Application of vitepofen in preparation of anti-colorectal cancer medicine

Abstract

The invention discloses an application of vitepofen in preparation of a medicine for resisting colorectal cancer. According to the research of the invention, the vinipofen has a remarkable treatment effect on colorectal cancer as a non-photosensitizer, the dose without remarkable toxicity and with the optimal curative effect is determined to be 60 mg / kg, and in addition, the specific action mechanism of the vinipofen is deeply explored. Therefore, the vinipofen can be used as a lead compound for treating the major disease of the colorectal cancer, has important development prospects and clinical application values, and also provides a scientific basis and an experimental basis for developing other anti-cancer drugs and exploring pathogenesis of the colorectal cancer and finding therapeutic markers.

Description

Technical Field

[0001] The invention belongs to the field of medical technology, and in particular relates to an application of verteporfin in the preparation of an anti-colorectal cancer drug. Background Art

[0002] Colorectal cancer (CRC) is a common digestive system malignancy in the world, and its incidence rate is increasing year by year. It is worth noting that early patients often lack obvious symptoms, and the proportion of patients with colorectal cancer in stage IV is as high as 18.3% at the first diagnosis. In addition, CRC tumor cells are highly invasive and can spread to tissues and organs throughout the body, with the liver and lungs being the most common sites of metastasis, resulting in a five-year survival rate of only 14%. In clinical practice, surgical resection combined with adjuvant therapy is an effective treatment strategy for colorectal cancer. Adjuvant therapy mainly includes chemotherapy, targeted therapy, and immunotherapy. The latest guidelines issued by the National Comprehensive Cancer Network (NCCN) recommend that FOLFOX (5-fluorouracil, oxaliplatin, folinic acid) and FOLFIRI (5-fluorouracil, irinotecan, folinic acid) are the two main treatment options for locally advanced colorectal cancer. Among them, 5-fluorouracil is an antimetabolite chemotherapy drug. It can block the synthesis of tumor cell DNA by replacing uracil in cells, thereby inhibiting tumor growth; oxaliplatin, an alkylating agent, can directly damage the DNA of tumor cells to prevent cell proliferation, and its DNA damage effect is not limited by the cell cycle; irinotecan, which is also an anti-metabolic chemotherapy drug, is a DNA topoisomerase I inhibitor that can inhibit the replication and transcription of tumor cell DNA, and its main effect is on the S phase and G2 phase of the cell cycle. For advanced patients, the treatment model of chemotherapy combined with EGFR / VEGF inhibitors has been widely recognized. Although there are currently a variety of treatments for CRC patients, the current CRC treatment situation still faces many obstacles, especially the problem of patient resistance.

[0003] Abnormal expression of Tankyrase (TNKS) is closely related to the occurrence and development of colorectal cancer. TNKS is a member of the poly (ADP-riose) polymerase (PARP) superfamily and has two subtypes: TNKS1 and TNKS2. TNKS is widely distributed in human tissues, and TNKS is abnormally expressed in many tumor tissues including CRC. Gelmini et al. found that the expression level of TNKS was negatively correlated with the patient's prognosis and survival. Moreover, the abnormally high expression of TNKS in tumor cells mediates its resistance to chemotherapeutic drugs. Therefore, targeted inhibition of TNKS is an effective means of treating colorectal cancer. TNKS mainly promotes the occurrence and development of tumors and mediates their resistance through interactions with binding substrate proteins. TNKS can recognize and degrade TRF1 to maintain the continuous proliferation of tumor cells, thereby mediating their resistance to telomerase inhibitors. Combining telomerase inhibitors and knocking down TNKS can enhance the efficacy and avoid the emergence of resistance problems. TNKS can also bind to and ubiquitinate PTEN for degradation. Inactivation of PTEN can induce CRC resistance to the first-line chemotherapy drug oxaliplatin by inhibiting ferroptosis. In addition, the deubiquitinase USP25 can also bind to TNKS to protect it from ubiquitination degradation. USP25 promotes the growth of colorectal cancer cell DLD-1 in a TNKS-dependent manner, while DLD-1 with USP25 knockdown exhibits anti-proliferative activity. Blocking the interaction between TNKS and USP25 can inhibit the Wnt signaling pathway to overcome drug resistance. More importantly, inhibiting TNKS combined with PI3K / AKT inhibitors, EGFR inhibitors, MEK inhibitors, immune checkpoint inhibitors (ICI), etc. has a synergistic therapeutic effect, and even combined treatment can inhibit the growth of tumor cells that are insensitive to these targeted inhibitors.

