Quadrivalent platinum autophagy inducer as well as preparation method and application thereof

Abstract

The invention discloses a tetravalent platinum autophagy inducer with a structure of a general formula I as well as a preparation method and application of the tetravalent platinum autophagy inducer. The compound disclosed by the invention has an induction effect on cell autophagy, and can be used for degrading immunosuppressive protein tenascin-C (Tenascinin-C) through autophagy so as to improve a tumor immunosuppressive microenvironment. The tetravalent platinum complex induces tumor cells to generate autophagic death through unique structural characteristics, promotes immune killer cells to infiltrate a tumor immune microenvironment, can be used for preparing novel chemical immunotherapy drugs with multiple anti-tumor mechanisms, and has broad application prospects. The problems that the traditional treatment effect is poor and a small molecule immunotherapeutic agent is lacked are solved. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and relates to a tetravalent platinum autophagy inducer and a preparation method and application thereof. Specifically, the tetravalent platinum autophagy inducer of the present invention has the function of degrading the immunosuppressive protein Tenascin-C. Background Art

[0002] The incidence and mortality of cancer are increasing rapidly, becoming a major public health problem threatening human health.

[0003] Due to tumor heterogeneity and the lack of biomarkers, treatment options are relatively limited. Platinum drugs represented by cisplatin are the main treatment options, but some patients still have poor prognosis and low survival rates for advanced patients, and these problems need to be solved urgently. In recent years, the development of immunotherapy has brought hope for breaking through the difficulties in curing tumors, but the results of single-agent immunotherapy are still insufficient to improve the long-term survival rate of patients. Chemotherapy and immunotherapy combination therapy has now entered clinical research and has shown acceptable safety and outstanding anti-tumor activity. Therefore, the development of platinum drugs that can effectively activate the immune system and work synergistically with chemotherapy has potential value for the treatment of tumors.

[0004] The immunosuppressive protein Tenascin-C (TNC) is an extracellular matrix glycoprotein that is almost not expressed in healthy tissues but highly expressed in tumor tissues, especially in tumors with low autophagy levels. Tenascin-C is secreted by tumor cells into the cell matrix and deposited in the matrix. It will bind to CD8 + T cells interact with and bind to tumor cells and retain them in the extracellular matrix, allowing CD8 + T cells cannot infiltrate tumor cells, which affects their function. The deposition of Tenascin-C not only provides an ideal microenvironment for cancer cell adhesion, migration, proliferation and angiogenesis, but also promotes the growth and development of tumor cells and reduces the effective delivery of drugs. In addition, the immunosuppressive effect of Tenascin-C on T cells has been verified in breast cancer and prostate cancer. On the one hand, Tenascin-C induces TLR4 and binds to CXCL12, which converts CD8 + T cells remain in the matrix, allowing CD8 +T cells cannot reach the target tumor site, which blocks their function and inhibits T cell-mediated tumor killing. On the other hand, Tenascin-C can also promote breast cancer lung metastasis through TLR4, polarize macrophages to M2 type, and form an immunosuppressive environment to support tumor development. Based on the function of Tenascin-C in tumors, it can be seen that Tenascin-C is a key immunosuppressive molecule regulated by autophagy, and has potential targeting value in reversing T cell immune tolerance of tumors.

[0005] Immunogenic cell death (ICD) is a special way of cancer cell death. When tumor cells die due to external stimuli, three major types of damage-associated molecular patterns (DAMPs) are produced, including exposure of calreticulin on the surface of dying cells, release of ATP molecules by cells, and secretion of high mobility group box-1 (HMGB1). At this time, dying tumor cells change from non-immunogenic to immunogenic, thus causing the body to produce an anti-tumor immune response.

[0006] Therefore, the development of chemotherapy drugs with ICD induction ability, in addition to playing the basic anti-cancer effect of controlling tumor growth and metastasis, can also trigger the immune response in the body, recruit "positive" immune cells to enrich in the microenvironment, transform immune "cold" tumors into "hot" tumors, and antagonize immune escape. It is of great significance for the long-term and effective fight against tumors and reducing the recurrence rate of tumors. This also provides a new strategy for clinical anti-tumor research. Summary of the invention

[0007] Purpose of the invention: The purpose of the present invention is to provide a tetravalent platinum autophagy inducer and its preparation method and application, especially its application in tumor immunotherapy.

[0008] The autophagy inducer of the present invention is a tetravalent platinum complex, which has the ability to induce autophagy in tumor cells, can degrade the immunosuppressive protein Tenascin-C through autophagy, and induce immunogenic cell death in tumors, showing excellent activity against triple-negative breast cancer cells. The tetravalent platinum complex of the present invention, through its unique structural advantages, combines the effects of inducing autophagy in cancer cells and inducing immune response, and can be used to prepare new chemotherapeutic immunotherapy drugs with multiple anti-tumor mechanisms to solve the problems of poor traditional treatment effects and lack of small molecule immunotherapy agents.

[0009] Technical solution: The purpose of the present invention is achieved through the following technical solution:

[0010] The present invention provides a tetravalent platinum autophagy inducer having a general formula I:

[0011]

[0012] in,

[0013] The structure is any of the following:

[0014]

[0015] The tetravalent platinum autophagy inducer of the present invention is preferably the following compound:

[0016]

[0017] The present invention also provides a method for preparing the tetravalent platinum autophagy inducer, comprising the following steps:

[0018] (1) Resuspend the divalent platinum complex in hydrogen peroxide H 2 O 2 The tetravalent platinum complex is prepared by the reaction;

[0019] Wherein, the divalent platinum complex is selected from any one of the following compounds:

[0020]

[0021] (2) reacting the tetravalent platinum complex obtained in step (1) with ketorolac CA O-Benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate (TBTU) and triethylamine (TEA) are dissolved in dimethylformamide (DMF) and stirred at room temperature until the reaction is completed:

[0022] (3) Centrifuge and concentrate the supernatant to obtain a concentrate; add an appropriate amount of methanol, mix it evenly with the concentrate, add it to isopropanol to precipitate a solid, centrifuge, discard the supernatant, wash the precipitate repeatedly with isopropanol and ether, and dry it to obtain the target product.

[0023] A preferred embodiment of the present invention is that in step (2), the tetravalent platinum complex prepared in step (1) and ketorolac CA are dissolved in DMF, TBTU and TEA are slowly added, stirred to dissolve, and reacted at room temperature for 48 hours.

