Use of fluphenazine in the preparation of a medicament for treating cancer with iron overload

Abstract

The application discloses application of fluphenazine in preparation of a medicine for treating cancer with iron overload, and relates to the technical field of biological medicine.The application provided can significantly inhibit growth of liver cancer cells in a mouse body by injecting FPZ or DFO alone compared with a physiological saline injection group, and has a stronger inhibiting effect on liver cancer by jointly injecting FPZ and DFO; the combined use of FPZ and DFO can significantly inhibit an IRP2 activation effect caused by DFO; FPZ can activate KLF14 to reduce expression of IRP2, thereby reducing iron pool level of liver cancer cells and inhibiting liver cancer; DFO can reduce iron pool level in the liver cancer cells by chelating intracellular iron, thereby inhibiting liver cancer, but can feedback increase expression of IRP2 and activate an IRPs-IRE system; and the combined use of FPZ and DFO can significantly inhibit the feedback effect, thereby further enhancing the inhibiting effect on liver cancer.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the use of fluphenazine in the preparation of medicaments for treating cancers with iron overload. Background Technology

[0002] Liver cancer lacks effective early diagnostic markers, resulting in high recurrence and mortality rates. Currently, surgical resection or liver transplantation are the most effective treatments for liver cancer. However, most liver cancer patients are diagnosed at an advanced stage, making surgery unsuitable. Therefore, discovering new therapeutic drugs or strategies is of significant clinical value. Liver cancer primarily originates from hepatocytes, typically developing from chronic liver disease accompanied by chronic inflammation and fibrosis, progressing to cirrhosis, and ultimately transforming into liver cancer. High-risk factors inducing liver cancer include hepatitis B / C virus, fatty liver disease, and alcoholic cirrhosis.

[0003] Iron overload is also a risk factor for liver cancer. In 1996, researchers discovered spontaneous hepatocellular carcinoma in a mouse model of iron overload, indicating a correlation between iron overload and liver cancer development. Epidemiological studies have found that one-third of patients with long-term cirrhosis have high iron load in their livers; this "cirrhosis-associated liver iron overload" can induce liver cancer. Follow-up studies of patients with alcoholic cirrhosis or chronic hepatitis B infection have found that patients with higher liver iron reserves have a higher risk of developing liver cancer than those with lower or normal iron reserves. Extensive research reports that the iron content in the liver tissue of patients with chronic hepatitis C is 2-5 times higher than that in normal liver tissue. Severe iron overload in the liver of patients with hereditary hemochromatosis can lead to cirrhosis, and patients with cirrhosis have a nearly 20-fold increased risk of developing liver cancer.

[0004] Intracellular iron overload has been reported to be associated with the development and progression of various tumors, including liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, and glioma. Drugs can inhibit the occurrence and development of iron overload-related tumors by inducing cellular iron deficiency. Among these methods, the use of iron chelators is a commonly used approach to induce cellular iron deficiency. Deferroamine (DFO, CAS Registry No. 70-51-9) can chelate intracellular iron ions, inducing cellular iron deficiency and thus inhibiting the growth of tumor cells, including liver cancer cells, breast cancer cells, colorectal cancer cells, and ovarian cancer cells, which are associated with iron overload. Thiosemicarbazone-24 (TSC24) significantly reduces the concentration of iron ions in tumor cells by blocking iron uptake and interfering with the normal regulation of cellular iron homeostasis, thereby inducing cellular iron deficiency, reducing tumor cell activity, and inhibiting tumor cell growth. However, after iron chelators act on cells, they will feedback to increase the expression of iron regulatory protein 2 (IRP2), thereby activating the iron regulatory protein-iron response element (IRP-IRE) mechanism that regulates cellular iron homeostasis, thus reducing their effect.