[0004] Verteporfin (VP) is a member of the porphyrin family and has been approved by the U.S. Food and Drug Administration (FDA) as a porphyrin photosensitizer in the clinic for the treatment of neovascular macular degeneration. In vivo studies have found that after intravenous injection, VP circulating in the body binds to low-density lipoprotein (LDL) and selectively aggregates in choroidal neovascularization. Subsequently, through non-thermal laser sparks, the activated VP is converted from the ground state to the excited triplet state and directly initiates the photochemical reaction by forming Yang free radicals, or indirectly initiates the photochemical reaction by transferring energy to highly reactive singlet oxygen and ground state oxygen.

[0005] Although there are many treatments for CRC patients, CRC is still facing an increasing mortality rate year by year. The main reasons for the failure of CRC treatment are:

[0006] (1) Clinical treatment options are limited, and chemotherapy drugs have significant toxic side effects

[0007] Chemotherapy drugs are widely used as first-line drugs in the treatment of CRC, but they can damage the patient's body while killing tumor cells, that is, they often have some toxic side effects on patients. More importantly, if standard treatment fails, the drugs available for follow-up treatment are very limited.

[0008] (2) Clinical patients face the important issue of chemotherapy drug resistance

[0009] Chemotherapy or radiotherapy and other related auxiliary treatments after surgical resection have been used in the treatment of CRC, but tumor recurrence and metastasis cannot be avoided, and patients will also develop resistance to chemotherapy drugs. Drug resistance can be divided into primary resistance (intrinsic resistance) and secondary resistance (acquired resistance). Some patients show insensitivity to chemotherapy drugs at the beginning of treatment, which leads to disease progression, while other patients gradually develop resistance to chemotherapy drugs during the treatment process, resulting in weakened or disappeared treatment effects. Even more unfortunately, there is currently a lack of treatments and methods to address this tumor resistance to help patients overcome resistance. Therefore, finding new and effective colorectal cancer treatment strategies remains a global public health problem. The development mechanism of colorectal cancer and the development of effective therapeutic drugs and treatment plans have become important focuses in the field of colorectal cancer research.

[0010] At present, a variety of TNKS inhibitors have been reported, including XAV939, IWR-1, G007-LK, NVP-TNKS656, RK-287107, etc., while the marketed Olaprib (AZD2281) and Niraparib (MK-4827) only have a weak inhibitory effect on TNKS, mainly as selective inhibitors of PARP1 and PARP2. Like other PARP inhibitors, the currently developed TNKS inhibitors mainly target the TNKS catalytic domain (PARP domain) to inhibit enzyme activity. XAV939 is the first TNKS inhibitor discovered, which blocks TNKS enzyme activity by binding to the nicotinamide pocket of TNKS. IWR-1 acts as a non-competitive inhibitor of TNKS to affect TNKS enzyme activity. Based on these two TNKS inhibitors, researchers have developed a series of TNKS inhibitors with different structures to achieve better targeting and selectivity for tumor treatment, but the mode of action of these TNKS inhibitors is also mainly to act on the PARP catalytic domain to inhibit the enzymatic catalytic activity of TNKS to exert anti-tumor effects. Therefore, although researchers continue to design and develop new TNKS inhibitors for TNKS targets, it is regrettable to find that these TNKS inhibitors are not always effective, at least they cannot inhibit the growth of all colorectal cancers with high TNKS expression, such as G007-LK cannot effectively inhibit the growth of colorectal cancer cells with different genetic backgrounds; colorectal cancer cells DLD-1 and RKO show resistance to RK-287197. As a promising drug target and biomarker, TNKS has high clinical application value, but the unique role of TNKS in tumor cells and tumor resistance has not been fully elucidated, and the development of TNKS inhibitors also faces many problems. Therefore, exploring new TNKS inhibitors to directly inhibit the expression level of TNKS and deeply analyzing its mechanism of action will be beneficial to the development and application of its target drugs, and may bring new treatment hope to clinical patients. Summary of the invention

[0011] The purpose of the present invention is to provide an application of verteporfin in the preparation of anti-colorectal cancer drugs.

[0012] In order to achieve the above object, the technical solution adopted by the present invention is as follows:

[0013] The first aspect of the present invention provides a use of verteporfin or a pharmaceutically acceptable salt thereof in the preparation of an anti-colorectal cancer drug.

[0014] The English name of verteporfin is Verteporfin (VP) (Chin Pharm J, October 2001, Vol. 36 No. 10 709-710), and the molecular formula is C 82 H 84N8O 16 , the molecular structure is as follows (compound 1 and compound 2 are mixed in a 1:1 ratio):

[0015]

[0016] The verteporfin inhibits tumor growth by inducing ferroptosis in colorectal cancer.

[0017] The verteporfin induces ferroptosis by inhibiting YAP / TNKS.

[0018] The verteporfin inhibits the synthesis of GSH by regulating the transcription of xCT through YAP / TNKS, ultimately triggering ferroptosis in colorectal cancer and achieving the effect of inhibiting tumor growth.

[0019] In the application, verteporfin or its pharmaceutically acceptable salt is used as the only active ingredient.