[0024] Preferably, in step (3), the drying method is vacuum drying.

[0025] The present invention also provides a pharmaceutical composition, comprising the tetravalent platinum autophagy inducer and a pharmaceutically acceptable carrier or excipient.

[0026] The pharmaceutical composition of the present invention can be administered in various known ways, such as orally, parenterally, by inhalation spray or via an implanted reservoir. The pharmaceutical composition of the present invention can be administered alone or in combination with other drugs. Oral compositions can be in any oral acceptable dosage form, including but not limited to tablets, capsules, emulsions, suspensions, dispersions and solutions.

[0027] Commonly used pharmaceutically acceptable carriers or excipients include stabilizers, diluents, surfactants, lubricants, antioxidants, adhesives, colorants, fillers, emulsifiers, and the like.

[0028] Sterile injectable compositions can be formulated according to techniques known in the art using suitable dispersants or wetting agents and suspending agents. Pharmaceutically acceptable carriers and solvents that can be used include water, mannitol, sodium chloride solution, and the like.

[0029] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention can be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The dosage level selected depends on a variety of factors, including the activity of the specific compound of the present invention or its salt used, the route of administration, the time of administration, the excretion rate of the specific composition used, the duration of treatment, other drugs, compounds and / or materials used in combination with the specific composition used, the age, sex, weight, general health and previous medical history of the patient being treated, and similar factors well known in the medical field.

[0030] The present invention also provides the use of the tetravalent platinum autophagy inducer in the preparation of anti-tumor drugs.

[0031] A specific preferred embodiment of the present invention is that the tetravalent platinum autophagy inducer is used to prepare tumor immunotherapy drugs.

[0032] The tumor is breast cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colorectal cancer, cervical cancer, prostate cancer, head and neck cancer or glioma.

[0033] The present invention also provides a chemical immune anticancer agent, the effective component of which is PCA.

[0034] Beneficial effects:

[0035] (1) The tetravalent platinum autophagy inducer prepared by the present invention has an immune activation effect, maintains low activity in plasma and normal tissues, and is reduced to a divalent platinum complex and ketorolac in the reducing environment of tumor cells, and exerts its efficacy. Studies have found that PCA can not only exert tumor killing activity through chemotherapy effects such as DNA damage, cell cycle arrest, and induction of apoptosis, but also induce immunogenic death of tumors. At the same time, by inducing autophagy of tumor cells to degrade the immunosuppressive protein Tenascin-C, it promotes the increase of cytotoxic T cells and M1 macrophages in the tumor immune microenvironment, and the decrease of M2 macrophages, thereby achieving tumor immunosuppression.

[0036] (2) The present invention obtains a tetravalent platinum complex by connecting ketorolac (CA) to a cisplatin derivative. The preparation method is simple and easy, and the synthetic raw materials are easily available.

[0037] (3) The tetravalent platinum autophagy inducer prepared by the present invention is a chemical immune anticancer agent with a novel mechanism of action dominated by autophagy, which has dual anti-tumor effects of chemotherapy and triggered immunity, and has great application prospects in combined immunotherapy. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 is the PCA in Example 1 of the present invention 1 H-NMR spectrum (DMSO-d 6 , 500MHz);

[0039] Figure 2 is the PCA in Example 1 of the present invention 13 C-NMR spectrum (DMSO-d 6 , 126MHz);

[0040] Figure 3 is the PCA in Example 1 of the present invention 195 Pt-NMR spectrum (DMSO-d 6 , 86MHz);

[0041] Figure 4 HR-ESI-MS (CH 3 OH) spectrum;

[0042] Figure 5 is the effect of PCA on the expression of DNA damage marker γ-H2AX in MDA-MB-231 cells; Figure 5 A is the effect diagram of the effect on γ-H2AX protein expression; Figure 5 B is the statistical graph of grayscale analysis of WB bands expressing γ-H2AX protein;

[0043] Figure 6The effect of PCA on the cell cycle arrest of MDA-MB-231 cells;

[0044] Figure 7 The effect of PCA on apoptosis of MDA-MB-231 cells;

[0045] Figure 8 The induction of death mode of MDA-MB-231 cells by PCA;

[0046] Fig. 9 The effect of PCA on autophagolysosomes and autophagosomes in the ultrastructure of MDA-MB-231 cells;

[0047] Fig.10 The effect of PCA on autophagy-related proteins LC3 and p62 in MDA-MB-231 cells; Fig.10 A is a strip diagram showing changes in protein expression; Fig.10 B is the grayscale analysis diagram of LC3-II protein expression. Fig.10 C is the grayscale analysis diagram of p62 protein expression.

[0048] Fig.11 PCA is used to investigate the effect of PCA on the autophagy-related protein LC3 in MDA-MB-231 cells. Fig.11 A) and p62( Fig.11 B) Immunofluorescence images of protein changes.

[0049] Fig.12 The effect of PCA on the immunogenic cell death molecular markers of MDA-MB-231 cells; Fig.12 A is the CRT immunofluorescence image on the cell surface after drug administration. Fig.12 B is the fluorescence distribution diagram of CRT flow cytometry detection on the cell surface; Fig.12 C is the statistical graph of the mean fluorescence intensity of CRT flow cytometry on the cell surface; Fig.12 D is the amount of ATP released in the culture medium; Fig.12 E is the detection of HMGB1 release in the culture medium;

[0050] Fig.13 The results of the in vivo tumor inhibition experiment of compound PCA are shown in Table 1. Fig.13 A is a picture of the subcutaneous transplanted tumor removed after drug treatment; Fig.13 B shows the changes in tumor volume; Fig.13 C is the difference in tumor weight; Fig.13 D is the weight change of mice;

[0051] Fig.14Effects of PCA on the expression of autophagy-related proteins LC3 and p62 and immunosuppressive protein Tenascin-C in 4T1 breast cancer transplanted tumor tissues of Balb / c mice ( Fig.14 A); After PCA administration, flow cytometry was used to detect the number of helper T cells (CD4 + T cells), cytotoxic T cells (CD8 + T cells), M1 macrophages (CD86 + ), M2 macrophages (CD206 + )Changes( Fig.14 B); Tenascin-C and CD8 in tumor tissues + Changes in fluorescence intensity of T cells ( Fig.14 C) and changes in serum inflammatory cytokines IFN-γ, IL-6, and IL-10 ( Fig.14 D)

[0052] Fig.15 Schematic diagram of the mechanism of action of PCA of the present invention. DETAILED DESCRIPTION

[0053] The technical solution of the present invention is described in detail below through specific embodiments, but the protection scope of the present invention is not limited to the embodiments.

