Anti-angiogenesis drug resistance marker and application thereof

Abstract

The invention provides a drug resistance marker of an anti-angiogenesis drug and application of the drug resistance marker, and particularly provides application of gamma-synuclein (SNCG) as the drug resistance marker of the anti-angiogenesis drug, especially bevacizumab.

Description

Technical Field

[0001] The present invention relates to a drug resistance marker for anti - angiogenic drugs and its application. Specifically, it relates to γ - synuclein (SNCG) as a drug resistance marker for anti - angiogenic drugs such as bevacizumab, sorafenib, and sunitinib, etc. Background Art

[0002] In the 1970s, Professor Folkman proposed the concept that tumor growth and metastasis depend on the formation of new blood vessels, and anti - angiogenesis may become an important strategy for tumor treatment. A large number of subsequent studies have also shown that the vascular status of tumor tissues, including vascular density, structure, etc., is closely related to the prognosis of tumor patients. At the same time, it has been found that tumor cells can promote the angiogenesis of tumor tissues by secreting angiogenesis - promoting substances such as vascular endothelial growth factor (VEGF), and thus promote the growth and metastasis of tumors. Among many angiogenesis - promoting substances, VEGF is the most important angiogenesis - promoting factor, and VEGF and its receptor (VEGFR) have become the most important targets for anti - angiogenesis.

[0003] In 2004, the first anti - angiogenic drug targeting VEGF, bevacizumab, was approved by the US FDA for the treatment of metastatic colorectal cancer in combination with chemotherapy. Currently, this drug has been widely used in the treatment of various tumors such as lung cancer, breast cancer, and renal cancer, and has become a star molecule of biopharmaceuticals (Literature: Hurwitz H, Fehrenbacher L, Novotny W, et al. Bevacizumab plus irinotecan, fluorouracil, and leucovorin for metastatic colorectal cancer. N Engl J Med. 2004. 350(23):2335 - 42.). In addition, small - molecule multi - target tyrosine kinase inhibitors such as sunitinib and sorafenib also have anti - angiogenic and anti - tumor activities and are widely used in the treatment of tumors such as liver cancer. At the same time, like many other targeted drugs, the use of bevacizumab and small - molecule multi - target tyrosine kinase inhibitors also has the problem of primary or acquired drug resistance, which reduces their therapeutic effects.

[0004] Therefore, screening and obtaining markers that can be used to predict the efficacy of anti - angiogenic drugs such as bevacizumab and how to overcome drug resistance are important problems that urgently need to be solved in clinical practice. Summary of the Invention

[0005] One object of the present invention is to provide a drug resistance marker for anti - angiogenic drugs.

[0006] Another object of the present invention is to provide the related applications of the anti-angiogenic drug resistance marker.

[0007] The present invention provides an anti-angiogenic drug resistance marker (or simply referred to as anti-angiogenic resistance marker), and this marker is synuclein γ (γ-synuclein, simply referred to as SNCG). SNCG is a small molecule synaptic protein composed of 127 amino acids, belonging to a class of intrinsically disordered proteins in terms of structure, and is normally only expressed in the brain and peripheral nerve tissues, playing an important role in cell stress. There is no report in the prior art on the correlation between SNCG and the efficacy of anti-angiogenic drugs.

[0008] According to one aspect of the present invention, the present invention provides the application of γ-synuclein (i.e., SNCG) as an anti-angiogenic drug resistance marker. In some specific embodiments of the present invention, the application can be for diagnostic or therapeutic purposes, or for non-diagnostic and non-therapeutic purposes.

[0009] According to the specific embodiments of the present invention, the present invention provides the application of γ-synuclein in the preparation of a preparation for promoting angiogenesis. The γ-synuclein can be used alone as an active component for promoting angiogenesis, or in combination with other angiogenesis-promoting substances. The other angiogenesis-promoting substances can be, for example, angiogenesis-promoting factors (such as VEGF). The preparation for promoting angiogenesis can be a reagent for scientific research. The promotion of angiogenesis includes promoting the growth / healing of vascular endothelial cells, and / or promoting angiogenesis and / or tubule formation. In some embodiments of the present invention, in the scratch healing experiment, chicken embryo chorioallantoic membrane angiogenesis experiment and mouse aortic arch sprouting experiment, adding SNCG protein can promote the scratch healing of vascular endothelial cells and angiogenesis and tubule formation in the chicken embryo chorioallantoic membrane angiogenesis experiment and mouse aortic arch sprouting experiment, and has a synergistic effect with VEGF.