[0020] The pharmaceutically acceptable salt is an acid addition salt formed by verteporfin and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

[0021] The second aspect of the present invention provides a pharmaceutical preparation, which is prepared from the verteporfin or a pharmaceutically acceptable salt thereof and a medically acceptable excipient.

[0022] The dosage form of the pharmaceutical preparation is selected from powders, tablets, granules, capsules, and suspensions.

[0023] The administration mode of the pharmaceutical preparation is intravenous injection.

[0024] Due to the adoption of the above technical solution, the present invention has the following advantages and beneficial effects:

[0025] The present invention provides a clinical drug, verteporfin, which has a significant therapeutic effect on colorectal cancer as a non-photosensitizer, and conducts pharmacodynamic evaluations in multiple models. Its mechanism of action is also disclosed: it induces ferroptosis of colorectal cancer by regulating YAP / TNKS / xCT to achieve the effect of treating colorectal cancer.

[0026] The present invention finds that verteporfin can be used as a lead compound for colorectal cancer, a major disease, and has great development potential and clinical application value, providing new ideas for further exploring the pathogenesis of colorectal cancer and developing new treatment methods.

[0027] Through the research of the present invention, it is found that verteporfin has a significant therapeutic effect on colorectal cancer as a non-photosensitizer, and it is determined that there is no significant toxicity ( Figure 5.G HE staining of important organs of mice shows that verteporfin has no obvious effect on important organs of mice, but has certain hepatotoxicity and renal toxicity after the dosage reaches 100 mg / kg; Figure 5 .H showed that verteporfin had no significant effect on mouse body weight) and the dose with the best therapeutic effect was 60 mg / kg. In addition, its specific mechanism of action was further explored ( Figure 6-Figure 10 It shows that verteporfin inhibits xCT transcription by regulating TNKS / YAP, thereby inducing ferroptosis in colorectal cancer). Therefore, verteporfin can be used as a lead compound for the treatment of colorectal cancer, a major disease, and has important development prospects and clinical application value. It also provides a scientific basis and experimental foundation for the development of other anticancer drugs and the exploration of the pathogenesis and treatment markers of colorectal cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a schematic diagram of high-throughput screening of cell viability for small molecule compounds.

[0029] Figure 2 Schematic diagram of verteporfin inhibiting TNKS protein expression and inducing ferroptosis in colorectal cancer cells.

[0030] Figure 3 This is a schematic diagram of the organoids constructed from tumor tissues of two patients.

[0031] Figure 4 Schematic diagram of verteporfin-induced ferroptosis in organoids.

[0032] Figure 5 It is a schematic diagram of the in vivo efficacy evaluation and safety evaluation of verteporfin.

[0033] Figure 6 Schematic diagram of verteporfin inhibiting YAP / TNKS protein levels.

[0034] Figure 7 Schematic diagram of verteporfin inducing ferroptosis by regulating YAP / TNKS.

[0035] Figure 8 Schematic diagram of the analysis of transcriptome sequencing results after verteporfin treatment.

[0036] Fig. 9 Schematic diagram of verteporfin inhibiting xCT expression level and GSH level in vivo and in vitro.

[0037] Fig.10 Schematic diagram of verteporfin regulating xCT-induced ferroptosis through YAP / TNKS. DETAILED DESCRIPTION

[0038] In order to explain the present invention more clearly, the present invention is further described below in conjunction with preferred embodiments. It should be understood by those skilled in the art that the following specific description is illustrative rather than restrictive, and should not be used to limit the scope of protection of the present invention.

[0039] Main materials: Verteporfin, Z-VAD-FMK, Necrostatin-1 (Nec-1), Deferoxamine (DFO), Ferrostatin1 (Fer-1), Disulfiram (DSF), TCEP, 3-Methyladenine (3-MA), Oxaliplatin (Oxa), 5-fluorouracil (5-Fu), Erastin were purchased from Shanghai Haoyuan Biotechnology Co., Ltd.; FerroOrange probe and Lipid Peroxidation Probe-BDP 581 / 591 C11-lipid peroxidation probe were purchased from Tongren Chemical Research Institute; Human 4-HNE ELISA detection kit was purchased from Shanghai Keaibo Biotechnology Co., Ltd.; CellTiter-Lumi Plus luminescent cell viability detection kit, GSH and GSSG detection kits were purchased from Shanghai Bio-Technology Co., Ltd.; siRNA and transfection reagents were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd., shRNA and transfection reagents were purchased from Shanghai Qihe Biotechnology Co., Ltd.; Co-IP magnetic bead kit and fluorescent secondary antibody were purchased from Thermo Fisher Scientific (China) Co., Ltd.; TNKS mouse and rabbit primary antibodies, YAP mouse primary antibody were purchased from Beijing Meirui Biotechnology Co., Ltd., xCT rabbit primary antibody was purchased from Abcam (Shanghai) Trading Co., Ltd., β-actin mouse primary antibody and rabbit / mouse secondary antibodies were purchased from Shanghai Pumai Biotechnology Co., Ltd.; total RNA extraction kit was purchased from Suzhou Xinsaimei Biotechnology Co., Ltd., and qPCR reaction kit was purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.