[0054] The starting materials, reaction reagents, etc. used in the specific embodiments of the present invention are all commercially available. The present invention can be prepared in the form of salt by using a salt-forming method commonly used in the art, for example, dissolving the compound in hydrochloric acid ethanol at room temperature to react to generate a hydrochloride; or adding benzenesulfonic acid to react to generate a benzenesulfonate.

[0055] The experimental methods in the examples of the present invention where specific conditions are not specified are generally carried out under conventional conditions or under conditions recommended by raw material or product manufacturers.

[0056] Example 1 Synthesis of tetravalent platinum complex PCA

[0057] (1) Place 1 g of cisplatin in a flask, add 40 ml of water and stir until dissolved, then slowly drop 50 ml of 30% hydrogen peroxide, heat to 75°C, stir for 5 h in a dark place, filter while hot, place in a refrigerator at 4°C for two days, and vacuum dry to obtain tetravalent Oxoplatin pale yellow crystals with a yield of 76%.

[0058]

[0059] (2) Weigh 100 mg of Oxoplatin, 229.24 mg of CA, and 289 mg of TBTU into a round-bottom flask, add about 20 mL of DMF to dissolve them, slowly drop 125 μL of TEA into the reaction system under stirring, and react at room temperature in the dark for 48 h.

[0060] (3) After the reaction is completed, the reaction liquid is centrifuged, and the supernatant is collected and subjected to rotary evaporation to remove the solvent DMF. When the liquid is concentrated to 1-2 mL, the rotary evaporation is stopped, and about 3 mL of methanol is added to disperse evenly, and then slowly dripped into 30 mL of isopropanol to precipitate gray-white flocs. The precipitate is retained by centrifugation, and a small amount of isopropanol and ether are added to wash it repeatedly for 3 times, and then vacuum dried. The gray solid obtained is the tetravalent platinum complex PCA, with a yield of 43%.

[0061] Tetravalent platinum complex PCA 1 H-NMR spectrum (DMSO-d6, 500MHz) Figure 1 , 13 C-NMR spectrum (DMSO-d6, 126MHz) Figure 2 , 195 Pt-NMR spectrum (DMSO-d6, 86MHz) Figure 3 , high resolution mass spectrometry (CH 3 OH) Figure 4 .

[0062] 1 H NMR (500 MHz, DMSO-d 6 )δ(ppm)7.77-7.75(d 4H),7.62-7.59(t,2H),7.54-7.51(t,4H),6.78-6.77(d,2H),6.56(s,6H),6.21 -6.20(d,2H),4.44 -4.42(m,2H),4.26-4.24(m,2H),4.20-4.17(t,2H),2.79-2.73(m,2H),2.71-2.67(m,2H).

[0063] 13 C-NMR (126 MHz, DMSO-d 6 , ppm):183.92,178.61,145.58,139.55,131.86,128.90,128.83,126.34,124.89,104.25,47.90,46.32,43.94,31.57,25.99,9.15.

[0064] 195 Pt-NMR (86 MHz, DMSO-d 6,ppm):1214.34.

[0065] ESI-MS(negative,m / z):804.09795,805.11215,806.11415,807.11249,808.11232,809.11225,810.11349,812.11544.

[0066] Example 2 Cytotoxic activity test of compound PCA

[0067] The compound PCA prepared in Example 1 of the present invention was subjected to a tumor cell proliferation inhibition test using the CCK-8 method.

[0068] Tumor cell lines MDA-MB-231, A549, A549 / DDP, Huh7, SGC7901, Panc-1, and normal cell line HK-2 were selected respectively. All cell lines were purchased from the Cell Bank of Type Culture Collection Committee of Chinese Academy of Sciences.

[0069] The culture conditions are as follows:

[0070] MDA-MB-231: 10% FBSDMEM complete medium;

[0071] A549: 10% FBS RPMI-1640 complete medium;

[0072] A549 / DDP: 10% FBS RPMI-1640 complete medium;

[0073] Huh7: 10% FBSDMEM complete medium;

[0074] SGC7901: 10% FBSDMEM complete medium;

[0075] Panc-1: 10% FBS RPMI-1640 complete medium;

[0076] HK-2: 10% FBSDMEM complete medium.

[0077] The fetal bovine serum and culture medium were purchased from Gibco.

[0078] PCA and cisplatin (CDDP) were used as the drugs to be tested. After the drugs were exposed to the cells for 48 hours, the survival of the cells was observed and the half inhibitory concentration of the test cells was calculated. The specific steps are as follows:

[0079] (1) Collect the above-mentioned logarithmic phase cells, resuspend them in the above-mentioned complete medium, and adjust the concentration of the cell suspension to 3×10 3 / mL, inoculated in a 96-well plate, 200 μL per well, set up 3 replicates;

[0080] (2) Place the above experimental cells in CO 2 The concentration was 5% in a cell culture incubator and cultured at 37°C for 24 h until the cells adhered to the wall;

[0081] (3) Preparation of drug solution: PCA and cisplatin were prepared as stock solutions with DMSO, respectively, and serially diluted with serum-free medium (Gibco) to concentrations of 200, 100, 50, 25, 12.5, 6.25, 3.125, 1.5625, 0.78125, and 0.390625 μM, and added to the above 96-well plate filled with cells, with 3 replicates for each concentration, and incubated for 48 h;

[0082] (4) CCK-8 reagent (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was diluted 10 times with serum-free medium, and 100 μL of medium containing 10 μL of CCK-8 reagent was added to each well and incubated for 1 h;

[0083] (5) Detect the absorbance of each well at 450 nm using an enzyme reader;

[0084] (6) Calculate the cell survival rate according to the following formula:

[0085]

[0086] Wherein, Abs(sample) is the absorbance value of cells in the sample group; Abs(blank) is the absorbance value of the liquid in the blank group containing no cells but only CCK-8 culture medium solution; Abs(control) is the absorbance value of cells in the control group that have not been treated with drugs;

[0087] (7) Draw a cell survival rate-concentration curve and calculate the half inhibitory concentration (IC) of the compound. 50 ), the results are shown in Table 1.