[0010] According to the specific embodiments of the present invention, the present invention provides the application of γ-synuclein in the preparation of a preparation for upregulating the phosphorylation of VEGF receptors. The γ-synuclein can be used alone as an active component for upregulating the phosphorylation of VEGF receptors, or in combination with other active substances. The VEGF receptor is the VEGFR protein. The other active substances can be, for example, angiogenesis-promoting factors (such as VEGF). The preparation for upregulating the phosphorylation of VEGF receptors can be a reagent for scientific research. In some embodiments of the present invention, after culturing human umbilical vein endothelial cells (HUVEC) overnight in serum starvation, SNCG and / or VEGF are added separately or in combination to treat the cells for 20 minutes, and then the total cell protein is extracted. After electrophoresis and membrane transfer, Western blot detection shows that SNCG can upregulate the phosphorylation of proteins such as VEGFR, and there is a significant synergistic effect when combined with VEGF.

[0011] According to specific embodiments of the present invention, the present invention provides the use of γ-synuclein in the preparation of an evaluation product for evaluating the efficacy of anti-angiogenic drugs. The anti-angiogenic drug is preferably an anti-angiogenic drug targeting VEGF, including but not limited to Bevacizumab, sunitinib, sorafenib, etc. The efficacy of the anti-angiogenic drug includes the inhibitory effect on the migration and / or invasion of cancer cells, and / or the inhibitory effect on tumor growth. The evaluation product can be, for example, an evaluation detection reagent composition, an evaluation detection kit, or an evaluation detection device (system). In some embodiments of the present invention, intestinal cancer cells were treated with different doses of Bevacizumab respectively, and the inhibitory effect on cell migration was observed. It was found that there were differences in the inhibitory effect of the antibody on the migration of different intestinal cancer cells; Western blot and ELISA analysis showed that this difference was related to the level of SNCG expression and secretion in cells; the inhibitory effect of Bevacizumab on cell migration was negatively correlated with the expression and secretion levels of SNCG. Thus, by detecting the cellular expression level and / or secretion level of SNCG, the efficacy of anti-angiogenic drugs can be predicted, and to a certain extent, the individual resistance to anti-angiogenic drugs can be reflected. The methods and reagents for detecting the cellular expression level and / or secretion level of SNCG can be the methods and reagents for detecting the cellular expression level and / or secretion level of SNCG in the prior art. For example, the double-antibody sandwich ELISA method can be used for detection, and the detection reagent can be an SNCG antibody in the prior art, such as the SNCG antibody disclosed in CN1752104B.

[0012] According to the specific embodiments of the present invention, the present invention provides the use of γ-synuclein as a target in screening and / or preparing anti-angiogenic reagents. The anti-angiogenic reagent is preferably an anti-angiogenic drug. The anti-angiogenesis includes an inhibitory effect on cancer cell migration and / or invasion, and / or an inhibitory effect on tumor growth. In some embodiments of the present invention, a cell line with high expression of SNCG was constructed by plasmid transfection. It was found that after the high expression of SNCG, the inhibitory effect of bevacizumab on cell migration could be down-regulated in the Transwell experiment; similarly, treating cells with the conditioned medium of cells with high expression of SNCG could also significantly down-regulate the inhibitory effect of bevacizumab on cell migration. Conversely, if the expression of SNCG was knocked out, the inhibitory effect of bevacizumab on cell migration could be up-regulated. Thus, by overexpressing SNCG (including increasing the activity of SNCG) and / or knocking down SNCG (including decreasing the activity of SNCG), and detecting the effect of the test substance on cell migration, anti-angiogenic reagents can be screened. The methods and reagents for overexpressing SNCG and knocking down SNCG can be the methods and reagents for overexpressing SNCG or knocking down SNCG in the prior art. For example, pcDNA-SNCG recombinant plasmid can be transfected to overexpress SNCG, and the gRNA Cas9 vector can be constructed by CRISPR-Cas9 technology to knock down SNCG.

[0013] In some embodiments of the present invention, tumor-bearing mouse models were established using DLD-1-C and DLD-1-SNCG intestinal cancer cells respectively. After tumor formation, bevacizumab was administered via the tail vein (40 μg or 100 μg per mouse, twice a week). Three weeks later, the mice were sacrificed, the tumor masses were dissected and weighed, and the contents of VEGF and SNCG in the mouse serum were detected. The results showed that the inhibitory effect of bevacizumab on the growth of DLD-1-SNCG tumors was significantly lower than that of the parental cell DLD-1-C; there was no significant difference in the VEGF levels in the mouse serum among different groups, and there was also no obvious correlation between the tumor inhibitory effect and the VEGF level. However, the SNCG level in the serum of DLD-1-SNCG tumor-bearing mice was significantly increased, and its SNCG level was significantly correlated with the tumor size.

