TWI504404B - Pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer and metrocarcinoma - Google Patents

Description

Pharmaceutical combination for inhibiting growth and metastasis of colorectal cancer cells and uterine epithelial cancer cells Object

The present invention relates to a pharmaceutical composition for inhibiting the growth and metastasis of colorectal cancer cells and uterine epithelial cancer cells. In particular, the present invention relates to inhibiting the growth of colorectal cancer cells by using a stromal interaction molecule 1 (STIM1) inhibitor or a pool-operated calcium channel (SOC) activity inhibitor. Transfer. In addition, in patients with colorectal cancer, the STIM1 expression was significantly positively correlated with the clinical molecular marker of cancer colorectal cancer, the cancer embryo antigen (CEA). Therefore, the amount of STIM1 in colorectal cancer tissue can be a molecular marker for potential prediction.

The most terrifying part of cancer is that the disease enters the stage of metastasis, and the cancer cells spread to other organs and tissues of the body. According to statistics, the most common cause of cancer deaths is cancer cells transferring vital organs such as lungs, liver, bones and brain. Less than 10% of them are caused by the first primary tumor. Cancer cell metastasis is a continuous process: cancer cells are exfoliated from the original tumor and propagated to the appropriate location by the blood vessels to multiply. It will build a vascular system in a new place to support its own growth, and grow up to the amount that will threaten life. Although methods such as chemotherapy can prolong the life of patients with cancer metastasis, because the medical community has always had limited knowledge of cancer cell metastasis, few drugs or therapies can really block the metastasis process and cure the disease. This is also everyone. A major cause of cancerous discoloration.

Changes in intracellular calcium ion levels can modulate various physiological functions. The stromal interaction molecule 1, referred to as STIM1) is an important calcium ion sensing molecule in the cell, which can detect calcium ion concentration changes 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 aggregate on the endoplasmic reticulum and regulates the calcium channel with the calcium pool on the cell membrane (Store-Operated Calcium). The secondary unit Orai1 of Channels (SOC) interacts to open the calcium pool to regulate calcium channels, allowing extracellular calcium ions to enter the cell (James WP, 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 Orai1 and STIM1 are involved in the metastasis of cancer cells. If the effects of Orai1 and STIM1 are inhibited, the metastasis of breast cancer cells in experimental mice can be reduced. However, the mechanism by which Orai1 and STIM1 cause cancer cell metastasis is unclear (Yang S. et al., Cancer Cell 2009 3; 15(2): 124-34), and clinically relevant research is also quite lacking.

The stimulation of epithelial growth factor (EGF) plays an important role in the regulation of cancer cell growth. The blocker of EGF receptor (EGFR) has also been an important drug for cancer treatment; on the other hand, EGF stimulation can be Indirect activation of the calcium pool regulates calcium channel. However, in the process of cancer cell proliferation and metastasis caused by epithelial growth factor, the role of calcium pool in regulating calcium channel is still unclear.

Summary of invention

An object of the present invention is to provide a pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer cells and uterine epithelial cancer cells, which is One less STIM1 inhibitor or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

Another object of the present invention is to provide a pharmaceutical composition for inhibiting proliferation and metastasis of colorectal cancer cells and uterine epithelial cancer cells, which comprises at least one SOC activity inhibitor or a pharmaceutically acceptable salt thereof and a medicine An acceptable carrier or diluent.

It is still another object of the present invention to provide a tumor molecular marker for predicting the recovery of colorectal cancer and uterine epithelial cancer patients.