[0088] Table 1 IC of PCA and cisplatin on different cells 50 Value (μM, 48h)

[0089]

[0090] The results in Table 1 show that PCA showed high toxicity to various tumor cell lines tested, and the effect was significantly better than cisplatin. For the human triple-negative breast cancer cell MDA-MB-231, which has weak cisplatin activity, PCA still showed high toxicity, about 26 times that of cisplatin. This shows that PCA may have the potential to treat triple-negative breast cancer. In addition, PCA has a certain selectivity and is less toxic to normal cells.

[0091] Example 3 Study on the Damage of Tumor Cell DNA by Compound PCA

[0092] Western blot technology was used to test the protein expression level of intracellular γ-H2AX and to detect the DNA damage caused by PCA to MDA-MB-231 tumor cells.

[0093] The specific implementation is as follows:

[0094] (1) Collect logarithmic phase cells and inoculate them into 6-well plates with approximately 200,000 cells per well. Incubate the plates at 37°C and 5% CO 2 Incubate overnight under the same conditions. Aspirate and discard the old culture medium, dilute CDDP to 2 μM and PCA to 0.5, 1, and 2 μM with serum-free culture medium, add 1 mL to each well, and continue incubating in the incubator for 24 hours;

[0095] (2) Protein extraction: cells were collected and total protein was extracted using protease inhibitors (Thermo Fisher Scientific, USA), phosphatase inhibitors (Thermo Fisher Scientific, USA) and RIPA lysis buffer (Biyuntian), lysed on ice for 30 min, centrifuged at 12,000 rpm for 15 min, and the supernatant was taken to determine the protein concentration using the BCA method;

[0096] (3) Sample preparation: The protein concentration of each sample was diluted to 1 μg / μL by adding water and 5× Loading buffer (Biyuntian), and incubated in a metal bath at 100°C for 15 min to obtain the protein sample;

[0097] (4) Perform electrophoresis in electrophoresis buffer (3.03 g Tris, 18.77 g glycine, 1 g SDS, fixed to 1000 mL deionized water), add 10 μL of each protein sample to each well, and perform electrophoresis under the conditions of 80 V for 30 min in the concentration phase and 120 V for 60 min in the separation phase. After the electrophoresis, transfer to the membrane is performed under the conditions of 0.32 A for 60 min.

[0098] (5) After blocking with blocking solution (5% BSA diluted, purchased from Nanjing Shengxing Biotechnology Co., Ltd.) at room temperature for 1 h, the PVDF strips were incubated with the corresponding primary antibody γ-H2AX (Wuhan Abotek Biotechnology Co., Ltd.) at 4°C overnight and washed three times with 1× TBST, 7 min each time;

[0099] (6) HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA) was used for incubation at room temperature for 1 h, washed three times with 1× TBST, and exposed to chemiluminescence. The results are shown in Figure 5 .

[0100] Depend on Figure 5The results showed that compared with the control group without drug treatment, PCA could cause a significant upregulation of γ-H2AX in MDA-MB-231 cells, and the degree of induction was significantly higher than that of CDDP, indicating that PCA caused significant damage to DNA and the DNA damage signaling pathway was being activated.

[0101] Example 4 Study on the effect of compound PCA on tumor cell cycle arrest

[0102] In this example, cell cycle arrest was tested by flow cytometry.

[0103] The specific implementation is as follows:

[0104] (1) MDA-MB-231 cells were cultured at 2×10 5 The cells were inoculated at a density of 100 cells / well on a 6-well plate. After the cells attached to the wall, the culture medium was discarded and CDDP was diluted to 2 μM and PCA was diluted to 0.5, 1, and 2 μM with serum-free culture medium (Gibco). 1 mL was added to each well and incubated at 37°C with 5% CO 2 Continue to culture for 24 h.

[0105] (2) After incubation, the drug-containing medium was discarded, the cells were washed twice with PBS, digested with trypsin (Gibco), centrifuged at 1000 rpm for 3 min, resuspended with PBS and washed twice with centrifugation. Then, 1 mL of pre-cooled 70% ethanol was added to each tube, and the cells were gently pipetted to make single cells and evenly suspended, and fixed at 4°C overnight (12 h).

[0106] (3) The fixed cells were centrifuged and washed twice with pre-cooled PBS. The PI reagent and RNase A were mixed in a volume ratio of 9:1 to prepare a working solution. 500 μL of the mixed working solution was added to each sample and incubated at 37°C in the dark for 30 min. The incubated samples were loaded onto the flow cytometer (the PI reagent, RNase A and working solution used were all from the cell cycle detection kit purchased from Jiangsu Keygene Biotech Co., Ltd.). The experimental results are shown in Figure 6 .

[0107] Depend on Figure 6 It can be seen that compared with the control group without drug treatment, 0.5, 1, and 2 μM PCA acted on MDA-MB-231, which produced a blocking effect on the cell cycle, reduced the number of cells in the G0 / G1 phase, and increased the number of cells in the S and M phases, that is, it mainly affected cell DNA replication and mitosis, and the degree of blocking was concentration-dependent.

[0108] Example 5 Study on the apoptosis of tumor cells induced by compound PCA

[0109] The apoptosis of tumor cells after drug administration was tested by flow cytometry using Annexin V-FITC / PI double staining method.

[0110] The specific implementation is as follows:

[0111] (1) MDA-MB-231 cells were cultured at 2×10 5 The cells were inoculated at a density of 100 cells / well on a 6-well plate. After the cells attached to the wall, the culture medium was discarded and CDDP was diluted to 2 μM and PCA was diluted to 0.5, 1, and 2 μM with serum-free culture medium (Gibco). 1 mL was added to each well and incubated at 37°C with 5% CO 2 Continue culturing for 48 h.

[0112] (2) After incubation, discard the drug-containing medium, wash twice with PBS, digest the cells with EDTA-free trypsin (Gibco), collect the cells after digestion, centrifuge at 1000 rpm and 4°C for 5 min, and discard the supernatant. Wash the cells twice with pre-cooled PBS, centrifuge at 1000 rpm and 4°C for 5 min each time, and discard the supernatant.

