An anti-tumor drug composition

Abstract

The invention discloses an anti-tumor pharmaceutical composition, belonging to the technical field of pharmaceuticals; the anti-tumor pharmaceutical composition provided by the invention consists of arsenic trioxide and gemcitabine; the pharmaceutical composition of the invention can enhance the chemotherapy sensitivity of pancreatic cancer and has a significant anti-tumor effect.

Description

Technical Field

[0001] The invention belongs to the field of pharmaceutical technology, and in particular relates to an anti-tumor pharmaceutical composition. Background Art

[0002] Pancreatic cancer is a highly malignant digestive system tumor with a very poor prognosis. It is prone to recurrence, metastasis, and drug resistance after surgery. The treatment of pancreatic cancer is still mainly surgical resection, combined with comprehensive treatments such as radiotherapy and chemotherapy. Since gemcitabine was approved as a first-line drug for the treatment of pancreatic cancer, various improved chemotherapy regimens have been proposed one after another, including gemcitabine combined with capecitabine, S-1, mFOLFIRINOX, etc. as first-line adjuvant chemotherapy for resectable pancreatic cancer, and gemcitabine combined with ernotinib or albumin paclitaxel, FOLFIRINOX, S-1, etc. as first-line treatment for advanced metastatic pancreatic cancer. At present, gemcitabine combined with capecitabine, albumin paclitaxel, etc. and FOLFIRINOX are standard chemotherapy for resectable or advanced metastatic pancreatic cancer.

[0003] Although there are many chemotherapy options for pancreatic cancer, pancreatic cancer has low chemotherapy sensitivity and is prone to drug resistance, which limits the effectiveness of chemotherapy. As a result, the patient's survival period has not been significantly extended. In addition, the disease is accompanied by many adverse reactions and toxic side effects that seriously affect the patient's quality of life. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a pharmaceutical composition that can effectively enhance the chemosensitivity of pancreatic cancer cells to gemcitabine, solve the problems of large toxic and side effects of gemcitabine chemotherapy and easy generation of chemotherapeutic resistance.

[0005] To achieve the above objectives, in a first aspect, the present invention provides the use of arsenic trioxide in the preparation of TIMP1 (tissue matrix metalloproteinase inhibitor 1) inhibitors.

[0006] Arsenic trioxide (ATO) is a basic drug for the treatment of acute promyelocytic leukemia and is also a therapeutic drug for various solid malignant tumors. The inventors further studied arsenic trioxide and found that it can downregulate the expression level of TIMP1 in cells, so it can be used to prepare TIMP1 inhibitors.

[0007] In a second aspect, the present invention provides an anti-tumor pharmaceutical composition, which consists of arsenic trioxide and gemcitabine.

[0008] The inventors have found that due to the obvious drug resistance problem in the current use of gemcitabine (GEM), by comparing pancreatic cancer cell lines and pancreatic cancer gemcitabine-resistant cell lines, it was found that the expression of TIMP1 in the pancreatic cancer gemcitabine-resistant cell lines was significantly upregulated, and the PI3K / AKT / mTOR pathway was activated; and the inventors found that arsenic trioxide can downregulate the expression level of TIMP1, so arsenic trioxide and gemcitabine are made into a composition, and the arsenic trioxide in the composition can downregulate the increasing trend of TIMP1 caused by gemcitabine, thereby playing a role in inhibiting the growth of pancreatic cancer.

[0009] As a preferred embodiment of the pharmaceutical composition of the present invention, the molar ratio of gemcitabine to arsenic trioxide is gemcitabine:arsenic trioxide=1:(0.01-8000).

[0010] The cell survival rate of pancreatic cancer cells after treatment with ATO and / or GEM was detected according to the CCK-8 method, and the combined action coefficient CI of the two drugs was calculated at the same time. It was found that when the molar ratio of gemcitabine to arsenic trioxide was gemcitabine: arsenic trioxide = 1: (0.01-8000), the obtained CI index was less than 1, that is, within this range, the two drugs acted synergistically.

[0011] Preferably, the molar ratio of gemcitabine to arsenic trioxide is gemcitabine:arsenic trioxide=1:(0.01-0.5).

[0012] More preferably, the molar ratio of gemcitabine to arsenic trioxide is gemcitabine:arsenic trioxide=1:0.013.

[0013] The inventors further found through experiments that when the molar ratio of gemcitabine to arsenic trioxide is gemcitabine: arsenic trioxide = 1: (0.01-0.5), arsenic trioxide can also significantly increase the chemosensitivity of gemcitabine in treating pancreatic cancer; considering the possible side effects caused by excessive drug use, the mass ratio of the two is further preferably gemcitabine: arsenic trioxide = 1: 0.013, which can achieve excellent chemosensitivity on the basis of minimizing the side effects caused by excessive drug use, and significantly increase the therapeutic effect of pancreatic cancer.

[0014] As a preferred embodiment of the pharmaceutical composition of the present invention, the tumor is selected from pancreatic cancer.

[0015] In a third aspect, the present invention provides a method for preparing the pharmaceutical composition, which comprises the following steps: mixing arsenic trioxide and gemcitabine in a molar ratio to obtain a pharmaceutical composition.

