CN117257811A - Use of fluphenazine in the preparation of a drug for the treatment of cancer associated with iron overload - Google Patents
Use of fluphenazine in the preparation of a drug for the treatment of cancer associated with iron overload Download PDFInfo
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Abstract
The invention discloses an application of fluphenazine in preparing medicaments for treating cancers accompanied with iron overload, and relates to the technical field of biological medicaments. Compared with the injection of normal saline, the application provided by the invention has the advantages that the single injection of FPZ or DFO can obviously inhibit the growth of liver cancer cells in mice, and the combined injection of FPZ and DFO has stronger inhibition effect on liver cancer; the FPZ and DFO combined drug can obviously inhibit the IRP2 activation effect caused by DFO; FPZ can activate KLF14 to reduce the expression of IRP2 so as to reduce the level of an iron pool of liver cancer cells and inhibit liver cancer; DFO reduces the level of an iron pool in a liver cancer cell to inhibit liver cancer by chelating iron in the cell, but can improve the expression level of IRP2 in a feedback way to activate an IRPs-IRE system, and the combined use of FPZ and DFO can obviously inhibit the feedback effect and further enhance the liver cancer inhibition effect.
Description
Technical Field
The invention relates to the technical field of biological medicine, in particular to application of fluphenazine in preparing medicaments for treating cancers accompanied by iron overload.
Background
The liver cancer lacks an early effective diagnosis marker, and the recurrence rate and death rate are high. Currently, surgical excision or liver transplantation is the most effective method for treating liver cancer, but most liver cancer patients are diagnosed at an advanced stage, and are not suitable for surgical treatment, so that more new therapeutic drugs or therapeutic strategies are found to have very important clinical value. Liver cancer mainly originates in liver parenchymal cells, i.e., hepatocytes, and usually develops from chronic liver diseases, accompanied by chronic inflammation and fibrosis, develops into cirrhosis, and finally is converted into liver cancer. High risk factors for inducing liver cancer include hepatitis B/C virus, fatty liver disease, alcoholic liver cirrhosis, etc.
Iron overload is also a risk factor for the induction of liver cancer. In 1996, researchers found spontaneous hepatocellular carcinoma in iron overload mouse model, indicating that iron overload is related to the occurrence of liver cancer. Epidemiological studies have found that three of patients with long-term cirrhosis develop liver high iron load, which "liver cirrhosis-associated liver iron overload" can induce liver cancer. Follow-up studies on patients with alcoholic cirrhosis or chronic hepatitis b have found that patients with higher iron reserves in the liver are at a higher risk of developing liver cancer than patients with lower or normal iron reserves. Extensive studies have reported that the iron content in liver tissue of patients with chronic hepatitis c is 2-5 times that in normal liver tissue, and that severe iron overload in the liver of patients with hereditary hemochromatosis can lead to cirrhosis, with nearly 20 times increased risk of liver cancer in patients with cirrhosis.
Intracellular iron overload has been reported to be associated with the development of a variety of tumors, including liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer, glioma, and the like. The medicine can inhibit the occurrence and development of tumor related to iron overload by inducing cell iron deficiency. Among them, the use of iron chelators is one of the commonly used methods for inducing iron deficiency in cells. Deferoxamine (DFO, CAS accession No. 70-51-9) can sequester intracellular iron ions to induce iron deficiency in cells and thereby inhibit the growth of tumor cells, including iron overload-related tumor cells such as liver cancer cells, breast cancer cells, colorectal cancer cells, ovarian cancer cells, etc., in vitro and in vivo. Thiosemicarbazone-24 (TSC 24) can remarkably reduce iron ion concentration in tumor cells to induce iron deficiency of the cells so as to reduce the activity of the tumor cells and inhibit the growth of the tumor cells by blocking iron uptake and interfering with normal regulation of cell iron steady state. However, the iron chelator acts on cells to feedback increase the expression of iron regulatory protein 2 (IRP 2) and thereby activate the mechanism of iron regulatory protein-iron response elements (IRP-IRES) that regulate cellular iron homeostasis, reducing the effect.