[0005] Fluphenazine (FPZ, CAS Registry No. 69-23-8) is a phenothiazine antipsychotic drug used to treat schizophrenia and bipolar disorder. It was approved for clinical use in 1972. Fluphenazine primarily works by antagonizing dopamine receptors (DA2) in the mesolimbic, substantia nigra, and tuberoinfundibular neural pathways. Inhibition of postsynaptic dopamine receptors in the mesolimbic pathway can have a positive effect on schizophrenia-related symptoms. In 1985, Hait et al. first discovered that FPZ could inhibit the proliferation of L1210 leukemia cells. This finding opened the door to research on FPZ in oncology. Subsequently, more and more studies have shown that FPZ has antitumor properties against breast cancer, colorectal cancer, neuroblastoma, and glioma. Specifically, FPZ can inhibit tumor cell growth by inhibiting the MAPK signaling pathway in breast cancer cells, reducing mitochondrial function in glioblastoma cells, and decreasing the activity and expression of cyclooxygenase (COX)-2 in colorectal cancer cells. The mechanism by which fluphenazine affects the growth of liver cancer cells remains unclear, and the relationship between fluphenazine and the regulation of intracellular iron ion levels has not yet been discovered or reported. The effective application of fluphenazine in the treatment of liver cancer still needs further research and development. Summary of the Invention

[0006] In view of the deficiencies in the prior art, the present invention provides the use of fluphenazine in the preparation of a medicament for treating cancers with iron overload.

[0007] The technical solution of the present invention is as follows:

[0008] This invention provides the use of fluphenazine in the preparation of a medicament for treating cancers with iron overload, wherein the fluphenazine is fluphenazine itself or a pharmaceutically acceptable salt.

[0009] Preferably, the cancer is liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, or glioma.

[0010] The present invention also provides the use of fluphenazine and deferoxamine in combination in the preparation of a medicament for treating cancers with iron overload, wherein the fluphenazine is fluphenazine itself or a pharmaceutically acceptable salt, and the deferoxamine is deferoxamine itself or a pharmaceutically acceptable salt.

[0011] Preferably, the cancer is liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, or glioma.

[0012] Preferably, the dosage form of the drug is a liquid injection, powder for injection, tablet, capsule, powder, pill, oral liquid, ointment, granule, or dressing. In this application, a liquid injection is used, which is injected intraperitoneally into nude mice when used in combination.

[0013] When the drug is in the form of a liquid injection, the dose of fluphenazine is 8 mg / kg and the dose of deferoxamine is 50 mg / kg.

[0014] Compared with the saline injection group, injection of FPZ or DFO alone can significantly inhibit the growth of liver cancer cells in mice, while the combined injection of FPZ and DFO has a stronger inhibitory effect on liver cancer.

[0015] The present invention also provides a pharmaceutical composition comprising fluphenazine or a pharmaceutically acceptable salt, and deferoxamine or a pharmaceutically acceptable salt.

[0016] The dosage form of the drug is liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, ointment, granule, or dressing. In this application, a liquid injection is used, which is injected intraperitoneally into nude mice when used in combination.

[0017] When the drug is in the form of a liquid injection, the dose of fluphenazine is 8 mg / kg and the dose of deferoxamine is 50 mg / kg.

[0018] The expression level of IRP2 in the DFO monotherapy group was significantly higher than that in the control group, while the expression level of IRP2 in the FPZ and DFO combination therapy group was significantly lower than that in the DFO monotherapy group. DFO monotherapy can significantly increase IRP2 expression, while FPZ and DFO combination therapy can significantly inhibit the IRP2 activation effect induced by DFO. This indicates that there is a synergistic effect between the two.

[0019] The beneficial effects of this invention are:

[0020] The application provided by this invention shows that, compared with the saline injection group, injection of FPZ or DFO alone can significantly inhibit the growth of liver cancer cells in mice, while the combined injection of FPZ and DFO has a stronger inhibitory effect on liver cancer. The combined use of FPZ and DFO can significantly inhibit the IRP2 activation effect induced by DFO. FPZ can activate KLF14 to reduce IRP2 expression, thereby reducing the iron pool level of liver cancer cells and inhibiting liver cancer. DFO inhibits liver cancer by reducing the iron pool level of liver cancer cells through chelation of intracellular iron, but it will feedback increase the expression of IRP2 and activate the IRPs-IRE system. The combined use of FPZ and DFO can significantly inhibit the feedback effect and further enhance the inhibitory effect on liver cancer. Attached Figure Description

[0021] Figure 1 Figure 1 shows the results of detecting the transcriptional activities of KLF14 and IRP2 by fluphenazine; where A: the results of detecting the transcriptional activity of KLF14 after HepG2 cells were treated with 10 μmol / L FPZ for 48 hours; B: the results of detecting the transcriptional activity of IRP2 after HepG2 cells were treated with 10 μmol / L FPZ for 48 hours; **P < 0.01.