[0016] In a fourth aspect, the present invention provides an anti-tumor pharmaceutical preparation, which comprises the anti-tumor pharmaceutical composition of the present invention and pharmaceutically acceptable excipients.

[0017] In a fifth aspect, the present invention provides an anti-tumor combination drug, which comprises arsenic trioxide and gemcitabine of the same or different specifications administered simultaneously or separately, and a pharmaceutically acceptable carrier.

[0018] As a preferred embodiment of the anti-tumor combination drug of the present invention, the molar ratio of gemcitabine to arsenic trioxide is gemcitabine: arsenic trioxide = 1: (0.01-8000), and the tumor is pancreatic cancer.

[0019] Preferably, the molar ratio of gemcitabine to arsenic trioxide is gemcitabine: arsenic trioxide = 1: (0.01-0.5), and more preferably, the molar ratio of gemcitabine to arsenic trioxide is gemcitabine: arsenic trioxide = 1: 0.013. Within the above preferred molar ratio range of gemcitabine to arsenic trioxide, while achieving excellent synergistic effects, the amount of drug used can be reduced as much as possible to avoid other side effects caused by excessive drug use.

[0020] In a sixth aspect, the present invention provides the use of a combination of arsenic trioxide and gemcitabine in the preparation of an anti-tumor drug.

[0021] As a preferred embodiment of the application of the present invention, the tumor is pancreatic cancer.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] The combined use of arsenic trioxide and gemcitabine in the anti-tumor drug composition provided by the present invention can effectively inhibit the growth of pancreatic cancer cells, increase the chemotherapy sensitivity of pancreatic cancer, and reverse the drug resistance of gemcitabine, thereby achieving a better pancreatic cancer treatment effect. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 This is a diagram showing the activation results of the PI3K / AKT / mTOR pathway in pancreatic cancer cells after knocking down TIMP1 in Example 1;

[0025] Figure 2 This is a diagram showing the activation results of the PI3K / AKT / mTOR pathway in pancreatic cancer cells after overexpression of TIMP1 in Example 2;

[0026] Figure 3 This is a diagram showing the results of the PI3K inhibitor ZSTK474 inhibiting the activation of the PI3K / AKT / mTOR pathway in pancreatic cancer cells (MIA PaCa-2) overexpressing TIMP1 in Example 3;

[0027] Figure 4 This is a graph showing the results of the PI3K inhibitor ZSTK474 inhibiting the activation of the PI3K / AKT / mTOR pathway in pancreatic cancer cells (PANC-1) overexpressing TIMP1 in Example 3;

[0028] Figure 5 This is a diagram showing the activation of the TIMP1 / PI3K / AKT / mTOR pathway in the pancreatic cancer cell line and the gemcitabine-resistant cell line in Example 4;

[0029] Figure 6 This is a diagram showing the activation of the PI3K / AKT / mTOR pathway after TIMP1 was knocked down in the pancreatic cancer resistant cell line in Example 5;

[0030] Figure 7 The graph is a result of the half-inhibitory concentration of ATO in pancreatic cancer cell lines and gemcitabine-resistant cell lines detected by CCK-8 cell proliferation in Example 6;

[0031] Figure 8 This is a graph showing the activation of the TIMP1 / PI3K / AKT / mTOR pathway at different time points of ATO treatment of pancreatic cancer cells in Example 7;

[0032] Fig. 9 This is a graph showing the results of flow cytometry detection of oxidative stress intensity of pancreatic cancer cells after treatment with ATO and / or NAC in Example 8;

[0033] Fig.10 This is a graph showing the results of using a fluorescent microplate reader to detect the oxidative stress intensity of pancreatic cancer cells after ATO and / or NAC treatment in Example 8;

[0034] Fig.11 This is a graph showing the results of fluorescence imaging to detect the oxidative stress intensity of pancreatic cancer cells after ATO and / or NAC treatment in Example 8;

[0035] Fig.12 The figure is the result of Western Blot and qRT-PCR detection of the change of TIMP1 expression level in pancreatic cancer cells after ATO and / or NAC treatment in Example 9;

[0036] Fig.13 The results of Western Blot detection of TIMP1 / PI3K / AKT / mTOR pathway activation and expression of epithelial-mesenchymal transition, apoptosis and other indicators after ATO and / or GEM treatment of pancreatic cancer cells in Example 10;

[0037] Fig.14The results of Western Blot detection of TIMP1 / PI3K / AKT / mTOR pathway activation and expression of epithelial-mesenchymal transition, apoptosis and other indicators after ATO and / or GEM treatment of pancreatic cancer cells in Example 10;

[0038] Fig.15 The CCK-8 method is used to detect the cell survival rate of pancreatic cancer cells after ATO and / or GEM are treated, and the combined action coefficient (CI) of the two drugs is calculated in Example 11;

[0039] Fig.16 This is a graph showing the results of flow cytometry detection of the proportion of apoptotic cells in pancreatic cancer cells after treatment with ATO and / or GEM in Example 12;

[0040] Fig.17 This is a graph showing the results of detecting the changes in migration and invasion ability of pancreatic cancer cells after treatment with ATO and / or GEM in the Transwell migration and invasion model in Example 14;

[0041] Fig.18 This is a result diagram of the plate clone formation experiment in Example 13 to detect the cell proliferation of pancreatic cancer cells after ATO and / or GEM treatment;

[0042] Fig.19 This is a graph showing the results of an in vivo study in Example 15 demonstrating that ATO synergizes with GEM to inhibit pancreatic cancer tumor growth;

[0043] Fig. 20 Schematic diagram of the mechanism of action of the combination of ATO and GEM. DETAILED DESCRIPTION

[0044] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0045] Unless otherwise specified, the reagents, methods and equipment used in the present invention are conventional reagents, methods and equipment in the art.