Fluphenazine (FPZ, CAS accession number 69-23-8) is a phenothiazine with antipsychotic properties for the treatment of patients with schizophrenia and bipolar disorder, approved for clinical use in 1972. Fluchenazine acts primarily by antagonizing dopamine receptors (DA 2) in the limbic, striatal and funneling pathways of the tuberosity, inhibiting dopamine receptors following synapses in the limbic pathway may positively affect symptoms associated with schizophrenia. In 1985, hait et al found for the first time that FPZ was able to inhibit proliferation of L1210 leukemia cells. This study results opened the door to the study of FPZ in terms of tumors. Subsequently, more and more studies have shown that FPZ has anti-tumor properties against breast cancer, colorectal cancer, human neuroblastoma and glioma. Wherein, FPZ can inhibit the growth of tumor cells by inhibiting MAPK signaling pathway in breast cancer cells, reducing mitochondrial function of human glioblastoma cells, and reducing Cyclooxygenase (COX) -2 activity and expression in colorectal cancer cells. The mechanism by which fluphenazine affects the growth of liver cancer cells has not yet been elucidated, and the relationship between fluphenazine and intracellular iron ion level regulation has not yet been found and reported, and the effective application of fluphenazine in liver cancer treatment has yet to be developed.
Disclosure of Invention
Based on the deficiencies of the prior art, the present invention provides the use of fluphenazine for the manufacture of a medicament for the treatment of cancer associated with iron overload.
The technical scheme of the invention is as follows:
the present invention provides the use of fluphenazine, either as fluphenazine itself or as a pharmaceutically acceptable salt, in the manufacture of a medicament for the treatment of cancer associated with iron overload.
Preferably, the cancer is liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer or glioma.
The invention also provides the use of fluphenazine, either as fluphenazine itself or a pharmaceutically acceptable salt, in combination with deferoxamine, either deferoxamine itself or a pharmaceutically acceptable salt, for the manufacture of a medicament for the treatment of cancer associated with iron overload.
Preferably, the cancer is liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer or glioma.
Preferably, the dosage form of the medicine is liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, paste, granule or dressing. In this application, the liquid injection is injected into the body of a nude mouse by intraperitoneal injection when combined.
When the dosage form of the drug is a liquid injection, the dosage of fluphenazine injection is 8mg/kg, and the dosage of deferoxamine injection is 50mg/kg.
Compared with the normal saline injection group, the FPZ or the DFO alone can remarkably inhibit the growth of liver cancer cells in mice, and the combined injection of the FPZ and the DFO has stronger inhibition effect on liver cancer.
The invention also provides a pharmaceutical composition comprising fluphenazine or a pharmaceutically acceptable salt, and deferoxamine or a pharmaceutically acceptable salt
The medicine is in the form of liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, paste, granule or dressing. In this application, the liquid injection is injected into the body of a nude mouse by intraperitoneal injection when combined.
When the dosage form of the drug is a liquid injection, the dosage of fluphenazine injection is 8mg/kg, and the dosage of deferoxamine injection is 50mg/kg.
The expression level of IRP2 in the DFO independent administration group is obviously higher than that in the control group, and the expression level of IRP2 in the FPZ and DFO combined administration group is obviously lower than that in the DFO independent administration group; DFO alone can result in a significant increase in IRP2 expression, whereas FPZ in combination with DFO can significantly inhibit the IRP2 activation effect induced by DFO. Indicating that the two have a synergistic effect.
The invention has the beneficial effects that:
compared with the injection of normal saline, the application provided by the invention has the advantages that the single injection of FPZ or DFO can obviously inhibit the growth of liver cancer cells in mice, and the combined injection of FPZ and DFO has stronger inhibition effect on liver cancer; the FPZ and DFO combined drug can obviously inhibit the IRP2 activation effect caused by DFO; FPZ can activate KLF14 to reduce the expression of IRP2 so as to reduce the level of an iron pool of liver cancer cells and inhibit liver cancer; DFO reduces the level of an iron pool in a liver cancer cell to inhibit liver cancer by chelating iron in the cell, but can improve the expression level of IRP2 in a feedback way to activate an IRPs-IRE system, and the combined use of FPZ and DFO can obviously inhibit the feedback effect and further enhance the liver cancer inhibition effect.