[0022] Figure 2 Figure 1 shows the results of detecting the expression levels of KLF14 and IRP2 in hepatocellular carcinoma cells treated with fluphenazine. A: qRT-PCR results of KLF14 and IRP2 in hepatocellular carcinoma cells treated with the drug; B: Western blot results and statistical graph of KLF14 and IRP2 in HepG2 cells treated with the drug; C: Western blot results and statistical graph of KLF14 and IRP2 in Huh7 cells treated with the drug; *P<0.05, **P<0.01, ***P<0.001.

[0023] Figure 3 The fluorescence intensity results of intracellular iron ion levels were obtained after FPZ treatment of liver cancer cells for 36 hours. A: Fluorescence intensity results of intracellular iron ion levels in HepG2 cells treated with FPZ; B: Fluorescence intensity results of intracellular iron ion levels in Huh7 cells treated with FPZ. **P < 0.01, ns indicates no statistical significance.

[0024] Figure 4 After treating liver cancer cells with 10 μmol / L FPZ for 24 hours, fluorescence imaging and statistical graphs of intracellular iron ion levels were displayed; where A: fluorescence imaging; B: statistical graph; **P < 0.01.

[0025] Figure 5Cell growth curves of hepatocellular carcinoma cells treated with 10 μmol / L FPZ; where A: cell growth curve of HepG2 cells after treatment with 10 μmol / L FPZ; B: cell growth curve of Huh7 cells after treatment with 10 μmol / L FPZ; ***P < 0.001.

[0026] Figure 6 The graphs show the growth curves of liver cancer cells after treatment with DFO and FPZ; where A: cell growth curve of HepG2 cells after treatment with DFO and FPZ; B: cell growth curve of Huh7 cells after treatment with DFO and FPZ; **P<0.01, ***P<0.001.

[0027] Figure 7 Figure 1 shows the effects of FPZ and DFO on subcutaneous tumorigenesis of liver cancer cells; where A: curve of subcutaneous tumor volume growth; B: schematic diagram of tumor tissue after removal in each group; C: weight detection results of tumor tissue after removal; *P<0.05, **P<0.01, ***P<0.001.

[0028] Figure 8 Figure 1 shows the qRT-PCR and Western blot results of KLF14 and IRP2 in subcutaneous tumor tissue. A: qRT-PCR results of KLF14 and IRP2 in tumor tissue of the FPZ monotherapy group; B: Western blot results of KLF14 and IRP2 in tumor tissue of the FPZ monotherapy group; C: Western blot results of KLF14 in tumor tissue of the FPZ monotherapy group and the DFO / FPZ combination therapy group; D: Western blot results of IRP2 in tumor tissue of the control group, FPZ monotherapy group, DFO monotherapy group, and DFO / FPZ combination therapy group. *P < 0.05, ***P < 0.001.

[0029] Figure 9 The results and statistical graphs of immunohistochemistry in subcutaneous tumor tissue are shown; where A: schematic diagram of Ki67 and Perl'Blue immunohistochemical staining in tumor tissue; B: statistical graph of immunohistochemical results; **P<0.01, ***P<0.001.

[0030] Figure 10 This diagram illustrates the mechanism by which fluphenazine and deferoxamine act on liver cancer. Detailed Implementation

[0031] Fluphenazine hydrochloride: The source is the National Standard Material Center, purchased from the distributor Henan Beina Biotechnology Testing and Inspection Co., Ltd., Cat. No. 100162.

[0032] Example 1

[0033] Fluphenazine activates KLF14 and inhibits IRP2 expression.

[0034] The effect of FPZ on the transcriptional activities of KLF14 and IRP2 in hepatocellular carcinoma cells was detected using a luciferase reporter gene assay. Luciferase reporter gene plasmids PGL4-KLF14-1uc and PGL4-IRP2-1uc were constructed. A 1kb fragment each upstream and downstream of the KLF14 / IRP2 transcription start site (-1000bp to +1000bp) was amplified from genomic DNA and then ligated into the pGL4-luciferase reporter vector (Promega, Cat. No. E4611). Primers used for plasmid construction are shown in Table 1 below.