Detailed description of the invention

STIM1 as used herein refers to a matrix action molecule 1; the SOC system described herein refers to a calcium pool regulating calcium channel; the EGFR described herein refers to an epidermal growth factor receptor; the Orai system described herein refers to a calcium pool regulating calcium. Subunit of ion channel (SOC), including Orai1, Orai2, and Orai3; EGF as used herein refers to epithelial growth factor, EGFR refers to epithelial growth factor receptor; 2-APB (2-aminoethoxydiphenyl borate) system described herein Refers to the calcium pool to regulate the 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 lipofection of the present invention uses Lipofectamine. 2000 (Invitrogen, Grand Island, NY, USA); the siSTIM1 small fragment nucleic acid system used in the present invention is a product of Invitrogen No. HSS186144 (SEQ ID NO. 1), and two thymines are additionally attached to the 3' end ( Tt).

The present invention relates to a method for inhibiting colorectal cancer cells and the uterus A pharmaceutical composition for the proliferation and metastasis of cutaneous cancer cells, comprising at least one inhibitor of stromal interaction molecule 1 (STIM1) or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or Thinner.

In one embodiment, the colorectal cancer cell and uterine epithelial cancer cell line are STIM1-sensitive cancer cells. In a preferred embodiment, the colorectal cancer cell line is derived from a tissue of a colorectal cancer patient; in another preferred embodiment, the colorectal cancer cell line is a HT29 or SW480 cell line; In a preferred embodiment, the uterine epithelial cancer cell line is an A431 cell line that overexpresses EGFR.

In the pharmaceutical composition of the present invention, the cancer cell proliferation and metastasis is caused by overexpression of the STIM1 gene, and the degree of expression of the STIM1 gene is related to the tumor molecular marker CEA. In a preferred embodiment, the overexpression of the STIM1 gene is positively correlated with the tumor molecular marker CEA. Therefore, the expression of STIM1 in colorectal cancer and uterine epithelial cancer tissue can be a molecular marker for potential prediction.

In the pharmaceutical composition of the present invention, wherein the STIM1 inhibitor comprises a small fragment nucleic acid molecule (siRNA) or a chemical 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 (SOC) activity. Or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier or diluent.

In one embodiment, the colorectal cancer cell and the uterine epithelial cancer cell line are STIM1-sensitive cancer cells, wherein the cancer cell proliferation and metastasis are The STIM1 gene is over-expressed, and the degree of expression of the STIM1 gene is positively correlated with the tumor molecular marker CEA. In a preferred embodiment, the colorectal cancer cell line is derived from a tissue of a colorectal cancer patient; in another preferred embodiment, the colorectal cancer cell line is a SW480 or HT29 cell line; In a preferred embodiment, the uterine epithelial cancer cell line is an A431 cell line that overexpresses EGFR.

In a preferred embodiment, the calcium pool-regulated calcium channel (SOC) activity inhibitor in the pharmaceutical composition of the invention is a calcium pool-regulated calcium channel blocker; in a more preferred embodiment, the calcium The pool-regulated calcium channel blocker is 2-APB.

The mode of action of the pharmaceutical composition of the present invention is shown in Fig. 12: Twelve A shows that in normal cells, the normal expression amount of STIM1 can maintain the normal intracellular calcium ion concentration, so that the cells can normally proliferate; Excessive expression of STIM1 in cancer cells causes excessive intracellular calcium concentration, which promotes abnormal proliferation and metastasis of cells, and inhibition of STIM1 by siSTIM1 or blocking of SOC by SOC blocker 2-APB can inhibit Abnormal proliferation and metastasis of cells.

The effects of the pharmaceutical compositions of the present invention will be described in the following examples, which are intended to serve as representative of the various aspects and features of the present invention and are not to be construed as limited to the invention. The content.

Example 1. Correlation analysis between STIM1 expression and colorectal tumors

Taking tumor tissue specimens from 24 patients with colorectal cancer, using instant determination A quantitative polymerase chain reaction (real-time PCR) was performed to analyze the STIM1 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 data (Figure 1), it was found that about 37% of the tumor tissues of colorectal cancer patients showed excessive expression of STIM1 gene. situation. Immunohistochemical staining of the tissue sections of the STIM1 gene overexpressed showed that the STIM1 protein expression of colorectal cancer cells was 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 performance. The clinical test values of these two groups were used to calculate the STIM1 gene expression and the tumor molecular marker CEA ( The level of Carcinoembronic Antigen, cancer embryo antigen, an important indicator of gastrointestinal tumors is significantly correlated (Figure 3).