Drawings
FIG. 1 is a graph showing the results of the assay of transcriptional activity of fluphenazine on KLF14 and IRP 2; wherein A: FPZ treatment of HepG with 10. Mu. Mol/L 2 A graph of the results of the detection of KLF14 transcriptional activity after 48 hours of cells; b: FPZ treatment of HepG with 10. Mu. Mol/L 2 After 48 hours of cells, IRP2 transcriptional activity was detected; * P < 0.01.
FIG. 2 is a graph showing the results of detection of expression levels of KLF14 and IRP2 in fluphenazine treated hepatoma cells; wherein A: qRT-PCR detection result diagram of medicine-treated liver cancer cells, KLF14 and IRP 2; b: drug treatment HepG 2 Western blot results and statistical plots of cells, KLF14 and IRP 2; c: western blot results and statistical plots of drug-treated Huh7 cells, KLF14 and IRP 2; * P < 0.05, P < 0.01, P < 0.001.
FIG. 3 is a graph showing the results of fluorescence intensity detection of intracellular iron ion levels after 36 hours of FPZ treatment of hepatoma cells; wherein A: FPZ treatment HepG 2 Cells, a graph showing the results of fluorescence intensity detection of intracellular iron ion levels; b: FPZ treatment of Huh7 cells showed a plot of fluorescence intensity measurements of intracellular iron levels; * P < 0.01, n.s represents no statistical significance.
FIG. 4 is a fluorescence imaging and statistical graph showing intracellular iron ion levels 24 hours after treatment of liver cancer cells with 10. Mu. Mol/L FPZ; wherein A: a fluorescence imaging map; b: a statistical chart; * P < 0.01.
FIG. 5 is a graph showing cell growth curve of 10. Mu. Mol/L FPZ-treated hepatoma cells; wherein A: FPZ treatment of HepG with 10. Mu. Mol/L 2 Graph of cell growth after cells; b: cell growth after 10. Mu. Mol/L FPZ treatment of Huh7 cellsA long graph; * P < 0.001.
FIG. 6 is a graph showing the growth of hepatoma cells after DFO and FPZ treatments; wherein A: DFO and FPZ treatment HepG 2 Graph of cell growth after cells; b: graph of cell growth after DFO and FPZ treatment of Huh7 cells; * P < 0.01, P < 0.001.
FIG. 7 is a graph showing the results of FPZ and DFO effects on subcutaneous tumor formation in hepatoma cells; wherein A: volume increase profile of subcutaneous tumor; b: schematic representation of each group of tumor tissue after removal; c: a weight detection result graph after tumor tissue is taken out; * P < 0.05, P < 0.01, P < 0.001.
FIG. 8 is a graph showing qRT-PCR and Western blotting detection results of KLF14 and IRP2 in subcutaneous tumor tissues; wherein A: qRT-PCR detection result graphs of KLF14 and IRP2 in tumor tissues of FPZ single administration group; b: western blot detection result graphs of KLF14 and IRP2 in tumor tissues of FPZ alone administration group; c: immunoblotting detection result diagram of KLF14 in tumor tissues of FPZ single administration group and DFO and FPZ combined administration group; d: a immunoblotting detection result graph of IRP2 in tumor tissues of a control group, a FPZ single administration group, a DFO single administration group and a DFO and FPZ combined administration group; * P < 0.05, P < 0.001.
FIG. 9 is a graph showing the results and statistics of immunohistochemistry of subcutaneous tumor tissue; wherein A: immunohistochemical staining of Ki67 and Perl' Blue in tumor tissue; b: a statistical result diagram of immunohistochemistry; * P < 0.01, P < 0.001.
Fig. 10 is a graph showing the mechanism of action of fluphenazine and deferoxamine administration on liver cancer.
Detailed Description
Fluphenazine hydrochloride: the source is the national standard substance center, purchased from the biological assay detection limited company, henan, inc., cat.No.100162.
Example 1
Fluphenazine activates KLF14 inhibiting IRP2 expression.