[0035] Table 1 Primers for constructing luciferase reporter gene plasmids

[0036]

[0037] HepG2 cells were seeded into 12-well plates and transfected with either the PGL4-KLF14-luc plasmid or the PGL4-IRP2-luc plasmid using a Lipofectamine 2000 (Life Technologies) at a cell density of 60%-70%. Twelve hours after transfection, cells were treated with fluphenazine hydrochloride (FPZ) at a final concentration of 10 μmol / L (the hydrochloride salt was used in this example, but will be referred to as FPZ in the following examples). After 48 hours of treatment, cells were lysed, and luciferase activity was measured using a luciferase reporter assay kit (Promega, Cat. No. E6110). Increased luciferase activity indicates enhanced transcriptional activity, while decreased activity indicates weakened activity.

[0038] HepG2 cells were seeded into 12-well plates. After 12 hours, FPZ (prepared with PBS) at final concentrations of 0.1 μmol / L, 1 μmol / L, and 10 μmol / L was added to treat the cells. After 48 hours of treatment, the cells were collected, and the expression levels of KLF14 and IRP2 in the cells were detected by qRT-PCR (Novizan, Cat. No. Q711-02) and Western blotting.

[0039] like Figure 1 The results show the detection results of fluphenazine on the transcriptional activity of KLF14 and IRP2. Figure 1 A represents the results of KLF14 transcriptional activity assay after HepG2 cells were treated with 10 μmol / L FPZ for 48 hours. Figure 1B represents the results of IRP2 transcriptional activity assay after treating HepG2 cells with 10 μmol / L FPZ for 48 hours. The results showed that after 48 hours of treatment with 10 μmol / L FPZ, KLF14 transcriptional activity increased more than twofold, while IRP2 transcriptional activity significantly decreased. Data are from at least three independent replicates; *P < 0.05, **P < 0.01, ***P < 0.001. Values ​​are presented as mean ± SD.

[0040] like Figure 2 The results show the detection of KLF14 and IRP2 expression levels in liver cancer cells treated with fluphenazine. Figure 2 Figure A shows the qRT-PCR detection results of KLF14 and IRP2; Figure 2 B represents the Western blot results of KLF14 and IRP2 after drug treatment; Figure 2 C represents the statistical results of Western blot analysis of KLF14 and IRP2 proteins. The results showed that treatment of hepatocellular carcinoma cells with 0.1 μmol / L and 1 μmol / L fluphenazine had no significant effect on the expression of KLF14 and IRP2, while 10 μmol / L FPZ significantly increased KLF14 expression by more than 2-fold and significantly decreased IRP2 expression. Data were obtained from at least three independent replicates; *P < 0.05, **P < 0.01, ***P < 0.001. Values ​​are presented as mean ± SD.

[0041] The results showed that 10 μmol / L FPZ could activate KLF14 in liver cancer cells and inhibit the expression of IRP2.

[0042] Example 2

[0043] Fluphenazine induces iron deficiency in liver cancer cells.

[0044] Intracellular iron levels were detected using Calcein-AM, a probe that exhibits green fluorescence upon entering the cell and has no significant impact on cell viability. It binds to intracellular iron ions to form a complex, quenching the fluorescence, and is minimally affected by other metal ions. Based on this mechanism, stronger green fluorescence indicates lower intracellular iron concentration, and vice versa. HepG2 and Huh7 cells were digested and counted at 3 × 10⁻⁶ cells per well. 5Cells were seeded at a density of [number] cells per well in 12-well plates and cultured in fresh complete culture medium (DMEM + 10% FBS, m / v). After 12 hours, cells were treated with FPZ (prepared with PBS) at final concentrations of 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, respectively, while the control group was treated with an equal volume of PBS. After 36 hours of treatment, the cell culture medium was removed, and the cells were washed twice with 1×PBS. Cells were digested with trypsin, collected, washed once with 1×PBS, and counted in each group. Calcein-AM working solution (Yeasen, Cat. No. 40719ES50) at a final concentration of 100 nmol / L was prepared using PBS. An equal number of cells were mixed with the working solution and incubated at 37°C for 30 min. Cells were collected, resuspended in 1×PBS, and placed in 96-well plates for fluorescence detection using a microplate reader (λex 488 nm, λem 518 nm).