[0040] Experimental animals: The experimental mice were provided by the Experimental Animal Center of Renji Hospital Affiliated to Shanghai Jiao Tong University. All mice were nude mice, SPF grade, 6 weeks old, with an average weight of about 18 g. They were raised in the Experimental Animal Center of Renji Hospital Affiliated to Shanghai Jiao Tong University, SPF grade, with free access to food and water. The bedding was changed once a week and feed and drinking water were supplemented. There were no pathogens.

[0041] Example 1

[0042] 1. Verteporfin has the effect of inhibiting the growth of colorectal cancer cells

[0043] A high-throughput cell viability screening of 150 candidate compounds revealed that five candidate drugs could simultaneously inhibit the growth of both HCT116 and SW480 colorectal cancer cells. Figure 1 As shown, Figure 1 This is a schematic diagram of high-throughput screening of small molecule compounds for cell viability. Then, the IC values ​​of five candidate drugs against two colorectal cancer lines, HCT116 and SW480, were tested. 50 The effects of values ​​and drugs on TNKS protein expression were investigated and it was found that verteporfin had a significant anti-cancer effect and down-regulated TNKS protein levels. The results are shown in Table 1 and Figure 2 As shown in A. Figure 2 Figure 1 is a schematic diagram of verteporfin-induced ferroptosis in colorectal cancer cells. A is the effect of five candidate compounds on TNKS protein levels after treating SW480 cells. The results show that compounds 6, 32, and 45 can inhibit the expression level of TNKS protein. Table 1 is the half-inhibitory concentration IC of two colorectal cancer cells after 72 hours of treatment with five candidate compounds. 50 The results showed that compound No. 45 had the best inhibitory effect on the two cell lines; therefore, compound No. 45, namely verteporfin, was screened out as a potential effective drug for the treatment of colorectal cancer.

[0044] Table 1

[0045]

[0046] 2. Verteporfin can induce ferroptosis in colorectal cancer cells

[0047] Colorectal cancer cells were treated with a combination of multiple death inhibitors (20 μM apoptosis inhibitor Z-VAD-FMK, 20 μM necrosis inhibitor Nec-1, 10 μM ferroptosis inhibitors DFO and Fer-1, 10 μM pyroptosis inhibitor DSF, 100 μM disulfide death inhibitor TCEP, 1 mM autophagy inhibitor 3-MA) ​​and 4 μM verteporfin for 72 hours, and the effect on cell viability was detected. It was found that the addition of ferroptosis inhibitors could reverse the inhibitory effect of verteporfin on tumor growth. The results are as follows Figure 2 As shown in B. B represents the cell viability results after verteporfin treatment and verteporfin combined with different cell death inhibitors. The results show that verteporfin can inhibit the growth of colorectal cancer cells. This inhibitory effect is partially reversed after combined with ferroptosis inhibitors, indicating that verteporfin can induce ferroptosis in colorectal cancer cells.

[0048] In addition, verteporfin treatment promoted lipid peroxidation levels in colorectal cancer cells and caused accumulation of ferrous ions, and this effect was partially reversed after combined with ferroptosis inhibitors, further confirming that verteporfin can induce ferroptosis in colorectal cells. Figure 2As shown in C-F. C is a schematic diagram showing the effect of different concentrations of verteporfin on the intracellular lipid peroxidation level after treatment, and the results show that verteporfin promotes the accumulation of lipid reactive oxygen species in colorectal cancer cells in a concentration-dependent manner; D is a schematic diagram showing the effect of different concentrations of verteporfin on the intracellular ferrous ion level after treatment, and the results show that verteporfin increases the level of ferrous ions in colorectal cancer cells in a concentration-dependent manner; E is a schematic diagram showing the effect of verteporfin combined with ferroptosis inhibitor on the intracellular lipid peroxidation level after treatment, and the results show that ferroptosis inhibitor can partially reverse the effect of verteporfin on promoting intracellular lipid oxidation; F is a schematic diagram showing the effect of verteporfin combined with ferroptosis inhibitor on the intracellular ferrous ion level after treatment, and the results show that ferroptosis inhibitor can partially reverse the effect of verteporfin on promoting the accumulation of ferrous ions in cells.

[0049] 3. Verteporfin can induce ferroptosis in organoids

[0050] Organoids derived from patient tumor tissues were constructed and the expression of TNKS was analyzed by immunohistochemical staining.