And (3) detecting the influence of FPZ on the transcription activities of KLF14 and IRP2 in liver cancer cells by using a luciferase reporter gene experiment. Construction of luciferase reporter plasmids PGL4-KLF14-1uc and PGL4-IRP2-1uc A1 kb fragment upstream/downstream of the KLF14/IRP2 transcription initiation site (-1000 bp to +1000 bp) was amplified from genomic DNA, and the amplified fragments were ligated into pGL 4-luciferase reporter vector (Promega Co., cat. No. E4611) and the plasmid construction primers were as shown in Table 1 below.
TABLE 1 primers for construction of luciferase reporter plasmids
HepG 2 Cells were inoculated into 12 wells and simultaneously transfected into HepG using Lipofectamine2000 (Life Technologies) at 60% -70% cell density 2 In cells. Cells were treated with fluphenazine hydrochloride (hydrochloride was purchased in the examples, but the content of the following examples was abbreviated as FPZ) at a final concentration of 10. Mu. Mol/L12 hours after transfection, and after drug treatment for 48 hours, the cells were lysed and luciferase activity was measured using a luciferase reporter assay kit (Promega Corp., cat. No. E6110). An increase in luciferase activity represents an increase in transcriptional activity and vice versa represents a decrease.
HepG 2 Cells were seeded in 12-well plates, treated with FPZ (PBS configuration) at final concentrations of 0.1. Mu. Mol/L, 1. Mu. Mol/L, and 10. Mu. Mol/L for 12 hours after plating, and cells were collected after drug treatment for 48 hours and examined for the expression levels of KLF14 and IRP2 in the cells by qRT-PCR (Norpran Corp., cat. No. Q711-02) and Western blotting experiments.
FIG. 1 shows the results of the assay of transcriptional activity of fluphenazine on KLF14 and IRP 2. FIG. 1A is a 10. Mu. Mol/L FPZ treated HepG 2 After 48 hours of cells, KLF14 transcriptional activity was measured; FIG. 1B is a 10. Mu. Mol/L FPZ treated HepG 2 After 48 hours of cells, IRP2 transcriptional activity was measured. The results showed that 10. Mu. Mol/L FPZ treated HepG 2 After 48 hours of cells, the transcription activity of KLF14 is improved by more than 2 times, and the transcription activity of IRP2 is obviously reduced. Data from at least three independent replicates, P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
FIG. 2 shows the results of detection of the expression levels of KLF14 and IRP2 in fluphenazine treated hepatoma cells. FIG. 2A is a graph of qRT-PCR detection results of KLF14 and IRP 2; FIG. 2B shows the results of western blotting of KLF14 and IRP2 after drug treatment; FIG. 2C shows the results of KLF14 and IRP2 western blotting. The results show that 0.1 mu mol/L and 1 mu mol/L of fluphenazine treated liver cancer cells have no obvious effect on the expression of KLF14 and IRP2, and 10 mu mol/L of FPZ can obviously improve the expression quantity of KLF14 by more than 2 times and obviously reduce the expression quantity of IRP 2. Data from at least three independent replicates P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
The result shows that the FPZ with the concentration of 10 mu mol/L can activate KLF14 of liver cancer cells and inhibit the expression of IRP 2.
Example 2
Fluphenazine induces iron deficiency in hepatoma cells.
The intracellular iron pool level is detected by using Calcein-AM, and the probe shows green fluorescence after entering cells, and has no obvious influence on the activity of the cells. It can combine with intracellular iron ions to form a complex, such that its fluorescence is quenched and other metal ions interfere less with it. Based on the principle of action, the stronger the intracellular green fluorescence intensity, the lower the intracellular iron ion concentration, and conversely, the weaker the intracellular green fluorescence intensity, the higher the intracellular iron ion concentration. HepG 2 Digestion counts with Huh7 cells at 3X 10 per well 5 The density of individual cells was inoculated into 12-well plates, and cell culture was performed by adding fresh complete medium (DMEM+10% FBS, m/v). After 12 hours, FPZ (PBS-formulated) treated cells were added to the medium at final concentrations of 0.1. Mu. Mol/L, 1. Mu. Mol/L, and 10. Mu. Mol/L, respectively, and the control group was added with an equal volume of PBS treated cells. After 36 hours of treatment, the cell culture medium was removed and the cells were washed twice with 1×pbs. Cells were trypsinized, collected, washed once with 1 XPBS, and counted for each group. The final 100nmol/L Calcein-AM working solution (Yeasen, cat. No.40719 ES50) was prepared using PBS, and the same number of cells and workers were takenThe culture solution is evenly mixed and placed in a cell incubator at 37 ℃ for incubation for 30min. Cells were collected, resuspended in 1 XPBS, and the cells were placed in 96-well plates and assayed for fluorescence using a microplate reader (λex 488nm, λem 518 nm).