[0045] like Figure 3 The image shows the fluorescence intensity results of intracellular iron ion levels after 36 hours of treatment with different concentrations of FPZ on liver cancer cells. Figure 3 A shows the fluorescence intensity results of intracellular iron ion levels in HepG2 cells treated with different concentrations of FPZ. Figure 3 B shows the fluorescence intensity results of Huh7 cells treated with different concentrations of FPZ, illustrating the intracellular iron ion levels. The results showed that after 36 hours of treatment with 0.1 μmol / L and 1 μmol / L FPZ, the fluorescence intensity of HepG2 and Huh7 cells did not change significantly compared to the untreated group, indicating no significant change in intracellular iron ion levels. However, after 36 hours of treatment with 10 μmol / L FPZ, the fluorescence intensity of HepG2 and Huh7 cells significantly increased, indicating a significant decrease in intracellular iron ion levels. These results indicate that 10 μmol / L FPZ can significantly reduce iron ion levels in liver cancer cells, inducing cellular iron deficiency. Data are from at least three independent replicates; *P < 0.05, **P < 0.01, ***P < 0.001. Values ​​are presented as mean ± SD.

[0046] like Figure 4 The image shows a fluorescence imaging pattern and statistical diagram of intracellular iron ion levels after 24 hours of treatment with 10 μmol / L FPZ on liver cancer cells. Figure 4 A represents the fluorescence image. The results show that after FPZ treatment of HepG2 and Huh7 cells for 24 hours, the intracellular fluorescence intensity was significantly enhanced. Figure 4B represents the statistical graph. The results showed that after 24 hours of treatment with 10 μmol / L FPZ in HepG2 cells, the intracellular iron level was significantly lower than that in the control group; similarly, after 24 hours of treatment with 10 μmol / L FPZ in Huh7 cells, the intracellular iron level was significantly lower than that in the control group. Data were obtained from at least three independent replicates; *P < 0.05, **P < 0.01, ***P < 0.001. Values ​​are presented as mean ± SD.

[0047] The above results indicate that 10 μmol / L FPZ can significantly reduce the iron ion level in liver cancer cells and induce cellular iron deficiency.

[0048] Example 3

[0049] Fluphenazine inhibits liver cancer.

[0050] HepG2 and Huh7 cells were digested and counted at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 12-well plates and cultured in fresh complete culture medium (DMEM + 10% FBS, m / v). After 12 hours, cells were treated with FPZ at a final concentration of 10 μmol / L, while the control group was treated with an equal volume of PBS. At designated time points (24, 48, 72, and 96 hours) after cell seeding, the cell culture medium was removed, cells were washed once with PBS, digested with 25% trypsin (containing EDTA), collected by centrifugation, and viable cell counts were recorded using a hemocytometer. Finally, a cell growth curve was plotted based on the number of viable cells at each time point.

[0051] like Figure 5 The image shows the cell growth curve of liver cancer cells treated with 10 μmol / L FPZ. Figure 5 A is the cell growth curve of HepG2 cells after treatment with 10 μmol / L FPZ. Figure 5 B shows the cell growth curve of Huh7 cells after treatment with 10 μmol / L FPZ. The results show that 10 μmol / L FPZ significantly inhibited the growth of liver cancer cells. These results indicate that 10 μmol / L FPZ can significantly inhibit the growth of liver cancer cells in vitro. Data are from at least three independent replicates; *P < 0.05, **P < 0.01, ***P < 0.001. Values ​​are presented as mean ± SD.

[0052] The above results indicate that fluphenazine can induce iron deficiency in liver cancer cells and inhibit their growth.

[0053] Example 4

[0054] The combined use of fluphenazine and deferoxamine (DFO) enhances the inhibitory effect on liver cancer.

[0055] HepG2 and Huh7 cells were digested and counted at 1 × 10⁻⁶ cells per well. 5 Cells were seeded at a density of [number] cells per well in 12-well plates and cultured in fresh complete culture medium (DMEM + 10% FBS, m / v). After 12 hours, cells were treated with 10 μmol / L FPZ (PBS preparation) and 100 μmol / L DFO (PBS / 10% DMSO preparation, v / v) (MCE, Cat.no.HY-B0988) to a final concentration. The control group was treated with an equal volume of PBS / 10% DMSO. At designated time points (24, 48, 72, 96 hours) after cell seeding, the cell culture medium was removed, cells were washed once with PBS, digested with 25% trypsin (containing EDTA, m / v), collected by centrifugation, and viable cell counts were recorded using a hemocytometer. Finally, a cell growth curve was plotted based on the number of viable cells at each time point.