[0014] In some embodiments of the present invention, a tumor-bearing model of NOD-SCID mice was established using intestinal cancer cells HT29 and intestinal cancer cells HT29 with SNCG knocked out (HT29GKO); after tumor formation, bevacizumab was administered via the tail vein (75 μg or 100 μg per mouse each time), once every 3 days, and tumor volume was measured; after 5 administrations, the drug was discontinued, and the mice were sacrificed on the 4th day after discontinuation, and the tumors were dissected and weighed. The results showed that for HT29 cells with high expression of SNCG, the tumor inhibition rates of 75 μg or 100 μg bevacizumab per mouse each time were 13.7% and 18.9% respectively, with no significant difference compared to the control group without drug administration; for HT29GKO cells with SNCG knocked out, the tumor inhibition rates were 40.3% and 43.4% respectively, showing a significant difference compared to the control group.

[0015] In some embodiments of the present invention, cells were continuously treated with bevacizumab for three months to establish bevacizumab-resistant cells HCT116-Bev. Bevacizumab could significantly inhibit the migration and invasion of parental cells HCT116-C, but the inhibitory ability of bevacizumab on the migration and invasion of resistant cells HCT116-Bev was significantly reduced. However, when an SNCG antibody was administered simultaneously, the sensitivity of the resistant cell line HCT116-Bev to bevacizumab could be significantly increased, and the inhibitory effect of bevacizumab on its migration and invasion could be enhanced.

[0016] In some embodiments of the present invention, the serum SNCG levels of 62 CRC patients before treatment with bevacizumab combined with chemotherapy were detected by double antibody sandwich ELISA method. Taking 5 ng / ml SNCG level as the Cutoff value, it was found that the progression-free survival (PFS, average 10.5 months) of SNCG-negative patients was significantly longer than that of SNCG-positive patients (average 6.2 months), and the difference between the two was significant (P = 0.005).

[0017] In some embodiments of the present invention, using intestinal cancer cells LOVO and LOVO-SNCG cells transfected with SNCG, the inhibitory effects of small molecule anti-angiogenic drugs sorafenib and sunitinib on cell migration and invasion ability were detected in these two types of cells respectively. The results showed that the inhibitory ability of sorafenib or sunitinib on the migration and invasion of LOVO-SNCG cells was significantly weaker than that on the migration and invasion of parental cells LOVO, and the difference between the two was significant.

[0018] According to another aspect of the present invention, the present invention also provides an evaluation device (evaluation system) for evaluating the efficacy of anti-angiogenic drugs in an individual, and the device includes:

[0019] A detection unit for detecting γ-synuclein in an individual sample to obtain a detection result of the expression level of γ-synuclein;

[0020] An analysis unit for analyzing the detection results of the detection unit and evaluating the efficacy of anti-angiogenic drugs in the individual to be tested.

[0021] According to a specific embodiment of the present invention, in the evaluation device of the present invention, the detection unit may include a specific detection reagent for detecting the level of gamma-synuclein in an individual sample, such as a detection kit. The detection sample may be a body fluid sample from the individual to be tested, such as serum.

[0022] According to a specific embodiment of the present invention, in the evaluation device of the present invention, the analysis unit evaluates the progression-free survival period of the individual to be tested under anti-angiogenic drug treatment according to the level of gamma-synuclein in the sample from the individual to be tested.

[0023] According to a specific embodiment of the present invention, in the evaluation device of the present invention, if the level of gamma-synuclein from the individual to be tested is low, the individual to be tested has a long progression-free survival period under anti-angiogenic drug treatment.

[0024] According to a specific embodiment of the present invention, in the evaluation device of the present invention, when the analysis unit conducts analysis and evaluation, it operates as follows:

[0025] Compare the gamma-synuclein level of the detection unit with the Cutoff value;

[0026] For individuals with a gamma-synuclein level below 5 ng / ml, they have a long progression-free survival period under anti-angiogenic drug treatment;

[0027] Preferably, the anti-angiogenic drug is an anti-angiogenic drug targeting VEGF, including but not limited to Bevacizumab, sunitinib, and sorafenib.

[0028] In summary, the present invention provides that SNCG can be used as a marker for anti-angiogenic drug resistance. Using this marker to screen patients who are going to receive anti-angiogenic treatment, if the SNCG expression is high, it indicates that the patient may be resistant to anti-angiogenic drug treatment, and other treatment options can be changed or anti-SNCG treatment can be combined, which can promote the development of precision cancer treatment. Description of the Drawings

[0029] Figure 1 It shows that SNCG can promote angiogenesis in different models and has a synergistic effect with VEGF. Among them, Picture A, scratch assay of human umbilical vein endothelial cells (HUVEC); Picture B, angiogenesis assay of mouse aortic ring; Picture C, angiogenesis assay of chick embryo chorioallantoic membrane.