[0046] The overall summary of Examples 1-15 of the present invention is as follows:

[0047] Since the applicant's research found that TIMP1 is related to the chemosensitivity of pancreatic cancer to gemcitabine, and compared with pancreatic cancer cell lines, the expression of TIMP1 in pancreatic cancer gemcitabine-resistant cell lines is significantly upregulated, the inventors set up Examples 1-5 to further illustrate the mechanism by which TIMP1 induces pancreatic cancer cells to gemcitabine chemotherapy resistance; for pancreatic cancer cell line PANC-1 and gemcitabine-resistant cell line PANC-1 R , pancreatic cancer cell line MIA PaCa-2 and gemcitabine-resistant cell line MIAPaCa-2 RThe applicant detected the expression level of TIMP1 and found that the expression level of TIMP1 in gemcitabine-resistant cell lines was upregulated, and the PI3K / AKT / mTOR pathway was activated. The TIMP1 gene was knocked down or overexpressed in pancreatic cancer cell lines PANC-1 and MIA PaCa-2, and the expression levels of TIMP1 and PI3K / AKT / mTOR pathway-related proteins were detected by WB. At the same time, the PI3K pathway inhibitor ZSTK474 can partially offset the activation of the PI3K / AKT / mTOR pathway caused by overexpression of TIMP1. In addition, in the pancreatic cancer gemcitabine-resistant cell line MIA PaCa-2, the expression level of TIMP1 and PI3K / AKT / mTOR pathway-related proteins was activated by WB. R When TIMP1 was knocked down, the expression of PI3K / AKT / mTOR pathway proteins was also downregulated; therefore, it is sufficient to indicate that the TIMP1 / PI3K / AKT / mTOR pathway is activated in pancreatic cancer resistant cell lines.

[0048] In combination with the regulation of gemcitabine resistance by the TIMP1 / PI3K / AKT / mTOR pathway in pancreatic cancer, we further explored whether ATO affects the activation of the TIMP1 / PI3K / AKT / mTOR pathway to exert a synergistic GEM anti-pancreatic cancer effect through Examples 6 and 7. First, the pancreatic cancer cell lines PANC-1 and PANC-1 R and MIA PaCa-2 and MIA PaCa-2 R Then, in Example 7, PANC-1 and PANC-1 were tested at different time points (0h, 24h, 48h, 72h). R and MIA PaCa-2 and MIA PaCa-2 R ATO was applied separately (ATO concentration: MIA PaCa-2 R and MIA PaCa-2: 0.5 μM, PANC-1 R and PANC-1: 1.5μM) to study whether it acts through the TIMP1 / PI3K / AKT / mTOR pathway; the results showed that TIMP1, PI3K, p-AKT / AKT, and p-mTOR / mTOR gradually decreased with the increase of ATO treatment time, and the effect was most obvious after 72h of ATO treatment; the above results indicate that ATO can downregulate the expression level of TIMP1, thereby affecting the activation of the PI3K / AKT / mTOR pathway.

[0049] In order to further clarify the mechanism by which ATO downregulates TIMP1 expression levels, Examples 8-9 were set up for research; wherein, ATO (2.5 μM, 5 μM, 10 μM) treatment for 1 h and 2 h resulted in a dose-dependent increase in the production of reactive oxygen species in pancreatic cancer parental cells and gemcitabine-resistant cells; the reactive oxygen species antagonist N-acetyl-l-cysteine ​​(NAC) eliminated the ATO-induced reactive oxygen species production; the oxidative stress intensity was detected by flow cytometry analysis, microplate reader fluorescence intensity detection, and fluorescence microscopy imaging; compared with control cells, ATO (2.5 μM, 5 μM, 10 μM) reduced the TIMP1 level in pancreatic cancer cells, and NAC could restore the downregulation of TIMP1 caused by ATO; that is, ATO downregulated the expression level of TIMP1 by inducing oxidative stress.

[0050] Based on Examples 1-9, it can be found that the TIMP1 / PI3K / AKT / mTOR pathway is activated in gemcitabine-resistant pancreatic cancer cells; ATO downregulates the expression level of TIMP1 by inducing oxidative stress, thereby inhibiting the activation of the PI3K / AKT / mTOR pathway, and further inhibiting the growth of pancreatic cancer, clarifying the role of single-agent ATO in inhibiting pancreatic cancer.