[0051] The operation of organoid construction is as follows: 1. Collect in situ tumor tissues from clinical patients after surgery, store them in precooled tissue preservation solution and retrieve them; 2. Wash the tumor tissues in saline containing double antibodies and wipe the blood with sterile gauze; 3. Cut the tumor tissues in a culture dish and place them in collagenase II preheated at 37°C, and digest them in a 37°C incubator for 40-60 minutes; 4. Add 1% FBS to stop digestion, take a cell filter with a pore size of less than 100nm, and sieve the digested samples. Then centrifuge to keep the precipitate, 500xg / 1000rpm, 4°C, 4-5min; 5. Red blood cell lysis: After centrifugation of tumor tissue samples, the precipitate often appears red and needs to be lysed with red blood cell lysis solution. Add 2ml of red blood cell lysis solution to the tumor precipitate obtained by centrifugation in the previous step, lyse in a refrigerator at 4℃ for 5 minutes, then centrifuge and discard the supernatant to obtain a white precipitate; 6. In order to completely remove collagenase and other substances, use basal culture medium to wash the tumor sample 2-3 times, and centrifuge at 500xg / 1000rpm, 4℃, 4-5min (basic culture medium: adDMEM / F12+10% double antibody); 7. Resuspend the obtained tumor and adjacent tissue precipitates in matrix gel and drop them into a 24-well plate, drop 30-40ul per well, then put the 24-well plate into the incubator and wait for about 20-30min for the matrix gel to solidify; 8. Add about 400ul of complete culture medium to the 24-well cell culture plate for organoid culture. The successfully constructed organoids were photographed under the microscope and subjected to drug sensitivity tests. In addition, the successfully constructed organoids were fixed in 4% paraformaldehyde and then subjected to immunohistochemical staining.

[0052] The results are as follows Figure 3 As shown, Figure 3 The following is a schematic diagram of organoids constructed from tumor tissues of two patients. The morphology of the successfully constructed organoids can be seen from the microscopic figure. The morphology of the organoids changed after the addition of verteporfin. In addition, verteporfin treatment had a significant inhibitory effect on the organoids, and the effect was better than the first-line chemotherapy drugs for colorectal cancer, oxaliplatin and 5-fluorouracil. The results are shown in Table 2. Table 2 shows that the two organoids are insensitive to oxaliplatin and 5-fluorouracil, but sensitive to verteporfin, indicating that verteporfin can inhibit the growth of organoids that are resistant to first-line chemotherapy drugs.

[0053] Table 2

[0054]

[0055] Verteporfin can promote lipid peroxidation and accumulation of ferrous ions in organoids, indicating that verteporfin promotes ferroptosis at the organoid level to achieve anti-cancer effects. Figure 4 As shown, Figure 4 Schematic diagram of verteporfin-induced ferroptosis in organoids. A is a schematic diagram of the FerriOrange probe incubation and observation of organoid morphology and ferrous ion levels under a confocal microscope after treatment with different concentrations of verteporfin. The results show that the organoids cannot maintain their normal cavity morphology after verteporfin treatment, and ferrous ions are significantly accumulated; B is a schematic diagram of organoid lipid peroxidation levels, and the results show that verteporfin treatment promotes the accumulation of lipid reactive oxygen in organoids; C is a schematic diagram of organoid ferrous ion levels, and the results show that verteporfin treatment increases the ferrous ion level in the organoid.

[0056] 4. Verteporfin inhibits the growth of xenograft tumors in mice without obvious toxicity and the optimal dosage is 60 mg / kg

[0057] Subcutaneous xenograft tumors were constructed in mice to evaluate the in vivo efficacy and safety of verteporfin.

[0058] The procedure for constructing a mouse tumor model is as follows: 7 The cells were directly injected subcutaneously into NOD-scid mice at a concentration of 100 SW480 cells / mouse to form tumors. The growth of the subcutaneous tumors in the mice was observed during the period. When the subcutaneous tumors grew to about 800 mm 3 When the volume was 1.57 × 10.87 × 10.67 × 10.77 × 10.6 ... 3When the volume was measured, the mice were randomly divided into 5 groups: Vehicle group (blank solvent group); 20 mg / kg Erastin group; 30 mg / kg VP group (low-dose group); 60 mg / kg VP group (medium-dose group); 100 mg / kg VP group (high-dose group), and the drugs were administered according to the experimental groups (intraperitoneal injection every other day, and the administration cycle was 3 weeks). The tumor volume and mouse body weight were recorded every other day. After the administration, the nude mice were killed by cervical dislocation and tumor tissues were obtained for subsequent experiments.