[0030] Figure 2It is shown that SNCG can activate the phosphorylation of multiple proteins in human umbilical vein endothelial cells (HUVECs) and has a synergistic effect with VEGF. Among them, in Figure A, SNCG and VEGF synergistically up-regulate the levels of p-FAK (Y 397 ,Y 925 ), p-VEGFR2 (Y 1175 ); in Figure B, SNCG can up-regulate the levels of p-Src (Y 416 ), p-Akt (S 473 ), NFκB p-P65 (S 536 ) alone or in combination with VEGF; in Figure C, SNCG can up-regulate the levels of p-VEGFR1 (Y 1213 ), p-VEGFR2 (Y 1175 ) alone or in combination with VEGF, showing a dose-dependent manner; in Figure D, SNCG can up-regulate the levels of p-VEGFR1 (Y 1213 ), p-VEGFR2 (Y 1175 ) alone or in combination with VEGF in a time-dependent manner.

[0031] Figure 3 It is shown that the endogenous SNCG expression and secretion level in colorectal cancer cell lines are negatively correlated with the ability of bevacizumab to inhibit tumor cell migration. Among them, in Figure A, the inhibition of the migration of colorectal cancer cell lines HCT116, DLD-1, RKO, LOVO by bevacizumab (Bev) is dose-dependent, while it seems to be resistant to SW480; in Figure B, the expression levels of SNCG, VEGF, VEGFR1, VEGFR2 in colorectal cancer cell lines. In Figure C, there is no correlation between the ability of bevacizumab to inhibit the migration of the above colorectal cancer cell lines and the VEGF level (P = 0.685); in Figure D, there is a significant correlation between the ability of bevacizumab to inhibit the migration of the above colorectal cancer cell lines and the SNCG level (r = -0.971, P = 0.006).

[0032] Figure 4Showing up - regulation or down - regulation of SNCG levels in tumor cells can inhibit or enhance the ability of bevacizumab (Bev) to inhibit the migration of tumor cells. Among them, in Figure A, LOVO - SNCG and DLD - 1 - SNCG are stable transfected cell lines with exogenous SNCG transfection, and LOVO - C and DLD - 1 - C are empty vector control cells respectively; in Figure B, the ability of Bev to inhibit the migration of SNCG - negative cell line LOVO - C is dose - dependent, while bevacizumab shows relative resistance to the migration of SNCG - overexpressing cells LOVO - SNCG; in Figure C, the ability of Bev to inhibit the migration of SNCG - negative cell line DLD - 1 - C is dose - dependent, while bevacizumab shows relative resistance to the migration of SNCG - overexpressing cell line DLD - 1 - SNCG; in Figure D, HT29 and HCT116 cells are endogenous SNCG - positive cell lines, and HT29GKO and HCT116GKO are cell lines after knocking out SNCG; in Figure E, the ability of bevacizumab to inhibit the migration of HCT116GKO cell line after knocking out SNCG is significantly more sensitive than that of SNCG - positive cell HCT116; in Figure F, due to the morphological characteristics of HT29 cells themselves, the migration experiment cannot be carried out. The conditioned culture supernatants of HT29 and HT29GKO cells are added to the migration of LOVO cells. The results show that the ability of bevacizumab to inhibit the migration of LOVO is significantly affected by SNCG.

[0033] Figure 5 Showing that compared with the parental cell DLD - 1, the inhibitory effect of bevacizumab on tumor growth of SNCG - overexpressing cells DLD - 1 - SNCG in mice is significantly reduced in vivo, and the tumor size is positively correlated with the SNCG level in mouse serum. Among them, in Figure A, bevacizumab significantly inhibits the growth of DLD - 1 tumors subcutaneously in mice and is dose - dependent, while the inhibitory effect on the growth of DLD - 1 - SNCG tumors is reduced; in Figure B, the serum VEGF level in tumor - bearing mice is almost undetectable and has nothing to do with the tumor size; in Figure C, the serum SNCG level in DLD - 1 - SNCG tumor - bearing mice is positively correlated with its tumor size (R = 0.888, p < 0.0001).

[0034] Figure 6 Showing that after knocking out the endogenous SNCG expression in HT29 tumor cells, the anti - tumor growth activity of bevacizumab against tumor - bearing mice can be significantly improved. Bevacizumab significantly inhibits the growth of HT29GKO tumors subcutaneously in mice and is dose - dependent, while the inhibitory effect on the growth of SNCG - overexpressing cells HT29 tumors shows relative resistance.

[0035] Figure 7 Showing that the serum SNCG level of patients is related to the progression - free survival (PFS) after treatment with bevacizumab. The progression - free survival of patients with high SNCG expression after treatment with bevacizumab is significantly lower than that of patients with negative SNCG expression.

[0036] Figure 8 It is shown that the ability of sorafenib and sunitinib to inhibit the migration of LOVO-SNCG cells is weaker than that of LOVO cells. Among them, Picture A shows the pictures of sorafenib and sunitinib inhibiting the migration of LOVO and LOVO-SNCG cells. Picture B shows the statistical data of the ability of sorafenib and sunitinib to inhibit the migration of LOVO and LOVO-SNCG cells.