[0056] Construction and drug treatment of a subcutaneous tumor model in nude mice. 4-6 week old BALB / c nude mice (Jiangsu Jicui Yaokang Biotechnology Co., Ltd.) were purchased and housed in a specific pathogen-free (SPF-grade) laboratory animal facility. The nude mice were randomly divided into groups of 6 mice each (n=6). Cells were digested, resuspended in pre-chilled 1×PBS, and placed on ice. Equal volumes of HepG2 cells (4×10⁻⁶) were then added to the subcutaneous tumor model. 6 The subcutaneous tumor was injected subcutaneously into the lower right groin area of ​​mice. Seven days later, the volume of the subcutaneous tumor was measured and calculated using calipers. The volume of the subcutaneous tumor was measured every three days. The formula for calculating the subcutaneous tumor volume is: Volume = Length (mm) × Width (mm). 2 / 2. Subcutaneous tumor volume reaches 100mm 3 In this study, FPZ (8 mg / kg) and DFO (50 mg / kg) were injected intraperitoneally into nude mice, while control mice were injected intraperitoneally with saline. Three weeks after administration, the mice were sacrificed, and tumors were surgically removed, weighed, and used for subsequent experiments. The extracted tumor tissue was divided into three parts: one part was used to extract RNA for qRT-PCR to detect the mRNA expression levels of KLF14 and IRP2 in the tumor tissue; another part was used to extract tissue proteins for immunoprecipitation to detect the protein expression levels of KLF14 and IRP2 in the tumor tissue; and the third part was fixed with 4% paraformaldehyde (m / v) for subsequent immunohistochemical experiments to detect the expression levels of growth markers Ki67 and IRP2 and intracellular iron ion concentration (Perl'Blue staining) in the tumor tissue. The animal experiments were approved by the Animal Ethics Committee of the Experimental Animal Center of Zhejiang University.

[0057] like Figure 6 The figure shows the growth curves of liver cancer cells after treatment with DFO and FPZ. Figure 6 A represents the cell growth curves of HepG2 cells after treatment with DFO and FPZ. Figure 6 B shows the cell growth curves of Huh7 cells after treatment with DFO and FPZ. The results showed that DFO treatment alone significantly inhibited the growth of hepatocellular carcinoma cells, while the combined action of DFO and FPZ further inhibited their growth. This indicates that the combined action of fluphenazine and deferoxamine in vitro can further inhibit the growth of hepatocellular carcinoma cells, exhibiting a stronger inhibitory effect compared to DFO alone. Data are from at least three independent replicates; *P < 0.05, **P < 0.01, ***P < 0.001. Values ​​are presented as mean ± SD.

[0058] like Figure 7 The results of FPZ and DFO in detecting subcutaneous tumorigenesis of liver cancer cells are shown. Figure 7 A represents the volume growth curve of the subcutaneous tumor. Figure 7 B is a comparison image of the tumor tissues removed from each group. Figure 7 C represents the weight of the tumor tissue after removal. Results showed that, compared with the saline injection group, injection of FPZ or DFO alone significantly inhibited the growth of liver cancer cells in mice, while combined injection of FPZ and DFO had a stronger inhibitory effect on liver cancer. n=5, *P<0.05, **P<0.01, ***P<0.001. Values ​​are presented as mean ± SD.

[0059] Next, we used qRT-PCR and Western blotting to detect the expression levels of KLF14 and IRP2 in subcutaneous tumor tissues. In the FPZ group, the mRNA and protein levels of KLF14 were significantly increased, while the mRNA and protein levels of IRP2 were significantly decreased, a result consistent with that of FPZ-treated hepatocellular carcinoma cells. Figure 8 A and Figure 8 B). Simultaneously, immunoblotting results also showed that the protein expression level of KLF14 was significantly increased in tumor tissues of both the FPZ group and the DFO / FPZ combination therapy group. Figure 8 C). These results indicate that FPZ can significantly activate KLF14 and reduce IRP2 expression. Immunoblotting results from four groups of tumor tissues showed that IRP2 expression was significantly higher in the DFO monotherapy group than in the control group, and IRP2 expression was significantly lower in the FPZ and DFO combination therapy group than in the DFO monotherapy group. Figure 8 D). These results indicate that DFO alone significantly increases IRP2 expression, while the combination of FPZ and DFO significantly inhibits the DFO-induced IRP2 activation effect.