[0059] The results are as follows Figure 5 As shown, Figure 5 This is a schematic diagram of the in vivo efficacy and safety evaluation of verteporfin. Verteporfin can inhibit the growth of tumors in mice by inducing ferroptosis and its effect is better than Erastin ( Figure 5 AF). Among them, A is a photographic diagram of the subcutaneous tumor of nude mice in each group, and the results show that both Erastin and Verteporfin can inhibit the growth of tumors in vivo, and Verteporfin has a better effect; B is a schematic diagram of the growth curve of tumors in each group, and the results show that Verteporfin inhibits the growth of tumors, and the inhibitory effects of the medium and high dose groups are equivalent; C is a statistical diagram of tumor tissues obtained from each group, and the results show that Verteporfin inhibits the growth of tumors, and the inhibitory effects of the medium and high dose groups are equivalent; Figure D is the level of 4-HNE in the tumor tissues obtained from each group, which is used to evaluate the occurrence of ferroptosis, and the results show that Verteporfin can induce ferroptosis of tumors in vivo; E is a HE staining diagram of the subcutaneous tumor tissues of mice obtained, and the results show that Verteporfin causes tumor necrosis and is better than Erastin; F is a statistical diagram of the T / C values ​​of tumors in each group, and the results show that Verteporfin inhibits the growth of tumors, and the inhibitory effects of the medium and high dose groups are equivalent.

[0060] At the same time, an in vivo safety evaluation was conducted (the weight of nude mice was observed and recorded every other day during the experiment, and the important organs of the mice were obtained for HE staining after the administration for in vivo safety evaluation). The results are as follows Figure 5 As shown in the GH, verteporfin had no obvious toxicity to the important organs of mice (heart, liver, spleen, lung, and kidney), and had no obvious effect on the body weight of mice. Finally, 60 mg / kg was determined to be the optimal dosage of verteporfin, at which the tumor inhibition effect was good and there was no obvious toxicity.

[0061] 5. Verteporfin can inhibit the protein expression of TNKS / YAP

[0062] Western blotting was used to detect the effect of verteporfin on TNKS protein levels. Figure 6Schematic diagram of verteporfin inhibiting YAP / TNKS protein levels. AB is a schematic diagram of TNKS protein levels in SW480 and HCT116 cells after verteporfin treatment for different time periods. The results show that verteporfin inhibits TNKS protein levels in a time-dependent manner, indicating that verteporfin can downregulate TNKS protein expression levels; C is a microscopic image of the immunofluorescence experiment at the cell level, and the results show that verteporfin inhibits the protein expression of TNKS and YAP in cells, but the inhibitory effect on YAP is weak; D is a microscopic image of the immunofluorescence experiment at the organoid level, and the results show that verteporfin inhibits the protein expression of TNKS and YAP in organoids, but the inhibitory effect on YAP is weak.

[0063] After colorectal cancer cells were treated with verteporfin and verteporfin combined with protein synthesis inhibitor CHX or proteasome inhibitor MG-132 or lysosome inhibitor Baf-A, cell proteins were extracted for western blotting to observe the protein expression level of TNKS. It was found that verteporfin could promote the ubiquitination degradation of TNKS protein. The results are as follows Figure 6 As shown in EG.

[0064] 6. Verteporfin induces ferroptosis in colorectal cancer cells by regulating TNKS / YAP

[0065] The role of TNKS / YAP protein in ferroptosis was verified by detecting the intracellular lipid peroxidation level and ferrous ion level after genetic knockdown of TNKS or YAP.

[0066] Gene knockdown experiments are mainly carried out through the following experiments:

[0067] The first step is to construct shRNA TNKS cell line by lentiviral transfection:

[0068] (1) The shRNA sequence lentiviral vector was constructed and packaged by Shanghai Heyuan Biotechnology Co., Ltd. Average virus titer: 1×10 8 TU / mL.

[0069] The shRNA sequence of the TNKS gene is:

[0070] #1106: sense: (SEQ ID NO.1);

[0071] antisense: (SEQ ID NO.2);

[0072] #1274: sense: (SEQ ID NO.3);

[0073] antisense: (SEQ ID NO.4);

[0074] (2) The steps for lentivirus infection of cells are as follows:

[0075] ① Cell inoculation: SW480 and HCT116 cells in the logarithmic growth phase were digested with 0.25% trypsin to prepare a single cell suspension; and inoculated in a 6-well plate at an appropriate concentration, cultured overnight at 37°C, and virus infection was started when the cell confluence was about 60%;

[0076] ②Infection: discard the old culture medium, take 100 μL of virus solution, 0.8 μL of ploybrene (about 8 μg / ml) and 900 μL of fresh culture medium and add them to a 6-well plate. After 8 hours, discard the culture medium containing the virus and replace it with normal fresh culture medium and continue to culture for 72 hours;

[0077] ③Puromycin resistance screening: Add 2 μL of 1 mg / mL puromycin to the cells infected with the lentivirus and the cells not infected with the virus, respectively. When all the cells not infected with the virus die and all the cells infected with the virus survive, the screening is completed (usually 4-7 days);

[0078] ④ Subculture and freeze the resistant cells, and perform Western blot to detect whether the TNKS protein expression is disturbed;

[0079] ⑤The successfully constructed TNKS knockdown cells were used for subsequent experiments.