[0051] Secondly, to further clarify the mechanism of action of ATO combined with GEM in treating pancreatic cancer, Example 10 was set up; drug-resistant pancreatic cancer cells and their parental cells were treated with ATO and / or GEM for 72 hours, and the inhibition of TIMP1 / PI3K / AKT / mTOR pathway was detected by Western blot; the results showed that compared with the ATO or GEM alone treatment group, the expression of TIMP1, PI3K, p-AKT / AKT and p-mTOR / mTOR in the ATO and GEM combination group was significantly reduced, and the TIMP1 / PI3K / AKT / mTOR pathway was significantly inhibited; at the same time, compared with the ATO or GEM monotherapy group, the protein levels of the metastatic factors N-cadherin (N-cad), Snail and TWIST1 in the ATO and GEM combination group were significantly reduced; on the contrary, when ATO was used in combination with GEM, the protein level of the anti-metastatic factor E-cadherin (N-cad) was significantly increased; the specific mechanism of action is as follows Fig. 20 shown.

[0052] In order to illustrate that ATO can cooperate with GEM in killing pancreatic cancer cells, Example 11 was set up, and ATO or a control compound was combined with the indicated concentration of GEM and applied to MIA PaCa-2, PANC-1 and gemcitabine-resistant cells thereof; the CCK-8 method was used to detect the cell survival rate and drug inhibition rate of pancreatic cancer cells and gemcitabine-resistant cells treated with ATO and GEM alone or in combination for 72 hours, and then the synergistic effect coefficient (combined Index, CI) was calculated using Calcusyn software; the existence of CI=1 indicated that the effects of the two drugs were only additive, CI greater than 1 indicated that the two drugs antagonized each other, and CI less than 1 indicated that the two drugs synergized each other; the results showed that the drug synergistic coefficient of pancreatic cancer parental cells and gemcitabine-resistant cells was less than 1, and the effect of ATO combined with GEM was significantly better than that of the GEM group alone or the ATO group alone.

[0053] In addition, the present invention illustrates that ATO enhances GEM-induced cell apoptosis by down-regulating Bcl-2 and up-regulating Bax; the flow cytometry analysis in Example 12 shows that the cell apoptosis in the ATO and GEM combination group is the most obvious; at the same time, in Example 13, ATO and GEM synergistically restrict tumor cell proliferation and inhibit plate clone formation, indicating that they affect long-term survival; based on these findings, it can be seen that ATO can significantly enhance GEM-induced pancreatic cancer cell apoptosis; Example 14 also reflects the effect of ATO and GEM combined to inhibit pancreatic cancer cell invasion. Compared with single-drug treatment, the number of cells that migrated and invaded in the ATO and GEM combination group was significantly reduced. In the cell experiments of Examples 10-14, the concentrations of ATO and / or GEM used were kept consistent. Specifically, for the cell lines MIA PaCa-2 and MIA PaCa-2 R The concentration of ATO used in the treatment was 0.5 μM. For the cell lines PANC-1 and PANC-1 R The concentration used for ATO treatment was 1.5 μM; for all cell lines, the concentration used for GEM treatment was 1 μM.

[0054] In addition to the above in vitro studies, the present invention also constructed a nude mouse pancreatic cancer orthotopic transplant tumor model, as shown in Example 15, and confirmed the synergistic effect of ATO and GEM in inhibiting the growth of pancreatic cancer cells and reversing GEM resistance through in vivo experiments; the results showed that compared with the ATO or GEM alone treatment group, ATO significantly increased the sensitivity of pancreatic cancer cells to GEM, and the combined use of ATO and GEM significantly inhibited tumor growth; the tumor specimens were subjected to TIMP1, p-mTOR, TWIST1, Bcl-2, Bax, and Ki-67 immunohistochemical staining, and the results showed that ATO and GEM combined to inhibit the epithelial-mesenchymal transition (EMT) of pancreatic cancer cells, inhibit proliferation, and promote apoptosis.

[0055] The specific embodiments are as follows:

[0056] Example 1

[0057] This example explores the activation of the PI3K / AKT / mTOR pathway in pancreatic cancer cells after knocking down TIMP1; the specific exploration steps are as follows:

[0058] 1.1 Experimental selection: pancreatic cancer cell lines MIA PaCa-2 and PANC-1, cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0059] 1.2 Experimental method: After the cells are stable, the cells are plated into six-well plates, with 15×10 4 cells;

[0060] 1.3 Grouped as follows:

[0061] 1.3.1 Control group: MIA PaCa-2-shcon, PANC-1-shcon;

[0062] 1.3.2 TIMP1 knockdown group: MIA PaCa-2-shTIMP1, PANC-1-shTIMP1;

[0063] 1.4 When the cells grew to 80-90%, extract the total protein by RIPA and perform Western blot detection. The detection indicators include TIMP1, PI3K, p-AKT / AKT, p-mTOR / mTOR, and GAPDH as the internal reference.

[0064] 1.5 Experimental results Figure 1 As shown, from Figure 1 It can be seen that after knocking down the TIMP1 gene expression level, the TIMP1 protein expression of pancreatic cancer cells MIA PaCa-2 and PANC-1 was downregulated, and the PI3K / AKT / mTOR pathway was inhibited.