[0037] Figure 9 It is shown that the ability of sorafenib and sunitinib to inhibit the invasion of LOVO-SNCG cells is weaker than that of LOVO cells in terms of migration inhibition. Among them, Picture A shows the pictures of sorafenib and sunitinib inhibiting the invasion of LOVO and LOVO-SNCG cells. Picture B shows the statistical data of the ability of sorafenib and sunitinib to inhibit the invasion of LOVO and LOVO-SNCG cells. Detailed implementation manners

[0038] For a clearer understanding of the technical features, objectives, and beneficial effects of the present invention, the technical solutions of the present invention will be described in detail below in conjunction with specific embodiments and accompanying drawings. Those skilled in the art know that the embodiments describe the present invention by way of example and are not intended to limit the scope claimed by the present invention. The technical features involved in various embodiments of the present invention can be combined with each other as long as they do not conflict with each other. All the published cases and other reference materials mentioned herein are incorporated herein by reference in their entirety. In the embodiments, all the original reagent materials can be obtained commercially. The experimental methods without specific conditions are the conventional methods and conventional conditions well known in the art, or the conditions recommended by the manufacturer. The reagents or instruments without indicating the manufacturer can be conventional products obtained through commercial purchase.

[0039] Example 1: SNCG can promote angiogenesis in different models and has a synergistic effect with VEGF

[0040] This example proves that SNCG can promote angiogenesis and has a synergistic effect with VEGF. The following experiments were carried out respectively:

[0041] A, Scratch wound healing assay: Human umbilical vein endothelial cells (HUVEC) were seeded in 24-well cell culture plates. After about 24 hours of cell culture, a straight line was scratched in the cell culture wells with a 100 μl pipette tip. The floating cells were washed away with PBS, and the culture medium containing or not containing SNCG, VEGF (R&D company), and SNCG + VEGF was added. After about 8 hours, the cell migration ability was photographed, and the area of scratch wound healing was calculated;

[0042] B, Mouse aortic ring angiogenesis assay: The thoracic aorta of C57 mice was taken, and the surrounding adipose and connective tissues were removed. The aorta was cut into aortic rings 1 mm thick, and the aortic rings were placed vertically on a 48-well cell culture plate pre-coated with Matrigel (Corning). Incubate at 37°C for 10 min, then add another 60 μl of Matrigel to cover the aortic rings. Incubate in an incubator at 37°C for 3 hours until fully solidified. Add culture medium with or without SNCG, VEGF, or SNCG + VEGF, 500 μl per well. Replace the fresh culture medium every two days. After about 7 days, take pictures according to the budding of aortic vascular endothelial cells and statistically analyze the budding area.

[0043] C, Chicken embryo chorioallantoic membrane angiogenesis assay: The purchased SPF-grade eggs (CE5, Merial Vetoquinol) were disinfected with 1% benzalkonium bromide solution and then transferred to an incubator at 37°C for incubation until the 4th day. The surface of the eggs was disinfected with 75% medical alcohol, and the eggshell was carefully opened. The chicken embryos were placed in a culture dish with the yolk sac facing up and centered to avoid affecting the growth and development of blood vessels. Incubate in the incubator for 24 h to allow the chicken embryos to gradually adapt to the culture environment. Place glass fiber filter papers containing SNCG, VEGF, or SNCG + VEGF on the appropriate blood vessel sites on the chicken embryo chorioallantoic membrane, and continue to incubate for 48 h. Carefully remove the glass fiber filter papers, observe the blood vessel development of the experimental sites and their surroundings under a dissecting microscope, take pictures, and statistically analyze the total number of large blood vessels and capillaries.

[0044] The results are shown in Figure 1 as follows. The results show that SNCG can promote angiogenesis in the above different models and has a synergistic effect with VEGF.

[0045] Example 2: SNCG can promote the phosphorylation of multiple proteins in human umbilical vein endothelial cells (HUVEC) and has a synergistic effect with VEGF

[0046] Based on the result of Example 1 that SNCG has an effect on promoting angiogenesis, this example further explores whether SNCG participates in the VEGF-VEGFR signaling pathway. The following experiments were conducted:

[0047] Human umbilical vein endothelial cells (HUVECs) of P3 - P5 (the 3rd to 5th generation) were seeded in a 12 - well cell culture plate. After about 24 hours of cell culture, the cells were starved for 16 h. Then, the cells were treated with VEGF, SNCG, or their combination at different time points (0, 5 min, 30 min, 90 min) or different doses. The cells were washed once with PBS, and 60 μl of RIPA lysis buffer was added to each well. The obtained samples were subjected to Western Blot experiment: that is, the above - mentioned protein lysis buffer was boiled with SDS - PSGE loading buffer for 5 - 10 min, and then SDS - PAGE electrophoresis was carried out; transfer the membrane under ice - bath conditions at 300 mA for about 3 h; take out the nitrocellulose membrane; block it with 5% non - fat milk - TBST at room temperature for 2 h; add the corresponding antibody and react overnight; react with the secondary antibody for 1 h; wash the membrane 3 times for 10 min each, and then perform luminescence detection.