[0080] Step 2: Small interfering transfection experiment to construct siRNA YAP cell line:

[0081] (1) siRNA sequences were purchased from Shanghai Jima Pharmaceutical Technology Co., Ltd.

[0082] The siRNA sequence of the YAP gene is:

[0083] NC:sense: (SEQ ID NO.5);

[0084] antisense: (SEQ ID NO.6);

[0085] #954: sense: (SEQ ID NO.7);

[0086] antisense: (SEQ ID NO.8);

[0087] #1475: sense: (SEQ ID NO.9);

[0088] antisense: (SEQ ID NO.10).

[0089] (2) The steps for small interfering cell infection are as follows:

[0090] ① Cell inoculation: Digest and count SW480 and HCT116 cells grown to the logarithmic phase. Dilute with culture medium to an appropriate concentration and add to a 24-well plate (for Topflash / Fopflash dual luciferase reporter gene assay, with 2×10 cells per well). 5 cells / well) or add to a 6-well plate (for Western blot detection, so that the number of cells per well is 8×10 5 The cells were cultured in a 37°C, 5% CO2 incubator for 24 hours until the cell confluence was about 60-70%.

[0091] ② Transfection: Dilute siRNA and GP-transfect Mate Reagent with opti-MEM medium, respectively. The dilution ratio is as follows for 24-well plates (50 µL opti-MEM medium mixed with 2 µL GP-transfect Mate Reagent; 50 µL opti-MEM medium mixed with 3 µL siRNA); for 6-well plates (200 µL opti-MEM medium mixed with 5 µL GP-transfect Mate Reagent; 200 µL opti-MEM medium mixed with 8 µL siRNA). After standing for 5 minutes, mix siRNA and GP-transfect Mate Reagent, continue to stand for 20 min, add to 6-well plates or 24-well plates, and culture for 24 h.

[0092] ③ Dosage treatment: Add drugs in groups according to the actual needs of each experiment, and conduct corresponding follow-up experiments.

[0093] The results are as follows Figure 7 As shown, Figure 7Schematic diagram of verteporfin inducing ferroptosis by regulating YAP / TNKS. Among them, A is a schematic diagram of the changes in intracellular ferrous ion levels after knocking down TNKS, and the results show that the intracellular ferrous ion level increases after knocking down TNKS; B is a schematic diagram of the changes in intracellular lipid reactive oxygen levels after knocking down TNKS, and the results show that the intracellular lipid reactive oxygen level increases after knocking down TNKS; C is a schematic diagram of the changes in intracellular 4-HNE levels after knocking down TNKS, and the results show that intracellular lipid oxidation increases after knocking down TNKS; D is a schematic diagram of the changes in intracellular ferrous ion levels after knocking down YAP, and the results show that the intracellular ferrous ion level increases after knocking down YAP; E is a schematic diagram of the changes in intracellular lipid reactive oxygen levels after knocking down YAP, and the results show that the intracellular lipid reactive oxygen level increases after knocking down YAP; These results indicate that both TNKS and YAP are involved in the regulation of ferroptosis, and verteporfin induces ferroptosis in colorectal cancer by inhibiting TNKS / YAP.

[0094] Through gene knockdown combined with WB experiment and immunoprecipitation Co-IP experiment, it was found that verteporfin could inhibit the interaction of YAP / TNK and promote the degradation of TNKS. Figure 7 As shown in FG. F is a schematic diagram of TNKS protein levels after YAP knockdown, and the results show that the protein level of TNKS is inhibited after YAP knockdown; G is the Co-IP result diagram, and the results show that there is an interaction between TNKS and YAP, and verteporfin can inhibit this effect; it shows that verteporfin triggers ferroptosis by inhibiting TNKS / YAP interaction and promoting TNKS degradation.

[0095] 7. Verteporfin inhibits xCT transcription and GSH levels to induce ferroptosis

[0096] After 6 h of treatment with or without verteporfin, total RNA of the two groups of cells was extracted and transcriptome sequencing was performed. Figure 8 As shown, Figure 8 Figure 1 is a schematic diagram of the analysis of transcriptome sequencing results after verteporfin treatment. A is a KEGG enrichment analysis of the two transcriptome sequencings, and the results show that verteporfin can cause gene changes in ferroptosis-related pathways, indicating that verteporfin induces ferroptosis in colorectal cancer cells; B is a differential gene analysis of the KEGG-enriched ferroptosis pathway, and the results show that 24 groups of differential genes are enriched, among which the transcription of the classic ferroptosis negative regulator xCT is downregulated.