[0113] (3) First add 100μl 1×Binding Buffer to each sample, then add 5μl Annexin V-FITC and 5μl PI Staining Solution, blow gently to mix, and incubate at room temperature in the dark for 10 minutes; finally add 400μl 1×Binding Buffer and mix gently. After staining, the samples were detected by flow cytometry within 1 hour (Annexin V-FITC, PIStaining Solution and 1×Binding Buffer used were all from the cell apoptosis detection kit, purchased from Nanjing Novozymes Biotech Co., Ltd.). The results are as follows Figure 7 shown.

[0114] Depend on Figure 7 The results showed that compared with the control group without drug treatment, cisplatin had a weaker ability to induce tumor cell apoptosis, with only 19% of cells undergoing apoptosis at 2μM. PCA was stronger than cisplatin in inducing cell apoptosis at low, medium and high concentrations, causing 30% of cells to apoptosis at 0.5μM, and the apoptosis rate increased with the increase of drug concentration, but the apoptosis rate was 39% at 2μM, which was lower than its IC 50 The concentration (0.9 μM) was inconsistent, suggesting that PCA also induced other death modes besides apoptosis.

[0115] Example 6 Study on the mode of tumor cell death induced by compound PCA

[0116] The CCK-8 method was used to investigate the survival of tumor cells after drug administration by adding different death mode inhibitors.

[0117] The specific implementation is as follows:

[0118] (1) MDA-MB-231 cells were cultured at 5×10 3 / well were inoculated in a 96-well plate, and the old culture medium was discarded after the cells adhered to the wall. A blank control group, a PCA group with a concentration of 2 μM, an inhibitor group, apoptosis inhibitor Z-VAD-FMK (Z-VAD, 10 μM), an autophagy inhibitor 3-methyladenine (3-MA, 100 μM), a ferroptosis inhibitor Ferrostatin-1 (Ferr-1, 10 μM), a necrosis inhibitor Necrostatin-1 (Necro-1, 10 μM), a pyroptosis inhibitor Disulfiram (DSF, 10 μM), and a PCA and inhibitor mixed group were set up.

[0119] (2) 100 μL of medium containing PCA, inhibitor, or a mixture of PCA and inhibitor was added to each well, and serum-free medium was added to the blank control group. After 48 h of drug action, the old medium was discarded. 100 μL of DMEM medium (Gibco, USA) containing 10 μL of CCK-8 reagent (purchased from Nanjing Novozyme Biotechnology Co., Ltd.) was added to each well and incubated for another 1 h;

[0120] (3) Detect the absorbance of each well at 450 nm using an ELISA reader;

[0121] (4) Calculate the cell survival rate according to the following formula:

[0122]

[0123] Among them, Abs(sample) is the absorbance value of cells in the sample group; Abs(blank) is the absorbance value of the liquid in the blank group (serum-free medium containing only CCK-8 but no cells); Abs(control) is the absorbance value of cells in the control group that have not been treated with drugs. The results are shown in Figure 8 .

[0124] Depend on Figure 8 The results showed that compared with the group treated with PCA alone, the cell survival rate increased from 56% to 93% after PCA combined with 3-MA ( Figure 8 B), 3-MA can effectively inhibit PCA-induced cell death, indicating that PCA affects tumor cell survival by regulating the autophagy signaling pathway. In addition, the cell survival rate of PCA combined with Z-VAD increased to 75% ( Figure 8A), indicating that PCA can also induce cell apoptosis. The other inhibitors of pyroptosis, necrosis, and ferroptosis had no significant effect on cell survival rate ( Figure 8 CE). Therefore, PCA-treated MDA-MB-231 cells undergo multiple types of death, mainly autophagic death and supplemented by apoptosis.

[0125] Example 7 Study on the Compound PCA Inducing Autophagy in Tumor Cells

[0126] After treating tumor cells with PCA prepared in Example 1 of the present invention, the level of intracellular autophagy was investigated, and the experimental methods adopted transmission electron microscopy, Western blot and immunofluorescence. The cell line selected was MDA-MB-231.

[0127] Transmission electron microscopy: MDA-MB-231 cells growing in the logarithmic phase were collected. After the cells adhered to the wall, the original culture medium was discarded and drug-containing culture medium containing 2 μM cisplatin or 2 μM PCA (Gibco) was added. A control group (serum-free culture medium) was set up and incubated at 37°C with 5% CO 2 The samples were washed twice with pre-cooled PBS, digested with trypsin, and then added with 1 mL of electron microscope fixative. After dehydration, infiltration, embedding, sectioning, and staining, the samples were observed under a transmission electron microscope. Fig. 9 .

[0128] Depend on Fig. 9 The results showed that a small amount of autophagolysosomes were produced in the control group and the cisplatin group, indicating that the level of autophagy in cells was low under normal conditions or after treatment with cisplatin, and cisplatin hardly induced cell autophagy; the mitochondrial morphology and structure in the cytoplasm were round or oval, with clear and flat cristae and neat arrangement, indicating that the mitochondrial structure was relatively normal and the damage was not obvious. In the cytoplasm of the PCA group, a large number of autophagolysosomes and autophagosomes were observed, indicating that a large amount of autophagy occurred in cells treated with PCA. In addition, the volume of most mitochondria in the cytoplasm was significantly reduced, showing spherical and rod-shaped, with obvious condensation, thickened cristae, reduced or even disappeared, and the intercrista cavity was significantly widened, indicating that PCA would cause mitochondrial damage.

[0129] Western blot method:

[0130] (1) MDA-MB-231 cells were cultured at 2×10 5 The cells were inoculated at a density of 100 cells / well on a 6-well plate. After the cells attached to the wall, the culture medium was discarded and CDDP was diluted to 2 μM and PCA was diluted to 0.5, 1, and 2 μM with serum-free culture medium (Gibco). 1 mL was added to each well and incubated at 37°C with 5% CO 2 Continue to culture for 6 h.

[0131] (2) Protein extraction: cells were collected and total protein was extracted using protease inhibitors (Thermo Fisher Scientific, USA), phosphatase inhibitors (Thermo Fisher Scientific, USA) and RIPA lysis buffer (Shanghai Biotech Biotechnology Co., Ltd.). The cells were lysed on ice for 30 min and centrifuged at 12,000 rpm for 15 min. The supernatant was taken and the protein concentration was determined by the BCA method.