[0048] The results are shown in Figure 2 as follows. The results showed that: during the 5 - 90 min period of treating HUVEC cells with SNCG and VEGF, SNCG could up - regulate the levels of p - Src (Y 416 ), p - Akt (S 473 ), NFκB p - P65 (S 536 ), p - FAK (Y 397 , Y 925 ), p - VEGFR2 (Y 1175 ) alone or synergistically with VEGF, and it was dose - and time - dependent.

[0049] Example 3: The endogenous SNCG expression and secretion level in colorectal cancer cell lines is negatively correlated with the ability of bevacizumab to inhibit tumor cell migration

[0050] This example demonstrated that the SNCG expression level in colorectal cancer cells is negatively correlated with the phenotype of bevacizumab (Bev) inhibiting colorectal cancer cell lines.

[0051] Transwell experiments were carried out, which proved that the ability of bevacizumab (Bev) to inhibit colorectal cancer cell lines is negatively correlated with the level of SNCG secreted by cells. Resuscitate colorectal cancer cell lines HCT116, DLD - 1, RKO, LOVO, SW480. When the cell confluence reached 80 - 90%, perform cell migration experiments. Add 800 μL of RPMI - 1640 conventional culture medium containing 10% FBS to the lower chamber of Transwell. Add 1×10 5 -1×10 6 cells / 200 μL of serum - free RPMI - 1640 culture medium to the upper chamber, culture in an incubator for 24 h, fix with ice - cold methanol for 10 min, stain with 0.1% crystal violet for 4 h, wipe off the non - migrated cells on the upper layer of the chamber with a cotton swab, seal the slide with neutral gum, take pictures, and count the number of migrated cells.

[0052] See the results in Figure 3 shown below. The results showed that bevacizumab inhibited the migration ability of colorectal cancer cells HCT116, DLD-1, RKO, and LOVO in a dose-dependent manner, and had a significant negative correlation with the SNCG level (r = -0.971, P = 0.006), while having no correlation with the VEGF level (P = 0.685).

[0053] Example 4: Upregulating or downregulating the SNCG level in tumor cells can inhibit or enhance the ability of bevacizumab (Bev) to inhibit the migration of tumor cells

[0054] In this example, a pcDNA-SNCG recombinant plasmid was constructed. The specific process was as follows: The SNCG gene sequence was amplified by PCR, and the SNCG gene fragment was inserted into the pcDNA3.0 vector (Invitrogen), and the pcDNA-SNCG recombinant plasmid was constructed. Six-well plates were seeded with SNCG-negative cell lines DLD-1 and LOVO cells. After 24 hours of seeding, the pcDNA-SNCG recombinant plasmid was transfected using lipo2000 liposomes (Invitrogen). The transfection process was carried out according to the operation instructions. After 24 hours of transfection, the cells were passaged, and screened with 400 μg / mL G418 for 2 weeks. Monoclonal cells were selected, and the SNCG expression was identified by Western Blot experiment to establish high-expression cell lines DLD-1-SNCG and LOVO-SNCG with stable SNCG expression; at the same time, the gRNA Cas9 vector (Gemma) was constructed using the CRISPR-Cas9 technology, transfected into HT29 and HCT116 cells according to the operation instructions, screened with 1 μg / mL Puromycin antibiotic for 2 weeks, single clones were picked, and after co-identification by ELISA and Western blot, cell lines HT29GKO and HCT116GKO with stable SNCG knockout were obtained.

[0055] Using the established SNCG high-expression cell lines and SNCG knockout cell lines, the effect of the SNCG level in tumor cells on the ability of bevacizumab to inhibit the migration of tumor cells was studied. The Transwell experiment method was the same as in Example 3. 800 μL of RPMI-1640 conventional culture medium containing 10% FBS was added to the lower chamber of Transwell. 1×10 5 -1×10 6 cells / 200 μL of serum-free RPMI-1640 culture medium was added to the upper chamber, cultured in an incubator for 24 h, fixed with ice methanol for 10 min, stained with 0.1% crystal violet for 4 h, the non-migrated cells on the upper layer of the chamber were wiped clean with a cotton swab, and after sealing with neutral gum, photos were taken and the number of migrated cells was counted.

[0056] See the results inFigure 4 As shown in the figure. The results showed that bevacizumab inhibited the migration ability of SNCG-negative cell lines LOVO-C and DLD-1-C in a dose-dependent manner, while the inhibitory effect of bevacizumab on the migration of SNCG-high expressing cell lines LOVO-SNCG and DLD-1-SNCG was relatively weak; bevacizumab inhibited the migration ability of HCT116GKO cells after knocking out SNCG significantly stronger than that of SNCG-positive cells HCT116; The results of cell migration experiments with LOVO cells added with conditioned culture supernatants of HT29 and HT29GKO cells showed that SNCG could antagonize the ability of bevacizumab to inhibit LOVO migration.

