much lower than that in the control group (Fig. 8E). 'It shows that transfection of siSTIM1 can inhibit the increase of intracellular calcium concentration caused by STIM1 gene expression, and suggests that this result may help inhibit the growth and metastasis of cancer cells. . Example 6 Calcium Pool Regulating Calcium Channel (s〇c) Blocker 2 Effect of ApB on Cancer Cell Growth and Metastasis The calcium pool regulating calcium channel blocker used in the present invention is 2-aminoethylene- Laminoethoxydiphenyl borate (2-APB), its chemical formula is shown in Figure 9.厶ΑρΒ inhibits inositol triphosphate (Diver JM β α/. 2001, Ce// 30(5), 323-32) and the calcium pool regulates the opening of calcium channels, blocking the entry of calcium ions. The experiment was carried out with the cancer cell line HT29 of the transgenic gene STIM1, and the cells were divided into untreated (control group), 25 ng/ml EGF treatment, 25 ku and 20 micromolar 2_na treatment and 25 ng/ The milliliter coffee dish. 100 micromoles 2-APB co-treated four groups, and observed the relative cell proliferation degree (compared with the control group) at the 24th hour (Fig. 10A) and / 201204369. In the 24th hour and the 48th hour, EGF promoted the effective treatment of both the group and the group treated with the micro-mole 2-APB (Figures 10A, 10B). In the 48th hour, the group with significant co-treatment was also divided into cell strains, and the cell curcum 2〇H 'Aplf, EGF treatment, 25 Ng/ ML 100 stalk ι ΐ Mo lang treatment and 25 ng / ml EGF and transfer test together for treatment of four groups, 48 hours of in vitro cells

- using the experimental procedure of Example 4 for EGF treatment 2: observation of calcium ion concentration, the experimental group and adding 100 micromolar 2: PB W (four) (four) _ sub-solution, (10) degrees far lower than the control ^ (Figure with ten treatment ^The amplitude of the increase in intracellular contact concentration of the cell = the pool whip _ subchannel, the simple diagram of the improvement caused by the improvement of the woven simple ion cleavage], Figure 1 is the statistical graph of the STIM1 expression of 24 cases. 12 201204369 The second line is a comparison of the expression of STIM1 protein in tumor tissue and normal tissues by immunofluorescence staining. Figure 3 shows the correlation between STIM1 expression and CAE. Figure 4 shows the experiment of transfecting STIM1 gene with SW480 cancer cell line. Results · Four A showed that cells transfected with STIM1 gene had higher STIM1 protein expression; Four B showed that excessive expression of STIM1 gene and EGF stimulation resulted in strong cell transfer ability; Four C showed STIM1 expression The increase in the amount of cells significantly promoted cell proliferation. Figure 5 shows the results of experiments with the A431 cancer cell line transfected with the STIM1 gene. Five A shows that the cells transfected with the STIM1 gene have higher STIM1 protein expression; five B shows STIM1 expression. Increase Addition significantly promoted cell proliferation. 'Five C shows that the overexpression of STIM1 gene will enhance the cell's invasive ability. The six lines are the SW4801 gene transfected with SW480 cell line and transfected with small fragment nucleic acid siSTIMl. Six a shows that the expression of STIM1 protein is decreased in cells transfected with siSTIM1; six b shows proliferation of siSTIM1 cells; six C and six D show that EGF enhances cell turnover, but siSTIM1 can inhibit EGF The cell transfer ability of the cell line showed that STIM1 over-aged cake cells (10) Linzi concentration increased. f ^ is the M31 cancer cell line transfected STIM1 gene, and transfected with small fragment two after the experiment results: eight A shows that the transfection of siSTIM1 can reduce the expression of βΓTM1 protein; human B shows the proliferation of siSTIM1's shoes; &quot;VC and eight D show that EGF will greatly enhance the cells and shift the force' but siSTIMl can Inhibition of cell-induced cell metastasis can increase the strength of 201204369; eight E shows that STIM1 over-expresses and stimulates the concentration of calcium in the cells under the stimulation of egf, but siSTIM1 can significantly reduce intracellular calcium ions. Concentration. 'Figure 9 is the structural formula of SOC blocker 2-APB. Figure 10 is the result of transfusion of STIM1 gene with HT29 cancer cell line, stimulation with EGF, and treatment with 2_APB: Ten A shows 24 hours of experiment After the '100 concentration of micro-molar 2-APB can effectively inhibit the cell proliferation caused by EGF; Ten B shows that after 2 hours of experiment, the concentration of 2 〇 micro-mole and 1 〇〇 micro-mole 2-APB can effectively inhibit Cell proliferation caused by EGF. Figure 11 shows the results of the experiment of transfecting STIM1 gene with A431 cancer cell line, giving EGF stimulation, and treating with 2-APB: _ a shows that 2-APB treatment can effectively slow down the cell metastasis caused by EGF; _ a b shows that after 48 hours of experiment, the concentration of 20 micromoles and 1 〇〇 micromolar 2_apb can effectively inhibit the cell proliferation caused by EGF; _c shows the concentration of 2 micromoles and 1 after 48 hours of experiment The 2-ApB胄 of micro-mole can effectively inhibit the cell transfer ability caused by EGF. Figure 12 is a schematic diagram of the mechanism of action of the present invention: Twelve A shows that in normal cells, the normal performance of STIMi can maintain normal intracellular! The concentration of the bow ion makes the cells proliferate normally. · Twelve B shows the excessive expression of STM1 in the cancer cells, causing the cell (10) ion concentration to be too high, causing the cells to abnormally proliferate and metastasize, and using siSTIMl to inhibit the performance of STIM1, or using s 〇c blocker 2-APB _ SOC' can inhibit abnormal proliferation and metastasis of cells. [Main component symbol description] 201204369 [Sequence table]