[0065] Example 2

[0066] This example explores the activation of the PI3K / AKT / mTOR pathway in pancreatic cancer cells after overexpression of TIMP1; the specific exploration steps are as follows:

[0067] 2.1 Experimental selection: Pancreatic cancer cell lines MIA PaCa-2 and PANC-1, cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0068] 2.2 Experimental method: After the cells are stable, the cells are plated into six-well plates, with 15×104 cells;

[0069] 2.3 Grouped as follows:

[0070] 2.3.1 Control group: MIA PaCa-2-LVcon, PANC-1-LVcon;

[0071] 2.3.2 TIMP1 overexpression group: MIA PaCa-2-LVTIMP1, PANC-1-LVTIMP1;

[0072] 2.4 When the cells grow to 80-90%, extract the total protein by RIPA and perform Western blot detection. The detection indicators include TIMP1, PI3K, p-AKT / AKT, p-mTOR / mTOR, and GAPDH as the internal reference.

[0073] 2.5 Experimental results Figure 2 As shown, from Figure 2 It can be seen that after overexpression of TIMP1 gene expression level, TIMP1 protein expression was upregulated in pancreatic cancer cells MIA PaCa-2 and PANC-1, and the PI3K / AKT / mTOR pathway was activated.

[0074] Example 3

[0075] This example explores the inhibition of PI3K inhibitor ZSTK474 on the activation of PI3K / AKT / mTOR pathway in pancreatic cancer cells that overexpress TIMP1. The specific exploration steps are as follows:

[0076] 3.1 Experimental selection: MIA PaCa-2-LVcon, MIA PaCa-2-LVTIMP1, PANC-1-LVcon, PANC-1-LVTIMP1. Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0077] 3.2 Experimental method: After the cells are stable, the cells are plated into six-well plates, with 15×10 4 cells;

[0078] 3.3 Grouped as follows:

[0079] 3.3.1 Control group: MIA PaCa-2-LVcon, PANC-1-LVcon;

[0080] 3.3.2 TIMP1 overexpression group: MIA PaCa-2-LVTIMP1, MIA PaCa-2-LVTIMP1;

[0081] 3.3.3 Overexpression of TIMP1+ZSTK474 inhibitor group: MIA PaCa-2-LVTIMP1+ZSTK474, PANC-1-LVTIMP1+ZSTK474;

[0082] 3.4 After the cells were plated, ZSTK474 was added to the TIMP1 overexpression group. When the cells grew to 80-90%, total protein was extracted by RIPA and Western blot was performed. The detection indicators were TIMP1, PI3K, p-AKT / AKT, p-mTOR / mTOR, and GAPDH was used as an internal reference.

[0083] 3.5 Experimental results Figure 3-4 As shown, from Figure 3-4 It can be seen that ZSTK474 can partially restore the activation of the PI3K / AKT / mTOR pathway caused by overexpression of the TIMP1 gene, further clarifying that upregulated TIMP1 expression in pancreatic cancer activates the PI3K / AKT / mTOR pathway.

[0084] Example 4

[0085] This example explores the activation of the TIMP1 / PI3K / AKT / mTOR pathway in pancreatic cancer cell lines and gemcitabine-resistant cell lines. The specific exploration steps are as follows:

[0086] 4.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0087] 4.2 Experimental method: After the cells are stable, the cells are plated into six-well plates, with 15×10 4 cells;

[0088] 4.3 Experimental Grouping:

[0089] 4.3.1 Control group: MIA PaCa-2, PANC-1;

[0090] 4.3.2 Drug-resistant group: MIA PaCa-2 R 、PANC-1 R ;

[0091] 4.4 When the cells grow to 80-90%, extract the total protein by RIPA and perform Western blot detection. The detection indicators include TIMP1, PI3K, p-AKT / AKT, p-mTOR / mTOR, and GAPDH as the internal reference.

[0092] 4.5 Experimental results Figure 5 As shown, from Figure 5 It can be seen that compared with wild-type pancreatic cancer cell lines (MIA PaCa-2, PANC-1), gemcitabine-resistant pancreatic cancer cell lines (MIA PaCa-2 R 、PANC-1 R )TIMP1 expression level was higher and PI3K / AKT / mTOR pathway activation was more significant.

[0093] Example 5

[0094] This example explores the activation of the PI3K / AKT / mTOR pathway after knocking down TIMP1 in pancreatic cancer resistant cell lines. The specific exploration steps are as follows:

[0095] 5.1 Experimental selection: pancreatic cancer gemcitabine-resistant cell line MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0096] 5.2 Experimental methods: Lentiviral transfection to knock down TIMP1 expression levels;

[0097] 5.3 Experimental Grouping:

[0098] 5.3.1 Control group: MIA PaCa-2 R -shcon, PANC-1 R -shcon;

[0099] 5.3.2 TIMP1 knockdown group: MIA PaCa-2 R -shTIMP1, PANC-1 R -shTIMP1;

[0100] 5.4 When the cells grow to 80-90%, extract the total protein by RIPA and perform Western blot detection. The detection indicators are TIMP1, PI3K, p-AKT / AKT, p-mTOR / mTOR, and GAPDH is used as the internal reference;

[0101] 5.5 Experimental results Figure 6 As shown, from Figure 6 It can be seen that after knocking down the TIMP1 gene, the gemcitabine-resistant pancreatic cancer cell line (MIA PaCa-2 R 、PANC-1 R )TIMP1 protein expression level was downregulated, and the PI3K / AKT / mTOR pathway was inhibited.