[0057] Example 5: The tumor growth inhibitory effect of bevacizumab on intestinal cancer cells DLD-1-SNCG with high SNCG expression in mice was significantly lower than that of the parental cell line DLD-1, and the tumor size was positively correlated with the SNCG level in the serum of mice.

[0058] In this example, the role of SNCG in antagonizing the inhibitory effect of bevacizumab on tumor cell growth was further verified in NOD-SCID mice, and the relationship between SNCG and bevacizumab resistance was verified.

[0059] Experimental method: The SNCG-high expressing cell line DLD-1-SNCG (using its empty vector transfected cell line DLD-1-C as a control) was transfected externally and subcutaneously inoculated into the right upper axilla of NOD-SCID mice at 3×10 6 cells / mouse, including a control group and bevacizumab treatment groups (two dose groups of 2 mg / kg and 5 mg / kg), a total of 6 groups, with 4-6 mice in each group; When the subcutaneous tumor volume reached about 100 mm 3 or so, bevacizumab was administered via the tail vein twice a week. The length and width of the tumor were measured and the body weight was weighed. When the maximum tumor volume reached about 1000 mm 3 or so, the experiment was terminated, the tumors were removed, weighed, and blood was taken. The tumor inhibition rate and the relationship between the serum SNCG level and the tumor size were statistically analyzed.

[0060] The results are shown in Figure 5 As shown in the figure. The results showed that bevacizumab significantly inhibited the growth of subcutaneous DLD-1 tumors in mice and was dose-dependent, while the inhibitory effect on the growth of DLD-1-SNCG tumors was reduced; The serum SNCG level of DLD-1-SNCG tumor-bearing mice was positively correlated with the tumor size (R = 0.888, p < 0.0001).

[0061] Example 6: After knocking out the endogenous SNCG expression in HT29 tumor cells, the anti-tumor growth activity of bevacizumab in tumor-bearing mice can be significantly improved.

[0062] Experimental method: The SNCG knockout cell line HT29GKO (using its parental cell line HT29 as a control) was inoculated subcutaneously into the right upper axilla of NOD-SCID mice at 2×10 6 cells / mouse, including a control group and bevacizumab treatment groups (two dose groups of 2 mg / kg and 5 mg / kg), a total of 6 groups, with 6 mice in each group; when the subcutaneous tumor volume reached about 100 mm 3 or so, bevacizumab was administered via the tail vein twice a week, and the tumor length and width were measured and the body weight was weighed. When the maximum tumor volume reached about 1000 mm 3 or so, the experiment was terminated, the tumors were removed and weighed. The tumor inhibition rate of each group was statistically analyzed.

[0063] The results are shown in Figure 6 the figure. The results showed that bevacizumab significantly inhibited the growth of subcutaneous HT29GKO tumors in mice in a dose-dependent manner, while the inhibitory effect on the growth of HT29 tumors with high SNCG expression was relatively weak.

[0064] Example 7: The serum SNCG level in colorectal cancer patients is negatively correlated with the therapeutic effect of bevacizumab

[0065] Experimental method: The SNCG level in the pre-treatment serum of 62 colorectal cancer (CRC) patients treated with bevacizumab combined with chemotherapy was detected by a double-antibody sandwich ELISA method. With a SNCG level of 5 ng / ml as the Cutoff value, it was found that the progression-free survival (PFS) of SNCG-negative patients was 10.5 months on average, significantly longer than that of SNCG-positive patients (6.2 months on average), and the difference between the two was significant (P = 0.005).

[0066] The results are shown in Figure 7 the figure. The results showed that there was a correlation between the SNCG expression level of patients and the therapeutic effect of bevacizumab. The progression-free survival period of patients with high SNCG expression after bevacizumab treatment was significantly lower than that of patients with low SNCG expression.

[0067] Example 8: SNCG reduces the ability of sorafenib and sunitinib to inhibit tumor cell migration

[0068] Experimental method: Resuscitate the colorectal cancer cell lines LOVO and LOVO-SNCG. When the cell confluence reached 80-90%, a cell migration experiment was carried out. 800 μL of RPMI-1640 conventional culture medium containing 10% FBS was added to the lower chamber of Transwell, and 1×10 5 -1×10 6Add cells / 200 μL of serum-free RPMI-1640 culture medium to the upper chamber, culture in an incubator for 24 h, fix with ice-cold methanol for 10 min, stain with 0.1% crystal violet for 4 h, wipe off the non-migrated cells on the upper layer of the chamber with a cotton swab, seal the slide with neutral gum, take pictures and count the number of migrated cells.