[0060] like Figure 9 The image shows the results and statistical chart of immunohistochemistry of subcutaneous tumor tissue. Figure 9 A shows the staining results of Ki67 and Perl'Blue in tumor tissue (magnification: 400×). Figure 9 B represents the statistical results of immunohistochemistry. The results showed that, compared with the control group, Ki67 expression was significantly downregulated in tumor tissues treated with FPZ or DFO alone, with no significant difference between the two groups treated alone. In tumor tissues treated with the combined administration of the two drugs, Ki67 expression was significantly downregulated compared to the control group and also significantly downregulated compared to the single-drug groups, indicating that the combined administration of FPZ and DFO can enhance the inhibitory effect on liver cancer. Perl'Blue staining results showed that the proportion of Perl'Blue-positive cells in tumor tissues treated with FPZ or DFO alone was significantly lower than that in the control group, with no significant difference between the two groups treated alone. In tumor tissues treated with the combined administration of the two drugs, the proportion of Perl'Blue-positive cells was significantly lower than that in the control group and lower than that in the single-drug groups, indicating that FPZ or DFO alone can reduce intracellular iron ion levels, while the combined use of the two drugs can further induce cellular iron deficiency. n=5, *P<0.05, **P<0.01, ***P<0.001. The values ​​are presented as mean ± SD.

[0061] like Figure 10 This diagram illustrates the mechanism by which fluphenazine and deferoxamine act on hepatocellular carcinoma (drawn using the BioRender online website). Fluphenazine activates KLF14, reducing IRP2 expression and thus lowering the iron pool level in hepatocellular carcinoma cells, thereby inhibiting hepatocellular carcinoma. Deferoxamine inhibits hepatocellular carcinoma by chelating intracellular iron, reducing the iron pool level in hepatocellular carcinoma cells, but it also feedback-increases IRP2 expression, activating the IRPs-IRE system. The combined use of fluphenazine and deferoxamine significantly inhibits this feedback effect, further enhancing the inhibitory effect on hepatocellular carcinoma.

[0062] The above results collectively indicate that the combined effect of fluphenazine and deferoxamine can significantly inhibit liver cancer. sequence list <110> Zhejiang University <120> Application of fluphenazine in the preparation of drugs for treating cancers with iron overload <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 20 <212> DNA <213> Artificial Sequence <400> 1 ccttcaattt tatcagaaat 20 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <400> 2 cgacatgctg ggaccgcccg 20 <210> 3 <211> twenty one <212> DNA <213> Artificial Sequence <400> 3 agcctgagat caagcaccct t 21 <210> 4 <211> 18 <212> DNA <213> Artificial Sequence <400> 4 tgcttttggg gcgtccat 18

Claims

1. The use of fluphenazine as the sole active ingredient in the preparation of a medicament for the treatment of liver cancer, wherein the fluphenazine is fluphenazine itself or a pharmaceutically acceptable salt thereof.

2. The use of fluphenazine and deferoxamine in combination in the preparation of a medicament for the treatment of liver cancer, wherein the fluphenazine is fluphenazine itself or a pharmaceutically acceptable salt, and the deferoxamine is deferoxamine itself or a pharmaceutically acceptable salt.

3. The application as described in claim 2, characterized in that, The dosage form of the drug is liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, ointment, granule or dressing.

4. The application as described in claim 3, characterized in that, When the drug is in the form of a liquid injection, the dose of fluphenazine is 8 mg / kg and the dose of deferoxamine is 50 mg / kg.

5. A pharmaceutical composition, characterized in that, This includes fluphenazine or pharmaceutically acceptable salts, as well as deferoxamine or pharmaceutically acceptable salts.

6. The drug as described in claim 5, characterized in that, When the dosage form of the drug is a liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, ointment, granule or dressing.

7. The drug as described in claim 6, characterized in that, When the drug is in the form of a liquid injection, the dose of fluphenazine is 8 mg / kg and the dose of deferoxamine is 50 mg / kg.

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