[0132] (3) Sample preparation: The protein concentration of each sample was diluted to 1 μg / μL by adding water and 5× Loading buffer (Biyuntian Biotechnology Co., Ltd., Shanghai, China) and incubated in a metal bath at 100°C for 15 min to obtain the protein sample;

[0133] (4) Perform electrophoresis in electrophoresis buffer (3.03 g Tris, 18.77 g glycine, 1 g SDS, fixed to 1000 mL deionized water), add 10 μL of each protein sample to each well, and perform electrophoresis under the conditions of 80 V for 30 min in the concentration phase and 120 V for 60 min in the separation phase. After the electrophoresis, perform membrane transfer under the conditions of 0.32 A for 60 min.

[0134] (5) After blocking with blocking solution (5% BSA dilution) at room temperature for 1 h, the PVDF strips were incubated with the corresponding primary antibodies LC3 (Wuhan Tri-Eagle Biotechnology Co., Ltd.) and p62 (Wuhan Tri-Eagle Biotechnology Co., Ltd.) at 4°C overnight, and washed three times with 1× TBST, 7 min each time;

[0135] (6) HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA) was used for incubation at room temperature for 1 h, washed three times with 1× TBST, and exposed to chemiluminescence. The results are shown in Fig.10 .

[0136] Depend on Fig.10 The results showed that compared with the control group, LC3-II in the cisplatin group increased slightly, and p62 was not significantly downregulated; while LC3-II in the PCA group increased significantly, and p62 was significantly downregulated. In order to amplify the changes in LC3 and p62 proteins, 30 μM chloroquine (CQ) was added to block autophagy, and it was found that the LC3-II protein on the autophagosome membrane induced by the PCA group increased slightly.

[0137] Immunofluorescence:

[0138] MDA-MB-231 cells were cultured at 8×10 4The cells were inoculated at a density of 1.54 × 10 / well on the slide. After the cells adhered to the wall, the original culture medium was discarded, and a control group with only serum-free culture medium and a drug-containing culture medium were set up. The cisplatin group had a concentration of 2 μM, and the PCA drug concentration was 2 μM. After 6 hours of drug treatment, the drug-containing culture medium was discarded, and the cells were washed with PBS for 3 times, each time for 5 minutes. 500 μL of 4% paraformaldehyde was added to each well for 15 minutes, and PBS was rinsed 3 times. 0.1% Triton-100 was added to break the membrane, and PBS was washed. 5% goat serum (Gibco, USA) was added and incubated at room temperature for 1 hour for blocking. The blocking solution was aspirated and the primary antibodies LC3 (Wuhan Sanying Biotechnology Co., Ltd.) and p62 (Wuhan Sanying Biotechnology Co., Ltd.) were incubated at 4°C overnight. After the incubation, the cells were washed with PBS 3 times, and the corresponding secondary antibodies (Goatanti-Rabbit IgG (H+L) Secondary Antibody, HRP purchased from Thermo, USA) were added and incubated for 1 hour. The secondary antibodies were washed with PBS, and DAPI (Shanghai Biyuntian Biotechnology Co., Ltd.) was stained for 5 minutes. Fluorescence was captured using a laser confocal microscope. The results are shown in Fig.11 .

[0139] Depend on Fig.11 The results showed that the immunofluorescence results showed that the fluorescence intensity of LC3 increased after PCA treatment ( Fig.11 A) p62 fluorescence intensity decreased ( Fig.11 B), indicating that PCA can induce autophagy.

[0140] Example 8 Detection of molecular markers of PCA-induced immunogenic death of tumor cells

[0141] The early molecular mechanism of immunogenic death after the cells were treated with PCA prepared in Example 1 of the present invention was studied, and the experimental methods used immunofluorescence, flow cytometry, and ELISA. The cell line was MDA-MB-231, and after PCA acted on the cells for 24 hours, the calreticulin (CRT) on the cell surface, the ATP secreted outside the cells, and the high mobility group protein B1 (HMGB1) content released outside the cells at a later stage were detected. The specific operation steps are as follows:

[0142] (1) MDA-MB-231 cells were inoculated on a 24-well plate and cultured at 37°C for 24 h. Drug-containing serum-free culture medium (Gibco) was added and cultured for another 24 h.

[0143] (2) After incubation, the cells were washed twice with cold PBS, fixed with 4% paraformaldehyde for 15 min, blocked with 5% goat serum albumin (Gibco) diluted with 3% BSA for 1 h, and CRT antibody (Abcam, USA) (1:50, diluted with 5% BSA) was added and incubated overnight at 4°C;

[0144] (3) The cells were washed three times with cold PBS, incubated with FITC fluorescent secondary antibody (Cell Signaling Technology) (1:500, TBST dilution) in the dark for 1 h, washed with PBS, stained with DAPI for 5 min, and washed with PBS. The cell fluorescence was photographed by laser scanning confocal microscopy. The results are shown in Fig.12 A.

[0145] (4) MDA-MB-231 cells were cultured at 2×10 5 / well were inoculated in 6-well plates and placed in an incubator. After the cells adhered to the wall, the original culture medium was discarded and the drug-containing culture medium was added. The control group (serum-free culture medium was added), the cisplatin group 8μM, and the PCA drug concentrations were 2μM, 4μM, and 8μM, respectively. After 24 hours of drug treatment, the drug-containing culture medium was discarded, the cells were washed twice with pre-cooled PBS, and the cells were digested with trypsin and collected in a centrifuge tube. Centrifuged at 800rpm for 5min, washed twice with PBS, and each tube was blocked with 1% BSA for 30min. The cells were washed twice with PBS, and CRT antibody (Abcam, USA) (1:50, 5% BSA dilution) was added and incubated at room temperature for 30min. The cells were immediately detected by flow cytometry. The results are shown in the figure. Fig.12 B. Fig.12 C.

[0146] (5) MDA-MB-231 cells were seeded in 6-well plates at a number of 2×10 5 After culturing for 24 hours, the culture medium was discarded and 1 mL of serum-free culture medium containing 8 μM cisplatin and 1 μM, 2 μM, 4 μM, and 8 μM PCA was added. The culture was continued for 24 hours. The supernatant was carefully collected and the ATP concentration in the supernatant was detected using an enhanced ATP detection kit (Shanghai Biotech Biotechnology Co., Ltd., S0027) according to the instructions provided by the manufacturer. The results are shown in Fig.12 D.