[0069] The results are shown in Figure 8 as follows. The results show that: at the concentrations of 10 μM and 20 μM of sorafenib and 5 μM and 20 μM of sunitinib, the ability of the drugs to inhibit the migration of LOVO-SNCG cells is significantly lower than that of inhibiting the migration of LOVO cells. It shows that SNCG can reduce the effects of sorafenib and sunitinib.

[0070] Example 9: SNCG reduces the ability of sorafenib and sunitinib to inhibit the invasion of tumor cells

[0071] Experimental method: Resuscitate the colorectal cancer cell lines LOVO and LOVO-SNCG. When the cell confluence reaches 80-90%, perform a cell invasion experiment. Add 800 μL of RPMI-1640 conventional culture medium containing 10% FBS to the lower chamber of the invasion plate, and add 5 -1×10 6 cells / 200 μL of serum-free RPMI-1640 culture medium to the upper chamber, culture in an incubator for 24 h, fix with ice-cold methanol for 10 min, stain with 0.1% crystal violet for 4 h, wipe off the non-migrated cells on the upper layer of the chamber with a cotton swab, seal the slide with neutral gum, take pictures and count the number of migrated cells.

[0072] The results are shown in Figure 9 as follows. The results show that: at the concentrations of 10 μM and 20 μM of sorafenib and 5 μM and 20 μM of sunitinib, the ability of the drugs to inhibit the invasion of LOVO-SNCG cells is significantly lower than that of inhibiting the invasion of LOVO cells. It shows that SNCG can reduce the anti-tumor cell invasion ability of sorafenib and sunitinib.

[0073] The descriptions presented in the above exemplary embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to be exhaustive, nor to limit the present invention to the precise forms described. Obviously, many changes and variations are possible for those of ordinary skill in the art according to the above teachings. The selection of exemplary embodiments and the description are for explaining the specific principles of the present invention and its practical applications, so that other technicians in the art can understand, implement and utilize various exemplary embodiments of the present invention and their various alternative forms and modified forms. The protection scope of the present invention is intended to be defined by the scope of the claims and their equivalent forms.

Claims

1. Use of γ-synuclein as a biomarker for resistance to anti-angiogenic drugs.

2. Use of γ-synuclein in the preparation of a preparation for promoting angiogenesis; Preferably, the γ-synuclein is used alone as an active component for promoting angiogenesis or in combination with other angiogenesis-promoting substances for the preparation of a preparation for promoting angiogenesis; Preferably, the angiogenesis promotion includes promoting the growth / healing of vascular endothelial cells and / or promoting angiogenesis and / or tubule formation.

3. Use of γ-synuclein in the preparation of a preparation for upregulating the phosphorylation of VEGF receptors; Preferably, the γ-synuclein is used alone as an active component for upregulating the phosphorylation of VEGF receptors or in combination with other active substances for the preparation of a preparation for upregulating the phosphorylation of VEGF receptors.

4. The use according to claim 2 or 3, wherein, the other active substance is an angiogenesis-promoting factor, such as VEGF.

5. Use of γ-synuclein in the preparation of an evaluation product for evaluating the efficacy of anti-angiogenic drugs; Preferably, the anti-angiogenic drug is a VEGF-targeted anti-angiogenic drug, including but not limited to Bevacizumab, sunitinib, sorafenib; Preferably, the efficacy of the anti-angiogenic drug includes an inhibitory effect on the migration and / or invasion of cancer cells and / or an inhibitory effect on tumor growth.

6. Use of γ-synuclein as a target in the screening and / or preparation of anti-angiogenic reagents; Preferably, the anti-angiogenic reagent is an anti-angiogenic drug; Preferably, the anti-angiogenesis includes an inhibitory effect on the migration and / or invasion of cancer cells and / or an inhibitory effect on tumor growth.

7. An evaluation device for evaluating the efficacy of anti-angiogenic drugs in an individual, the device comprising: a detection unit for detecting γ-synuclein in an individual sample to obtain a detection result of the expression level of γ-synuclein; an analysis unit for analyzing the detection result of the detection unit to evaluate the efficacy of the anti-angiogenic drug in the individual to be tested.

8. The evaluation device according to claim 7, wherein, the analysis unit evaluates the progression-free survival period of the anti-angiogenic drug treatment in the individual to be tested according to the level of γ-synuclein in the sample from the individual to be tested.

9. The evaluation device according to claim 8, wherein, a low level of γ-synuclein from the individual to be tested indicates a long progression-free survival period of the anti-angiogenic drug treatment in the individual to be tested.

10. The evaluation device according to any one of claims 7-9, wherein, when the analysis unit performs analysis and evaluation, it operates as follows: comparing the γ-synuclein level of the detection unit with a Cutoff value; for individuals with a γ-synuclein level below 5 ng / ml, they have a longer progression-free survival period of anti-angiogenic drug treatment; Preferably, the anti-angiogenic drug is a VEGF-targeted anti-angiogenic drug, including but not limited to Bevacizumab, sunitinib, sorafenib.

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