[0102] Example 6

[0103] This example explores the half inhibitory concentration (IC50) of ATO in pancreatic cancer cell lines and gemcitabine-resistant cell lines detected by CCK-8 cell proliferation. The specific exploration steps are as follows:

[0104] 6.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0105] 6.2 Experimental method: After the cells have grown stably, they are plated in a 96-well plate with 8×10 3 , 200 μL complete medium per well;

[0106] 6.3 Grouped as follows:

[0107] 6.3.1 Control group: remove the old culture medium and replace with 200 μL complete culture medium;

[0108] 6.3.2 ATO group: Remove the old culture medium and use complete culture medium to prepare 200 μL of ATO at concentrations of 0.5, 1, 2, and 4 μM to treat PANC-1 and MIA PaCa-2; use complete culture medium to prepare 200 μL of ATO at concentrations of 1, 2, 4, and 8 μM to treat PANC-1 R and MIA PaCa-2 R ;

[0109] 6.4 After administration, the cells were cultured in an incubator for 72 hours, the old culture medium was removed, 10% CCK-8 / DMEM was added and incubated in the dark for 3 hours, the absorbance at 450 nm was measured by an enzyme reader, and the blank group was used as the zero. The measured data were used to calculate the IC50 of ATO using Graphpad 8.0 software;

[0110] 6.5 Experimental results Figure 7 shown.

[0111] Example 7

[0112] This example explores the activation of the TIMP1 / PI3K / AKT / mTOR pathway at different time points of ATO treatment of pancreatic cancer cells. The specific exploration steps are as follows:

[0113] 7.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0114] 7.2 Experimental grouping: ATO was treated at different time points (0h, 24h, 48h, 72h), and total cell protein was collected for Western Blot detection. The detection indicators were: TIMP1, PI3K, p-AKT / AKT, p-mTOR / mTOR, and GAPDH was used as an internal reference;

[0115] 7.3 Experimental results Figure 8 As shown, from Figure 8 It can be seen that TIMP1, PI3K, p-AKT / AKT, and p-mTOR / mTOR gradually decreased with the increase of ATO treatment time, and the effect was most obvious after 72 hours of ATO treatment; that is, ATO can downregulate the expression level of TIMP1, thereby affecting the activation of the PI3K / AKT / mTOR pathway.

[0116] Example 8

[0117] This example explores the intensity of oxidative stress in pancreatic cancer cells after ATO and / or NAC treatment by flow cytometry, fluorescence microplate reader, and fluorescence imaging. The specific exploration steps are as follows:

[0118] 8.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0119] 8.2 Experimental groups: control group, ATO (2.5 μM) group, ATO (2.5 μM) + NAC (5 mM) group, ATO (5 μM) group, ATO (5 μM) + NAC (5 mM) group;

[0120] 8.3 Experimental methods: After stable cell growth, the cells were plated. The DHE probe was used to evaluate the generation of reactive oxygen species in cells after ATO and / or NAC treatment. After ATO and / or NAC treatment, the cells were washed three times with PBS and incubated with the DHE probe at 37°C in the dark for 30 min. The cells were then observed under a fluorescence microscope and photographed. The ROS fluorescence intensity was measured by a flow cytometry system and a microplate reader (Bio-Tek SYNERGY H1).

[0121] 8.4 Experimental results Figure 9-11 As shown, from Figure 9-11 It can be seen that ATO can induce enhanced oxidative stress in pancreatic cancer cells and increase the generation of reactive oxygen species.

[0122] Example 9

[0123] This example explores the changes in TIMP1 expression levels in pancreatic cancer cells after treatment with ATO and / or NAC using Western Blot and qRT-PCR. The specific exploration steps are as follows:

[0124] 9.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0125] 9.2 Experimental groups: 1h control group, 1h ATO (2.5μM) group, 1h ATO (2.5μM) + NAC (5mM) group, 2h control group, 2h ATO (2.5μM) group, 2h ATO (2.5μM) + NAC (5mM) group;

[0126] 9.3 Experimental methods: After the above cell lines grew stably, they were inoculated into 6-well plates. When the cells grew to 80-90%, they were treated with ATO and / or NAC for 1h and 2h respectively. The total protein and total RNA of the cells were collected. The changes in TIMP1 expression levels were detected by Western blot and qRT-PCR, and GAPDH was used as an internal reference.

[0127] 9.4 Experimental results Fig.12 As shown, from Fig.12 It can be seen that ATO inhibits the expression level of TIMP1 by inducing the oxidative stress pathway.