Example 2. Correlation analysis between the expression of STIM1 and the proliferation and metastasis ability of colorectal tumor and uterine epithelial cancer cells

Using the cancer cell line SW480, the STIM1 gene was transfected into cells (the control group was incubated into an empty vector) and cultured for 24 hours, followed by administration of EGF stimulation at a concentration of 25 Ng/ml for comparison with the control group. And performed a 48-hour in vitro migration assay ( in vitro migration assay, wound-healing assay). Based on the group that did not give EGF and colonized the empty vector, the relative metastasis ability and proliferation of the cells were compared. It was found that the STIM1 expression increased in the group without EGF (Fig. 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).

The cancer cell line A431 was used for the experiment, and the STIM1 gene was transfected into the cells (the control group was cultured into an empty vector) and cultured for 24 hours, followed by administration of EGF stimulation at a concentration of 25 Ng/ml for comparison with the control group. And in vitro matrix 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 proliferation 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 cell proliferation is also significantly increased (Figure 5B). Comparing the degree of cell invasion at 12 hours and 48 hours in each group, it was found that the group transfected with STIM1 gene had obvious invasion status, and the group administered with EGF had stronger invasion ability (Fig. 5C).

These results show 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.

Example 3. Inhibitory analysis of small fragment nucleic acid siSTIM1 on growth and metastasis of cancer cells

The lipofecting 20 nanomolecule to 100 nanomolar small fragment nucleic acid sequence SEQ ID No. 1 to the transfected STIM1 gene cancer cell line SW480, inhibiting the expression of the STIM1 gene, and then 25 ng The cells were treated with /ml of EGF for 48 hours of in vitro cell transfer assay and the amount of STIM1 protein was measured.

As shown in Figure 6A, the amount 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 did not transfect siSTIM1 and did not receive EGF, the relative metastasis ability and proliferation of each group were compared. It was found that the degree of cell proliferation was observed in the group transfected with siSTIM1 with or without EGF treatment. Significant decline (Figure 6 B), and transfection of siSTIM1 can effectively reduce the cell transfer capacity caused by EGF (Fig. 6C, 6D).

Example 4. Measurement of calcium pool regulation of calcium ion channel (SOC) activity

The calcium ion channel signal was recorded in a calcium imaging experiment 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, and the cells were first added with 1 micromolar Fluo-4 followed by a calcium 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 145 millimoles of sodium chloride, 5.4 millimoles of potassium chloride, 2.0 millimoles of calcium chloride, 2 millimoles of magnesium sulfate, 20 millimoles of HEPES, 5.5 millimoles of D-glucose. , sodium hydroxide, pH 7.4.

The cancer cell line A431 transfected with the STIM1 gene was compared with the control group of the transgenic vector, and 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 intracellular calcium concentration and concentration increased. The group was much higher, showing that the number of cells or the permeability of the calcium channel-regulated calcium channel (SOC) was much higher in cells transfected with the STIM1 gene. Further, Examples 2 and 3 show that the overexpression of the STIM1 gene is associated with the growth and metastasis of colorectal cancer, which also suggests an increase in the intracellular calcium ion concentration associated with colorectal cancer.

Example 5. Effect of small fragment nucleic acid siSTIM1 on calcium channel regulated calcium channel (SOC) activity

Experiment with the cancer cell line A431 of the transgenic gene STIM1, experimental group The small fragment nucleic acid siSTIM1 was transfected, subjected to an in vitro cell transfer assay for 48 hours, and the amount of STIM1 protein expression was measured. As shown in Figure VIIIA, 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 did not transfect siSTIM1 and did not receive EGF, the relative metastasis ability and proliferation of each group were compared. It was found that the degree of cell proliferation was observed in the group transfected with siSTIM1 with or without EGF treatment ( Figure 8 B) and cell transfer capacity (Figures 8C, 8D) were significantly reduced.