[0147] (6) MDA-MB-231 cells were cultured at 2×10 5 The cells were seeded at a density of 100 cells / well on a 6-well plate. After the cells adhered to the wall, the culture medium was discarded and 1 mL of serum-free culture medium containing 8 μM cisplatin and 1 μM, 2 μM, 4 μM, and 8 μM PCA was added. The culture was continued for 36 h, and the supernatant was collected. According to the instructions provided by the manufacturer, the HMGB1 in the supernatant was detected using an ELISA detection kit (Shenzhen Xinbosheng Biotechnology Co., Ltd., NOV-NB-S11133). The results are shown in Fig.12 E.

[0148] Fig.12The results showed that compared with the control group without drug treatment, after PCA acted on MDA-MB-231 cells, there was a more obvious CRT exposure, and CRT was spotted and unevenly distributed on the surface of the cells, and the CRT on the cell membrane surface increased significantly (see Fig.12 AC); released ATP ( Fig.12 D) and HMGB1( Fig.12 E) significantly increased. Through the verification of these markers, it was further confirmed that PCA has a good potential to induce ICD.

[0149] Example 9 In vivo tumor inhibition experiment of compound PCA

[0150] (1) Subcutaneous tumor transplantation: Mouse breast cancer 4T1 cells in the logarithmic growth phase (purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) were digested with 0.25% trypsin and the cell concentration was adjusted to 2×10 6 / mL, 0.1mL of cell suspension was injected subcutaneously under the left armpit of Balb / c mice (female, 6 weeks old, 18-20g, purchased from Hangzhou Ziyuan Experimental Animal Technology Co., Ltd.). The tumor growth was observed, and the transplanted tumor model was successfully established when the tumor grew to 5mm×5mm. The length and width of the tumor were measured daily, and the tumor volume was calculated according to volume = length*width*width / 2.

[0151] (2) When the tumor volume reaches 100 mm 3 Afterwards, the above 18 mice were randomly divided into 3 groups (Control group, CDDP group and PCA group). The two drug-treated groups were injected with 5 mg / kg cisplatin and 13.5 mg / kg PCA through the tail vein, respectively, and the control group was given an equal volume of normal saline, injected once every two days. After the drug administration, the tumor was removed, weighed and photographed, and the volume of the subcutaneous tumor during the drug administration period was recorded and observed.

[0152] (3) Data processing: Data were expressed as mean ± standard deviation (Mean ± SD) and analyzed using Graphpad Prime 8.0 software; unpaired two-sided t-test was used for comparison between the two groups. Fig.13 .

[0153] Depend on Fig.13 It can be seen that compared with the control group, after administration of cisplatin, the tumor volume decreased, but the degree of decrease was smaller, and the tumor growth inhibition rate was 13.88%, indicating that cisplatin has a weak therapeutic effect on triple-negative breast cancer; after PCA treatment, the tumor weight of mice was significantly reduced, and the tumor growth inhibition rate was 57.71%, which is about 4.2 times that of cisplatin, and the therapeutic effect is significantly better than cisplatin.

[0154] Example 10 Compound PCA induces autophagy in vivo to promote immune killer cell infiltration

[0155] Western blot was used to detect the changes of autophagy-related proteins LC3 and p62 and immunosuppressive protein Tenascin-C in the tumors of mice after administration; flow cytometry was used to detect the changes of cytotoxic T cells (CD8 + T cells), helper T cells (CD4 + T cells), M1 macrophages (CD86 + cells), M2 macrophages (CD206 + Immunofluorescence was used to detect the changes of Tenascin-C and CD8 + T expression was detected; the levels of inflammatory cytokines (IFN-γ, IL-6, IL-10) in mouse serum were detected by ELISA technology.

[0156] The specific implementation plan is as follows:

[0157] (1) The Balb / c mice in Example 9 were killed, and the tumors were collected and added with 500 μL RIPA lysis buffer (Shanghai Biyuntian Biotechnology Co., Ltd.), protease inhibitors (Thermo Corporation, USA), and phosphatase inhibitors (Thermo Corporation, USA). After grinding with a homogenizer, the tumors were lysed on ice for 30 min, and centrifuged at 12000 rpm for 15 min. The supernatant was taken and the protein concentration was determined by the BCA method;

[0158] (2) Sample preparation: The protein concentration of each sample was diluted to 5 μg / μL by adding water and 5× Loading buffer (Biyuntian Biotechnology Co., Ltd., Shanghai, China) and incubated in a metal bath at 100°C for 15 min to obtain the protein sample;

[0159] (3) Perform electrophoresis in electrophoresis buffer (3.03 g Tris, 18.77 g glycine, 1 g SDS, fixed to 1000 mL deionized water), add 10 μL of each protein sample to each well, and perform electrophoresis under the conditions of 80 V for 30 min in the concentration phase and 120 V for 60 min in the separation phase. After the electrophoresis, transfer to the membrane under the conditions of 0.32 A for 60 min.

[0160] (4) After blocking with blocking solution (5% BSA dilution) at room temperature for 1 h, the PVDF strips were incubated with the corresponding primary antibodies LC3 (Wuhan Tri-Eagle Biotechnology Co., Ltd.) and p62 (Wuhan Tri-Eagle Biotechnology Co., Ltd.) and Tenascin-C (Wuhan Tri-Eagle Biotechnology Co., Ltd.) at 4°C overnight, and washed three times with 1× TBST, 7 min each time;

[0161] (4) HRP-labeled goat anti-rabbit secondary antibody (Abcam, USA) was used for incubation at room temperature for 1 h, washed three times with 1× TBST, and exposed to chemiluminescence. The results are shown in Fig.14 A.

[0162] (5) Prepare the tumor into a single-cell suspension. Cut the tumor into pieces and digest it with an enzyme mixture (type I collagenase, hyaluronidase, DNase I, Sigma) at 37°C for 1 h. Invert and mix every 15 min during the digestion process. After digestion, gently grind the mixture and filter it through a 200-mesh sieve to obtain a single-cell suspension.

[0163] (6) Red blood cells in the single-cell suspension were removed using red blood cell lysis buffer (Sigma), and the cells were washed twice with 1× PBS.