&lt;110&gt; Kaohsiung Medical University /KAOHSIUNGMEDICALUNIVERSITY

&lt;120&gt; PHARMACEUTICAL COMPOSITION FOR INHIBITING PROLIFERATION AND METASTASIS OF COLORECTAL CANCER AND METROCARCINOMA

&lt;130> 1258-KMU-TW &lt;160〉 1 &lt;170〉 Patentln version 3.5 &lt;210&gt; 1 &lt;211&gt; 21 &lt;212&gt; RNA &lt;213&gt; Artificial Sequence &lt;220&gt;&lt;223&gt; siSTIM1 siRNA &lt;220&gt;&lt;221&gt; misc RNA &lt;222> (1)..(21) &lt;400&gt; 1 aaggcucugg auacagugcu c 21 15

Claims (1)

201204369 VII. Scope of application: 1. A pharmaceutical composition for inhibiting the proliferation and metastasis of colon cancer cells and uterine epithelial cancer cells, comprising at least one matrix action molecule 1 (strIM) An inhibitor or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent. 2. The pharmaceutical composition according to the invention of claim 2, wherein the colorectal cancer cell and the uterine epithelial cancer cell line are STIM1-sensitive colorectal cancer cells and sero-epithelial cancer cells. 3. The pharmaceutical composition according to claim 4, wherein the colorectal cancer cell and uterine epithelial cancer cell proliferation and metastasis are caused by excessive overexpression of the STIM1 gene. 4. The pharmaceutical composition according to claim 3, wherein the overexpression of the STIM1 gene is positively correlated with the tumor molecular marker CEA. 5. The pharmaceutical composition according to claim 1, wherein the STIM1 inhibitor comprises a small fragment of a nucleic acid molecule (siRNA) or a chemical inhibitor. 6. The pharmaceutical composition according to claim 5, wherein the small fragment nucleic acid molecule is SEQ ID NO: 1. 7. A pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer cells and uterine epithelial cancer cells, comprising at least one season of Calcium Channel-Controlled Calcium Channels (SOC) activity inhibitors or Bean doctor, pharmaceutically acceptable salt and a pharmaceutically acceptable carrier or diluent. 201204369 Cell and uterine epithelial cancer cell lines according to item 7 of the patent application and uterine epithelial cancer cells. A pharmaceutical composition in which the colorectal cancer, STIM1-sensitive colorectal cancer cells 9. = The patent and the uterine epithelial cancer cell sigma are derived from the colorectal cancer. STIM1 gene over-expression A pharmaceutical composition according to item 9, wherein the overexpression of the STIM1 gene is positively correlated with the tumor molecule labeled CEA. 11. According to the application of the fourth paragraph (4) of the Patent Model®, the simple ion-regulated mother ion channel activity inhibitor is a calcium pool-regulated calcium channel blocker. 12. The pharmaceutical composition according to item n of the scope of the patent application, wherein the calcium channel regulating I bow ion channel blocker is 2-aminoethoxydiphenyl borate (2-aminoethoxydiphenyl borate, referred to as 2-ΑΡΒ).