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 the cells transfected with siSTIM1 had an increase in intracellular calcium ion concentration after administration of 2 mmol of calcium ion solution. The amplitude was much lower than that of the control group (Fig. 8E), indicating that transfection of siSTIM1 inhibited the increase in intracellular calcium concentration caused by STIM1 gene expression, suggesting that this result may help inhibit cancer cell growth and metastasis.

Example 6. Effect of calcium pool regulating calcium channel (SOC) blocker 2-APB on cancer cell growth and metastasis

The calcium pool regulating calcium channel blocker used in the present invention is 2-aminoethoxydiphenyl borate (2-APB), and its chemical formula is shown in FIG. 2-APB inhibit inositol triphosphate (IP ₃₎ receptor (Diver JM et al. 2001, Cell Calcium, 30 (5), 323-32) and an open cell calcium regulation of calcium channel blocking calcium ions into.

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 ng/ml EGF and 20 micromolar 2-APB co-treated and 25 Nike/ml EGF and 100 micromoles 2-APB co-treat four groups at 24 hours (Figure 10A) and The relative degree of cell proliferation was observed at 48 hours (Fig. 10B) (compared to the control group). As a result, it was found that EGF promoted cancer cell proliferation both at 24 hours and 48 hours, and the group treated with 100 micromolar 2-APB was effective in inhibiting cancer cell proliferation (Fig. 10A). , 10 B), and in the 48th hour, the group treated with 20 micromolar 2-APB also had a significant decrease in the degree of cancer cell proliferation.

The experiment was carried out with the cancer cell line A431 of the transgenic gene STIM1, and the cells were divided into untreated (control group), 25 ng/ml EGF treatment, 25 ng/ml EGF and 20 micromolar 2-APB co-treated and 25 Nike/ml EGF was co-treated with 100 micromolar 2-APB for 48 hours in vitro cell transfer experiments. The relative metastatic ability and proliferation of the cells were compared. The experimental results are shown in Figure XI. The results showed that EGF can promote the proliferation and metastasis of cancer cells, and the group treated with 20 micromoles or 100 micromoles 2-APB can effectively inhibit the proliferation and metastasis of cancer cells (Fig. 11B, 11C). ).

The intracellular calcium ion concentration of EGF-treated A431 cells was observed using the experimental procedure as in Example 4. The experimental group was treated with 100 micromolar 2-APB and found to be administered with 2 millimoles of calcium ion solution. The intracellular calcium concentration of cells co-treated with 100 micromolar 2-APB was much lower than that of the control group (Fig. 11D).

The above results show that the addition of 2-APB can also effectively inhibit the growth and metastasis of cancer cells caused by STIM1 gene expression, which inhibits the calcium channel by blocking the calcium pool and inhibits the increase of intracellular calcium concentration. of.

Figure 1 shows the STIM1 performance statistics for 24 cases.

Figure 2 is a comparison of the immunofluorescence staining of STIM1 protein expression in tumor tissues and normal tissues.

Figure 3 is a correlation analysis between STIM1 performance and CAE.

Figure 4 shows the results of experiments with the ST4801 gene transfected with the SW480 cancer cell line: Four A shows that the cells transfected with the STIM1 gene have higher STIM1 protein expression; and four B shows that the STIM1 gene is overexpressed and the EGF stimulation is Will make the cells have a strong ability to metastasize; four C shows that the increased expression of STIM1 will significantly promote cell proliferation.

Figure 5 shows the results of experiments with the A431 cancer cell line transfected with the STIM1 gene: five A shows that cells transfected with the STIM1 gene have higher STIM1 protein expression; five B shows that increased STIM1 expression significantly promotes cell proliferation; Five C shows that the excessive expression of the STIM1 gene enhances the invasive ability of the cells.