[0164] (7) The collected cells were blocked with anti-mouse CD16 / 32 (BioLegend, USA), and APC anti-mouse CD3 (CD3-APC), FITC anti-mouse CD4 (CD4-FITC), PE anti-mouse CD8a (CD8a-PE), and Brilliant Violet 421 according to the manufacturer's instructions. TM Anti-mouse CD86 (CD86-Brilliant Violet 421 TM ), PE anti-mouse CD206 (CD206-PE), PE / Cyanine7 anti-mouse F4 / 80 (F4 / 80-PE / Cyanine7) (BioLegend, USA) were used for staining. Finally, the stained cells were analyzed by flow cytometry. The results are shown in Fig.14 B.

[0165] (8) The frozen sections of the tumor were placed at room temperature for 30 minutes, fixed with 4% paraformaldehyde for 10 minutes, washed with PBS three times, 5 minutes each time, and blocked with blocking solution (5% BSA diluted) at room temperature for 1 hour. Tenascin-C (Wuhan Sanying Biotechnology Co., Ltd.) and CD8 + T (BioLegend, USA) was incubated at 4°C overnight, washed 3 times with PBS, and the liquid was aspirated. AlexaFluor TM The slides were incubated with 647 goat anti-mouse IgG2a (Thermo Company, USA) for 1 h, washed 3 times with PBS, and sealed with DAPI anti-fluorescence quencher (Shanghai Bio-Tech Co., Ltd.). The fluorescence was observed under a confocal microscope. The results are shown in Fig.14 C.

[0166] (9) The eyeballs of the mice were removed and blood was collected. The blood was placed at room temperature for 1 h, allowed to coagulate and stratify, and then centrifuged at 3500 rpm for 15 min. The upper serum was carefully collected and the levels of cytokines IL-6, IFN-γ, and IL-10 in the serum were detected using an ELISA kit (Shenzhen Xinbosheng Biotechnology Co., Ltd.). The results are as follows: Fig.14 As shown in D.

[0167] Fig.14 The results showed that at the end of treatment, compared with the control group injected with normal saline, the conversion of LC3-I to LC3-II in the cisplatin group increased slightly, and p62 and Tenascin-C decreased, but none of them were significant. In the PCA group, LC3-II in tumor tissue increased significantly, and the expression of p62 and Tenascin-C proteins decreased significantly, indicating that after PCA treatment, a large amount of autophagy occurred in the tumor tissue, resulting in the degradation of Tenascin-C ( Fig.14 A). After PCA treatment, the tumor immune microenvironment in mice changed significantly. First, CD8 + T and CD4 + The proportion of T cells increased significantly, and CD4 + T increased 5 times, CD8 + T increased by 8 times, which shows that PCA can increase the infiltration of immune cells into tumors in vivo to achieve a synergistic effect of killing tumors. Secondly, M1 macrophages increased by about 2 times; M2 macrophages decreased by 5 times, indicating that PCA promoted the polarization of macrophages from M2 to M1 ( Fig.14 B) Compared with the control group, the CD8 + There was no significant change in the fluorescence intensity of T cell surface markers CD8 and Tenascin-C. In the PCA treatment group, CD8 fluorescence was enhanced and Tenascin-C fluorescence was weakened, indicating that after a period of PCA administration, PCA can stimulate the reduction of Tenascin-C in the tumor site of mice, thereby increasing CD8 + T cell infiltration, thereby playing a better anti-cancer role ( Fig.14 C). In addition, the pro-inflammatory cytokines IL-6 and IFN-γ in serum increased, while the anti-inflammatory cytokine IL-10 decreased, further proving that Pt-FFA can activate a strong immune response ( Fig.14 D).

[0168] In summary, if Fig.15 As shown in Figure 2, PCA has multiple anti-tumor activities. PCA can induce autophagy, which leads to more degradation of Tenascin-C, thereby increasing CD4 + T, CD8 +The infiltration of T cells promotes the transformation of M2 macrophages to M1, reshapes the tumor immune microenvironment, and can also induce tumor immunogenic death, with synergistic anti-tumor properties of chemotherapy-immunotherapy. Therefore, the tetravalent platinum autophagy inducer provided by the present invention can be used to prepare anti-tumor drugs, and has great application prospects in the development of chemotherapy-immune anticancer drugs.

[0169] As described above, although the present invention has been shown and described with reference to specific preferred embodiments, it should not be construed as limiting the present invention itself. Various changes in form and details may be made without departing from the spirit and scope of the present invention as defined in the appended claims.

Claims

1. A tetravalent platinum autophagy inducer having the general formula I: in, The structure is any of the following:

2. The tetravalent platinum autophagy inducer according to claim 1, characterized in that Selected from:

3. A method for preparing the tetravalent platinum autophagy inducer according to any one of claims 1 to 2, characterized in that: The following steps are involved: (1) reacting a divalent platinum complex with hydrogen peroxide H2O2 to obtain a tetravalent platinum complex; Wherein, the divalent platinum complex is selected from any one of the following compounds: (2) reacting the tetravalent platinum complex obtained in step (1) with ketorolac CA O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate TBTU and triethylamine are dissolved in dimethylformamide DMF and stirred at room temperature until the reaction is completed; (3) After centrifugation, the supernatant is concentrated to obtain a concentrated solution; methanol is added, the concentrated solution is mixed evenly, and then the solution is added to isopropanol to precipitate a solid; the solid is centrifuged, the supernatant is discarded, and the precipitate is repeatedly washed with isopropanol and ether, and dried to obtain the target product.

4. A pharmaceutical composition, characterized in that It comprises the tetravalent platinum autophagy inducer according to any one of claims 1 to 2 and a pharmaceutically acceptable carrier or excipient.

5. Use of the tetravalent platinum autophagy inducer according to any one of claims 1 to 2 in the preparation of anti-tumor drugs.

6. The use according to claim 5, characterized in that: The tetravalent platinum autophagy inducer is used for preparing tumor immunotherapy drugs.

7. The use according to claim 5, characterized in that: The tumor is breast cancer, lung cancer, liver cancer, gastric cancer, pancreatic cancer, ovarian cancer, colorectal cancer, cervical cancer, prostate cancer, head and neck cancer or glioma.

8. A chemoimmunoassay anticancer agent, characterized in that: Its active ingredient is PCA.

Images