[0128] Example 10

[0129] This example explores the activation of the TIMP1 / PI3K / AKT / mTOR pathway and the expression of indicators such as epithelial-mesenchymal transition and apoptosis after pancreatic cancer cells are treated with ATO and / or GEM by Western Blot. The specific exploration steps are as follows:

[0130] 10.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0131] 10.2 Experimental groups: control group, ATO group, GEM group, ATO+GEM group;

[0132] 10.3 Experimental method: After the cells have grown stably, they are inoculated in 6-well plates, with 15×10 4 After the cells adhered to the wall, they were treated with ATO and / or GEM for 72 hours. The total protein of the cells was collected by RIPA and detected by Western blot. The detection indicators included TIMP1, PI3K, p-AKT / AKT, p-mTOR / mTOR, GAPDH as an internal reference, and N-Cadherin, E-Cadherin, TWIST1, Snail, Bax, and Bcl2.

[0133] 10.4 Experimental results are as follows Figure 13-14 As shown in the data, compared with the ATO or GEM alone treatment group, the expressions of TIMP1, PI3K, p-AKT / AKT and p-mTOR / mTOR in the ATO and GEM combination group were significantly decreased, and the TIMP1 / PI3K / AKT / mTOR pathway was significantly inhibited; at the same time, compared with the ATO or GEM alone treatment group, the protein levels of the pro-metastatic factors N-cadherin (N-cad), Snail and TWIST1 in the ATO and GEM combination group were significantly decreased; on the contrary, when ATO was used in combination with GEM, the protein level of the anti-metastatic factor E-cadherin (E-cad) was significantly increased.

[0134] Embodiment 11

[0135] This example explores the CCK-8 method to detect the cell survival rate after ATO and / or GEM treatment of pancreatic cancer cells, and simultaneously calculates the combined effect coefficient (CI) of the two drugs. The specific steps are as follows:

[0136] 11.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0137] 11.2 Experimental grouping: After different concentrations of ATO or GEM acted on pancreatic cancer cells or pancreatic cancer resistant cells for 72 hours, the CCK-8 cytotoxicity test was used to detect the drug inhibition rate;

[0138] 11.3 Experimental method: Calculate the combined index (CI); CI = D1 / (Dx)1+D2 / (Dx)2, D1 represents the dose of drug 1 required to produce X% effect when the drug is used in combination, (Dx)1 represents the dose of drug 1 required to produce X% effect when drug 1 is used alone, CI = 1 indicates that the effects of the two drugs are simply additive, CI>1 indicates that the two drugs have antagonistic effects, and CI<1 indicates that the two drugs have synergistic effects;

[0139] 11.4 Experimental results are as follows Fig.15 As shown, from Fig.15 It can be seen that the combined effect coefficients CI of ATO and GEM are less than 1, so the combined effect of ATO and GEM is a synergistic effect.

[0140] Example 12

[0141] In this example, flow cytometry was used to detect the proportion of apoptotic cells in pancreatic cancer cells after ATO and / or GEM were treated, which specifically included the following steps:

[0142] 12.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0143] 12.2 Experimental groups: control group, ATO group, GEM group, ATO+GEM group;

[0144] 12.3 Experimental method: After the cells have grown stably, they are inoculated in 6-well plates, with 15×10 4 After the cells adhered, they were treated with ATO and / or GEM for 72 hours, the adhered cells were digested and resuspended into a cell suspension, washed twice with PBS, centrifuged at 1000 rpm for 5 minutes, the supernatant was discarded, the cells were resuspended with 500 μL binding buffer, 10 μL Annexin V-FITC reagent was added, incubated at room temperature in the dark for 15 minutes, 5 μL PI reagent was added, and the cell apoptosis rate was detected by flow cytometry;

[0145] 12.4 Experimental results are as follows Fig.16 As shown, from Fig.16 It can be seen that compared with the control group, ATO alone group, and GEM alone group, ATO combined with GEM can promote the apoptosis of pancreatic cancer cells, thereby promoting the killing effect of GEM on pancreatic cancer.

[0146] Embodiment 13

[0147] In this example, a plate clone formation experiment is used to detect the cell proliferation of pancreatic cancer cells after ATO and / or GEM treatment, which specifically includes the following steps:

[0148] 13.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0149] 13.2 Experimental groups: control group, ATO group, GEM group, ATO+GEM group;

[0150] 13.3 Experimental method: After the cells grow stably, they are inoculated in a 6-well plate with 1000 cells per well. After 8 days of culture in an incubator, a large number of clones visible to the naked eye are observed. The old culture medium is discarded, and the cells are washed twice with PBS and fixed with 2 mL / well of pure methanol. After 15 minutes, the fixative is removed, and crystal violet is added for staining for 30 minutes. Finally, the staining solution is slowly washed off with running water, and the cells are placed in air to dry. The results are observed with the naked eye, and clone images are taken with a camera.

[0151] 13.4 Experimental results are as follows Fig.18 As shown, compared with the control group and the single drug group, ATO combined with GEM significantly inhibited the cloning ability of pancreatic cancer cells, inhibited cell apoptosis and long-term survival.