Figure 6 shows the results of experiments after transfecting STIM1 gene with SW480 cancer cell line and transfecting small fragment nucleic acid siSTIM1: Six A shows that the expression of STIM1 protein is decreased in cells transfected with siSTIM1; six B shows that siSTIM1 can be Inhibition of cell proliferation; six C and six D show that EGF enhances cell metastatic ability, but siSTIM1 can inhibit EGF-induced cell metastasis.

Figure 7 shows that excessive expression of STIM1 causes a significant increase in intracellular calcium ion concentration.

Figure 8 shows the results of experiments in which the STIM1 gene was transfected with the A431 cancer cell line and transfected with the small fragment nucleic acid siSTIM1. Eight A showed that the expression of STIM1 protein was decreased in cells transfected with siSTIM1; eight B showed that siSTIM1 could be Inhibition of cell proliferation; eight C and eight D show that EGF can significantly enhance cell metastatic ability, but siSTIM1 can inhibit cell transfer energy caused by EGF The force is increased; eight E shows that STIM1 over-expresses and stimulates the concentration of calcium in the cells under the stimulation of EGF, but siSTIM1 can greatly reduce the intracellular calcium concentration.

Figure 9 is the structural formula of the SOC blocker 2-APB.

Figure 10 shows the results of the experiment of transfecting STIM1 gene with HT29 cancer cell line, giving EGF stimulation, and treating with 2-APB: Ten A shows that after 2 hours of experiment, 2-APB at 100 micromolar can effectively inhibit EGF. Cell proliferation; Ten B shows that after 48 hours of experiment, 20 μmol and 100 μmol 2-APB can effectively inhibit cell proliferation induced 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: 11A shows that treatment with 2-APB can effectively slow down the cell metastasis caused by EGF; One B showed that after 48 hours of experiment, 20 μmol and 100 μmol 2-APB could effectively inhibit cell proliferation induced by EGF; Both 2-APB in the ear 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, normal expression of STIM1 maintains normal intracellular calcium ion concentration, allowing cells to proliferate normally; Twelve B shows STIM1 of cancer cells Excessive performance, resulting in excessive intracellular calcium concentration, promotes abnormal proliferation and metastasis of cells, and inhibition of STIM1 by siST1M1, or blocking of SOC by SOC blocker 2-APB, can inhibit abnormal cells. Hyperplasia and metastasis.

<110> Kaohsiung Medical University/KAOHSIUNG MEDICAL UNIVERSITY

<120> Pharmaceutical composition for inhibiting growth and metastasis of colorectal cancer and uterine epithelial cancer/PHARMACEUTICAL COMPOSITION FOR INHIBITING PROLIFERATION AND METASTASIS OF COLORECTAL CANCER AND METROCARCINOMA

<130> 1258-KMU-TW

<160> 1

<170> PatentIn version 3.5

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<212> RNA

<213> Artificial Sequence

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<223> siSTIM1 siRNA

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<221> misc_RNA

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Claims (5)

Use of at least one inhibitor of stromal interaction molecule 1 (STIM1) for the preparation of a medicament for inhibiting proliferation and metastasis of colorectal cancer cells and uterine epithelial cancer cells, wherein the colorectal cancer cells and uterine epithelial cancer cells Proliferation and metastasis are caused by overexpression of the STIM1 gene. According to the use of the first aspect of the patent application, the colorectal cancer cell and the uterine epithelial cancer cell line are STIM1-sensitive colorectal cancer cells and uterine epithelial cancer cells. According to the use of the first aspect of the patent application, wherein the overexpression of the STIM1 gene is positively correlated with the tumor molecular marker CEA. The use according to claim 1, wherein the STIM1 inhibitor comprises a small fragment of a nucleic acid molecule (siRNA) or a chemical inhibitor. The use according to item 4 of the patent application, wherein the small fragment nucleic acid molecule is SEQ ID NO: 1.