FIG. 3 shows fluorescence intensity detection results of iron ion levels in liver cancer cells treated with FPZ at different concentrations for 36 hours. FIG. 3A shows different concentrations of FPZ treated HepG 2 Cells, which display the fluorescence intensity detection results of intracellular iron ion levels; FIG. 3B shows fluorescence intensity measurements of intracellular iron ion levels for different concentrations of FPZ treated Huh7 cells. The results showed that O.1. Mu. Mol/L and 1. Mu. Mol/L FPZ treated HepG 2 After 36 hours from Huh7 cells, there was no significant change in fluorescence intensity compared to untreated group cells, indicating no significant change in intracellular iron ion levels. Whereas 10. Mu. Mol/L FPZ treated HepG 2 After 36 hours from Huh7 cells, the fluorescence intensity of the cells was significantly increased, indicating a significant decrease in intracellular iron ion levels. The results show that the FPZ with the concentration of 10 mu mol/L can obviously reduce the level of iron ions in liver cancer cells and induce the iron deficiency of the cells. Data from at least three independent replicates P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
FIG. 4 shows a fluorescence imaging and statistical graph of intracellular iron ion levels 24 hours after treatment of liver cancer cells with 10. Mu. Mol/L FPZ. Fig. 4A is a fluorescence diagram. The results showed that FPZ treated HepG 2 After 24 hours with Huh7 cells, the fluorescence intensity in the cells was significantly enhanced. Fig. 4B is a statistical diagram. The results showed that 10. Mu. Mol/L FPZ treated HepG 2 After 24 hours of cells, intracellular levels of iron ions were significantly reduced compared to control cells; after 24 hours of treatment of Huh7 cells with 10. Mu. Mol/L FPZ, the intracellular iron ion levels were significantly reduced compared to control cells. Data from at least three independent replicates P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
The results show that the FPZ with the concentration of 10 mu mol/L can obviously reduce the level of iron ions in liver cancer cells and induce the iron deficiency of the cells.
Example 3
Fluphenazine inhibits liver cancer.
HepG 2 Digestion counts with Huh7 cells at 1X 10 per well 5 The density of individual cells was inoculated into 12-well plates, and cell culture was performed by adding fresh complete medium (DMEM+10% FBS, m/v). After 12 hours, FPZ-treated cells were added to the medium at a final concentration of 10. Mu. Mol/L, respectively, and the control group was added with an equal volume of PBS-treated cells. Cell culture medium was removed at designated time points (24, 48, 72, 96 hours) after cell plating, cells were washed once with PBS, cells were digested with 25% pancreatin (EDTA-containing), cells were collected by centrifugation, viable cells were counted and recorded using a blood cell counting plate, and finally a cell growth curve was drawn according to the number of viable cells at each time point.
FIG. 5 shows a graph of cell growth curve of 10. Mu. Mol/L FPZ-treated hepatoma cells. FIG. 5A is a 10. Mu. Mol/L FPZ treated HepG 2 Graph of cell growth after cells. FIG. 5B is a graph of cell growth after 10. Mu. Mol/L FPZ treatment of Huh7 cells. The results show that 10 mu mol/L FPZ can significantly inhibit the growth of liver cancer cells. The above results indicate that 10. Mu. Mol/L FPZ can significantly inhibit the growth of hepatoma cells in vitro. Data from at least three independent replicates P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
The results show that the fluphenazine can induce iron deficiency of liver cancer cells and inhibit the growth of the liver cancer cells.
Example 4
The combination of fluphenazine and Deferoxamine (DFO) enhances liver cancer inhibition.