[0097] Fig. 9Schematic diagram of verteporfin inhibiting xCT expression level and GSH level in vivo and in vitro. Among them, A is a schematic diagram of the effect of verteporfin treatment on xCT protein expression after different time periods, and the results show that verteporfin inhibits the protein level of Xct in a time-dependent manner; B is a schematic diagram of the results of verteporfin treatment of xCT mRNA levels, and the results show that verteporfin inhibits the transcription of xCT in HCT116 and SW480 cell lines; C is a schematic diagram of the results of verteporfin treatment of colorectal cancer cells GSH levels, and the results show that verteporfin can inhibit the synthesis of intracellular GSH; D is a schematic diagram of in situ hybridization staining of xenograft tumor tissues constructed from subcutaneous SW480 in mice, yellow fluorescence represents xCT RNA levels, and blue fluorescence represents cell nuclei, and the results show that verteporfin inhibits the transcription of xCT in vivo; E is a schematic diagram of the xCT mRNA levels of in vivo transplanted tumor tissues collected by qRT-PCR experiments, and the results show that verteporfin inhibits the transcription of xCT in vivo; F is a schematic diagram of the detection of GSH and GSSG levels of in vivo transplanted tumor tissues, and the results show that verteporfin inhibits the synthesis of GSH in vivo without affecting GSSG.

[0098] 8. Verteporfin regulates xCT-induced ferroptosis through YAP / TNKS

[0099] Fig.10 Figure 1 is a schematic diagram of verteporfin regulating xCT-induced ferroptosis through YAP / TNKS. First, we performed gene knockdown experiments on TNSK and YAP to verify that the two jointly regulated the transcription and protein levels of xCT. The results are shown in Figure 1. Fig.10 As shown in AB and C, F. Among them, A is a schematic diagram of xCT mRNA level after gene knockdown of TNKS, and the results show that xCT mRNA level is reduced after knockdown of TNKS; B is a schematic diagram of xCT mRNA level after gene knockdown of YAP, and the results show that xCT mRNA level is reduced after knockdown of YAP; C is a schematic diagram of xCT protein level after gene knockdown of TNKS, and the results show that xCT protein level is reduced after knockdown of TNKS; F is a schematic diagram of xCT protein level after gene knockdown of YAP, and the results show that xCT protein level is reduced after knockdown of YAP; These results prove that in vitro verteporfin regulates the transcription of xCT by inhibiting TNKS / YAP and ultimately triggers ferroptosis. Secondly, the collected in vivo transplanted tumor tissues were subjected to immunohistochemical staining of TNSK\YAP\xCT ( Fig.10 As shown in E), the results showed that verteporfin inhibited the TNKS / YAP interaction, causing the degradation of TNKS, ultimately inhibiting xCT transcription and ultimately triggering ferroptosis. These results collectively indicate that verteporfin triggers colorectal cancer ferroptosis by co-regulating xCT through YAP / TNKS, thereby achieving a therapeutic effect on colorectal cancer.

[0100] As a photosensitizer, verteporfin is approved by the FDA for photodynamic therapy to treat age-related macular degeneration. Its application is limited to improving vision by damaging blood vessels with lasers. The present invention finds that verteporfin, as a non-photosensitizer, has a good therapeutic effect in the treatment of colorectal cancer. It can trigger ferroptosis of colorectal cancer by regulating the YAP / TNKS / xCT axis. It may be used as a new drug for this major disease, providing new strategies for clinical treatment and providing an experimental basis and scientific basis for the development of new anticancer drugs.

[0101] The above is only a preferred embodiment of the present invention, and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment, it is not used to limit the present invention. Any technician familiar with this patent can make some changes or modify the technical contents suggested above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any simple modification, equivalent change and modification made to the above embodiments according to the technical essence of the present invention without departing from the content of the technical solution of the present invention still fall within the scope of the solution of the present invention.

Claims

1. Use of verteporfin or a pharmaceutically acceptable salt thereof in the preparation of an anti-colorectal cancer drug.

2. The use of verteporfin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-colorectal cancer drug, characterized in that: The molecular structure of verteporfin is shown below, which is a mixture of compound 1 and compound 2 in a ratio of 1:

1. composition: 。 3. The use of verteporfin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-colorectal cancer drug, characterized in that: In the application, verteporfin or its pharmaceutically acceptable salt is used as the only active ingredient.

4. The use of verteporfin or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of an anti-colorectal cancer drug, characterized in that: The pharmaceutically acceptable salt is an acid addition salt formed by verteporfin and the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, lactic acid, citric acid, phosphoric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, tartaric acid, pyruvic acid, acetic acid, maleic acid or succinic acid, fumaric acid, salicylic acid, phenylacetic acid or mandelic acid.

5. A pharmaceutical preparation, characterized in that The invention is prepared from verteporfin or its pharmaceutically acceptable salt and medically acceptable auxiliary materials.

6. The pharmaceutical preparation according to claim 5, characterized in that The dosage form of the pharmaceutical preparation is selected from powders, tablets, granules, capsules, and suspensions.

7. The pharmaceutical preparation according to claim 5, characterized in that The administration mode of the pharmaceutical preparation is intravenous injection.

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