[0152] Embodiment 14

[0153] This example uses the Transwell migration and invasion model to detect changes in the migration and invasion abilities of pancreatic cancer cells after treatment with ATO and / or GEM, specifically including the following steps:

[0154] 14.1 Experimental selection: Pancreatic cancer cell lines: MIA PaCa-2, PANC-1, pancreatic cancer gemcitabine-resistant cell line: MIA PaCa-2 R 、PANC-1 R ; Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0155] 14.2 Experimental groups: control group, ATO group, GEM group, ATO+GEM group;

[0156] 14.3 Experimental methods:

[0157] (1) Invasion assay: 4 hours in advance, spread Matrigel (250 μg / mL) on the upper chamber of the Transwell chamber and incubate at 37°C until the gel solidifies.

[0158] (2) The adherent cells were digested into a cell suspension, centrifuged at 1000 rpm for 5 minutes, and resuspended in serum-free DMEM medium. The cell number was 2×10 4 / well was inoculated in the upper chamber of the Transwell chamber, and 500 μL of DMEM complete culture medium containing 10% FBS was added to the lower chamber of the chamber;

[0159] (3) After culturing in the cell culture incubator for 24 h (migration assay) or 48 h (invasion assay), remove the chamber, gently swab the cells on the upper chamber surface with a cotton swab, and dry at room temperature;

[0160] (4) Fix the cells on the lower chamber surface with pure methanol for 15 min at room temperature;

[0161] (5) Wash three times with PBS, wipe the upper chamber surface dry, and invert to dry at room temperature;

[0162] (6) Stain the cells on the lower chamber surface with crystal violet staining solution for 30 minutes, then wash three times with ultrapure water and fully dry at room temperature;

[0163] (7) Observe and photograph under a microscope, take 6 fields of view in each chamber at 100× magnification, count the cells and make statistics;

[0164] 14.5 Experimental results are as follows Fig.17 As shown, compared with the control group and the single drug group, ATO combined with GEM significantly inhibited the migration and invasion of pancreatic cancer cells.

[0165] Embodiment 15

[0166] This example uses in vivo animal experiments to prove that ATO cooperates with GEM to inhibit the proliferation and metastasis of pancreatic cancer cells, which specifically includes the following steps:

[0167] 15.1 Experimental Selection: MIA PaCa-2 Cells and MIA PaCa-2 R Drug-resistant cells. Cell culture conditions: complete medium (DMEM containing 10% fetal bovine serum), 37°C, 5% CO2, saturated humidity cell culture incubator;

[0168] 15.2 Experimental groups: control group, ATO group, GEM group, ATO+GEM group;

[0169] 15.3 Experimental methods: Establishment of nude mouse pancreatic cancer orthotopic transplantation tumor model; 2×10 6 MIA PaCa-2 transfected with luciferase gene Ror its parental cell MIA PaCa-2, were injected into the pancreas of 4-week-old female BALB / c nude mice. Before administration, the fluorescent images of tumor-bearing mice were taken using a small animal in vivo fluorescence imaging system to observe the growth of the tumor. When the in vivo fluorescence imaging showed the tumor (about 21 days after cell implantation), the mice were randomly divided into a control group, an ATO group, a GEM group, and an ATO+GEM group. The mice were intraperitoneally injected with ATO (1 mg / kg), GEM (100 mg / kg), ATO+GEM (ATO was 1 mg / kg and GEM was 100 mg / kg) or PBS (control group) twice a week for 3 consecutive weeks. The body weight of the mice was measured every day, and the tumor size was measured by the in vivo imaging system. The mice were killed on the 21st day after injection, and the in situ tumor tissue was obtained to calculate the tumor volume (mm 3 ) = (length in mm) × (width in mm) 2 / 2Tumors, lungs, and livers were dissected, fixed, paraffin-embedded, and sectioned, and immunohistochemical staining was performed to observe the expression of EMT indicators, proliferation, and apoptosis indicators;

[0170] 15.4 Experimental results are as follows Fig.19 As shown in the figure, compared with the control group and the single drug group, ATO significantly increased the sensitivity of pancreatic cancer cells to GEM, and the combination of ATO and GEM significantly inhibited tumor growth; the molar ratio of GEM to ATO here is GEM: ATO = 1: 0.013, and the molar concentration of ATO used is much lower than the molar concentration used in the in vitro experiment, but ATO still plays the effect of enhancing gemcitabine, further verifying the synergistic effect of ATO and GEM. Immunohistochemistry results showed that the combination of ATO and GEM inhibited the epithelial-mesenchymal transition (EMT) of pancreatic cancer cells, inhibited proliferation, and promoted apoptosis.

[0171] Finally, it should be noted that the above embodiments are intended to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. An anti-tumor pharmaceutical composition, characterized in that: The pharmaceutical composition consists of arsenic trioxide and gemcitabine; The molar ratio of gemcitabine to arsenic trioxide is 1:0.013; The preparation method of the pharmaceutical composition comprises the following steps: mixing arsenic trioxide and gemcitabine in a molar ratio to obtain the pharmaceutical composition; and the tumor is pancreatic cancer.

2. An anti-tumor drug preparation, characterized in that: The pharmaceutical preparation comprises the pharmaceutical composition according to claim 1 and pharmaceutically acceptable excipients; and the tumor is pancreatic cancer.

3. Use of the anti-tumor pharmaceutical composition according to claim 1 in the preparation of anti-tumor drugs, characterized in that: The tumor is pancreatic cancer.

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