HepG 2 Digestion counts with Huh7 cells at 1X 10 per well 5 The density of individual cells was inoculated into 12-well plates, and cell culture was performed by adding fresh complete medium (DMEM+10% FBS, m/v). After 12 hours, the cells were treated by adding FPZ (PBS configuration) and DFO (PBS/10% DMSO configuration, v/v) (MCE Co., cat. No. HY-B0988) to the medium at a final concentration of 10. Mu. Mol/L, respectively. Control groups were treated with an equal volume of PBS/10% DMSO. Cell culture medium was removed at designated time points (24, 48, 72, 96 hours) after cell plating, cells were washed once with PBS, 25% pancreatin (EDTA-containing)M/v) digesting the cells, centrifuging to collect the cells, counting and recording the living cells by using a blood cell counting plate, and finally drawing a cell growth curve according to the number of the living cells at each time point.
Construction of nude mice subcutaneous tumor model and drug treatment. BALB/c nude mice (Jiangsu Jiugang Biotech Co., ltd.) purchased at 4-6 weeks of age were bred in laboratory animal houses free of specific pathogens (SPF grade). The nude mice were randomly grouped, with 6 nude mice per group (n=6). Cells were digested, resuspended in pre-chilled 1 XPBS, and placed on ice. Equal amount of HepG 2 Cells (4X 10) 6 ) The subcutaneous injection was performed to the subcutaneous side of the lower inguinal right of the mice. After 7 days, the volume of the subcutaneous tumor was measured and calculated using vernier calipers. The subcutaneous tumor volume of the mice was measured every 3 days and the formula for calculating the subcutaneous tumor volume: volume = length (mm) x width (mm) 2 /2. Subcutaneous tumor volume up to 100mm 3 In this case, FPZ (8 mg/kg) and DFO (50 mg/kg) were intraperitoneally injected into nude mice, and control nude mice were intraperitoneally injected with physiological saline. Mice were sacrificed 3 weeks after dosing, tumors were surgically stripped, weighed and subjected to subsequent experiments. The taken tumor tissue is divided into three parts, and one part is used for extracting RNA to carry out qRT-PCR experiments so as to detect mRNA expression levels of KLF14 and IRP2 in the tumor tissue; a portion of the protein is used for extracting tissue protein to carry out immunoprecipitation experiments so as to detect protein expression levels of KLF14 and IRP2 in tumor tissues; the third fraction was fixed with 4% paraformaldehyde (m/v) for later performed immunohistochemical experiments to detect the expression levels of the growth markers Ki67 and IRP2 and intracellular iron ion concentration in tumor tissue (Perl' Blue staining). Animal experiments were approved by the animal ethics committee of the laboratory animal center, university of Zhejiang.
FIG. 6 shows the growth curve of liver cancer cells treated with DFO and FPZ. FIG. 6A shows DFO and FPZ treated HepG 2 Cell growth curve after cells. FIG. 6B is a graph showing the cell growth curve of Huh7 cells treated with DFO and FPZ. The results show that the DFO alone treated cells can significantly inhibit the growth of liver cancer cells, and the DFO and FPZ can further inhibit the growth of liver cancer cells when acting together. The results indicate that the fluphenazine and deferoxamine can act together in vitroCan further inhibit the growth of liver cancer cells, and has stronger inhibition effect compared with the single action of DFO. Data from at least three independent replicates P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
FIG. 7 shows the results of the detection of subcutaneous tumor formation of liver cancer cells by FPZ and DFO. Fig. 7A is a volume increase curve of subcutaneous tumors. Fig. 7B is a comparison of the tumor tissue of each group after removal. Fig. 7C is the weight after tumor tissue removal. The results show that FPZ or DFO alone can significantly inhibit the growth of liver cancer cells in mice compared with normal saline injection group, and that FPZ and DFO combined injection has stronger inhibition effect on liver cancer. n=5, P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
Next, we used qRT-PCR and immunoblotting experiments to detect the expression levels of KLF14 and IRP2 in subcutaneous tumor tissues. In the tumor tissues of the FPZ group, the mRNA and protein levels of KLF14 were significantly increased, while the mRNA and protein levels of IRP2 were significantly decreased, which was consistent with the results of FPZ treatment of hepatoma cells (fig. 8A and 8B). Meanwhile, the immunoblotting experiment results also show that the protein expression level of KLF14 is obviously increased in tumor tissues of the FPZ group and the combined use of DFO and FPZ (FIG. 8C). These results indicate that FPZ is able to significantly activate KLF14, reducing IRP2 expression. Immunoblotting results of four tumor tissues showed that the expression level of IRP2 was significantly higher in the DFO alone group than in the control group, and that the expression level of IRP2 was significantly lower in the FPZ and DFO combination group than in the DFO alone group (fig. 8D). The results indicate that DFO alone can significantly increase IRP2 expression, whereas FPZ in combination with DFO can significantly inhibit the IRP2 activation effect induced by DFO.
Results and statistical graphs of immunohistochemistry of subcutaneous tumor tissue are shown in fig. 9. FIG. 9A shows the results of Ki67 and Perl' Blue staining (magnification: 400×) of tumor tissue. Fig. 9B is a statistical result of immunohistochemistry. The results showed that the expression level of Ki67 was significantly down-regulated in tumor tissues injected with FPZ or DFO alone compared to the control group, and there was no significant difference between the two groups administered alone; in tumor tissues with combined administration of the two drugs, the expression level of Ki67 is significantly reduced compared with the control group, and is significantly reduced compared with the single administration group, which indicates that the combined administration of FPZ and DFO can strengthen the inhibition effect on liver cancer. The staining results of Perl 'Blue showed that the proportion of cells positively stained by Perl' Blue was significantly reduced in tumor tissues injected with FPZ or DFO alone compared to the control group, and there was no significant difference between the two groups administered alone; the proportion of cells positively stained by Perl' Blue in tumor tissue with the combination of the two drugs showed a more significant decrease compared to the control group and was lower than that of the control group, indicating that FPZ or DFO alone could reduce intracellular iron ion levels, whereas the combination of the two drugs could further induce cellular iron deficiency. n=5, P < 0.05, P < 0.01, P < 0.001. Values are presented as mean±sd.
FIG. 10 is a graph showing the mechanism of fluphenazine and deferoxamine in liver cancer (drawn using the BioRender online website). Fluphenazine can activate KLF14 to reduce the expression of IRP2 so as to reduce the level of an iron pool of liver cancer cells and inhibit liver cancer; the deferoxamine reduces the level of an iron pool in a liver cancer cell to inhibit liver cancer by chelating iron in the cell, but can improve the expression level of IRP2 in a feedback way to activate an IRPs-IRE system, and the combination of fluphenazine and deferoxamine can obviously inhibit the feedback effect and further enhance the liver cancer inhibition effect.
The results comprehensively show that the combined action of the fluphenazine and the deferoxamine can obviously inhibit liver cancer.
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Claims (9)
1. Use of fluphenazine, either as such or as a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of cancer associated with iron overload.
2. The use of claim 1, wherein the cancer is liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer or glioma.
3. Use of fluphenazine, either as fluphenazine itself or a pharmaceutically acceptable salt, in combination with deferoxamine, either deferoxamine itself or a pharmaceutically acceptable salt, for the manufacture of a medicament for the treatment of cancer associated with iron overload.
4. The use according to claim 3, wherein the cancer is liver cancer, colorectal cancer, breast cancer, lung cancer, ovarian cancer or glioma.
5. The use according to claim 3, wherein the medicament is in the form of a liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, paste, granule or dressing.
6. The use of claim 5, wherein when said medicament is in the form of a liquid injection, the dosage of fluphenazine is 8mg/kg and the dosage of deferoxamine is 50mg/kg.
7. A pharmaceutical composition comprising fluphenazine or a pharmaceutically acceptable salt, and deferoxamine or a pharmaceutically acceptable salt.
8. The medicament of claim 7, wherein when the medicament is in the form of liquid injection, powder injection, tablet, capsule, powder, pill, oral liquid, paste, granule or dressing.
9. The medicament of claim 8, wherein when the medicament is in the form of a liquid injection, the dosage of fluphenazine is 8mg/kg and the dosage of deferoxamine is 50mg/kg.
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Cited By (1)
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| CN120789273A (en) * | 2025-07-24 | 2025-10-17 | 中山大学附属第一医院 | Application of YAP inhibitor combined with DRD2 inhibitor in preparation of breast cancer and/or liver cancer treatment drugs |
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